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Posts Tagged ‘regenerative medicine’


Fat Cells Reprogrammed to Make Insulin

Curator: Larry H. Bernstein, MD, FCAP

 

A New Use for Love Handles, Insulin-Producing Beta Cells

http://www.genengnews.com/gen-news-highlights/a-new-use-for-love-handles-insulin-producing-beta-cells/81252612/

http://www.genengnews.com/Media/images/GENHighlight/112856_web9772135189.jpg

 

Scientists at the Swiss Federal Institute of Technology (ETH) in Zurich have found an exciting new use for the cells that reside in the undesirable flabby tissue—creating pancreatic beta cells. The ETH researchers extracted stem cells from a 50-year-old test subject’s fatty tissue and reprogrammed them into mature, insulin-producing beta cells.

The findings from this study were published recently in Nature Communications in an article entitled “A Programmable Synthetic Lineage-Control Network That Differentiates Human IPSCs into Glucose-Sensitive Insulin-Secreting Beta-Like Cells.”

The investigators added a highly complex synthetic network of genes to the stem cells to recreate precisely the key growth factors involved in this maturation process. Central to the process were the growth factors Ngn3, Pdx1, and MafA; the researchers found that concentrations of these factors change during the differentiation process.

For instance, MafA is not present at the start of maturation. Only on day 4, in the final maturation step, does it appear, its concentration rising steeply and then remaining at a high level. The changes in the concentrations of Ngn3 and Pdx1, however, are very complex: while the concentration of Ngn3 rises and then falls again, the level of Pdx1 rises at the beginning and toward the end of maturation.

Senior study author Martin Fussenegger, Ph.D., professor of biotechnology and bioengineering at ETH Zurich’s department of biosystems science and engineering stressed that it was essential to reproduce these natural processes as closely as possible to produce functioning beta cells, stating that “the timing and the quantities of these growth factors are extremely important.”

The ETH researchers believe that their work is a real breakthrough, in that a synthetic gene network has been used successfully to achieve genetic reprogramming that delivers beta cells. Until now, scientists have controlled such stem cell differentiation processes by adding various chemicals and proteins exogenously.

“It’s not only really hard to add just the right quantities of these components at just the right time, but it’s also inefficient and impossible to scale up,” Dr. Fussenegger noted.

While the beta cells not only looked very similar to their natural counterparts—containing dark spots known as granules that store insulin—the artificial beta cells also functioned in a very similar manner. However, the researchers admit that more work needs to be done to increase the insulin output.

“At the present time, the quantities of insulin they secrete are not as great as with natural beta cells,” Dr. Fussenegger stated. Yet, the key point is that the researchers have for the first time succeeded in reproducing the entire natural process chain, from stem cell to differentiated beta cell.

In future, the ETH scientists’ novel technique might make it possible to implant new functional beta cells in diabetes sufferers that are made from their adipose tissue. While beta cells have been transplanted in the past, this has always required subsequent suppression of the recipient’s immune system—as with any transplant of donor organs or tissue.

“With our beta cells, there would likely be no need for this action since we can make them using endogenous cell material taken from the patient’s own body,” Dr. Fussenegger said. “This is why our work is of such interest in the treatment of diabetes.”

A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

Pratik SaxenaBoon Chin HengPeng BaiMarc FolcherHenryk Zulewski & Martin Fussenegger
Nature Communications7,Article number:11247
         doi:10.1038/ncomms11247

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid, we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells, whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine.

Cell-fate decisions during development are regulated by various mechanisms, including morphogen gradients, regulated activation and silencing of key transcription factors, microRNAs, epigenetic modification and lateral inhibition. The latter implies that the decision of one cell to adopt a specific phenotype is associated with the inhibition of neighbouring cells to enter the same developmental path. In mammals, insights into the role of key transcription factors that control development of highly specialized organs like the pancreas were derived from experiments in mice, especially various genetically modified animals1, 2, 3, 4. Normal development of the pancreas requires the activation of pancreatic duodenal homeobox protein (Pdx1) in pre-patterned cells of the endoderm. Inactivating mutations of Pdx1 are associated with pancreas agenesis in mouse and humans5, 6. A similar cell fate decision occurs later with the activation of Ngn3 that is required for the development of all endocrine cells in the pancreas7. Absence of Ngn3 is associated with the loss of pancreatic endocrine cells, whereas the activation of Ngn3 not only allows the differentiation of endocrine cells but also induces lateral inhibition of neighbouring cells—via Delta-Notch pathway—to enter the same pancreatic endocrine cell fate8. This Ngn3-mediated cell-switch occurs at a specific time point and for a short period of time in mice9. Thereafter, it is silenced and becomes almost undetectable in postnatal pancreatic islets. Conversely, Pdx1-positive Ngn3-positive cells reduce Pdx1 expression, as Ngn3-positive cells are Pdx1 negative10. They re-express Pdx1, however, as they go on their path towards glucose-sensitive insulin-secreting cells with parallel induction of MafA that is required for proper differentiation and maturation of pancreatic beta cells11. Data supporting these expression dynamics are derived from mice experiments1, 11, 12. A synthetic gene-switch governing cell fate decision in human induced pluripotent stem cells (hIPSCs) could facilitate the differentiation of glucose-sensitive insulin-secreting cells.

In recent years, synthetic biology has significantly advanced the rational design of synthetic gene networks that can interface with host metabolism, correct physiological disturbances13 and provide treatment strategies for a variety of metabolic disorders, including gouty arthritis14, obesity15 and type-2 diabetes16. Currently, synthetic biology principles may provide the componentry and gene network topologies for the assembly of synthetic lineage-control networks that can programme cell-fate decisions and provide targeted differentiation of stem cells into terminally differentiated somatic cells. Synthetic lineage-control networks may therefore provide the missing link between human pluripotent stem cells17 and their true impact on regenerative medicine18, 19, 20. The use of autologous stem cells in regenerative medicine holds great promise for curing many diseases, including type-1 diabetes mellitus (T1DM), which is characterized by the autoimmune destruction of insulin-producing pancreatic beta cells, thus making patients dependent on exogenous insulin to control their blood glucose21, 22. Although insulin therapy has changed the prospects and survival of T1DM patients, these patients still suffer from diabetic complications arising from the lack of physiological insulin secretion and excessive glucose levels23. The replacement of the pancreatic beta cells either by pancreas transplantation or by transplantation of pancreatic islets has been shown to normalize blood glucose and even improve existing complications of diabetes24. However, insulin independence 5 years after islet transplantation can only be achieved in up to 55% of the patients even when using the latest generation of immune suppression strategies25, 26. Transplantation of human islets or the entire pancreas has allowed T1DM patients to become somewhat insulin independent, which provides a proof-of-concept for beta-cell replacement therapies27, 28. However, because of the shortage of donor pancreases and islets, as well as the significant risk associated with transplantation and life-long immunosuppression, the rational differentiation of stem cells into functional beta-cells remains an attractive alternative29, 30. Nevertheless, a definitive cure for T1DM should address both the beta-cell deficit and the autoimmune response to cells that express insulin. Any beta-cell mimetic should be able to store large amounts of insulin and secrete it on demand, as in response to glucose stimulation29, 31. The most effective protocols for the in vitro generation of bonafide insulin-secreting beta-like cells that are suitable for transplantation have been the result of sophisticated trial-and-error studies elaborating timely addition of complex growth factor and small-molecule compound cocktails to human pancreatic progenitor cells32, 33, 34. The differentiation of pancreatic progenitor cells to beta-like cells is the most challenging part as current protocols provide inconsistent results and limited success in programming pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells35, 36, 37. One of the reasons for these observations could be the heterogeneity in endocrine differentiation and maturation towards a beta cell phenotype. Here we show that a synthetic lineage-control network programming the dynamic expression of the transcription factors Ngn3, Pdx1 and MafA enables the differentiation of hIPSC-derived pancreatic progenitor cells to glucose-sensitive insulin-secreting beta-like cells (Supplementary Fig. 1).

 

Vanillic acid-programmable positive band-pass filter

The differentiation pathway from pancreatic progenitor cells to glucose-sensitive insulin-secreting pancreatic beta-cells combines the transient mutually exclusive expression switches of Ngn3 (OFF-ON-OFF) and Pdx1 (ON-OFF-ON) with the concomitant induction of MafA (OFF-ON) expression10,11. Since independent control of the pancreatic transcription factors Ngn3, Pdx1 and MafA by different antibiotic transgene control systems responsive to tetracycline, erythromycin and pristinamycin did not result in the desired differential control dynamics (Supplementary Fig. 2), we have designed a vanillic acid-programmable synthetic lineage-control network that programmes hIPSC-derived pancreatic progenitor cells to specifically differentiate into glucose-sensitive insulin-secreting beta-like cells in a seamless and self-sufficient manner. The timely coordination of mutually exclusive Ngn3 and Pdx1 expression with MafA induction requires the trigger-controlled execution of a complex genetic programme that orchestrates two overlapping antagonistic band-pass filter expression profiles (OFF-ON-OFF and ON-OFF-ON), a positive band-pass filter for Ngn3 (OFF-ON-OFF) and a negative band-pass filter, also known as band-stop filter, for Pdx1 (ON-OFF-ON), the ramp-up expression phase of which is linked to a graded induction of MafA (OFF-ON).

The core of the synthetic lineage-control network consists of two transgene control devices that are sensitive to the food component and licensed food additive vanillic acid. These devices are a synthetic vanillic acid-inducible (ON-type) signalling cascade that is gradually induced by increasing the vanillic acid concentration and a vanillic acid-repressible (OFF-type) gene switch that is repressed in a vanillic acid dose-dependent manner (Fig. 1a,b). The designer cascade consists of the vanillic acid-sensitive mammalian olfactory receptor MOR9-1, which sequentially activates the G protein Sα (GSα) and adenylyl cyclase to produce a cyclic AMP (cAMP) second messenger surge38 that is rewired via the cAMP-responsive protein kinase A-mediated phospho-activation of the cAMP-response element-binding protein 1 (CREB1) to the induction of synthetic promoters (PCRE) containing CREB1-specific cAMP response elements (CRE; Fig. 1a). The co-transfection of pCI-MOR9-1 (PhCMV-MOR9-1-pASV40) and pCK53 (PCRE-SEAP-pASV40) into human mesenchymal stem cells (hMSC-TERT) confirmed the vanillic acid-adjustable secreted alkaline phosphatase (SEAP) induction of the designer cascade (>10nM vanillic acid; Fig. 1a). The vanillic acid-repressible gene switch consists of the vanillic acid-dependent transactivator (VanA1), which binds and activates vanillic acid-responsive promoters (for example, P1VanO2) at low and medium vanillic acid levels (<2μM). At high vanillic acid concentrations (>2μM), VanA1 dissociates from P1VanO2, which results in the dose-dependent repression of transgene expression39 (Fig. 1b). The co-transfection of pMG250 (PSV40-VanA1-pASV40) and pMG252 (P1VanO2-SEAP-pASV40) into hMSC-TERT corroborated the fine-tuning of the vanillic acid-repressible SEAP expression (Fig. 1b).

Figure 1: Design of a vanillic acid-responsive positive band-pass filter providing an OFF-ON-OFF expression profile.

Design of a vanillic acid-responsive positive band-pass filter providing an OFF-ON-OFF expression profile.

http://www.nature.com/ncomms/2016/160411/ncomms11247/images_article/ncomms11247-f1.jpg

a) Vanillic acid-inducible transgene expression. The constitutively expressed vanillic acid-sensitive olfactory G protein-coupled receptor MOR9-1 (pCI-MOR9-1; PhCMV-MOR9-1-pA) senses extracellular vanillic acid levels and triggers G protein (Gs)-mediated activation of the membrane-bound adenylyl cyclase (AC) that converts ATP into cyclic AMP (cAMP). The resulting intracellular cAMP surge activates PKA (protein kinase A), whose catalytic subunits translocate into the nucleus to phosphorylate cAMP response element-binding protein 1 (CREB1). Activated CREB1 binds to synthetic promoters (PCRE) containing cAMP-response elements (CRE) and induces PCRE-driven expression of human placental secreted alkaline phosphatase (SEAP; pCK53, PCRE-SEAP-pA). Co-transfection of pCI-MOR9-1 and pCK53 into human mesenchymal stem cells (hMSC-TERT) grown for 48h in the presence of increasing vanillic acid concentrations results in a dose-inducible SEAP expression profile. (b) Vanillic acid-repressible transgene expression. The constitutively expressed, vanillic acid-dependent transactivator VanA1(pMG250, PSV40-VanA1-pA, VanA1, VanR-VP16) binds and activates the chimeric promoter P1VanO2 (pMG252, P1VanO2-SEAP-pA) in the absence of vanillic acid. In the presence of increasing vanillic acid concentrations, VanA1 is released from P1VanO2, and transgene expression is shut down. Co-transfection of pMG250 and pMG252 into hMSC-TERT grown for 48h in the presence of increasing vanillic acid concentrations results in a dose-repressible SEAP expression profile. (c) Positive band-pass expression filter. Serial interconnection of the synthetic vanillic acid-inducible signalling cascade (a) with the vanillic acid-repressible transcription factor-based gene switch (b) by PCRE-mediated expression of VanA1 (pSP1, PCRE-VanA1-pA) results in a two-level feed-forward cascade. Owing to the opposing responsiveness and differential sensitivity to vanillic acid, this synthetic gene network programmes SEAP expression with a positive band-pass filter profile (OFF-ON-OFF) as vanillic acid levels are increased. Medium vanillic acid levels activate MOR9-1, which induces PCRE-driven VanA1 expression. VanA1remains active and triggers P1VanO2-mediated SEAP expression in feed-forward manner, which increases to maximum levels. At high vanillic acid concentrations, MOR9-1 maintains PCRE-driven VanA1 expression, but the transactivator dissociates from P1VanO2, which shuts SEAP expression down. Co-transfection of pCI-MOR9-1, pSP1 and pMG252 into hMSC-TERT grown for 48h in the presence of increasing vanillic acid concentrations programmes SEAP expression with a positive band-pass profile (OFF-ON-OFF). Data are the means±s.d. of triplicate experiments (n=9).

The opposing responsiveness and differential sensitivity of the control devices to vanillic acid are essential to programme band-pass filter expression profiles. Upon daisy-chaining the designer cascade (pCI-MOR9-1; PhCMV-MOR9-1-pASV40; pSP1, PCRE-VanA1-pASV40) and the gene switch (pSP1, PCRE-VanA1-pASV40; pMG252, P1VanO2-SEAP-pASV40) in the same cell, the network executes a band-pass filter SEAP expression profile when exposed to increasing concentrations of vanillic acid (Fig. 1c). Medium vanillic acid levels (10nM to 2μM) activate MOR9-1, which induces PCRE-driven VanA1 expression. VanA1 remains active within this concentration range and, in a feed-forward amplifier manner, triggers P1VanO2-mediated SEAP expression, which gradually increases to maximum levels (Fig. 1c). At high vanillic acid concentrations (2μM to 400μM), MOR9-1 maintains PCRE-driven VanA1 expression, but the transactivator is inactivated and dissociates from P1VanO2, which results in the gradual shutdown of SEAP expression (Fig. 1c).

Vanillic acid-programmable lineage-control network

For the design of the vanillic acid-programmable synthetic lineage-control network, constitutive MOR9-1 expression and PCRE-driven VanA1 expression were combined with pSP12 (pASV40-Ngn3cm←P3VanO2right arrowmFT-miR30Pdx1g-shRNA-pASV40) for endocrine specification and pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40) for maturation of developing beta-cells (Fig. 2a,b). ThepSP12-encoded expression unit enables the VanA1-controlled induction of the optimized bidirectional vanillic acid-responsive promoter (P3VanO2) that drives expression of a codon-modified Ngn3cm, the nucleic acid sequence of which is distinct from its genomic counterpart (Ngn3g) to allow for quantitative reverse transcription–PCR (qRT–PCR)-based discrimination. In the opposite direction, P3VanO2 transcribes miR30Pdx1g-shRNA, which exclusively targets genomicPdx1 (Pdx1g) transcripts for RNA interference-based destruction and is linked to the production of a blue-to-red medium fluorescent timer40 (mFT) for precise visualization of the unit’s expression dynamics in situ. pSP17 contains a dicistronic expression unit in which the modified high-tightness and lower-sensitivity PCREm promoter (see below) drives co-cistronic expression of Pdx1cm andMafAcm, which are codon-modified versions producing native transcription factors that specifically differ from their genomic counterparts (Pdx1g, MafAg) in their nucleic acid sequence. After individual validation of the vanillic acid-controlled expression and functionality of all network components (Supplementary Figs 2–9), the lineage-control network was ready to be transfected into hIPSC-derived pancreatic progenitor cells. These cells are characterized by high expression of Pdx1g and Nkx6.1 levels and the absence of Ngn3g and MafAg production32, 33, 34 (day 0:Supplementary Figs 10–16).

 

Figure 2: Synthetic lineage-control network programming differential expression dynamics of pancreatic transcription factors.

Synthetic lineage-control network programming differential expression dynamics of pancreatic transcription factors.

 

http://www.nature.com/ncomms/2016/160411/ncomms11247/images/ncomms11247-f2.jpg

(a) Schematic of the synthetic lineage-control network. The constitutively expressed, vanillic acid-sensitive olfactory G protein-coupled receptor MOR9-1 (pCI-MOR9-1; PhCMV-MOR9-1-pA) senses extracellular vanillic acid levels and triggers a synthetic signalling cascade, inducing PCRE-driven expression of the transcription factor VanA1 (pSP1, PCRE-VanA1-pA). At medium vanillic acid concentrations (purple arrows), VanA1 binds and activates the bidirectional vanillic acid-responsive promoter P3VanO2 (pSP12, pA-Ngn3cm←P3VanO2right arrowmFT-miR30Pdx1g-shRNA-pA), which drives the induction of codon-modified Neurogenin 3 (Ngn3cm) as well as the coexpression of both the blue-to-red medium fluorescent timer (mFT) for precise visualization of the unit’s expression dynamics and miR30pdx1g-shRNA (a small hairpin RNA programming the exclusive destruction of genomic pancreatic and duodenal homeobox 1 (Pdx1g) transcripts). Consequently, Ngn3cm levels switch from low to high (OFF-to-ON), and Pdx1g levels toggle from high to low (ON-to-OFF). In addition, Ngn3cm triggers the transcription of Ngn3g from its genomic promoter, which initiates a positive-feedback loop. At high vanillic acid levels (orange arrows), VanA1 is inactivated, and both Ngn3cm and miR30pdx1g-shRNA are shut down. At the same time, the MOR9-1-driven signalling cascade induces the modified high-tightness and lower-sensitivity PCREm promoter that drives the co-cistronic expression of the codon-modified variants of Pdx1 (Pdx1cm) and V-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafAcm; pSP17, PCREm-Pdx1cm-2A-MafAcm-pA). Consequently, Pdx1cm and MafAcm become fully induced. As Pdx1cm expression ramps up, it initiates a positive-feedback loop by inducing the genomic counterparts Pdx1g and MafAg. Importantly, Pdx1cm levels are not affected by miR30Pdx1g-shRNA because the latter is specific for genomic Pdx1g transcripts and because the positive feedback loop-mediated amplification of Pdx1gexpression becomes active only after the shutdown of miR30Pdx1g-shRNA. Overall, the synthetic lineage-control network provides vanillic acid-programmable, transient, mutually exclusive expression switches for Ngn3 (OFF-ON-OFF) and Pdx1 (ON-OFF-ON) as well as the concomitant induction of MafA (OFF-ON) expression, which can be followed in real time (Supplementary Movies 1 and 2). (b) Schematic illustrating the individual differentiation steps from human IPSCs towards beta-like cells. The colours match the cell phenotypes reached during the individual differentiation stages programmed by the lineage-control network shown in a.

