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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/

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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 Saxena, Boon Chin Heng, Peng Bai, Marc Folcher, Henryk 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 animals^{1, 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 humans^{5, 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 experiments^{1, 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 medicine^{18, 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 glucose^{21, 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 strategies^{25, 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 therapies^{27, 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 alternative^{29, 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 stimulation^{29, 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 cells^{32, 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 cells^{35, 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) expression^10,11. 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 (G_Sα) 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 (P_CRE) containing CREB1-specific cAMP response elements (CRE; Fig. 1a). The co-transfection of pCI-MOR9-1 (P_hCMV-MOR9-1-pA_SV40) and pCK53 (P_CRE-SEAP-pA_SV40) into human mesenchymal stem cells (hMSC-TERT) confirmed the vanillic acid-adjustable secreted alkaline phosphatase (SEAP) induction of the designer cascade (>10 nM 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, P_1VanO2) at low and medium vanillic acid levels (<2 μM). At high vanillic acid concentrations (>2 μM), VanA₁ dissociates from P_1VanO2, which results in the dose-dependent repression of transgene expression³⁹ (Fig. 1b). The co-transfection of pMG250 (P_SV40-VanA₁-pA_SV40) and pMG252 (P_1VanO2-SEAP-pA_SV40) into hMSC-TERT corroborated the fine-tuning of the vanillic acid-repressible SEAP expression (Fig. 1b).

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; P_hCMV-MOR9-1-pA_SV40; pSP1, P_CRE-VanA₁-pA_SV40) and the gene switch (pSP1, P_CRE-VanA₁-pA_SV40; pMG252, P_1VanO2-SEAP-pA_SV40) 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 (10 nM to 2 μM) activate MOR9-1, which induces P_CRE-driven VanA₁ expression. VanA₁ remains active within this concentration range and, in a feed-forward amplifier manner, triggers P_1VanO2-mediated SEAP expression, which gradually increases to maximum levels (Fig. 1c). At high vanillic acid concentrations (2 μM to 400 μM), MOR9-1 maintains P_CRE-driven VanA₁ expression, but the transactivator is inactivated and dissociates from P_1VanO2, 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 P_CRE-driven VanA₁ expression were combined with pSP12 (pA_SV40-Ngn3_cm←P_3VanO2mFT-miR30Pdx1_g-shRNA-pA_SV40) for endocrine specification and pSP17(P_CREm-Pdx1_cm-2A-MafA_cm-pA_SV40) 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 (P_3VanO2) that drives expression of a codon-modified Ngn3_cm, the nucleic acid sequence of which is distinct from its genomic counterpart (Ngn3_g) to allow for quantitative reverse transcription–PCR (qRT–PCR)-based discrimination. In the opposite direction, P_3VanO2 transcribes miR30Pdx1_g-shRNA, which exclusively targets genomicPdx1 (Pdx1_g) 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 P_CREm promoter (see below) drives co-cistronic expression of Pdx1_cm andMafA_cm, which are codon-modified versions producing native transcription factors that specifically differ from their genomic counterparts (Pdx1_g, MafA_g) 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 Pdx1_g and Nkx6.1 levels and the absence of Ngn3_g and MafA_g production^{32, 33, 34} (day 0:Supplementary Figs 10–16).

 

Figure 2: 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; P_hCMV-MOR9-1-pA) senses extracellular vanillic acid levels and triggers a synthetic signalling cascade, inducing P_CRE-driven expression of the transcription factor VanA₁ (pSP1, P_CRE-VanA₁-pA). At medium vanillic acid concentrations (purple arrows), VanA₁ binds and activates the bidirectional vanillic acid-responsive promoter P_3VanO2 (pSP12, pA-Ngn3_cm←P_3VanO2mFT-miR30Pdx1_g-shRNA-pA), which drives the induction of codon-modified Neurogenin 3 (Ngn3_cm) 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 miR30pdx1_g-shRNA (a small hairpin RNA programming the exclusive destruction of genomic pancreatic and duodenal homeobox 1 (Pdx1_g) transcripts). Consequently, Ngn3_cm levels switch from low to high (OFF-to-ON), and Pdx1_g levels toggle from high to low (ON-to-OFF). In addition, Ngn3_cm triggers the transcription of Ngn3_g from its genomic promoter, which initiates a positive-feedback loop. At high vanillic acid levels (orange arrows), VanA₁ is inactivated, and both Ngn3_cm and miR30pdx1_g-shRNA are shut down. At the same time, the MOR9-1-driven signalling cascade induces the modified high-tightness and lower-sensitivity P_CREm promoter that drives the co-cistronic expression of the codon-modified variants of Pdx1 (Pdx1_cm) and V-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafA_cm; pSP17, P_CREm-Pdx1_cm-2A-MafA_cm-pA). Consequently, Pdx1_cm and MafA_cm become fully induced. As Pdx1_cm expression ramps up, it initiates a positive-feedback loop by inducing the genomic counterparts Pdx1_g and MafA_g. Importantly, Pdx1_cm levels are not affected by miR30Pdx1_g-shRNA because the latter is specific for genomic Pdx1_g transcripts and because the positive feedback loop-mediated amplification of Pdx1_gexpression becomes active only after the shutdown of miR30Pdx1_g-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 (P_hCMV-MOR9-1-pA_SV40), pSP1 (P_CRE-VanA₁-pA_SV40), pSP12 (pA_SV40-Ngn3_cm←P_3VanO2mFT-miR30Pdx1_g-shRNA-pA_SV40) and pSP17(P_CREm-Pdx1_cm-2A-MafA_cm-pA_SV40) 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 (2 μM) and then 7 days at high (400 μM) 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.

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 Ngn3_cm/g, Pdx1_cm/g and MafA_cm/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). (c–g) Immunocytochemistry of pancreatic transcription factors Ngn3_cm/g, Pdx1_cm/g and MafA_cm/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 (P_hCMV-MOR9-1-pA), pSP1 (P_CRE-VanA₁-pA), pSP12 (pA-Ngn3_cm←P_3VanO2mFT-miR30Pdx1_g-shRNA-pA) and pSP17 (P_CREm-Pdx1_cm-2A-MafA_cm) 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, 100 μm.

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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 time^{19, 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 challenging^{29, 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 pathways^{31, 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 factors^{32, 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 interventions^{37, 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 glucose^{32, 33, 34}. This observation suggests that functional beta-like cells can eventually be derived from hIPSCs^{32, 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 lines^{35, 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 diseases^{14, 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 drugs^{30, 65}, engineering or cell-based approaches^{66, 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/