Following the co-transfection of pCI-MOR9-1 (PhCMV-MOR9-1-pASV40), pSP1 (PCRE-VanA1-pASV40), pSP12 (pASV40-Ngn3cm←P3VanO2right arrowmFT-miR30Pdx1g-shRNA-pASV40) and pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40) into hIPSC-derived pancreatic progenitor cells, the synthetic lineage-control network should override random endogenous differentiation activities and execute the pancreatic beta-cell-specific differentiation programme in a vanillic acid remote-controlled manner. To confirm that the lineage-control network operates as programmed, we cultivated network-containing and pEGFP-N1-transfected (negative-control) cells for 4 days at medium (2μM) and then 7 days at high (400μM) vanillic acid concentrations and profiled the differential expression dynamics of all of the network components and their genomic counterparts as well as the interrelated transcription factors and hormones in both whole populations and individual cells at days 0, 4, 11 and 14 (Figs 2 and 3 and Supplementary Figs 11–17).

 

Figure 3: Dynamics of the lineage-control network.

Dynamics of the lineage-control network.

http://www.nature.com/ncomms/2016/160411/ncomms11247/images/ncomms11247-f3.jpg

(a,b) Quantitative RT–PCR-based expression profiling of the pancreatic transcription factors Ngn3cm/g, Pdx1cm/g and MafAcm/g in hIPSC-derived pancreatic progenitor cells containing the synthetic lineage-control network at days 4 and 11. Data are the means±s.d. of triplicate experiments (n=9). (cg) Immunocytochemistry of pancreatic transcription factors Ngn3cm/g, Pdx1cm/g and MafAcm/g in hIPSC-derived pancreatic progenitor cells containing the synthetic lineage-control network at days 4 and 11. hIPSC-derived pancreatic progenitor cells were co-transfected with the lineage-control vectors pCI-MOR9-1 (PhCMV-MOR9-1-pA), pSP1 (PCRE-VanA1-pA), pSP12 (pA-Ngn3cm←P3VanO2right arrowmFT-miR30Pdx1g-shRNA-pA) and pSP17 (PCREm-Pdx1cm-2A-MafAcm) and immunocytochemically stained for (c) VanA1 and Pdx1 (day 4), (d) VanA1 and Ngn3 (day 4), (e) VanA1 and Pdx1 (day 11), (f) MafA and Pdx1 (day 11) as well as (g) VanA1 and insulin (C-peptide) (day 11). The cells staining positive for VanA1 are containing the lineage-control network. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 100μm.

…….

Multicellular organisms, including humans, consist of a highly structured assembly of a multitude of specialized cell phenotypes that originate from the same zygote and have traversed a preprogrammed multifactorial developmental plan that orchestrates sequential differentiation steps with high precision in space and time19, 51. Because of the complexity of terminally differentiated cells, the function of damaged tissues can for most medical indications only be restored via the transplantation of donor material, which is in chronically short supply52.

Despite significant progress in regenerative medicine and the availability of stem cells, the design of protocols that replicate natural differentiation programmes and provide fully functional cell mimetics remains challenging29, 53. For example, efforts to generate beta-cells from human embryonic stem cells (hESCs) have led to reliable protocols involving the sequential administration of growth factors (activin A, bone morphogenetic protein 4 (BMP-4), basic fibroblast growth factor (bFGF), FGF-10, Noggin, vascular endothelial growth factor (VEGF) and Wnt3A) and small-molecule compounds (cyclopamine, forskolin, indolactam V, IDE1, IDE2, nicotinamide, retinoic acid, SB−431542 and γ-secretase inhibitor) that modulate differentiation-specific signalling pathways31, 54, 55. In vitro differentiation of hESC-derived pancreatic progenitor cells into beta-like cells is more challenging and has been achieved recently by a complex media formulation with chemicals and growth factors32, 33, 34.

hIPSCs have become a promising alternative to hESCs; however, their use remains restricted in many countries56. Most hIPSCs used for directed differentiation studies were derived from a juvenescent cell source that is expected to show a higher degree of differentiation potential compared with older donors that typically have a higher need for medical interventions37, 57, 58. We previously succeeded in producing mRNA-reprogrammed hIPSCs from adipose tissue-derived mesenchymal stem cells of a 50-year-old donor, demonstrating that the reprogramming of cells from a donor of advanced age is possible in principle59.

Recent studies applying similar hESC-based differentiation protocols to hIPSCs have produced cells that release insulin in response to high glucose32, 33, 34. This observation suggests that functional beta-like cells can eventually be derived from hIPSCs32, 33. In our hands, the growth-factor/chemical-based technique for differentiating human IPSCs resulted in beta-like cells with poor glucose responsiveness. Recent studies have revealed significant variability in the lineage specification propensity of different hIPSC lines35, 60 and substantial differences in the expression profiles of key transcription factors in hIPSC-derived beta-like cells33. Therefore, the growth-factor/chemical-based protocols may require further optimization and need to be customized for specific hIPSC lines35. Synthetic lineage-control networks providing precise dynamic control of transcription factor expression may overcome the challenges associated with the programming of beta-like cells from different hIPSC lines.

Rather than exposing hIPSCs to a refined compound cocktail that triggers the desired differentiation in a fraction of the stem cell population, we chose to design a synthetic lineage-control network to enable single input-programmable differentiation of hIPSC-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells. In contrast with the use of growth-factor/chemical-based cocktails, synthetic lineage-control networks are expected to (i) be more economical because of in situ production of the required transcription factors, (ii) enable simultaneous control of ectopic and chromosomally encoded transcription factor variants, (iii) tap into endogenous pathways and not be limited to cell-surface input, (iv) display improved reversibility that is not dependent on the removal of exogenous growth factors via culture media replacement, (v) provide lateral inhibition, thereby reducing the random differentiation of neighbouring cells and (vi) enable trigger-programmable and (vii) precise differential transcription factor expression switches.

The synthetic lineage-control network that precisely replicates the endogenous relative expression dynamics of the transcription factors Pdx-1, Ngn3 and MafA required the design of a new network topology that interconnects a synthetic signalling cascade and a gene switch with differential and opposing sensitivity to the food additive vanillic acid. This differentiation device provides different band-pass filter, time-delay and feed-forward amplifier topologies that interface with endogenous positive-feedback loops to orchestrate the timely expression and repression of heterologous and chromosomally encoded Ngn3, Pdx1 and MafA variants. The temporary nature of the engineering intervention, which consists of transient transfection of the genetic lineage-control components in the absence of any selection, is expected to avoid stable modification of host chromosomes and alleviate potential safety concerns. In addition, the resulting beta-cell mass could be encapsulated inside vascularized microcontainers28, a proven containment strategy in prototypic cell-based therapies currently being tested in animal models of prominent human diseases14, 15, 16, 61, 62 as well as in human clinical trials28.

The hIPSC-derived beta-like cells resulting from this trigger-induced synthetic lineage-control network exhibited glucose-stimulated insulin-release dynamics and capacity matching the human physiological range and transcriptional profiling, flow cytometric analysis and electron microscopy corroborated the lineage-controlled stem cells reached a mature beta-cell phenotype. In principle, the combination of hIPSCs derived from the adipose tissue of a 50-year-old donor59 with a synthetic lineage-control network programming glucose-sensitive insulin-secreting beta-like cells closes the design cycle of regenerative medicine63. However, hIPSCs that are derived from T1DM patients, differentiated into beta-like cells and transplanted back into the donor would still be targeted by the immune system, as demonstrated in the transplantation of segmental pancreatic grafts from identical twins64. Therefore, any beta-cell-replacement therapy will require complementary modulation of the immune system either via drugs30, 65, engineering or cell-based approaches66, 67 or packaging inside vascularizing, semi-permeable immunoprotective microcontainers28.

Capitalizing on the design principles of synthetic biology, we have successfully constructed and validated a synthetic lineage-control network that replicates the differential expression dynamics of critical transcription factors and mimicks the native differentiation pathway to programme hIPSC-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells that compare with human pancreatic islets at a high level. The design of input-triggered synthetic lineage-control networks that execute a preprogrammed sequential differentiation agenda coordinating the timely induction and repression of multiple genes could provide a new impetus for the advancement of developmental biology and regenerative medicine.

Other related articles published in this Open Access Online Scientific Journal include the following:

Adipocyte Derived Stroma Cells: Their Usage in Regenerative Medicine and Reprogramming into Pancreatic Beta-Like Cells

Curator: Evelina Cohn, Ph.D.

https://pharmaceuticalintelligence.com/2016/03/03/adipocyte-derived-stroma-cells-their-usage-in-regenerative-medicine-and-reprogramming-into-pancreatic-beta-like-cells/

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Brain Biobank and studies of disease structure correlates

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Unveiling Psychiatric Diseases

Researchers create neuropsychiatric cellular biobank

Image: iStock/mstroz
Image: iStock/mstroz
Researchers from Harvard Medical School and Massachusetts General Hospital have completed the first stage of an important collaboration aimed at understanding the intricate variables of neuropsychiatric disease—something that currently eludes clinicians and scientists.

The research team, led by Isaac Kohane at HMS and Roy Perlis at Mass General, has created a neuropsychiatric cellular biobank—one of the largest in the world.

It contains induced pluripotent stem cells, or iPSCs, derived from skin cells taken from 100 people with neuropsychiatric diseases such as schizophrenia, bipolar disorder and major depression, and from 50 people without neuropsychiatric illness.

In addition, a detailed profile of each patient, obtained from hours of in-person assessment as well as from electronic medical records, is matched to each cell sample.

As a result, the scientific community can now for the first time access cells representing a broad swath of neuropsychiatric illness. This enables researchers to correlate molecular data with clinical information in areas such as variability of drug reactions between patients. The ultimate goal is to help treat, with greater precision, conditions that often elude effective management.

The cell collection and generation was led by investigators at Mass General, who in collaboration with Kohane and his team are working to characterize the cell lines at a molecular level. The cell repository, funded by the National Institutes of Health, is housed at Rutgers University.

“This biobank, in its current form, is only the beginning,” said Perlis, director of the MGH Psychiatry Center for Experimental Drugs and Diagnostics and HMS associate professor of psychiatry. “By next year we’ll have cells from a total of four hundred patients, with additional clinical detail and additional cell types that we will share with investigators.”

A current major limitation to understanding brain diseases is the inability to access brain biopsies on living patients. As a result, researchers typically study blood cells from patients or examine post-mortem tissue. This is in stark contrast with diseases such as cancer, for which there are many existing repositories of highly characterized cells from patients.

The new biobank offers a way to push beyond this limitation.

 

A Big Step Forward

While the biobank is already a boon to the scientific community, researchers at MGH and the HMS Department of Biomedical Informatics will be adding additional layers of molecular data to all of the cell samples. This information will include whole genome sequencing and transcriptomic and epigenetic profiling of brain cells made from the stem cell lines.

Collaborators in the HMS Department of Neurobiology, led by Michael Greenberg, department chair and Nathan Marsh Pusey Professor of Neurobiology,  will also work to examine characteristics of other types of neurons derived from these stem cells.

“This can potentially alter the entire way we look at and diagnose many neuropsychiatric conditions,” said Perlis.

One example may be to understand how the cellular responses to medication correspond to the patient’s documented responses, comparing in vitro with in vivo. “This would be a big step forward in bringing precision medicine to psychiatry,” Perlis said.

“It’s important to recall that in the field of genomics, we didn’t find interesting connections to disease until we had large enough samples to really investigate these complex conditions,” said Kohane, chair of the HMS Department of Biomedical Informatics.

“Our hypothesis is that here we will require far fewer patients,” he said. “By measuring the molecular functioning of the cells of each patient rather than only their genetic risk, and combining that all that’s known of these people in terms of treatment response and cognitive function, we will discover a great deal of valuable information about these conditions.”

Added Perlis, “In the early days of genetics, there were frequent false positives because we were studying so few people. We’re hoping to avoid the same problem in making cellular models, by ensuring that we have a sufficient number of cell lines to be confident in reporting differences between patient groups.”

The generation of stem cell lines and characterization of patients and brain cell lines is funded jointly by the the National Institute of Mental Health, the National Human Genome Research Institute and a grant from the Centers of Excellence in Genomic Science program.

 

On C.T.E. and Athletes, Science Remains in Its Infancy

Se Hoon ChoiYoung Hye KimMatthias Hebisch, et al.

http://www.nature.com/articles/nature13800.epdf

Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles1. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau2, 3. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology4, 5. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles6, 7, 8, 9, 10, 11. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

 

 

Figure 2: Robust increases of extracellular amyloid-β deposits in 3D-differentiated hNPCs with FAD mutations.close

Robust increases of extracellular amyloid-[bgr] deposits in 3D-differentiated hNPCs with FAD mutations.

a, Thin-layer 3D culture protocol. HC, histochemistry; IF, immunofluorescence; IHC, immunohistochemistry. b, Amyloid-β deposits in 6-week differentiated control and FAD ReN cells in 3D Matrigel (green, GFP; blue, 3D6; scale bar, …

 

Stem Cell-Based Spinal Cord Repair Enables Robust Corticospinal Regeneration

 

Novel use of EPR spectroscopy to study in vivo protein structure

http://www.news-medical.net/whitepaper/20160315/Novel-use-of-EPR-spectroscopy-to-study-in-vivo-protein-structure.aspx

α-synuclein

α-synuclein is a protein found abundantly throughout the brain. It is present mainly at the neuron ends where it is thought to play a role in ensuring the supply of synaptic vesicles in presynaptic terminals, which are required for the release of neurotransmitters to relay signals between neurons. It is critical for normal brain function.

However, α-synuclein is also the primary protein component of the cerebral amyloid deposits characteristic of Parkinson’s disease and its precursor is found in the amyloid plaques of Alzheimer’s disease. Although α-synuclein is present in all areas of the brain, these disease-state amyloid plaques only arise in distinct areas.

Alpha-synuclein protein. May play role in Parkinson’s and Alzheimer’s disease.  © molekuul.be / Shutterstock.com

Imaging of isolated samples of α-synuclein in vitro indicate that it does not have the precise 3D folded structure usually associated with proteins. It is therefore classed as an intrinsically disordered protein. However, it was not known whether the protein also lacked a precise structure in vivo.

There have been reports that it can form helical tetramers. Since the 3D structure of a biological protein is usually precisely matched to the specific function it performs, knowing the structure of α-synuclein within a living cell will help elucidate its role and may also improve understanding of the disease states with which it is associated.

If α-synuclein remains disordered in vivo, it may be possible for the protein to achieve different structures, and have different properties, depending on its surroundings.

Techniques for determining protein structure

It has long been known that elucidating the structure of a protein at an atomic level is fundamental for understanding its normal function and behavior. Furthermore, such knowledge can also facilitate the development of targeted drug treatments. Unfortunately, observing the atomic structure of a protein in vivo is not straightforward.

X-ray diffraction is the technique usually adopted for visualizing structures at atomic resolution, but this requires crystals of the molecule to be produced and this cannot be done without separating the molecules of interest from their natural environment. Such processes can modify the protein from its usual state and, particularly with complex structures, such effects are difficult to predict.

The development of nuclear magnetic resonance (NMR) spectroscopy improved the situation by making it possible for molecules to be analyzed under in vivo conditions, i.e. same pH, temperature and ionic concentration.

More recently, increases in the sensitivity of NMR and the use of isotope labelling have enabled determinations of the atomic level structure and dynamics of proteins to be determined within living cells1. NMR has been used to determine the structure of a bacterial protein within living cells2 but it is difficult to achieve sufficient quantities of the required protein within mammalian cells and to keep the cells alive for NMR imaging to be conducted.

Electron paramagnetic resonance (EPR) spectroscopy for determining protein structure

Recently, researchers have managed to overcome these obstacles by using in-cell NMR and electron paramagnetic resonance (EPR) spectroscopy. EPR spectroscopy is a technique that is similar to NMR spectroscopy in that it is based on the measurement and interpretation of the energy differences between excited and relaxed molecular states.

In EPR spectroscopy it is electrons that are excited, whereas in NMR signals are created through the spinning of atomic nuclei. EPR was developed to measure radicals and metal complexes, but has also been utilized to study the dynamic organization of lipids in biological membranes3.

EPR has now been used for the first time in protein structure investigations and has provided atomic-resolution information on the structure of α-synuclein in living mammalians4,5.

Bacterial forms of the α-synuclein protein labelled with 15N isotopes were introduced into five types of mammalian cell using electroporation. Concentrations of α-synuclein close to those found in vivo were achieved and the 15N isotopes allowed the protein to be clearly defined from other cellular components by NMR. The conformation of the protein was then determined using electron paramagnetic resonance (EPR).

The results showed that within living mammalian cells α-synuclein remains as a disordered and highly dynamic monomer. Different intracellular environments did not induce major conformational changes.

Summary

The novel use of EPR spectroscopy has resolved the mystery surrounding the in vivo conformation of α-synuclein. It showed that α-synuclein maintains its disordered monomeric form under physiological cell conditions. It has been demonstrated for the first time that even in crowded intracellular environments α-synuclein does not form oligomers, showing that intrinsic structural disorder can be sustained within mammalian cells.

References

  1. Freedberg DI and Selenko P. Live cell NMR Annu. Rev. Biophys. 2014;43:171–192.
  2. Sakakibara D, et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 2009;458:102–105.
  3. Yashroy RC. Magnetic resonance studies of dynamic organisation of lipids in chloroplast membranes. Journal of Biosciences 1990;15(4):281.
  4. Alderson TA and Bax AD. Parkinson’s Disease. Disorder in the court. Nature 2016; doi:10.1038/nature16871.
  5. Theillet FX, et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 2016; doi:10.1038/nature16531.

 

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Essential for Rehabilitation

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Cellular Rehab

Physical therapy and exercise are critical to the success of cell therapies approaching the clinic.

By Elie Dolgin |  Scientist  Magazine Dec 1, 2015     http://www.the-scientist.com/?articles.view/articleNo/44605/title/Cellular-Rehab/

http://www.the-scientist.com/Dec2015/feature1.jpg

Ron Strang lay on his back and bent his left leg. “I could feel the difference right away,” recalls the 31-year-old ex-Marine.

The day before, Strang had undergone an experimental surgery to help repair a deep gouge in his quadriceps. He’d been injured in April 2010 while on foot patrol in Afghanistan’s Helmand Province, when a crude roadside bomb sent shrapnel tearing through his upper thigh. Ten soldiers were wounded in the blast, Strang the most grievously. A year later, even after numerous surgeries and skin grafts, he still couldn’t walk without his knee buckling. So he signed up to receive an experimental regenerative therapy.

In July 2011, Stephen Badylak, a tissue-engineering specialist at the University of Pittsburgh, transplanted a thin sheet of extracellular matrix (ECM) derived from pig bladders into Strang’s leg. The fibrous material was intended not only to provide structural support for the muscle, but also, by releasing signaling proteins, to recruit and coax stem cells in the body to differentiate into new tissue.

Physical forces

Researchers have long recognized the influence of physical forces on molecular and cellular function. Nearly 40 years ago, Judah Folkman, a cancer biologist at Harvard Medical School, and his undergraduate assistant Anne Moscona, now an infectious-disease researcher at Weill Cornell Medicine in New York City, grew cells in petri dishes and found that as cells stretched out and flattened more and more on the plate, their rate of DNA synthesis and cell division increased.4 This revelation led to an explosion of interest in how squeezes, tugs, pushes, and pulls mold the architecture of the cell and, in turn, influence molecular processes within, such as gene expression.

For the most part, however, the field of mechanobiology has been stuck in the laboratory, with few physicians thinking about how physical stresses at the cellular level might affect clinical outcomes, and even fewer physical therapists considering the molecular milieu. As Christopher Evans, director of the Rehabilitation Medicine Research Center at the Mayo Clinic in Rochester, Minnesota, puts it: “The people doing the stem cell work have been largely ignorant of rehabilitation, and the rehabilitation medicine community hasn’t been thinking in terms of cell and molecular biology.”

With stem cell therapies and tissue engineering nearing medical prime time, that’s starting to change. A growing number of scientists, clinicians, and physical therapists are now taking an interdisciplinary approach to rehabilitation, pairing exercise with technologies that regenerate bone, muscle, cartilage, ligaments, nerves, and other tissues. They call it regenerative rehabilitation.

“This is a new future,” says Carmen Perez-Terzic, a cardiovascular disease researcher at the Mayo Clinic. “This is an area that’s going to explode in the next 5 or 10 years.”

Fusion approach

The first public call for stem cell biologists and physical therapists to integrate regenerative medicine and rehabilitation science came in a 2010 editorial by Fabrisia Ambrosio, director of the University of Pittsburgh’s Cellular Rehabilitation Laboratory, and Alan Russell, then director of Pitt’s McGowan Institute for Regenerative Medicine.5“Regenerative rehabilitation is difficult but inevitable,” Ambrosio and Russell wrote, “and now is the time to prepare specific, science-based protocols.”

Ambrosio trained as a physical therapist before earning her PhD with rehabilitation medicine specialist Michael Boninger at Pitt, where she studied how wheelchair design affects strength in people with spinal cord injuries and degenerative conditions such as multiple sclerosis. When Ambrosio started her own research group at Pitt in 2005, she began to investigate how mechanical and electrical stimulation might promote healing following stem cell transplantation.

 

http://www.the-scientist.com/Dec2015/feature1_2.jpg

NEW NEURONS: In the dentate gyrus of the hippocampus, mice injected with stretched muscle stem cells show an approximately threefold greater increase in immature neurons (black, left) than mice injected with untreated stem cells (right), likely as a result of growth and neurotrophic factors released into the bloodstream by the stretched cells. JENNIFER MERRITT

 

She transplanted muscle-derived stem cells into bruised hind limbs of mice, then ran the animals on treadmills every weekday for five weeks. The active mice developed more new muscle cells than sedentary controls.6 Ambrosio’s team later demonstrated that applying low-level electrical pulses to muscles injected with stem cells improved strength and reduced fatigue in mice that experienced progressive muscle degeneration characteristic of Duchenne muscular dystrophy.7 “Using very noninvasive, clinically relevant protocols, we can actually dictate the behavior of stem cells,” she says. And that got her thinking: “All of this should lay the groundwork for how we see regenerative medicine therapies being applied in the clinic.”

Starting in 2011, Ambrosio and Boninger launched an annual Symposium on Regenerative Rehabilitation; they held the fourth conference in September at the Mayo Clinic in Minnesota. Last year, the duo also started the International Consortium for Regenerative Rehabilitation, a coalition of eight participating institutions from the U.S., Japan, and Italy that is now developing a strategic agenda for the field. And a few months ago, they secured funding to create the Alliance for Regenerative Rehabilitation Research & Training, which includes four US universities and hospitals (Pitt, Stanford University, Mayo, and the University of California, San Francisco) and will support webinars, minisabbaticals, seed grants, and more.

“This is about getting more people doing this work, understanding this work, and translating this field,” says Boninger, who is leading the alliance together with Stanford stem-cell biologist Thomas Rando. Just adding exercise to a stem cell therapy is “easy,” Boninger notes. “Doing the basic science to evaluate that is a little more challenging.”

The science may still be in its infancy, but Ambrosio says her efforts in community building are beginning to pay off. “I can see such a difference in the way people receive some of these ideas of regenerative rehab,” she says. “It was really kind of novel as recently as 2010, whereas now it’s actually part of our vernacular.”

(Re)Generating interest

Rehabilitation regimens are now being integrated into the preclinical development of regenerative treatments for heart disease, bone fractures, and even brain injuries. In Japan, for example, researchers at Hiroshima University have shown that running directs neural stem cells to properly differentiate when transplanted into mice with experimentally induced brain damage.8 “Combining cell therapy and rehabilitation is needed to correct the neural network and achieve a functional recovery,” says study author Takeshi Imura, who presented the research at Japan’s first-ever Workshop on Regenerative Rehabilitation in Kyoto last March. And earlier this year, muscle biologist Marni Boppart and her colleagues at the University of Illinois at Urbana-Champaign reported that stem cells only enhance muscle repair and growth in mice when coupled with weight-training exercise.9

In addition to exercising recipients of cell therapies, scientists are also looking to give the cells themselves a workout, by stretching stem cells in a dish ahead of transplantation. “In effect, we’re exercising the stem cells without exercising the animal,” says Boppart. In unpublished work, Boppart’s team found that old mice injected with muscle stem cells taken from young mice and stretched before injection exhibited improved blood flow, stronger muscles, and more new neurons in the brain’s hippocampus, thanks to the release of growth, neurotrophic, and immunomodulatory factors brought on by the mechanical stimulus. Stem cells not given the laboratory workout provided no such benefits.

At the Mayo Clinic, Perez-Terzic is also applying physical pressure in vitro to improve the differentiation of stem cells. Her goal is to develop new regenerative treatments for heart disease, and she is hoping to find more-efficient ways of coaxing embryonic stem cells to become heart muscle cells for transplantation. The results are preliminary, Perez-Terzic says, but so far it looks like “if you put some pressure into the system, the differentiation is much better.”

Boppart is hopeful that translating such therapies to the clinic will help patients who are unable to exercise, such as some elderly individuals or those with extreme muscle weakness. “This type of alternative stem cell therapy may provide the boost in strength necessary for someone to transition from disability to regain of function,” she says.

Richard Shields, an applied physiologist at the University of Iowa’s Carver College of Medicine, has another solution, one that doesn’t require any sort of cellular calisthenics in the laboratory. He has invented a device that can deliver different kinds of mechanical loads directly to the lower leg, even for patients confined to a wheelchair. A compression system covers the knee, while the foot rests on a vibrating platform. A doctor or physical therapist can then deliver therapeutic loads in a safe and quantifiable manner. (See illustration below.)

After testing the device on eight people with complete paralysis,10 Shields and his colleagues wondered whether delivering a controlled dose of vibration would improve bone architecture in spinal cord injury patients, many of whom eventually develop severe osteoporosis. After 12 months of regular vibration therapy, however, bone health continued to decline in all six study participants.11 “This means that people with long-term paralysis are very resistant to change [in bone density] or that the dose was not high enough,” says Shields, who is now working to refine the training regimen for better results.

Once he and his colleagues work out the kinks, Shields says he hopes that the setup will be useful to more patients than just those who are incapable of exercise. The limb-loading system offers greater control of the degree and target of stimulation than that afforded by running or weight lifting, he says—precision that could have utility for all manner of regenerative cellular treatments. “How you dose these mechanical loads is not just all or none,” he says. “The stresses have to be applied in opportune doses.”

Stretch goals

http://www.the-scientist.com/Dec2015/NovDolginThumb_310px.jpg

CELLULAR WORKOUT: Regenerative rehabilitation promises to enhance the potential of cell- and gene-based techniques by incorporating principles of physical therapy.
See full infographic here: WEB | PDF
© KIMBERLY BATTISTA

 

Martin Childers, who is collaborating with San Francisco–based Audentes Therapeutics, says he hopes it could also improve outcomes following a gene therapy in children with a rare and fatal muscle weakness disorder called X-linked myotubular myopathy.

The disorder is caused by mutations in the MTM1gene that encodes an enzyme needed for the development and maintenance of muscle cells. Children with the condition suffer from extreme muscle weakness, generally lacking the strength needed to move air in and out of their lungs without mechanical assistance. Audentes’s therapy will deliver a good copy of the MTM1 gene into the blood and hopefully help affected individuals respire without assistance. But after treatment, “you can’t just turn the ventilator off,” Childers says. “There’s going to have to be some rehabilitation therapies.”

Specifically, Childers plans to couple the gene therapy with breathing training. In addition to helping patients wean themselves off the ventilator, pulmonary exercise might enhance the expression of the introduced gene, he says. For now, this is only a hunch. But Audentes is preparing for the launch of a Phase 1 trial next year, and Childers is studying human tissue samples in the lab to answer this question.

Other gene therapy trials already incorporate physical therapy into their recovery protocols. At Nationwide Children’s Hospital in Columbus, Ohio, for example, Jerry Mendell and his colleagues are testing a gene-correction treatment on six- to nine-month-old babies with a severe form of spinal muscular atrophy, a genetic disease that involves the degeneration of motor neurons. The infants are born with tight joints; their legs are often fixed in a splayed, frog-like position. Mendell’s team strongly encourages parents to massage their children’s stiffened limbs on a daily basis after the gene transfer—a necessity, Mendell says, for the gene-corrected motor neurons to interface properly with the weakened muscles. Without it, “you’re not going to be able to improve function,” he says.

Mendell is also incorporating bicycle training into a small gene-therapy trial for children with Duchenne muscular dystrophy. Although mutations in the gene that codes for a muscle-associated protein called dystrophin are responsible for this disorder, Mendell is not delivering a working copy of that particular gene back into patients. Instead, he is using a gene therapy product he developed in collaboration with Cleveland-based Milo Biotechnology that includes the gene for follistatin, a protein that helps release the brakes on muscle growth and could thus prove beneficial for a variety of muscle diseases.

In an earlier trial that tested the same gene therapy on six adults with Becker muscular dystrophy, a milder condition also caused by mutations in the gene for dystrophin, Mendell noticed that the participant who had the most active lifestyle—on account of his job at a garden center—exhibited the most dramatic improvement in how far he could walk.12 “He was one of the highest responders to the follistatin gene therapy,” Mendell says. Hoping to re-create the success, Mendell is now having the kids in his ongoing Duchenne muscular dystrophy trial ride stationary bikes for 15-minute sessions three times per week.

One umbrella

http://www.the-scientist.com/Dec2015/feature1_3.jpg

WHEELCHAIR CALISTHENICS: The University of Iowa’s Richard Shields developed a system to assess the effects of mechanical loading, vibration, and electrical stimulation on bone, muscle, nerve, and cell signaling on the lower leg. The goal is to apply optimal combinations of the three forces to enhance regenerative medicine treatments. COURTESY OF RICHARD SHIELDS

“In the end,” says Boninger, regenerative rehabilitation “is about improving function in patients. So, being able to look someone in the eye and say, ‘This is why we’re doing this exercise program, and this will add to your recovery and function when you’re done’ is where I really want to get to.”

To help make that happen, several academic institutions have been shuffling departmental structures to bring stem cell scientists and rehabilitation doctors under the same administrative umbrella. These include the University of Pittsburgh and the Mayo Clinic, both leaders in the nascent hybrid discipline, though the first place to do so was Columbia University Medical Center in New York City, where the hospital renamed its rehab division the Department of Rehabilitation and Regenerative Medicine in 2010.

Five years on, however, efforts to bridge the two disciplines “remain to some degree aspirational,” admits Joel Stein, a rehabilitation specialist who chairs the Columbia department. Regenerative rehabilitation “is becoming more popular within the field as a vision for the future,” he says. “But has it translated into good, hard science that’s led to definitive new therapies? No, not yet, and it might take a while.”

“We need to be dedicating more efforts to thinking about dosing, intensity, and protocols,” Ambrosio agrees. “That means we have a lot of work ahead for us.”

In the meantime, proponents of regenerative rehabilitation continue to look to success stories like Strang’s for inspiration. At one point, Strang was unsure that he’d ever be able to walk normally again. Now a police officer at the Veterans Affairs hospital in Pittsburgh, Strang is on his feet constantly, moving about easily and pain-free. Just last month, in fact, he married his longtime girlfriend at a church outside Pittsburgh—and he walked down the aisle with no problems.

Elie Dolgin is a news editor at STAT in Boston.

References

  1. V.J. Mase, Jr., et al., “Clinical application of an acellular biologic scaffold for surgical repair of a large, traumatic quadriceps femoris muscle defect,” Orthopedics, 33:511, doi:10.3928/01477447-20100526-24, 2010.
  2. N.E. Gentile et al., “Targeted rehabilitation after extracellular matrix scaffold transplantation for the treatment of volumetric muscle loss,” Am J Phys Med Rehabil, 93:S79-S87, 2014.
  3. B.M. Sicari et al., “An acellular biologic scaffold promotes skeletal muscle formation in mice and humans with volumetric muscle loss,” Sci Transl Med, 6:234ra58, 2014.
  4. J. Folkman, A. Moscona, “Role of cell shape in growth control,” Nature, 273:345-49, 1978.
  5. F. Ambrosio, A. Russell, “Regenerative rehabilitation: a call to action,” J Rehabil Res Dev, 47:xi-xv, 2010.
  6. F. Ambrosio et al., “The synergistic effect of treadmill running on stem-cell transplantation to heal injured skeletal muscle,” Tissue Eng Part A, 16:839-49, 2010.
  7. G. Distefano et al., “Neuromuscular electrical stimulation as a method to maximize the beneficial effects of muscle stem cells transplanted into dystrophic skeletal muscle,” PLOS ONE, 8:e54922, 2013.
  8. T. Imura et al., “Interactive effects of cell therapy and rehabilitation realize the full potential of neurogenesis in brain injury model,” Neurosci Lett, 555:73-78, 2013.
  9. K. Zou et al., “Mesenchymal stem cells augment the adaptive response to eccentric exercise,” Med Sci Sports Exerc, 47:315-25, 2015.
  10. C.L. McHenry et al., “Potential regenerative rehabilitation technology: Implications of mechanical stimuli to tissue health,” BMC Res Notes, 7:334, 2014.
  11. S. Dudley-Javoroski et al., “Bone architecture adaptations after spinal cord injury: impact of long-term vibration of a constrained lower limb,” Osteoporos Int, doi:10.1007/s00198-015-3326-4, 2015.
  12. J.R. Mendell et al., “A phase 1/2a follistatin gene therapy trial for Becker muscular dystrophy,” Mol Ther, 23:192-201, 2015.

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Previously unseen immune reaction identified for stem cell transplants.

Reporter: Stephen J. Williams, Ph.D.

Reposted from at http://health-innovations.org/2014/11/21/previously-unseen-immune-reaction-identified-in-stem-cell-transplants/

 

Mouse cells and tissues created through nuclear transfer can be rejected by the body because of a previously unknown immune response to the cell’s mitochondria, according to an international study in mice by researchers at the Stanford University, MIT and colleagues in Germany and England.  The findings reveal a likely, but surmountable, hurdle if such therapies are ever used in humans, the researchers said.  The opensource study is published in Cell Stem Cell.

Stem cell therapies hold vast potential for repairing organs and treating disease. The greatest hope rests on the potential of pluripotent stem cells, which can become nearly any kind of cell in the body. One method of creating pluripotent stem cells is called somatic cell nuclear transfer, and involves taking the nucleus of an adult cell and injecting it into an egg cell from which the nucleus has been removed.

The promise of the SCNT method is that the nucleus of a patient’s skin cell, for example, could be used to create pluripotent cells that might be able to repair a part of that patient’s body.  One attraction of SCNT has always been that the genetic identity of the new pluripotent cell would be the same as the patient’s, since the transplanted nucleus carries the patient’s DNA.

The hope has been that this would eliminate the problem of the patient’s immune system attacking the pluripotent cells as foreign tissue, which is a problem with most organs and tissues when they are transplanted from one patient to another.

Stanford University have raised the possibility in the past that the immune system of a patient who received SCNT-derived cells might still react against the cells’ mitochondria, which act as the energy factories for the cell and have their own DNA. This reaction could occur because cells created through SCNT contain mitochondria from the egg donor and not from the patient, and therefore could still look like foreign tissue to the recipient’s immune system.

That hypothesis was never tested until the team took up the challenge.  There was a thought that because the mitochondria were on the inside of the cell, they would not be exposed to the host’s immune system.  The current study found that this was not the case.

The team used cells that were created by transferring the nuclei of adult mouse cells into enucleated eggs cells from genetically different mice. When transplanted back into the nucleus donor strain, the cells were rejected although there were only two single nucleotide substitutions in the mitochondrial DNA of these SCNT-derived cells compared to that of the nucleus donor.  The team were surprised to find that just two small differences in the mitochondrial DNA was enough to cause an immune reaction.

Until recently, researchers were able to perform SCNT in many species, but not in humans. When scientists at the Oregon Health and Science University announced success in performing SCNT with human cells last year, it reignited interest in eventually using the technique for human therapies. Although many stem cell researchers are focused on a different method of creating pluripotent stem cells, called induced pluripotent stem cells, there may be some applications for which SCNT-derived pluripotent cells are better suited.

The immunological reactions reported in the new paper will be a consideration if clinicians ever use SCNT-derived stem cells in human therapy, but such reactions should not prevent their use.   This research informs the medical community of the margin of safety that would be required if, in the distant future, researchers need to use SCNT to create pluripotent cells to treat someone.  In that case, clinicians would likely be able to handle the immunological reaction using the immunosuppression methods that are currently available.

In the future, scientists might also lessen the immune reaction by using eggs from someone who is genetically similar to the recipient, such as a mother or sister.

Source:  Stanford University School of Medicine

The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies.  SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts.  Schrepfer et al 2014.

The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies. SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts. Schrepfer et al 2014.

SCNT (somatic cell nuclear transfer)

scnt

Possible ways to generate immune-compatible derivatives of pluripotent cells. From Nature Reviews

From the following article: Derive and conquer: sourcing and differentiating stem cells for therapeutic applications

In genetics and developmental biology, somatic cell nuclear transfer (SCNT) is a laboratory technique for creating an ovum with a donor nucleus. It can be used in embryonic stem cell research, or in regenerative medicine where it is sometimes referred to as “therapeutic cloning.”

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FDA Guidance On Source Animal, Product, Preclinical and Clinical Issues Concerning the Use of Xenotranspantation Products in Humans – Implications for 3D BioPrinting of Regenerative Tissue

Reporter: Stephen J. Williams, Ph.D.

 

The FDA has submitted Final Guidance on use xeno-transplanted animal tissue, products, and cells into human and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.

This document is to provide guidance on the production, testing and evaluation of products intended for use in xenotransplantation. The guidance includes scientific questions that should be addressed by sponsors during protocol development and during the preparation of submissions to the Food and Drug Administration (FDA), e.g., Investigational New Drug Application (IND) and Biologics License Application (BLA). This guidance document finalizes the draft guidance of the same title dated February 2001.

For the purpose of this document, xenotransplantation refers to any procedure that involves the transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs. For the purpose of this document, xenotransplantation products include live cells, tissues or organs used in xenotransplantation. (See Definitions in section I.C.)

This document presents issues that should be considered in addressing the safety of viable materials obtained from animal sources and intended for clinical use in humans. The potential threat to both human and animal welfare from zoonotic or other infectious agents warrants careful characterization of animal sources of cells, tissues, and organs. This document addresses issues such as the characterization of source animals, source animal husbandry practices, characterization of xenotransplantation products, considerations for the xenotransplantation product manufacturing facility, appropriate preclinical models for xenotransplantation protocols, and monitoring of recipients of xenotransplantation products. This document recommends specific practices intended to prevent the introduction and spread of infectious agents of animal origin into the human population. FDA expects that new methods proposed by sponsors to address specific issues will be scientifically rigorous and that sufficient data will be presented to justify their use.

Examples of procedures involving xenotransplantation products include:

  • transplantation of xenogeneic hearts, kidneys, or pancreatic tissue to treat organ failure,
  • implantation of neural cells to ameliorate neurological degenerative diseases,
  • administration of human cells previously cultured ex vivo with live nonhuman animal antigen-presenting or feeder cells, and
  • extracorporeal perfusion of a patient’s blood or blood component perfused through an intact animal organ or isolated cells contained in a device to treat liver failure.

The guidance addresses issues such as:

  1. Clinical Protocol Review
  2. Xenotransplantation Site
  3. Criteria for Patient Selection
  4. Risk/Benefit Assessment
  5. Screening for Infectious Agents
  6. Patient Follow-up
  7. Archiving of Patient Plasma and Tissue Specimens
  8. Health Records and Data Management
  9. Informed Consent
  10. Responsibility of the Sponsor in Informing the Patient of New Scientific Information

A full copy of the PDF can be found below for reference:

fdaguidanceanimalsourcesxenotransplatntation

An example of the need for this guidance in conjunction with 3D printing technology can be understood from the below article (source http://www.geneticliteracyproject.org/2015/09/03/pig-us-xenotransplantation-new-age-chimeric-organs/)

Pig in us: Xenotransplantation and new age of chimeric organs

David Warmflash | September 3, 2015 | Genetic Literacy Project

Imagine stripping out the failing components of an old car — the engine, transmission, exhaust system and all of those parts — leaving just the old body and other structural elements. Replace those old mechanical parts with a brand new electric, hydrogen powered, biofuel, nuclear or whatever kind of engine you want and now you have a brand new car. It has an old frame, but that’s okay. The frame wasn’t causing the problem, and it can live on for years, undamaged.

When challenged to design internal organs, tissue engineers are taking a similar approach, particularly with the most complex organs, like the heart, liver and kidneys. These organs have three dimensional structures that are elaborate, not just at the gross anatomic level, but in microscopic anatomy too. Some day, their complex connective tissue scaffolding, the stroma, might be synthesized from the needed collagen proteins with advanced 3-D printing. But biomedical engineering is not there yet, so right now the best candidate for organ scaffolding comes from one of humanity’s favorite farm animals: the pig.

Chimera alarmists connecting with anti-biotechnology movements might cringe at the thought of building new human organs starting with pig tissue, but if you’re using only the organ scaffolding and building a working organ from there, pig organs may actually be more desirable than those donated by humans.

How big is the anti-chimerite movement?

Unlike anti-GMO and anti-vaccination activists, there really aren’t too many anti-chemerites around. Nevertheless, there is a presence on the web of people who express concern about mixing of humans and non-human animals. Presently, much of their concern is focussed on the growing of human organs inside non-human animals, pigs included. One anti-chemerite has written that it could be a problem for the following reason:

Once a human organ is grown inside a pig, that pig is no longer fully a pig. And without a doubt, that organ will no longer be a fully human organ after it is grown inside the pig. Those receiving those organs will be allowing human-animal hybrid organs to be implanted into them. Most people would be absolutely shocked to learn some of the things that are currently being done in the name of science.

The blog goes on to express alarm about the use of human genes in rice and from there morphs into an off the shelf garden variety anti-GMO tirade, though with an an anti-chemeric current running through it. The concern about making pigs a little bit human and humans a little bit pig becomes a concern about making rice a little bit human. But the concern about fusing tissues and genes of humans and other species does not fit with the trend in modern medicine.

Utilization of pig tissue enters a new age 

pigsinus

A porcine human ear for xenotransplantation. source: The Scientist

For decades, pig, bovine and other non-human tissues have been used in medicine. People are walking around with pig and cow heart valves. Diabetics used to get a lot of insulin from pigs and cows, although today, thanks to genetic engineering, they’re getting human insulin produced by microorganisms modified genetically to make human insulin, which is safer and more effective.

When it comes to building new organs from old ones, however, pig organs could actually be superior for a couple of reasons. For one thing, there’s no availability problem with pigs. Their hearts and other organs also have all of the crucial components of the extracellular matrix that makes up an organ’s scaffolding. But unlike human organs, the pig organs don’t tend to carry or transfer human diseases. That is a major advantage that makes them ideal starting material. Plus there is another advantage: typically, the hearts of human cadavers are damaged, either because heart disease is what killed the human owner or because resuscitation efforts aimed at restarting the heart of a dying person using electrical jolts and powerful drugs.

Rebuilding an old organ into a new one

How then does the process work? Whether starting with a donated human or pig organ, there are several possible methods. But what they all have in common is that only the scaffolding of the original organ is retained. Just like the engine and transmission of the old car, the working tissue is removed, usually using detergents. One promising technique that has been applied to engineer new hearts is being tested by researchers at the University of Pittsburgh. Detergents pumped into the aorta attached to a donated heart (donated by a human cadaver, or pig or cow). The pressure keeps the aortic valve closed, so the detergents to into the coronary arteries and through the myocardial (heart muscle) and endocardial (lining over the muscle inside the heart chambers) tissue, which thus gets dissolved over the course of days. What’s left is just the stroma tissue, forming a scaffold. But that scaffold has signaling factors that enable embryonic stem cells, or specially programed adult pleuripotent cells to become all of the needed cells for a new heart.

Eventually, 3-D printing technology may reach the point when no donated scaffolding is needed, but that’s not the case quite yet, plus with a pig scaffolding all of the needed signaling factors are there and they work just as well as those in a human heart scaffold. All of this can lead to a scenario, possibly very soon, in which organs are made using off-the-self scaffolding from pig organs, ready to produce a custom-made heart using stem or other cells donated by new organ’s recipient.

David Warmflash is an astrobiologist, physician, and science writer. Follow @CosmicEvolution to read what he is saying on Twitter.

And a Great Article in The Scientist by Dr. Ed Yong Entitled

Replacement Parts

To cope with a growing shortage of hearts, livers, and lungs suitable for transplant, some scientists are genetically engineering pigs, while others are growing organs in the lab.

By Ed Yong | August 1, 2012

Source: http://www.the-scientist.com/?articles.view/articleNo/32409/title/Replacement-Parts/

.. where Joseph Vacanti and David Cooper figured that using

“engineered pigs without the a-1,3-galactosyltransferase gene that produces the a-gal residues. In addition, the pigs carry human cell-membrane proteins such as CD55 and CD46 that prevent the host’s complement system from assembling and attacking the foreign cells”

thereby limiting rejection of the xenotransplated tissue.

In addition to issues related to animal virus transmission the issue of optimal scaffolds for organs as well as the advantages which 3D Printing would have in mass production of organs is discussed:

To Vacanti, artificial scaffolds are the future of organ engineering, and the only way in which organs for transplantation could be mass-produced. “You should be able to make them on demand, with low-cost materials and manufacturing technologies,” he says. That is relatively simple for organs like tracheas or bladders, which are just hollow tubes or sacs. Even though it is far more difficult for the lung or liver, which have complicated structures, Vacanti thinks it will be possible to simulate their architecture with computer models, and fabricate them with modern printing technology. (See “3-D Printing,” The Scientist, July 2012.) “They obey very ordered rules, so you can reduce it down to a series of algorithms, which can help you design them,” he says. But Taylor says that even if the architecture is correct, the scaffold would still need to contain the right surface molecules to guide the growth of any added cells. “It seems a bit of an overkill when nature has already done the work for us,” she says.

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New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue

Reporter: Stephen J. Williams, Ph.D.

The FDA recently came out with a Draft Guidance on use of human cells, tissues and cellular and tissue-based products (HCT/P) {defined in 21 CFR 1271.3(d)} and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.

A full copy of the PDF can be found here for reference but the following is a summary of points of the guidance.FO508ver – 2015-373 HomologousUseGuidanceFinal102715

In 21 CFR 1271.10, the regulations identify the criteria for regulation solely under section 361 of the PHS Act and 21 CFR Part 1271. An HCT/P is regulated solely under section 361 of the PHS Act and 21 CFR Part 1271 if it meets all of the following criteria (21 CFR 1271.10(a)):

  • The HCT/P is minimally manipulated;
  • The HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent;
  • The manufacture of the HCT/P does not involve the combination of the cells or tissues with another article, except for water, crystalloids, or a sterilizing, preserving, or storage agent, provided that the addition of water, crystalloids, or the sterilizing, preserving, or storage agent does not raise new clinical safety concerns with respect to the HCT/P; and
  • Either:
  1. The HCT/P does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function; or
  2. The HCT/P has a systemic effect or is dependent upon the metabolic activity of living cells for its primary function, and:
  3. Is for autologous use;
  4. Is for allogeneic use in a first-degree or second-degree blood relative; or
  5. Is for reproductive use.

If an HCT/P does not meet all of the criteria in 21 CFR 1271.10(a), and the establishment that manufactures the HCT/P does not qualify for any of the exceptions in 21 CFR 1271.15, the HCT/P will be regulated as a drug, device, and/or biological product under the Federal Food, Drug and Cosmetic Act (FD&C Act), and/or section 351 of the PHS Act, and applicable regulations, including 21 CFR Part 1271, and pre-market review will be required.

1 Examples of HCT/Ps include, but are not limited to, bone, ligament, skin, dura mater, heart valve, cornea, hematopoietic stem/progenitor cells derived from peripheral and cord blood, manipulated autologous chondrocytes, epithelial cells on a synthetic matrix, and semen or other reproductive tissue. The following articles are not considered HCT/Ps: (1) Vascularized human organs for transplantation; (2) Whole blood or blood components or blood derivative products subject to listing under 21 CFR Parts 607 and 207, respectively; (3) Secreted or extracted human products, such as milk, collagen, and cell factors, except that semen is considered an HCT/P; (4) Minimally manipulated bone marrow for homologous use and not combined with another article (except for water, crystalloids, or a sterilizing, preserving, or storage agent, if the addition of the agent does not raise new clinical safety concerns with respect to the bone marrow); (5) Ancillary products used in the manufacture of HCT/P; (6) Cells, tissues, and organs derived from animals other than humans; (7) In vitro diagnostic products as defined in 21 CFR 809.3(a); and (8) Blood vessels recovered with an organ, as defined in 42 CFR 121.2 that are intended for use in organ transplantation and labeled “For use in organ transplantation only.” (21 CFR 1271.3(d))

Contains Nonbinding Recommendations
Draft – Not for Implementation

Section 1271.10(a)(2) (21 CFR 1271.10(a)(2)) provides that one of the criteria for an HCT/P to be regulated solely under section 361 of the PHS Act is that the “HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.” As defined in 21 CFR 1271.3(c), homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor. This criterion reflects the Agency’s conclusion that there would be increased safety and effectiveness concerns for HCT/Ps that are intended for a non-homologous use, because there is less basis on which to predict the product’s behavior, whereas HCT/Ps for homologous use can reasonably be expected to function appropriately (assuming all of the other criteria are also met).2 In applying the homologous use criterion, FDA will determine what the intended use of the HCT/P is, as reflected by the the labeling, advertising, and other indications of a manufacturer’s objective intent, and will then apply the homologous use definition.

FDA has received many inquiries from manufacturers about whether their HCT/Ps meet the homologous use criterion in 21 CFR 1271.10(a)(2). Additionally, transplant and healthcare providers often need to know this information about the HCT/Ps that they are considering for use in their patients. This guidance provides examples of different types of HCT/Ps and how the regulation in 21 CFR 1271.10(a)(2) applies to them, and provides general principles that can be applied to HCT/Ps that may be developed in the future. In some of the examples, the HCT/Ps may fail to meet more than one of the four criteria in 21 CFR 1271.10(a).

III. QUESTIONS AND ANSWERS

  1. What is the definition of homologous use?

Homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor (21 CFR 1271.3(c)), including when such cells or tissues are for autologous use. We generally consider an HCT/P to be for homologous use when it is used to repair, reconstruct, replace, or supplement:

  • Recipient cells or tissues that are identical (e.g., skin for skin) to the donor cells or tissues, and perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor; or,
  • Recipient cells that may not be identical to the donor’s cells, or recipient tissues that may not be identical to the donor’s tissues, but that perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor.3

2 Proposed Approach to Regulation of Cellular and Tissue-Based Products, FDA Docket. No. 97N-0068 (February. 28, 1997) page 19. http://www.fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/tissue/ ucm062601.pdf.

3“Establishment Registration and Listing for Manufacturers of Human Cellular and Tissue-Based Products” 63 FR 26744 at 26749 (May 14, 1998).

Contains Nonbinding Recommendations
Draft – Not for Implementation

1-1. A heart valve is transplanted to replace a dysfunctional heart valve. This is homologous use because the donor heart valve performs the same basic function in the donor as in the recipient of ensuring unidirectional blood flow within the heart.

1-2. Pericardium is intended to be used as a wound covering for dura mater defects. This is homologous use because the pericardium is intended to repair or reconstruct the dura mater and serve as a covering in the recipient, which is one of the basic functions it performs in the donor.

Generally, if an HCT/P is intended for use as an unproven treatment for a myriad of

diseases or conditions, the HCT/P is likely not intended for homologous use only.4

  1. What does FDA mean by repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues?

Repair generally means the physical or mechanical restoration of tissues, including by covering or protecting. For example, FDA generally would consider skin removed from a donor and then transplanted to a recipient in order to cover a burn wound to be a homologous use. Reconstruction generally means surgical reassembling or re-forming. For example, reconstruction generally would include the reestablishment of the physical integrity of a damaged aorta.5 Replacement generally means substitution of a missing tissue or cell, for example, the replacement of a damaged or diseased cornea with a healthy cornea or the replacement of donor hematopoietic stem/progenitor cells in a recipient with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. Supplementation generally means to add to, or complete. For example, FDA generally would consider homologous uses to be the implantation of dermal matrix into the facial wrinkles to supplement a recipient’s tissues and the use of bone chips to supplement bony defects. Repair, reconstruction, replacement, and supplementation are not mutually exclusive functions and an HCT/P could perform more than one of these functions for a given intended use.

  1. What does FDA mean by “the same basic function or functions” in the definition of homologous use?

For the purpose of applying the regulatory framework, the same basic function or functions of HCT/Ps are considered to be those basic functions the HCT/P performs in the body of the donor, which, when transplanted, implanted, infused, or transferred, the HCT/P would be expected to perform in the recipient. It is not necessary for the HCT/P in the recipient to perform all of the basic functions it performed in the donor, in order to

4 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).

5 “Current Good Tissue Practice for Human Cell, Tissue, and Cellular and Tissue-Based Product Establishments; Inspection and Enforcement” 69 FR 68612 at 68643 (November 24, 2004) states, “HCT/Ps with claims for “reconstruction or repair” can be regulated solely under section 361 of the PHS Act, provided the HCT/P meets all the criteria in § 1271.10, including minimal manipulation and homologous use.”

Contains Nonbinding Recommendations
Draft – Not for Implementation

meet the definition of homologous use. However, to meet the definition of homologous use, any of the basic functions that the HCT/P is expected to perform in the recipient must be a basic function that the HCT/P performed in the donor.

A homologous use for a structural tissue would generally be to perform a structural function in the recipient, for example, to physically support or serve as a barrier or conduit, or connect, cover, or cushion.

A homologous use for a cellular or nonstructural tissue would generally be a metabolic or biochemical function in the recipient, such as, hematopoietic, immune, and endocrine functions.

3-1. The basic functions of hematopoietic stem/progenitor cells (HPCs) include to form and to replenish the hematopoietic system. Sources of HPCs include cord blood, peripheral blood, and bone marrow.6

  1. HPCs derived from peripheral blood are intended for transplantation into an individual with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. This is homologous use because the peripheral blood product performs the same basic function of reconstituting the hematopoietic system in the recipient.
  2. HPCs derived from bone marrow are infused into an artery with a balloon catheter for the purpose of limiting ventricular remodeling following acute myocardial infarction. This is not homologous use because limiting ventricular remodeling is not a basic function of bone marrow.
  3. A manufacturer provides HPCs derived from cord blood with a package insert stating that cord blood may be infused intravenously to differentiate into neuronal cells for treatment of cerebral palsy. This is not homologous use because there is insufficient evidence to support that such differentiation is a basic function of these cells in the donor.

3-2. The basic functions of the cornea include protecting the eye by forming its outermost layer and serving as the refracting medium of the eye. A corneal graft is transplanted to restore sight in a patient with corneal blindness. This is homologous use because a corneal graft performs the same basic functions in the donor as in the recipient.

3-3. The basic functions of a vein or artery include serving as a conduit for blood flow throughout the body. A cryopreserved vein or artery is used for arteriovenous access during hemodialysis. This is homologous use because the vein or artery is supplementing the vessel as a conduit for blood flow.

3-4. The basic functions of amniotic membrane include covering, protecting, serving as a selective barrier for the movement of nutrients between the external and in utero

6 Bone marrow meets the definition of an HCT/P only if is it more than minimally manipulated; intended by the manufacturer for a non-homologous use, or combined with certain drugs or devices.

Contains Nonbinding Recommendations
Draft – Not for Implementation

environment, and to retain fluid in utero. Amniotic membrane is used for bone tissue replacement to support bone regeneration following surgery to repair or replace bone defects. This is not a homologous use because bone regeneration is not a basic function of amniotic membrane.

3-5. The basic functions of pericardium include covering, protecting against infection, fixing the heart to the mediastinum, and providing lubrication to allow normal heart movement within chest. Autologous pericardium is used to replace a dysfunctional heart valve in the same patient. This is not homologous use because facilitating unidirectional blood flow is not a basic function of pericardium.

  1. Does my HCT/P have to be used in the same anatomic location to perform the same basic function or functions?

An HCT/P may perform the same basic function or functions even when it is not used in the same anatomic location where it existed in the donor.7 A transplanted HCT/P could replace missing tissue, or repair, reconstruct, or supplement tissue that is missing or damaged, either when placed in the same or different anatomic location, as long as it performs the same basic function(s) in the recipient as in the donor.

4-1. The basic functions of skin include covering, protecting the body from external force, and serving as a water-resistant barrier to pathogens or other damaging agents in the external environment. The dermis is the elastic connective tissue layer of the skin that provides a supportive layer of the integument and protects the body from mechanical stress.

  1. An acellular dermal product is used for supplemental support, protection, reinforcement, or covering for a tendon. This is homologous use because in both anatomic locations, the dermis provides support and protects the soft tissue structure from mechanical stress.
  2. An acellular dermal product is used for tendon replacement or repair. This is not homologous use because serving as a connection between muscle and bone is not a basic function of dermis.

4-2. The basic functions of amniotic membrane include serving as a selective barrier for the movement of nutrients between the external and in utero environment and to retain fluid in utero. An amniotic membrane product is used for wound healing of dermal ulcers and defects. This is not homologous use because wound healing of dermal lesions is not a basic function of amniotic membrane.

4-3. The basic functions of pancreatic islets include regulating glucose homeostasis within the body. Pancreatic islets are transplanted into the liver through the portal vein,

7 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).

6

Contains Nonbinding Recommendations
Draft – Not for Implementation

for preservation of endocrine function after pancreatectomy. This is homologous use because the regulation of glucose homeostasis is a basic function of pancreatic islets.

  1. What does FDA mean by “intended for homologous use” in 21 CFR 1271.10(a)(2)?

The regulatory criterion in 21 CFR 1271.10(a)(2) states that the HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.

Labeling includes the HCT/P label and any written, printed, or graphic materials that supplement, explain, or are textually related to the product, and which are disseminated by or on behalf of its manufacturer.8 Advertising includes information, other than labeling, that originates from the same source as the product and that is intended to supplement, explain, or be textually related to the product (e.g., print advertising, broadcast advertising, electronic advertising (including the Internet), statements of company representatives).9

An HCT/P is intended for homologous use when its labeling, advertising, or other indications of the manufacturer’s objective intent refer to only homologous uses for the HCT/P. When an HCT/P’s labeling, advertising, or other indications of the manufacturer’s objective intent refer to non-homologous uses, the HCT/P would not meet the homologous use criterion in 21 CFR 1271.10(a)(2).

  1. What does FDA mean by “manufacturer’s objective intent” in 21 CFR 1271.10(a)(2)?

A manufacturer’s objective intent is determined by the expressions of the manufacturer or its representatives, or may be shown by the circumstances surrounding the distribution of the article. A manufacturer’s objective intent may, for example, be shown by labeling claims, advertising matter, or oral or written statements by the manufacturer or its representatives. It may be shown by the circumstances that the HCT/P is, with the knowledge of the manufacturer or its representatives, offered for a purpose for which it is neither labeled nor advertised.

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Keeping Stem Cells in Check

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Researchers track gene that keeps stem cells in check      

“Prkci” influences whether stem cells self-renew or differentiate into more specialized cell types

http://news.usc.edu/88168/researchers-probe-a-gene-that-keeps-stem-cells-in-check/

Prkci gene

http://news.usc.edu/files/2015/10/nestin_tuj_ratreated_escells_WEB-824×549.jpg

When it comes to stem cells, too much of a good thing isn’t wonderful: Producing too many new stem cells may lead to cancer; producing too few inhibits the repair and maintenance of the body.

In a paper published in Stem Cell Reports, USC researcher In Kyoung Mah, who works in the lab of Francesca Mariani, and colleagues at the University of California, San Diego, describe a key gene that maintains this critical balance. Called Prkci, the gene influences whether stem cells self-renew to produce more stem cells or differentiate into more specialized cell types, such as blood or nerves.

In their experiments, the team grew mouse embryonic stem cells, which lacked Prkci, into embryo-like structures in the lab. Without Prkci, the stem cells favored self-renewal, generating large numbers of stem cells and, subsequently, an abundance of secondary structures.

Upon closer inspection, the stem cells lacking Prkci had many activated genes typical of stem cells, and some activated genes typical of neural, cardiac and blood-forming cells. Therefore, the loss of Prkci can also encourage stem cells to differentiate into the progenitor cells that form neurons, heart muscle and blood.

Follow the pathway

Prkci achieves these effects by activating or deactivating a well-known group of interacting genes that are part of the “Notch signaling pathway.” In the absence of Prkci, the Notch pathway produces a protein that signals to stem cells to make more stem cells. In the presence of Prkci, the Notch pathway remains silent, and stem cells differentiate into specific cell types.

These findings have implications for developing patient therapies. Even though Prkci can be active in certain skin cancers, inhibiting it might lead to unintended consequences, such as tumor overgrowth. However, for patients with certain injuries or diseases, it could be therapeutic to use small molecule inhibitors to block the activity of Prkci, thus boosting stem cell production.

“We expect that our findings will be applicable in diverse contexts and make it possible to easily generate stem cells that have typically been difficult to generate,” said Mariani, principal investigator at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC.

Additional co-authors on the study include Rachel Soloff and Stephen Hedrick from UCSD. The research was supported by USC and the Robert E. and May R. Wright Foundation.

PRKCI protein kinase C, iota [ Homo sapiens (human) ]

http://www.ncbi.nlm.nih.gov/gene/5584

Official Symbol PRKCIprovided by HGNCOfficial
Full Name protein kinase C, iota , provided by HGNC

Primary source HGNC:HGNC:9404
See relatedEnsembl:ENSG00000163558; HPRD:02105; MIM:600539; Vega:OTTHUMG00000150214
Gene type protein coding RefSeq status

REVIEWED

Organism Homo sapiens
Lineage – Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo
Also known asPKCI; DXS1179E; nPKC-iota
Summary: This gene encodes a member of the protein kinase C (PKC) family of serine/threonine protein kinases. The PKC family comprises at least eight members, which are differentially expressed and are involved in a wide variety of cellular processes. This protein kinase is calcium-independent and phospholipid-dependent. It is not activated by phorbolesters or diacylglycerol. This kinase can be recruited to vesicle tubular clusters (VTCs) by direct interaction with the small GTPase RAB2, where this kinase phosphorylates glyceraldehyde-3-phosphate dehydrogenase (GAPD/GAPDH) and plays a role in microtubule dynamics in the early secretory pathway. This kinase is found to be necessary for BCL-ABL-mediated resistance to drug-induced apoptosis and therefore protects leukemia cells against drug-induced apoptosis. There is a single exon pseudogene mapped on chromosome X. [provided by RefSeq, Jul 2008]

A Prkci gene keeps stem cells in check

Date:
October 30, 2015
Source:
University of Southern California – Health Sciences
Summary:
When it comes to stem cells, too much of a good thing isn’t wonderful: producing too many new stem cells may lead to cancer; producing too few inhibits the repair and maintenance of the body. Medical researchers now describe a key gene in maintaining this critical balance between producing too many and too few stem cells.

Newly-discovered gene controls stem cell production

|

A scientific team from the University of Southern California (USC) and the University of California, San Diego have described an important gene that maintains a critical balance between producing too many and too few stem cells. Called Prkci, the gene influences whether stem cells self-renew to produce more stem cells, or differentiate into more specialized cell types, such as blood or nerves.

When it comes to stem cells, too much of a good thing isn’t necessarily a benefit: producing too many new stem cells may lead to cancer; making too few inhibits the repair and maintenance of the body.

In their experiments, the researchers grew mouse embryonic stem cells, which lacked Prkci, into embryo-like structures in the laboratory. Without Prkci, the stem cells favored self-renewal, generating large numbers of stem cells and, subsequently, an abundance of secondary structures.

Upon closer inspection, the stem cells lacking Prkci had many activated genes typical of stem cells, and some activated genes typical of neural, cardiac, and blood-forming cells. Therefore, the loss of Prkci can also encourage stem cells to differentiate into the progenitor cells that form neurons, heart muscle, and blood.

Prkci achieves these effects by activating or deactivating a well-known group of interacting genes that are part of the Notch signaling pathway. In the absence of Prkci, the Notch pathway produces a protein that signals to stem cells to make more stem cells. In the presence of Prkci, the Notch pathway remains silent, and stem cells differentiate into specific cell types.

These findings have implications for developing patient therapies. Even though Prkci can be active in certain skin cancers, inhibiting it might lead to unintended consequences, such as tumor overgrowth. However, for patients with certain injuries or diseases, it could be therapeutic to use small molecule inhibitors to block the activity of Prkci, thus boosting stem cell production.

“We expect that our findings will be applicable in diverse contexts and make it possible to easily generate stem cells that have typically been difficult to generate,” said Francesca Mariani, Ph.D., principal investigator at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC.

Their study (“Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway”) was published in a Stem Cell Reports.

Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway
In Kyoung Mah,1 Rachel Soloff,2,3 Stephen M. Hedrick,2 and Francesca V. Mariani1, *
*Correspondence: fmariani@usc.edu    http://dx.doi.org/10.1016/j.stemcr.2015.09.021

Mah et al., Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.09.021

The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.

The control of asymmetric versus symmetric cell division in stem and progenitor cells balances self-renewal and differentiation to mediate tissue homeostasis and repair and involves key proteins that control cell polarity. In the case of excess symmetric division, too many stem-cell-like daughter cells are generated that can lead to tumor initiation and growth. Conversely, excess asymmetric cell division can severely limit the number of cells available for homeostasis and repair (Go´mez-Lo´pez et al., 2014; Inaba and Yamashita, 2012). The Notch pathway has been implicated in controlling stem cell self-renewal in a number of different contexts (Hori et al., 2013). However, how cell polarity, asymmetric cell division, and the activation of determinants ultimately impinges upon the control of stem cell expansion and maintenance is not fully understood. In this study, we examine the role of an atypical protein kinase C (aPKC), PRKCi, in stem cell self-renewal and, in particular, determine whether PRKCi acts via the Notch pathway.

PKCs are serine-threonine kinases that control many basic cellular processes and are typically classified into three subgroups—conventional, novel, and the aPKCs iota and zeta, which, in contrast to the others, are not activated by diacylglyceride or calcium. The aPKC proteins are best known for being central components of an evolutionarily conserved Par3-Par6-aPKC trimeric complex that controls cell polarity in C. elegans, Drosophila, Xenopus, zebrafish, and mammalian cells (Suzuki and Ohno, 2006).

Before Notch influences stem cell self-renewal, the regulation of cell polarity, asymmetric versus symmetric cell division, and the segregation of cell fate determinants such as NUMB may first be required (Knoblich, 2008). For example, mutational analysis in Drosophila has demonstrated that the aPKC-containing trimeric complex is required for maintaining polarity and for mediating asymmetric cell division during neurogenesis via activation and segregation of NUMB (Wirtz-Peitz et al., 2008). NUMB then functions as a cell fate determinant by inhibiting Notch signaling and preventing self-renewal (Wang et al., 2006). In mammals, the PAR3-PAR6-aPKC complex also can bind and phosphorylate NUMB in epithelial cells and can regulate the unequal distribution of Numb during asymmetric cell division (Smith et al., 2007). During mammalian neurogenesis, asymmetric division is also thought to involve the PAR3-PAR6-aPKC complex, NUMB segregation, and NOTCH activation (Bultje et al., 2009).

Mice deficient in Prkcz are grossly normal, with mild defects in secondary lymphoid organs (Leitges et al., 2001). In contrast, deficiency of the Prkci isozyme results in early embryonic lethality at embryonic day (E)9.5 (Seidl et al., 2013; Soloff et al., 2004). A few studies have investigated the conditional inactivation of Prkci; however, no dramatic changes in progenitor generation were detected in hematopoietic stem cells (HSCs) or the brain (Imai et al., 2006; Sengupta et al., 2011), although one study found evidence of a role for Prkci in controlling asymmetric cell division in the skin (Niessen et al., 2013). Analysis may be complicated by functional redundancy between the iota and zeta isoforms and/or because further studies perturbing aPKCs in specific cell lineages and/or at specific developmental stages are needed. Therefore, a complete picture for the requirement of aPKCs at different stages of mammalian development has not yet emerged.

Here, we investigate the requirement of Prkci in mouse cells using an in vitro system that bypasses early embryonic lethality. Embryonic stem (ES) cells are used to make embryoid bodies (EBs) that develop like the early post-implantation embryo in terms of lineage specification and morphology and can also be maintained in culture long enough to observe advanced stages of cellular differentiation (Desbaillets et al., 2000). Using this approach, we provide genetic evidence that inactivation of Prkci signaling leads to enhanced generation of pluripotent cells and some types of multipotent stem cells, including cells with primordial germ cell (PGC) characteristics. In addition, we provide evidence that aPKCs ultimately regulate stem cell fate via the Notch pathway

Figure 1. Prkci/ EBs Contain Cells with Pluripotency Characteristics (A and A0 ) Day (d) 12 heterozygous EBs have few OCT4/E-CAD+ cells, while null EBs contain many in clusters at the EB periphery. Inset: OCT4 (nucleus)/E-CAD (cytoplasm) double-positive cells. (B and B0 ) Adjacent sections in a null EB show that OCT4+ cells are likely also SSEA1+. (C) Dissociated day-12 Prkci/ EBs contain five to six times more OCT4+ and approximately three times more SSEA1+ cells than heterozygous EBs (three independent experiments). (D and D0 ) After 2 days in ES cell culture, no colonies are visible in null SSEA1 cultures while present in null SSEA1+ cultures (red arrows). (E–E00) SSEA1+ sorted cells can be maintained for many passages, 27+. (E) Prkci+/ sorted cells make colonies with differentiated cells at the outer edges (n = 27/35). (E0 ) Null cells form colonies with distinct edges (n = 39/45). (E00) The percentage of undifferentiated colonies is shown. ***p < 0.001.
(F) Sorted null cells express stem cell and differentiation markers at similar levels to normal ES cells (versus heterozygous EBs) (three independent experiments). (G) EBs made from null SSEA1+ sorted cells express germ layer marker genes at the indicated days. Error bars indicate mean ± SEM, three independent experiments. Scale bars, 100 mm in (A, D, and E); 25 mm in (B). See also Figure S1.

Prkci/ Cultures Have More Pluripotent Cells Even under Differentiation Conditions First, we compared Prkci null EB development to that of Prkci/ embryos. Consistent with another null allele (Seidl et al., 2013), both null embryos and EBs fail to properly cavitate (Figures S1A and S1B). The failure to cavitate is unlikely to be due to the inability to form one of the three germ layers, as null EBs express germ-layer-specific genes (Figure S1E). A failure of cavitation could alternatively be caused by an accumulation of pluripotent cells. For example, EBs generated from Timeless knockdown cells do not cavitate and contain large numbers of OCT4-expressing cells (O’Reilly et al., 2011). In addition, EBs generated with Prkcz isoform knockdown cells contain OCT4+ cells under differentiation conditions (Dutta et al., 2011; Rajendran et al., 2013). Thus, we first evaluated ES colony differentiation by alkaline phosphatase (AP) staining. After 4 days without leukemia inhibitory factor (LIF), Prkci/ ES cell colonies retained crisp boundaries and strong AP staining. In contrast, Prkci+/ colonies had uneven colony boundaries with diffuse AP staining (Figures S1F–S1F00). To definitively detect pluripotent cells, day-12 EBs were assayed for OCT4 and E-CADHERIN (E-CAD) protein expression. Prkci+/ EBs had very few OCT4/E-CAD double-positive cells (Figure 1A); however, null EBs contained large clusters of OCT4/E-CAD double-positive cells, concentrated in a peripheral zone (Figure 1A0 ). By examining adjacent sections, we found that OCT4+ cells could also be positive for stage-specific embryonic antigen 1 (SSEA1) (Figures 1B and 1B0 ). Quantification by fluorescence-activated cell sorting (FACS) analysis showed that day-12 Prkci/ EBs had more OCT4+ and SSEA1+ cells than Prkci+/ EBs (Figure 1C). We did not find any difference between heterozygous and wild-type cells with respect to the number of OCT4+ or SSEA1+ cells or in their levels of expression for Oct4, Nanog, and Sox2 (Figures S1I, S1I0 and S1J). However, we did find that Oct4, Nanog, and Sox2 were highly upregulated in OCT4+ null cells (Figure S1G). Thus, together, these data indicate that Prkci/ EBs contain large numbers of pluripotent stem cells, despite being cultured under differentiation conditions.

Functional Pluripotency Tests If primary EBs have a pluripotent population with the capacity to undergo self-renewal, they can easily form secondary EBs (O’Reilly et al., 2011). Using this assay, we found that more secondary EBs could be generated from Prkci/ versus Prkci+/ EBs, especially at days 6, 10, and 16; even when plated at a low density to control for aggregation (Figure S1H). To test whether SSEA1+ cells could maintain pluripotency long term, FACS-sorted Prkci/ SSEA1+ and SSEA1 cells were plated at a low density and maintained under ES cell culture conditions. SSEA1 cells were never able to form identifiable colonies and could not be maintained in culture (Figure 1D). SSEA1+ cells, however, formed many distinct colonies after 2 days of culture, and these cells could be maintained for over 27 passages (Figures 1D0 , 1E0 , and 1E00). Prkci+/ SSEA1+ cells formed colonies that easily differentiated at the outer edge, even in the presence of LIF (Figure 1E). In contrast Prkci/ SSEA1+ cells maintained distinct round colonies (Figure 1E0 ). Next, we determined whether null SSEA1+ cells expressed pluripotency and differentiation markers similarly to normal ES cells. Indeed, we found that Oct4, Nanog, and Sox2 were upregulated in both null SSEA1+ EB cells and heterozygous ES cells. In addition, differentiated markers (Fgf5, T, Wnt3, and Afp) and tissue stem/progenitor cell markers (neural: Nestin, Sox1, and NeuroD; cardiac: Nkx2-5 and Isl1; and hematopoietic: Gata1 and Hba-x) were downregulated in both SSEA1+ cells and heterozygous ES cells (Figure 1F). SSEA1+ cells likely have a wide range of potential, since EBs generated from these cells expressed markers for all three germ layers (Figure 1G). In addition, as expected, EBs made from null SSEA1+ cells were (F) Sorted null cells morphologically abnormal, similar to the EBs made from unsorted Prkci/ ES cells (Figure S1G0 ). Thus, taken together, several assays indicate that the OCT4 and SSEA1+ populations enriched in null EBs represent pluripotent stem cells that can self-renew and have broad differentiation capacity.

ERK1/2 Signaling during EB Development Stem cell self-renewal has been shown to require the activation of the JAK/STAT3 and PI3K/AKT pathways and the inhibition of ERK1/2 and GSK3 pathways (Kunath et al., 2007; Niwa et al., 1998; Sato et al., 2004; Watanabe et al., 2006). We found that both STAT3 and phosphorylated STAT3 levels were not grossly altered and that the p-STAT3/STAT3 ratio was similar between heterozygous and null ES cells and EBs (Figures S2A and S2B). In addition we did not see any difference in AKT, pAKT, or b-CATENIN levels when comparing heterozygous to null ES cells or EBs (Figures S2A and S2C). Thus, the effects observed by the loss of Prkci are unlikely to be due to a significant alteration in the JAK/STAT3, PI3K/AKT, or GSK3 pathways.

Next, we investigated ERK1/2 expression and activation. Consistent with other studies showing ERK1/2 activation to be downstream of Prkci in some mammalian cell types (Boeckeler et al., 2010; Litherland et al., 2010), pERK1/2 was markedly inactivated in Prkci null versus heterozygous ES cells. In addition, during differentiation, null EBs displayed strong pERK1/2 inhibition early (until day 6). Later, pERK1/2 was activated strongly, as the EB began differentiating (Figures 2A and 2B). By immunofluorescence, pERK1/2 was strongly enriched in the columnar epithelium of control EBs, while overall levels were much lower in Prkci/ EBs (Figure 2C). In addition, high OCT4 expression correlated with a marked inactivation of pERK1/2 (Figure 2C). Next, we examined Prkci/ SSEA1+ cells by western blot. We found that SSEA1+ cells isolated from day-12 null EBs had pSTAT3 expression levels similar to whole EBs, while pERK1/2 levels were low (Figure 2D). Thus, these experiments indicate that the higher numbers of pluripotent cells in null EBs correlate with a strong inactivation of ERK1/2.

Figure 2. Prkci and Pluripotency Pathways (A) ERK1/2 phosphorylation (Y202/Y204) is reduced in null ES cells and early day (d)-6 null EBs compared to heterozygous EBs and strongly increased at later stages. The first lane shows ES cells activated (A) by serum treatment 1 day after serum depletion. (B) Quantification of pERK1/2 normalized to non-phosphorylated ERK1/2 (three independent experiments; mean ± SEM; **p < 0.01). (C) pERK1/2 Y202/Y204 is strongly expressed in the columnar epithelium of heterozygous EBs that have just cavitated. Null EBs have lower expression. OCT4 and pERK1/2 expression do not co-localize. Scale bar, 100 mm. (D) pERK1/2Y202/Y204 levels are lower in null SSEA1+ sorted cells than in heterozygous or in null day-12 EBs that have undergone further differentiation. pSTAT3 and STAT levels are unchanged. See also Figure S2.

Neural Stem Cell Fate Is Favored in Prkci/ EBs It is well known that ERK/MEK inhibition is not sufficient for pluripotent stem cell maintenance (Ying et al., 2008); thus, other pathways are likely involved. Therefore, we used a TaqMan Mouse Stem Cell Pluripotency Panel (#4385363) on an OpenArray platform to investigate the mechanism of Prkci action. Day 13 and day 20 Prkci/ EBs expressed high levels of pluripotency and stemness markers versus heterozygous EBs, including Oct4, Utf1, Nodal, Xist, Fgf4, Gal, Lefty1, and Lefty2. However, interestingly, EBs also expressed markers for differentiated cell types and tissue stem cells, including Sst, Syp, and Sycp3 (neural-related genes), Isl1 (cardiac progenitor marker), Hba-x, and Cd34 (hematopoietic markers). Based on this first-pass test, we sought to determine whether loss of Prkci might favor the generation of neural, cardiac, and hematopoietic cell types and/or their progenitors.

First, we found that null EBs contained many more NESTIN- and PAX6-positive cells than heterozygous EBs (Figures 3A and 3B; Figures S3A and S3B) (neural stem A and progenitor markers) (Sansom et al., 2009; Tohyama et al., 1992). In addition, quantification of PAX6 immuno- fluorescence (easier to quantify because of its nuclear localization) using a pixel count method (Fogel et al., 2012) revealed more abundant PAX6+ cells in null EBs versus heterozygous EBs. This difference was no longer evident at day 16, presumably because most of the new neural progenitors had differentiated (Figure 3D). Indeed, differentiated neuronal markers MAP2 and TUJ1 could be expressed in null cell cultures (Figures 3C and 3C0 ). Retinoic acid (RA) treatment both in EBs and ES cells promotes neurogenesis (Xu et al., 2012). We found that, even under RA induction, null cultures contained a larger population of NESTIN+ and a smaller population of TUJ1+ cells when compared to heterozygous cultures (Figures 3E and 3F). Again, null neural progenitors were capable of undergoing some differentiation, since we could find cells expressing NEUROD, NEUN, and MAP2 (Figures 3F0 –3F000). We also assessed neurogenesis in monolayer culture, using media that promotes neural stem cell generation supplemented with a low concentration of RA (Xu et al., 2012). Similar to the EB assay, we found that null ES cells generated a larger NESTIN+ and smaller TUJ1+ population compared to heterozygous ES cells (Figures S3C and S3D). Like in EBs, MAP2- and TUJ1-positive cells could still be found in the null cultures (Figure S3D0 ). Thus, using several different neural-induction assays, we found that the absence of Prkci correlates with the production of more neural progenitors and that, although these cells may favor self-renewal, they are still capable of progressing toward differentiation.

Figure 3. Neural Stem Cell Populations Are Increased in Null EBs (A–C0 ) Prkci/ EBs (B) have more NESTINpositive cells than Prkci+/ EBs (A). (C and C0 ) MAP2 and TUJ1 are expressed in null EBs, similarly to heterozygous EBs (data not shown). (D) EBs were assessed for PAX6 expression, and the images were used for quantification (Figures S3A and S3B). The pixel count ratio of PAX6+ cells in null EBs (green) is substantially higher than that found in heterozygous EBs (black) (three independent experiments; mean ± SEM; *p < 0.05). (E–F000) Day 4 after RA treatment, Prkci/ EBs have more NESTIN- than TUJ1-positive neurons (E and F). However, null cells can still terminally differentiate into NEUROD-, NEUN-, and MAP2-positive cells (F0 –F000). Scale bars, 25 mm in (A and C) and 50 mm in (E). See also Figure S3.

The Generation of Cardiomyocyte and Erythrocyte Progenitors Is Also Favored Next, we examined ISL1 expression (a cardiac stem cell marker) by immunofluorescence and found that Prkci/ EBs contained larger ISL1 clusters compared with Prkci+/ EBs; this was confirmed using an image quantification assay (Figures 4A, 4A0 , and 4C). Differentiated cardiac cells and ventral spinal neurons can also express ISL1 (Ericson et al., 1992); therefore, we also examined Nkx2-5 expression, a better stem cell marker and regulator of cardiac progenitor determination (Brown et al., 2004), by RT-PCR and immunofluorescence. In null EBs, Nkx2-5 was upregulated (Figure 4D). In addition, in response to RA, which can promote cardiac fates in vitro (Niebruegge et al., 2008), cells expressing NKX2-5 were more prevalent in null versus heterozygous EBs (Figures 4B and 4B0 ).The abundant cardiac progenitors found in null EBs were still capable of undergoing differentiation (Figures 4E–4F0 ). Indeed, more cells exhibited the striated pattern characteristic of a-ACTININ in null versus heterozygous EBs with RA induction (Figures 4F and 4F0 ). In addition, many more Prkci/ EBs were beating after days 6 and 12 of culture (Figure 4G).

Figure 4. Cardiomyocyte and Erythrocyte Progenitors Are Increased in Prkci/ EBs (A–F0 ) In (A, A0 , E, and E0 ), Prkci/ EBs cultured without LIF have more ISL1 (cardiac progenitor marker) and a-ACTININ-positive cells compared to heterozygous EBs. (C) At day (d) 9, the pixel count ratio for ISL1 expression indicates that null EBs (green) have larger ISL1 populations than heterozygous EBs (black) (three independent experiments, n = 20 heterozygous EBs, 21 null EBs total; mean ± SEM; *p < 0.05). In (B, B0 , D, F, and F0 ), RA treatment induces more NKX2-5 (both nuclear and cytoplasmic) and a-ACTININ expression in null EBs. Arrows point to fibers in (F0 ). (G) Null EBs (green) generate more beating EBs with RA treatment compared to heterozygous EBs (black) (four independent experiments; mean ± SEM; *p < 0.05, ***p < 0.001). (H) Dissociated null EBs of different stages (green) generate more erythrocytes in a colony-forming assay (CFU-E) (four independent experiments; mean ± SEM; **p < 0.01). (I) Examples of red colonies. (J) Gene expression for primitive HSC markers is upregulated in null EBs (relative to heterozygous EBs) (three independent experiments; mean ± SEM). Scale bars, 50 mm in (A, B, and E); 100 mm in (F), and 25 mm in (I). See also Figure S4.

Hba-x expression is restricted to yolk sac blood islands and primitive erythrocyte populations (Lux et al., 2008; Trimborn et al., 1999). Cd34 is also a primitive HSC marker (Sutherland et al., 1992). Next, we determined whether the elevated expression of these markers observed with OpenArray might represent higher numbers of primitive hematopoietic progenitors. Using a colony-forming assay (Baum et al., 1992), we found that red colonies (indicative of erythrocyte differentiation; examples in Figure 4I) were produced significantly earlier and more readily from cells isolated from null versus heterozygous EBs (Figure 4H). By quantitative real-time PCR, upregulation of Hba-x and Cd34 genes confirmed the OpenArray results (Figure 4J). In addition, we found Gata1, an erythropoiesis-specific factor, and Epor, an erythropoietin receptor that mediates erythroid cell proliferation and differentiation (Chiba et al., 1991), to be highly upregulated in null versus heterozygous EBs (Figure 4J). These data suggest that the loss of Prkci promotes the generation of primitive erythroid progenitors that can differentiate into erythrocytes.

To determine whether the aforementioned tissue stem cells identified were represented in the OCT4+ population that we described earlier, we examined the expression of PAX6, ISL1, and OCT4 in adjacent EB sections. We found that cells expressing OCT4 appeared to represent a distinct population from those expressing PAX6 and ISL1 (although some cells were PAX6 and ISL1 double-positive) (Figures S4A–S4C).

Prkci/ Cells Are More Likely to Inherit NUMB/aNOTCH1 Symmetrically The enhanced production of both pluripotent and tissue stem cells suggests that the mechanism underlying the action of Prkci in these different contexts is fundamentally similar. Because the Notch pathway controls stem cell self-renewal in many contexts (Hori et al., 2013), and because previous studies implicated a connection between PRKCi function and the Notch pathway (Bultje et al., 2009; Smith et al., 2007), we examined the localization and activation of a key player in the Notch pathway, NUMB, (Inaba and Yamashita, 2012). Differences in NUMB expression were first evident in whole EBs, where polarized expression was evident in the ectodermal and endodermal epithelia of heterozygous EBs, while Prkci/ EBs exhibited a more even distribution (Figures 5A–5B0 ). To more definitively determine the inheritance of NUMB during cell division, doublets undergoing telophase or cytokinesis were scored for symmetric (evenly distributed in both cells) or asymmetric (unequally distributed) NUMB localization (examples: Figures 5C and 5C0 ). In dissociated day-10 EBs, Prkci+/ doublets displayed somewhat less symmetric versus asymmetric inheritance, while Prkci/ doublets exhibited nearly four times more symmetric versus asymmetric inheritance (Figure 5D). Although individual cells from null EBs that were OCT4+ or PAX6+ more likely to exhibit non-polarized NUMB distribution (Figures S5A and S5B), we decided to use an assay that allowed for FACS purifi- cation, followed by the more stringent doublet assay. Therefore, we chose CD24 (heat-stable antigen; BA-1), a cell-surface marker that is highly expressed in pre-differentiated neurons and neuroblasts (Pruszak et al., 2009), and tested this marker as a method to enrich for cells destined to differentiate into neurons (see Supplemental Experimental Procedures). To assess NUMB localization, FACSsorted CD24 cells isolated from the RA-treated EBs were then put in culture for 24 hr, and doublets were scored. Both Prkci/ CD24high and CD24low doublets exhibited more symmetric versus asymmetric NUMB localization when compared to Prkci+/ doublets (Figure 5E) (>23 more was observed for CD24low doublets; 1.5 ± 0.25 [null] versus 0.67 ± 0.2 [heterozygous]). Thus, in summary, loss of Prkci favors the generation of cells with symmetric NUMB distribution, even during EB differentiation. In addition, in situations where neurogenesis is stimulated (RA treatment), loss of Prkci favors symmetric NUMB distribution in both the CD24high/low subpopulations.

Because NUMB can be directly phosphorylated by aPKCs (both PRKCi and PRKCz) (Smith et al., 2007; Zhou et al., 2011), loss of Prkci might be expected to lead to decreased NUMB phosphorylation. Three NUMB phosphorylation sites—Ser7, Ser276, and Ser295—could be aPKC mediated (Smith et al., 2007). By immunofluorescence, we found that one of the most well-characterized sites (Ser276), was strongly inactivated in null versus heterozygous EBs, especially in the core (Figures 5F and 5G). Western analysis also confirmed that the levels of pNUMB (Ser276) were decreased in null versus heterozygous EBs (Figure S5F). Thus, genetic inactivation of Prkci leads to a marked decrease in the phosphorylation status of NUMB. Notch pathway inhibition by NUMB has been observed in flies and mammals (Berdnik et al., 2002; French et al., 2002). Therefore, we investigated whether reduced Numb activity in Prkci/ EBs might lead to enhanced NOTCH1 activity and the upregulation of the downstream transcriptional readouts (Meier-Stiegen et al., 2010). An overall increase in NOTCH1 activation was supported by western blot analysis showing that the level of activated NOTCH1 (aNOTCH1) was strongly increased in day 6 and day 10 null versus heterozygous EBs (Figure S5G). This was supported by immunofluorescence in EBs, where widespread strong expression of aNOTCH1 was seen in most null cells (Figures 5I and 5I0 ), while in heterozygous EBs, this pattern was observed only in the OCT4+ cells (Figures 5H and 5H0 ).

Figure 5. Prkci/ Cells Preferentially Inherit Symmetric Localization of NUMB and aNOTCH1 and Notch Signaling Is Required for Stem Cell Self-Renewal in Null Cells (A–B0 ) In (A and B), day (d)-7 heterozygous EBs have polarized NUMB localization within epithelia and strong expression in the endoderm, while null EBs have a more even distribution. (A0 and B0 ) Enlarged views. (C and C0 ) Asymmetric and symmetric NUMB expression examples. (D) Doublets from day-10 null EBs have more symmetric inheritance when compared to day-10 heterozygous doublets (three independent experiments; mean ± SEM; **p < 0.01). A red line indicates a ratio of 1 (equal percent symmetric and asymmetric). (E) CD24 high null doublets exhibited more symmetric NUMB inheritance than CD24 high heterozygous doublets (three independent experiments; mean ± SEM; *p < 0.05). A red line indicates where the ratio is 1. (F and G) Decreased pNUMB (Ser276) is evident in the core of null versus heterozygous EBs (n = 10 of each genotype). (H–I0 ) In (H and I), aNOTCH1 is strongly expressed in heterozygous EBs, including both OCT4+ and OCT4 cells, while strong aNOTCH1 expression is predominant in OCT4+ cells of null EBs (n = 10 of each genotype)). (H0 and I0 ) Enlarged views of boxed regions. OCT4+ cells are demarcated with dotted lines. (J and J0 ) OCT4+ cells express HES5 strongly in the nucleus (three independent experiments). (K) Null doublets from dissociated EBs have more symmetric aNOTCH1 inheritance compared to heterozygous doublets (three independent experiments; mean ± SEM; **p < 0.01). A red line indicates where the ratio is 1. (L) CD24high Prkci/ doublets exhibit more symmetric aNOTCH1 than CD24high heterozygous doublets (three independent experiments; mean ± SEM; *p < 0.05). A red line indicates where the ratio is 1. (M and M0 ) Examples of asymmetric and symmetric aNOTCH1 localization. (N and O) Day-3 DMSO-treated null ES colonies show strong AP staining all the way to the colony edge in (N). Treatment with 3 mM DAPT led to more differentiation in (O). (P–R) OCT4 is strongly expressed in day-4 DMSO-treated null ES cultures (P). With DAPT (Q,R), OCT4 expression is decreased. (S) Working model: In daughter cells that undergo differentiation, PRKCi can associate with PAR3 and PAR6. NUMB is recruited and directly phosphorylated. The activation of NUMB then leads to an inhibition in NOTCH1 activation and stimulation of a differentiation/maintenance program. In the absence of Prkci, the PAR3/PAR6 complex cannot assemble (although it may do so minimally with Prkcz). NUMB asymmetric localization and phosphorylation is reduced. Low levels of pNUMB are not sufficient to block NOTCH1 activation, and activated NOTCH1 preserves the stem cell self-renewal program. We suggest that PRKCi functions to drive differentiation by pushing the switch from an expansion phase that is symmetric to a differentiation and/or maintenance phase that is predominantly asymmetric. In situations of low or absent PRKCi, we propose that the expansion phase is prolonged. Scale bars, 50 mm in (A, B, F, G, H, I, J, J0 , P–R); 200 mm in (A0 and B0 ); 25 mm in (C, C0 , M, and M0 ); and 100 mm in (H0 , I0 , N, and O). See also Figure S5.

To examine the localization of aNOTCH1 and to better quantify the results seen in Figures 5H and 5I, doublets from dissociated EBs were scored. As seen with NUMB localization, null doublets were more likely to have symmetric localization of aNOTCH1 in comparison to heterozygous doublets (Figure 5K; examples in Figures 5M and 5M0 ). In addition, both CD24high and CD24low doublets from RA-treated null EBs were more likely to exhibit symmetric aNOTCH1 distribution versus doublets from RA-treated heterozygous EBs (Figure 5L; 3.46 ± 0.8 [null] versus 0.59 ± 0.06 [heterozygous] in CD24low doublets). In addition, by RT-PCR, the expression of Notch downstream genes Hes1, Hes5, Hey1, and Hey2 was increased in null versus heterozygous EBs (Figure S5I). Furthermore, HES5 by immunofluorescence was broadly expressed at similar levels in both null and heterozygous cells (Figures 5J and 5J0 ; Figures S5H and S5H0 ) but more strongly expressed in null OCT4+ cells (Figures 5J and 5J0 ). Thus, loss of Prkci is associated with NOTCH1 activation, aNOTCH1 symmetric localization, and the upregulation of Hes/Hey downstream genes in several assays.

To determine whether Notch pathway activation is required in the absence of Prkci, we examined AP activity and OCT4 expression while blocking the Notch pathway using DAPT to inhibit g-secretase (Sastre et al., 2001). DMSO-treated null ES cells stayed undifferentiated (sharp-edged colonies, strong AP staining); however, treatment of null ES cells with 3 mM DAPT led to more differentiation (AP-negative cells with cellular extensions) (Figures 5N, 5O, and S5J). In addition, OCT4 is strongly expressed in day-4 control ES cell cultures; however, in the presence of DAPT, OCT4 expression is much decreased both in monolayer culture (Figures 5P–5R) and in null EBs (48% lower OCT4+ signal versus DMSO controls, pixel counting on EB sections; data not shown). These results support the idea that activated Notch signaling is required in the absence of Prkci to see enhanced pluripotency.

Taken together, the combined effects of decreased NUMB activation, favored symmetric distribution of NUMB and aNOTCH1 and increased NOTCH1 activity support a model whereby loss of Prkci leads to sustained generation of pluripotent and some tissue stem cell populations (Figure 5S; and see Discussion).

Additional Loss of PRKCz Activity Boosts the Number of OCT4-, SSEA1-, and STELLA-Positive Cells The generation and maintenance of pluripotent stem cells from new sources or tissue stem cells for basic or translational research can be challenging, and there is need for new in vitro strategies. A PKC inhibitor (Go¨6983) that inhibits PKCa, -b, -g, -d, and -z has been used to help maintain mouse and rat ES cells in the absence of LIF (Dutta et al., 2011; Rajendran et al., 2013). Thus, we hypothesized that treating null cells with Go¨6983 might lead to better stem cell expansion compared to loss of just Prkci. In our hands, we found that, under differentiation conditions (no LIF), heterozygous ES cells treated with the inhibitor for 4 days still underwent differentiation (Figure 6A), while treated null ES cells largely stayed undifferentiated (Figure 6A0 ; Figure S6A). Drug treatment of heterozygous EBs boosted the generation of OCT4-expressing cells (Figure 6B), while treatment of null EBs resulted in an even larger OCT4+ population (Figure 6B0 ). NUMB localization was also moderately affected (Figure S6B). By cell sorting, we found that drug treatment significantly increased the percentage of OCT4+ cells in both Prkci+/ and Prkci/ EBs (Figures 6C and 6C0 ; Figures S6C and S6C0 ). Interestingly, Go¨6983 treatment also boosted the generation of SSEA1+ cells in both null and heterozygous EBs (Figures 6D and 6D0 ; Figures S6D and S6D0 ).

SSEA1 is expressed in BLIMP1-positive PGCs derived from mouse epiblast stem cells (Hayashi and Surani, 2009). Also, PGC-like cells can be derived from isolated SSEA1+/OCT4+ EB cells (Geijsen et al., 2004). Therefore, we speculated that the increase in SSEA1 and OCT4 due to Go¨6983 treatment could represent an increase in the generation of PGC-like cells instead of undifferentiated ES cells. Therefore, we examined the expression of STELLA (a PGC marker). As expected, heterozygous EBs contain small clusters of STELLA+ cells similar to EBs made of wild-type cells (Figure 6E) (Payer et al., 2006). The addition of Go¨6983 to Prkci+/ EBs induced a modest increase in the number of STELLA+ cells present in the clusters (Figure 6F). Without drug treatment, null EBs contained more clusters, and the clusters contained more STELLA+ cells when compared to heterozygous EBs (Figures 6E and 6G). Interestingly, when Prkci/ EBs were treated with Go¨6983, the generation of STELLA+ cells was strongly enhanced (Figure 6G versus Figure 6H). Because undifferentiated ES cells can still express STELLA (Payer et al., 2006), we co-stained Prkc EBs for VASA (a more differentiated PGC marker). We found many cells that were double positive (a little less than half) (Figure 6K) but also cells that expressed VASA only and STELLA only (23 more than VASA only) (Figures 6I–6K, red/green arrows). Therefore, the combined effect of loss of Prkci and PKC inhibition via Go¨6983 treatment leads to the production of STELLA and VASA+ PGC-like cells.

Figure 6. Additional Inhibition of PRKCz Results in an Even Higher Percentage of OCT4-, SSEA1-, and STELLA-Positive Cells (A and A0 ) After day 4 without LIF, heterozygous ES cells undergo differentiation in the presence of Go¨6983, while null ES cells stay as distinct colonies in (A0 ). (B and B0 ) Go¨6983 stimulates an increase in OCT4+ populations in heterozygous EBs and an even larger OCT4+ population in null EBs in (B0 , insets: green and red channels separately). (C–D0 ) An even higher percentage of cells are OCT4+ (C and C0 ) and SSEA1+ (D and D0 ) with Go¨6983 treatment (day 12, three independent experiments). (E and F) More STELLA+ clusters containing a larger number of cells are present in drugtreated heterozygous EBs. (G and H) Null EBs also have more STELLA+ clusters and cells. Drug-treated null EBs exhibit a dramatic increase in the number of STELLA+ cells. (I–K) Some cells are double positive for STELLA and VASA in drug-treated null EBs (yellow arrows). There are also VASAonly (green arrows) and STELLA-only cells (red arrows) (three independent experiments). (L–P) Treatment with ZIP results in an increase in OCT4+ and STELLA+ cells. ZIP treatment also results in more cells that are VASA+ (three independent experiments); n = 11 for Prkci+/, and n = 13 for Prkci+/ + ZIP; n = 14 for Prkci/, and n = 20 for Prkci/ + ZIP; eight EBs assayed for both STELLA and VASA expression). Scale bars, 100 mm in (A and A0 ); 50 mm in (B and B0 ); and 25 mm in (E, I, and L). See also Figure S6. 10 S.

Next, we examined whether the more specific aPKC inhibitor, ZIP, a myristolated aPKC pseudosubstrate with competitive binding to p62, had similar effects (Price and Ghosh, 2013; Tsai et al., 2015; Yao et al., 2013). We found that both heterozygous and null EBs treated with ZIP contained more OCT4+ cells compared to un-treated EBs (Figures 6L–6O). In addition, like Go¨6983, ZIP treatment resulted in a modest increase in the percentage of SSEA1+ cells found in heterozygous EBs and a strong increase in the percentage of SSEA1+ cells in null EBs (Figures S6E– S6F0 ). Furthermore, like Go¨6983, both STELLA+ and VASA+ populations were increased with ZIP treatment (Figure 6P). Thus, both pluripotent and PGC-like cells can be abundantly generated with Go¨6983 or ZIP treatment, suggesting that strategies that inhibit both PRKCi and/or PRKCz may be useful to maintain stem cell self-renewal and/or generate abundant PGC-like cells.

DISCUSSION In this report, we suggest that Prkci controls the balance between stem cell expansion and differentiation/maintenance by regulating the activation of NUMB, NOTCH1, and Hes /Hey downstream effector genes. In the absence of Prkci, the pluripotent cell fate is favored, even without LIF, yet cells still retain a broad capacity to differentiate. In addition, loss of Prkci results in enhanced generation of tissue progenitors such as neural stem cells and cardiomyocyte and erythrocyte progenitors. In contrast to recent findings on Prkcz (Dutta et al., 2011), loss of Prkci does not appear to influence STAT3, AKT, or GSK3 signaling but results in decreased ERK1/2 activation. We hypothesize that, in the absence of Prkci, although ERK1/2 inhibition may be involved, it is the decreased NUMB phosphorylation and increased NOTCH1 activation that promotes stem and progenitor cell fate. Thus, we conclude that PRKCi, a protein known to be required for cell polarity, also plays an essential role in controlling stem cell fate and generation via regulating NOTCH1 activation.

Notch Activation Drives the Decision to Self-Renew versus Differentiate Notch plays an important role in balancing stem cell selfrenewal and differentiation in a variety of stem cell types and may be one of the key downstream effectors of Prkci signaling. Sustained Notch1 activity in embryonic neural progenitors has been shown to maintain their undifferentiated state (Jadhav et al., 2006). Similarly, sustained constitutive activation of NOTCH1 stimulates the proliferation of immature cardiomyocytes in the rat myocardium (Collesi et al., 2008). In HSCs, overexpression of constitutively active NOTCH1 in hematopoietic progenitors and stem cells supports both primitive and definitive HSC selfrenewal (Stier et al., 2002). Together, these studies suggest that activation and/or sustained Notch signaling can lead to an increase in certain tissue stem cell populations. Thus, a working model for how tissue stem cell populations are favored in the absence of Prkci involves a sequence of events that ultimately leads to Notch activation. Recent studies have shown that aPKCs can be found in a complex with NUMB in both Drosophila and mammalian cells (Smith et al., 2007; Zhou et al., 2011); hence, in our working model (Figure 5S), we propose that the localization and phosphorylation of NUMB is highly dependent on the activity of PRKCi. When Prkci is downregulated or absent (as shown here), cell polarity is not promoted, leading to diffuse distribution and decreased phosphorylation of NUMB. Without active NUMB, NOTCH1 activation is enhanced, Hes/Hey genes are upregulated, and stem/progenitor fate generation is favored. To initiate differentiation, polarization could be stochastically determined but could also be dependent on external cues such as the presentation of certain ligands or extracellular matrix (ECM) proteins (Habib et al., 2013). When PRKCi is active and the cell becomes polarized, a trimeric complex is formed with PRKCi, PAR3, and PAR6. Numb is then recruited and phosphorylated, leading to Notch inactivation, the repression of downstream Hes/Hey genes, and differentiation is favored (see Figure 5S). Support for this working model comes from studies in Drosophila showing that the aPKC complex is essential for Numb activation and asymmetric localization (Knoblich, 2008; Smith et al., 2007; Wang et al., 2006). Additional studies on mouse neural progenitors show that regulating Numb localization and Notch activation is critical for maintaining the proper number of stem/progenitor cells in balance with differentiation (Bultje et al., 2009). Thus, an important function for PRKCi may be to regulate the switch between symmetric expansion of stem/progenitor cells to an asymmetric differentiation/maintenance phase. In situations of low or absent PRKCi, we propose that the expansion phase is favored. Thus, temporarily blocking either, or both, of the aPKC isozymes may be a powerful approach for expanding specific stem/progenitor populations for use in basic research or for therapeutic applications.

These studies, together with data presented here, provide genetic evidence that evolutionarily conserved polarity pathways may play a central role in NOTCH1 activation. and stem cell self-renewal in mammals. Further genetic studies using Cre transgenes that are specific for progenitors in the neural plate, primitive erythrocytes, cardiomyocytes, and other progenitors to ablate aPKC function will be needed to determine how generally this mechanism is used in diverse tissues.

Although we do not see changes in the activation status of the STAT3, AKT, or GSK3 pathway, loss of Prkci results in an inhibition of ERK1/2 (Figures 2A and 2B). This result is consistent with the findings that ERK1/2 inhibition is both correlated with and directly increases ES cell selfrenewal (Burdon et al., 1999). Modulation of ERK1/2 activity by Prkci has been observed in cancer cells and chondrocytes (Litherland et al., 2010; Murray et al., 2011). Although it is not clear whether a direct interaction exists between Prkci and ERK1/2, Prkcz directly interacts with ERK1/2 in the mouse liver and in hypoxia-exposed cells (Das et al., 2008; Peng et al., 2008). The Prkcz isozyme is still expressed in Prkci null cells but evidently cannot suf- ficiently compensate and activate the pathway normally. Furthermore, knocking down Prkcz function in ES cells does not result in ERK1/2 inhibition, suggesting that this isozyme does not impact ERK1/2 signaling in ES cells (Dutta et al., 2011). Therefore, although PRKCi may interact with ERK1/2 and be directly required for its activation, ERK1/2 inhibition could also be a readout for cells that are more stem-like. Further studies will be needed to address this question.

Utility of Inhibiting aPKC Function Loss of Prkci resulted in EBs that contained slightly more STELLA+ cells than EBs made from +/ cells. Furthermore, inhibition of both aPKC isozymes by treating Prkci null cells with the PKC inhibitor Go¨6983 or the more specific inhibitor, ZIP, strongly promoted the generation of large clusters of STELLA+ and VASA+ cells, suggesting that inhibition of both isozymes is important for PGC progenitor expansion (Figure 6). It is unclear what the mechanism for this might be; however, one possibility is that blocking both aPKCs is necessary to promote NOTCH1 activation in PGCs or in PGC progenitor cells that may ordinarily have strong inhibitions to expansion (Feng et al., 2014). Regardless of mechanism, the ability to generate PGC-like cells in culture is notoriously challenging, and our results provide a method for future studies on PGC specification and differentiation.

Expansion of stem/progenitor pools may not be desirable in the context of cancer. Prkci has been characterized as a human oncogene, a useful prognostic cancer marker, and a therapeutic target for cancer treatment. Overexpression of Prkci is found in epithelial cancers (Fields and Regala, 2007), and Prkci inhibitors are being evaluated as candidate cancer therapies (Atwood et al., 2013; Mansfield et al., 2013). However, because our results show that Prkci inhibition leads to enhanced stem cell production in vitro, Prkci inhibitor treatment as a cancer therapy might lead to unintended consequences (tumor overgrowth), depending on the context and treatment regimen. Thus, extending our findings to human stem and cancer stem cells is needed. In summary, here, we demonstrate that loss of Prkci leads to the generation of abundant pluripotent cells, even under differentiation conditions. In addition, we show that tissue stem cells such as neural stem cells, primitive erythrocytes, and cardiomyocyte progenitors can also be abundantly produced in the absence of Prkci. These increases in stem cell production correlate with decreased NUMB activation and symmetric NUMB localization and require Notch signaling. Further inhibition of Prkcz may have an additive effect and can enhance the production of PGC-like cells. Thus, Prkci (along with Prkcz) may play key roles in stem cell self-renewal and differentiation by regulating the Notch pathway. Furthermore, inhibition of Prkci and or Prkcz activity with specific small-molecule inhibitors might be a powerful method to boost stem cell production in the context of injury or disease.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures, six figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr. 2015.09.021.

REFERENCES

Atwood, S.X., Li, M., Lee, A., Tang, J.Y., and Oro, A.E. (2013). GLI activation by atypical protein kinase C i/l regulates the growth of basal cell carcinomas. Nature 494, 484–488.

Baum, C.M., Weissman, I.L., Tsukamoto, A.S., Buckle, A.M., and Peault, B. (1992). Isolation of a candidate human hematopoietic stem-cell population. Proc. Natl. Acad. Sci. USA 89, 2804–2808.

Berdnik, D., To¨ro¨k, T., Gonza´lez-Gaita´n, M., and Knoblich, J.A. (2002). The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila.

Dev. Cell 3, 221–231. Boeckeler, K., Rosse, C., Howell, M., and Parker, P.J. (2010). Manipulating signal delivery – plasma-membrane ERK activation in aPKC-dependent migration. J. Cell Sci. 123, 2725–2732.

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USC offers a summer of stem cells for local high school students

The teens boost their scientific IQ by conducting research in USC labs

The goal of these unique programs is to educate bright young minds at the stage where they’re still formulating ideas and still open and receptive to new discoveries.

Andrew McMahon

Twenty-three local high school students spent their summer vacations in a very unusual place: the Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC.

The students celebrated their graduations this month from the USC Early Investigator High School (EiHS) and the USC CIRM Science, Technology and Research (STAR) programs. These are the only programs that offer comprehensive training in stem cell research to high school students.

“The goal of these unique programs is to educate bright young minds at the stage where they’re still formulating ideas and still open and receptive to new discoveries, and introduce them to the wonder and inspirational power of stem cell biology,” said Andrew McMahon, director of USC’s stem cell research center and the Department of Stem Cell Biology and Regenerative Medicine, and head of the university-wide USC Stem Cell initiative uniting more than 100 researchers from all disciplines.

Stem cells, ethics and public policy

Over the course of the summer, the high school students participated in either a 10-day training course or eight-week research internship, working with human stem cells in USC’s world-class laboratories. 

Under the mentorship of USC faculty and graduate students, the students learned about the latest advances in regenerative medicine and explored stem cells, ethics and public policy.

Roberta Diaz Brinton, director of the CIRM STAR program, paid tribute to the accomplishments of the students.

“We’re very impressed by the caliber of science and more impressed by the caliber of young minds. These young scientists are generating the new knowledge from which stem cell biology and stem cell therapies will progress in the future,” said Brinton, professor at the USC School of Pharmacy, the USC Viterbi School of Engineering and the Keck School of Medicine of USC, and an executive committee member of USC Stem Cell.

True teamwork

Victoria Fox, director of the EiHS program, extended her thanks to everyone who contributed to the experience.

“The EiHS program was made possible by a team of very incredible people that starts with my laboratory staff and includes donors, the students, the administrators of the stem cell research center and the mentors who take the students in their laboratories,” she said. “I’m very grateful to all of these people.”

This year’s participants were selected from Harvard-Westlake School, Lifeline Education Charter School, Chadwick School and Bravo Medical Magnet High School, and many received scholarships.

“The program has motivated our students to be college-ready by giving them the opportunity to work in a university setting,” said Obed Nartey, principal of Lifeline Education Charter School. “Many of these students are the first generation to graduate from high school. For these students, college was seen as being out of reach until they met and worked with Dr. Fox and her team.”

On graduation day, the students shared their transformative summer experiences with their mentors, friends, parents and teachers by presenting scientific posters and by contributing articles to the program’s new EiHS Journal, which will publish its first issue in October.

“Being able to contribute to a scientific project that can play an important role in someone’s life is an amazing opportunity, and I would not trade it for the world,” said Marialuisa Flores, a student from Lifeline Education Charter School. “It was a very enjoyable learning experience, which has made a great impact on my life and future career.”

“Being able to contribute to a scientific project that can play an important role in someone’s life is an amazing opportunity, and I would not trade it for the world,” said Marialuisa Flores, a student from Lifeline Education Charter School. “It was a very enjoyable learning experience, which has made a great impact on my life and future career.”

A retreat from everything but stem cells

BY Cristy Lytal

It wasn’t the pristine 27-hole course that drew more than 120 stem cell researchers from USC and beyond to the Desert Princess Golf Resort near Palm Springs, Calif. It was the sixth annual retreat for the Eli and Edy the Broad Center for Regenerative Medicine and Stem Cell Research at USC, which took place on Oct. 20-­21.

The two-day, overnight retreat featured a plenary lecture by Clive Svendsen, director of the Regenerative Medicine Institute at Cedars-Sinai Medical Center, about the contribution of induced pluripotent stem (iPS) cells to regenerative medicine, particularly to studying and developing treatments for neurological disorders.

The retreat also included presentations by winners of the first Regenerative Medicine Initiative (RMI) Awards, which provide up to two years of seed funding for multi-investigator research collaborations that harness the full potential of USC-affiliated faculty members. The three winning teams are using various stem/progenitor cells that might lead to future therapies for certain forms of deafness, bone defects and pediatric leukemia.

Many other principal investigators, postdoctoral and graduate students shared innovative research advancing several key areas of regenerative medicine.

Rong Lu, who will leave Stanford University to join USC’s stem cell research center as a principal investigator in January, talked about her new cellular “tracking system” for hematopoietic, or blood-forming stem cells. The system allows for the more effective study of blood and other types of cancers.

Min Yu, who will leave Massachusetts General Hospital at Harvard Medical School to accept a joint appointment as a principal investigator at USC’s stem cell research center and the USC Norris Comprehensive Cancer Center in January, discussed how to filter out circulating cancer stem cells from billions of other blood cells to understand and stop cancer’s spread.

USC research associate Hu Zhao and research assistants Yichen Li and Yingxiao Shi gave presentations.

Postdoctoral students who presented research included Mohamed Hammad, Lori O’Brien, Sandeep Paul and Saaket Varma.

PhD student presenters included Wen-Hsuan Chang, Guanyi Huang, Sapna Jain, Erin Moran, Marie Rippen and Yuki Yamaguchi.

The retreat also showcased the USC stem cell research center’s core facilities for stem cell sorting, derivation, culture, iPS programming, imaging and therapeutic screening.

During the cocktail hour, guests exchanged new ideas while voting on their favorite posters, which introduced research opportunities related to the Development, Stem Cells, and Regenerative Medicine PhD program.

Retreat sponsors included the California Institute for Regenerative Medicine Amgen, Sanofi, Zeiss, Leica Microsystems, Fluidigm, Lonza and Novogenix Laboratories LLC.

“This year’s retreat was a great success,” said Andrew McMahon, who spearheads the USC Stem Cell initiative and directs the Broad Center. “It helped solidify USC Stem Cell as an interactive scientific community and build relationships with our colleagues at the university and beyond.”

$1.5M Goes to Stem Cell Research

$6.4M for Stem Cell Labs to USC, CHLA

$25 million Broad Foundation gift creates stem cell institute at USC

McMahon discusses central role of stem cell biology in medicine of the future

Andrew McMahon is a Provost Professor and inaugural holder of the W.M. Keck Professorship of Stem Cell Biology and Regenerative Medicine at USC. (Photo/Philip Channing)

McMahon installed as chair of stem cell biology

Army Research Laboratory selects USC institute as base for breakthroughs in science and technology

Brainpower applied to understanding of neural stem cells

Cristy Lytal
BY Cristy Lytal   OCTOBER 24, 2013

How do humans and other mammals get so brainy? USC researcher Wange Lu and his colleagues shed new light on this question in a paper published in the journal Cell Reports on Oct. 24.

The researchers donned their thinking caps to explain how neural stem and progenitor cells differentiate into neurons and related cells called glia. Neurons transmit information through electrical and chemical signals; glia surround, support and protect neurons in the brain and throughout the nervous system. Glia do everything from holding neurons in place to supplying them with nutrients and oxygen to protect them from pathogens.

By studying the embryo neural stem cells of mice in a petri dish, Lu and his colleagues discovered that a protein called SMEK1 promotes the differentiation of neural stem and progenitor cells. At the same time, SMEK1 keeps these cells in check by suppressing their uncontrolled proliferation.

The researchers also determined that SMEK1 doesn’t act alone: It works in concert with Protein Phosphatase 4 to suppress the activity of PAR3, a third protein that discourages neurogenesis — the birth of new neurons. With PAR3 out of the picture, neural stem cells and progenitors are free to differentiate into new neurons and glia.

“These studies reveal the mechanisms of how the brain keeps the balance of stem cells and neurons when the brain is formed,” said Wange Lu, associate professor of biochemistry and molecular biology at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC. “If this process goes wrong, it leads to cancer or mental retardation or other neurological diseases.”

neural stem cells

Neural stem and progenitor cells offer tremendous promise as a future treatment for neurodegenerative disorders, and understanding their differentiation is the first step toward harnessing the cells’ therapeutic potential. This could offer new hope for patients with Alzheimer’s, Parkinson’s and many other currently incurable diseases.

Co-authors from the Broad Center included Vicky Yamamoto, Si Ho Choi and Zhong Wei. Co-authors Hee-Ryang Kim and Choun-Ki Joo work at the Catholic University of Korea in Seoul, and first author Jungmook Lyu is affiliated with both institutions.

Funding for the study came from the National Institutes of Health (grant number 5R01NS067213).

Protein phosphatase 4 and Smek complex negatively regulate Par3 and promote neuronal differentiation of neural stem/progenitor cells.
Cell Rep. 2013 Nov 14;5(3):593-600. http://d.doi.org:/10.1016/j.celrep.2013.09.034. Epub 2013 Oct 24.
Neural progenitor cells (NPCs) are multipotent cells that can self-renew and differentiate into neurons and glial cells. However, mechanisms that control their fate decisions are poorly understood. Here, we show that Smek1, a regulatory subunit of the serine/threonine protein phosphatase PP4, promotes neuronal differentiation and suppresses the proliferative capacity of NPCs. We identify the cell polarity protein Par3, a negative regulator of neuronal differentiation, as a Smek1 substrate and demonstrate that Smek1 suppresses its activity. We also show that Smek1, which is predominantly nuclear in NPCs, is excluded from the nucleus during mitosis, allowing it to interact with cortical/cytoplasmic Par3 and mediate its dephosphorylation by the catalytic subunit PP4c. These results identify the PP4/Smek1 complex as a key regulator of neurogenesis.

Neural stem and progenitor cells located in the ventricular zone (VZ) of the embryonic neocortex are mitotically active, self-renewing cells with the potential to produce differentiated cell types (Temple, 2001). During cortical development, postmitotic neurons generated from NPCs migrate radially out of the VZ and form the cortical plate (CP) in an “inside-out pattern,” eventually establishing a six-layered cortex (Kriegstein et al., 2006). The timing of neuronal differentiation determines the size of the progenitor pool, the final number of neurons, and cortical thickness. However, the molecular mechanisms that control the switch from proliferation to neuronal differentiation of NPCs remain incompletely understood.

Studies of Drosophila neuroblasts show that the serine/threonine protein phosphatase 2A (PP2A) inhibits self-renewal and promotes neuronal differentiation by regulating the phosphorylation status of cell fate determinants, including Numb (Wang et al., 2009). Bazooka, a key component of the Par protein complex, is a well-characterized PP2A substrate in Drosophila neuroblasts (Krahn et al., 2009; Ogawa et al., 2009). PP2A antagonizes phosphorylation of Bazooka by Par1 kinase to control its subcellular localization. In mammals, a protein called Partitioning-defective 3 (Par3), the ortholog of Bazooka, accumulates at the tip of a growing axon in neurons and controls axon specification (Shi et al., 2003). Recently, it has been shown that Par3, which is enriched in the apical domain of NPCs of the VZ (Imai et al., 2006), critically regulates proliferation versus differentiation during cortical development (Bultje et al., 2009; Costa et al., 2008).

PP4, which belongs to the PP2A family, is a protein complex comprised of a catalytic subunit PP4c plus regulatory subunits (Gingras et al., 2005). Smek (also termed PP4R3) has been identified as a PP4 regulatory subunit and implicated in activities as diverse as regulation of MEK (Mendoza et al., 2005), insulin/IGF-1 signaling (Wolff et al., 2006), H2AX phosphorylation (Chowdhury et al., 2008), and histone H3 and H4 acetylation (Lyu et al., 2011). A recent study reported that Falafel (Flfl), the Drosophila homolog of Smek, mediates localization of the adaptor protein Miranda and the cell fate determinant Prospero in neuroblasts (Sousa-Nunes et al., 2009). However, the direct substrate of Smek remains unclear. Here we identify Par3 as a direct substrate of the PP4/Smek1 complex in NPCs and report a novel role for Smek1 in regulating neuronal differentiation.

Smek1 is required for neuronal differentiation of NPCs

During mouse cortical development, Smek1 is expressed in a distinct temporal and spatial pattern. At E11.5, we observed that Smek1 protein is expressed in most NPCs at the apical side of the forebrain VZ (Figure 1Aand S1A). At E14.5, Smek1 protein was detectable primarily in CP neurons (Figure 1B and S1B), while weak Smek1 expression was seen in some NPCs undergoing mitosis at the ventricle surface (Figure 1B, boxes). Interestingly, VZ neurons that migrate to the CP also expressed Smek1 protein (Figure 1B, arrows). In postnatal forebrain, Smek1 protein expression remained detectable in cortical layers I-IV (Figure S1C). Moreover, E14 cortices of Smek1-depleted mice (Smek1gt/gt) exhibited an increase in the number of Pax6-positive cells (an NPC marker) and a decrease in the number of Tbr1-positive cells (a marker of cortical neurons) as compared to E14 cortices of wild-type (Smek1+/+) mice (Figure 1C and S1D).

Figure 1

Smek1 regulates neuronal differentiation in the early phase of NPC differentiation

To assess Smek1’ function in neurogenesis, we employed an in vitro culture system using NPCs isolated from the E11.5 mouse forebrain neocortex. NPCs transduced with lentivirus expressing shRNA againstSmek1 or control shRNA under control of a doxycycline-inducible promoter (Figure S1E) were cultured in medium containing doxycycline for 6 days under differentiating conditions and then assessed for neurogenesis using TUJ1 (a marker of immature neurons) or MAP2 (a marker of mature neurons). The number of TUJ1- or MAP2-positive cells significantly decreased in Smek1 knockdown cultures compared to cultures expressing control shRNA (Figure 1D), indicating a neuronal differentiation defect. A decrease in number of neurons can be caused by a defect in NPC proliferation or neuronal apoptotic cell death. While no significant difference in the number of apoptotic cell death (as determined by TUNEL staining) was observed between control and Smek1 knockdown cells cultured under differentiation condition (data not shown),Smek1 knockdown NPCs grown under proliferation conditions underwent hyperproliferation (Figure S1F and G). We then asked whether Smek1 regulated the transition of NPCs from proliferative to differentiation states by knocking down Smek1 in NPCs prior to placing them in differentiating culture conditions. Western blotting of cells expressing Smek1 shRNA showed decreased levels of TUJ1 protein relative to controls by day 1 of culture (Figure S1H). At this time point, we found that the percentage of undifferentiated NPCs expressing both Nestin (an NPC marker) and Ki67 (a marker of proliferation) or Pax6 increased in cultures expressing Smek1 shRNA compared to control cultures, while the percentage of TUJ1-positive cells significantly decreased (Figure 1E and F). These findings suggest that Smek1 is required for neuronal differentiation and suppression of NPC proliferative capacity at an early phase of differentiation.

Smek1 recruits PP4c to promote neuronal differentiation

To determine Smek1 as a regulatory subunit of PP4 in neurogenesis, we asked whether Smek1 binds to the catalytic subunit PP4c in NPCs using co-immunoprecipitation. Western blot analysis revealed PP4c in Smek1 but not control immunoprecipitates, indicating that Smek1 physically interacts with PP4c. Such interactions did not change during differentiation (Figure 2A). To examine whether PP4c functions in neurogenesis, NPCs were exposed to lentivirus expressing PP4c or control shRNA and cultured as described in Figure 1D. PP4c knockdown led to changes similar to those accompanying Smek1 knockdown: relative to control cultures TUJ1 expression and the number of TUJ1-positive neurons decreased while Pax6-positive NPCs increased (Figure 2B and S2A and B). We next mapped Smek1 domains required for PP4c interaction. Smek contains four conserved domains: an N-terminal Ran-binding domain (RanBD), a domain of unknown function 625 (DUF625), an armadillo (Arm) repeat region, and a C-terminal nuclear localization sequence (NLS). We constructed a series of Flag-tagged deletion mutants, including Smek1ΔRanBD (lacking amino acid (aa) 2–100), ΔDUF625 (lacking aa 162-355), ΔArm (lacking 350-653), and ΔNLS (lacking aa 809-820) (Figure 2C, top) and introduced them or a wild-type construct into NPCs. PP4c was not be detected in anti-Flag immunoprecipitates from NPCs expressing Flag-Smek1ΔArm (Figure 2C, bottom) but was detected in cells expressing wild-type or other deletion mutants, suggesting that PP4c/Smek1 complex formation requires the Arm repeats. We also found that, while expression of wild-type Smek1 or corresponding ΔNLS mutant in cultures lacking endogenous Smek1 rescued the neuronal differentiation defect, the other mutants did not (Figure 2D and S2C and D). These results indicate that Smek1 regulates neuronal differentiation via its Arm repeats region through PP4c and suggest that both RanBD and DUF625 domains also participate in neurogenesis.

Figure 2

PP4c is required for neuronal differentiation   
Smek1 binds to and mediates Par3 dephosphorylation

To identify PP4 substrates regulated by Smek1 in NPCs, we employed affinity purification to purify proteins interacting with Smek1. Mass spectrometry analysis identified potential Smek1-binding proteins, including Par3, Kinesin-like protein, coiled-coil domain-containing protein 30 (CCDC 30), heat shock protein 90 (HSP90), PKC lambda, and HDAC1 Figure S3A. Among these, Par3, an intrinsic regulator of neurogenesis, is a particularly attractive candidate (Bultje et al., 2009; Costa et al., 2008). Using an antibody that detects the major isoforms (180, 150, and 100 kDa) of Par3, Western blot analysis revealed the predominant expression of two isoforms, 180 and 100 kDa forms, in NPCs, and that only the 180 kDa Par3 was detectable in Smek1 immunoprecipitates (Figure 3A). To determine whether Smek1/Par3 binding was direct, we performed an in vitro pull-down assay using purified Flag-Smek1 and His-fused Par3 fragments, the latter containing the CR1 domain (aa 1-338), the PDZ domain (aa 343-733), the aPKC-BR domain (aa 711-1054), or the C-terminal coiled-coil region (aa 1055-1334) (Figure S3B). Western blot analysis revealed that Flag-Smek1 pulled down only the Par3 coiled-coil region (Figure 3B), indicating direct binding through that region. Moreover, Par3 was detected in Flag immunoprecipitates derived from NPCs transduced with lentivirus expressing Myc-Par3 plus lentivirus expressing Flag-Smek1 wild-type or Smek1ΔRanBD, Smek1ΔArm, or Smek1ΔNLS constructs but not from NPCs expressing Smek1ΔDUF625 (Figure 3C). This result indicates that Smek1 DUF625 domain is required for Smek1/Par3 interaction.

Figure 3

Smek1 interacts with Par3 and inhibits its function in neuronal differentiation

To assess potential dephosphorylation of Par3 by Smek1, we phosphorylayed Myc-Par3 protein in vitro by incubating it with an NPC lysate and 32P-ATP and then treated it with a complex containing Flag-Smek1 proteins (Figure 3D). 32P-labeling of Par3 was significantly decreased when Par3 protein was incubated with a complex containing wild-type Flag-Smek1 protein and PP4c (Figure 3D). By contrast, treatment with a Flag-Smek1ΔArm protein complex lacking PP4c binding significantly reduced Par3 dephosphorylation. Moreover, Western blot analysis of Par3 immunoprecipitates with an anti-phospho-serine/threonine antibody confirmed that Smek1 and PP4c regulate Par3 phosphorylation through serine/threonine residues (Figure S3C). Since the DUF625 and Arm repeats regions of Smek1 are required for binding to Par3 and PP4c respectively, we examined the Par3 phosphorylation state in NPCs expressing Flag-tagged wild-type or mutant Smek1 together with Myc-tagged Par3. Western blot analysis of Myc-Par3 immunoprecipitates using anti-phospho-serine/threonine antibody showed that overexpression of wild-type Smek1 or Smek1ΔNLS significantly decreased Par3 phosphorylation levels compared to controls, whereas overexpression of Smek1ΔRanBD, ΔDUF625, or ΔArm did not (Figure 3E). These results suggest that, in addition to the DUF625 and Arm, the RanBD domain of Smek1 participates in regulation of Par3 phosphorylation at serine/threonine residues.

Smek1 negatively regulates Par3 in neurogenesis

Next we asked whether Par3 is required for Smek1-mediated neurogenesis. To this end, we assessed the effect of Smek1 loss-or gain-of function on neuronal differentiation in the presence or absence of Par3. NPCs expressing either Smek1 shRNA or wild-type Smek1 were transduced with lentivirus expressing Par3 or control shRNA (Figure S3D). At day 1 after differentiation, in the presence of Par3, knockdown of Smek1 led to a decrease in the number of TUJ1-positive neurons and an increase in the number of Nestin/Ki67 double-positive NPCs, while overexpression of Smek1 had the opposite effect (Figure 3F and S3E). In the absence of Par3 by using shRNA, the number of TUJ1-positve cell was increased and the number of Nestin/Ki67 double positive NPCs was decreased. However, in these cultures knockdown or overexpression of Smek1 did not significantly alter the number of neurons or undifferentiated NPCs. In addition, we also observed increased expression of mRNAs encoding the Notch targets Hes1 and Hes5 in cells expressing Smek1 shRNA compared to control cells (Figure S3F). Moreover, analysis of Notch reporter gene activity revealed that wild-type Smek1 inhibited Notch signaling activity induced by Par3 overexpression, while Smek1ΔDUF625 did not (Figure S3G). Given that Par3 activates Notch signaling (Bultje et al., 2009), these results suggest that Smek1 acts upstream of Par3 to negatively regulate its activity in neurogenesis.

Par3 loss of function promotes neuronal differentiation (Costa et al., 2008), consistent with the effect seen following Smek1 overexpression (Figure 3F). To confirm that Smek1 promotes neurogenesis by suppressing Par3 function, we transduced NPCs with lentiviruses expressing Par3 alone or Par3 together with wild-type Smek1 or Smek1ΔDUF625, cultured them under differentiation conditions, and then neuronal differentiation was quantified by determining the percentage of TUJ1-positive and Nestin/Ki67 double-positive cells one day later. Par3 overexpression decreased the number of TUJ1-positive neurons and increased the number of Nestin/Ki67-positive undifferentiated NPCs compared with control cells (Figure 3G and S3H). As expected, wild-type Smek1 negated the effect of Par3 overexpression, as determined by comparing the percentage of TUJ1-positive and Nestin/Ki67 double-positive cells in cultures expressing both Smek1 and Par3 to cultures expressing Par3 alone. In comparison with wild-type Smek1, no significant change was seen in cultures transduced with Smek1ΔDUF625, which cannot bind Par3. These experiments further confirm that Smek1 negatively regulates Par3 in NPC differentiation.

Dynamic changes in Smek1 subcellular localization facilitate targeting of PP4 to Par3

Par3 localizes to the apical cortex of NPCs (Bultje et al., 2009), while Smek1 is predominantly nuclear (Figure 1A and C). To determine if changes in Smek1 subcellular localization occur in NPCs during neurogenesis, coronal sections from E11.5 forebrain were immunostained with anti-Smek1 and -α-tubulin (a cytoplasmic marker) antibodies. Smek1 co-localized with α-tubulin in cells on the ventricular surface (Figure 4A, arrows), indicating a cytoplasmic/cortical localization in mitotic NPCs. In mitotic cells, Par3 showed a similar localization (Figure S4A and B). Moreover, immunostaining of NPC cultures with anti-Smek1 and -α-tubulin antibodies showed that Smek1 undergoes dynamic changes in subcellular localization during mitosis. While Smek1 was nuclear in interphase and prophase cells, it showed a cytoplasmic/cortical localization from prometaphase to anaphase (Figure 4B and S4C). Metaphase and anaphase cells also showed Smek1 enrichment at spindle microtubules.

Figure 4

Smek1 regulates subcellular localization of PP4c but not Par3

The RanBD motif of the Dictyostelium discoideum Smek homolog is reportedly critical for its cytoplasmic/cortical localization (Mendoza et al., 2005). To test whether this was the case for mammalian Smek1, Smek1-depleted NPCs were transduced with constructs encoding Flag-tagged wild-type Smek1 or its deletion mutants and immunostained with anti-Flag and anti-phospho-histone H3 (a marker of mitosis and chromatin condensation). Consistent with results reported in Dictyostelium discoideum, the Smek1ΔRanBD mutant failed to localize to the cytoplasm/spindle during mitosis but rather localized in the nucleus and remained there in interphase (Figure 4C). The subcellular localization of other mutants tested resembled that of wild-type Smek1, with the exception of Smek1ΔNLS, which was expressed in both the nucleus and cytoplasm of interphase cells. Smek1ΔRanBD contains domains that can bind Par3 and PP4c, as shown by immunoprecipitation (Figure 2C and and3C).3C). We thus asked whether ectopic expression of Smek1ΔRanBD promoted mislocalization of Par3 and PP4c during mitosis. When we expressed Smek1ΔRanBD ectopically in Smek1-depleted NPCs, cytoplasmic/cortical Par3 remained unchanged while Smek1ΔRanBD was nuclear (Figure S4D). In addition, no difference in localization of Par3 between Smek1-depleted and wild-type Smek1 re-expressing cells was observed, suggesting that Smek1 does not alter Par3 localization. To evaluate PP4c subcellular localization, chromosome-associated and cytosolic protein fractions were isolated from M phase-synchronized NPCs and compared by Western analysis using indicated antibodies (Figure 4D). Interestingly, PP4c protein levels increased in the chromosomal fraction from cells expressing Flag-Smek1ΔRanBD compared to control cells or wild-type Flag-Smek1, while in the cytosolic fraction the level of PP4c protein decreased, indicating altered localization of cytoplasmic PP4c to the nucleus. Taken together, these results demonstrate that PP4c subcellular localization depends on Smek1 localization during mitosis and suggest that cytoplasmic/cortical localization of Smek1 targets PP4 to Par3.

DISCUSSION

Neural stem and progenitor cells have been suggested as potential therapeutics for neurodegenerative disorders. However, understanding molecular and cellular mechanisms underlying their differentiation is a prerequisite to manipulating stem cell behavior. We show that Smek1, an evolutionarily conserved regulatory subunit of PP4, regulates neuronal differentiation and reveal an unreported function of PP4 in mammalian neurogenesis. Moreover, identification of Par3 as a novel Smek1-interacting protein and characterization of its conserved domains reveals a molecular mechanism by which Smek1 targets PP4 to Par3 during mitosis and negatively regulates Par3 function in neurogenesis.

In this study we identify Par3 as a PP4 substrate. We propose that Smek1, through its DUF625 domain, binds directly to the Par3 C-terminus. In NPCs Par3 is primarily cytoplasmic in interphase and mitosis. Thus, nuclear export of Smek1 to the cytoplasm is required for its interaction with Par3. We show dynamic changes in Smek1 subcellular localization in NPCs. While Smek1 localizes exclusively to the nucleus in interphase, during mitosis it becomes cytoplasmic. The RanBD of several proteins reportedly recognizes GTP-bound Ran (RanGTP), which directs assembly of spindle microtubules allowing chromosomal segregation and cytokinesis in mitosis (Carazo-Salas et al., 2001). Smek1 enrichment at spindle microtubules in metaphase and anaphase cells suggests that its RanBD may function in a RanGTP-dependent pathway during mitosis. Notably, nuclear export of Smek1 to the cytoplasm was observed from prometaphase cells when microtubules invade the nuclear space, and deletion of the Smek1 RanBD abolished this effect, as seen by nuclear localization. Thus our data suggest that Smek1 subcellular localization is regulated through the RanBD and that this activity may depend on microtubule dynamics functioning in a Ran-dependent pathway.

Most Smek homologs physically interact with the catalytic subunit PP4c (Gingras et al., 2005; Chowdhury et al., 2008), suggesting that the PP4 complex is evolutionarily conserved. We show that PP4c recognizes the Arm repeats region of Smek1 and its subcellular localization depends on Smek1 localization. Thus, nuclear export of Smek1 during mitosis facilitates dephosphorylation of Par3. This idea is supported by our observation that, while expression of Smek1 induced Par3 dephosphorylation in NPCs, expression of Smek1 mutants lacking RanBD and Arm repeats region did not. Interestingly, studies of Drosophila neuroblasts previously revealed that cell fate specification is tightly linked with phosphorylation status of bazooka protein (Betschinger et al., 2003; Krahn et al., 2009). However, it is now yet clear whether Par3 dephosphorylation directly regulates NPC neurogenesis. Although we could not identify specific phosphorylation sites targeted by PP4, our data defines three conserved domains of Smek1, namely RanBD, DUF625, and Arm repeats, necessary to target PP4 to its substrate Par3 and provides insight into the molecular mechanism by Smek1 to regulate PP4 function in NPCs.

We here show that Smek1 suppresses Par3, a negative regulator of neuronal differentiation. Par3 acts upstream of Notch signaling (Bultje et al., 2009), which critically regulates cell fate decision of NPCs in cortical development (Gaiano and Fishell, 2002). Notch gain-of-function activity inhibits neuronal differentiation (Nye et al., 1994), an effect similar to Smek1 loss-of-function. Moreover, Smek1 inhibits Par3-induced Notch reporter gene activity. Although it remains unclear whether Smek1 inhibits Par3’s ability to activate Notch signaling during mitosis, ensuring a neuronal fate, our data demonstrate Smek1 as a negative regulator of Par3 in regulating neuronal differentiation and suggest a novel role for PP4 in mammalian neurogenesis.

Supplementary Material   Click here to view.(13M, pdf)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855259/bin/NIHMS530096-supplement-01.pdf

Acknowledgments   We thank the USC Transgenic Core Facility for generating mutant mice. This research is funded by a NIH grant to W.L (5R01NS067213) and an NRF grant (NRF-2011-35B-E00015) to J.L.

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