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Adipose Cells to Insulin-Secreting Cells

 

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

Curator: Evelina Cohn, Ph.D.

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

 

Adipocyte Derived Stem Cell: Review of Methodologies

Curator: Dr. Williams

 

Reference	Molecular Biology of the Cell Vol. 16, 1131–1141, March 2005 Andrew C. Boquest http://www.ncbi.nlm.nih.gov/pmc/articles/PMC551479/pdf/00161131.pdf	Shigeki Sugiia,b, 3558–3563 | PNAS | February 23, 2010 | vol. 107 | no. 8 http://www.pnas.org/content/107/8/3558	Citation: Zhang Y, Wei C, Zhang P, Li X, Liu T, et al. (2014) Efficient Reprogramming of Naïve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System. PLoS ONE 9(1): e85089. doi:10.1371/journal.pone.0085089	Saxena, P. et al. A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells. Nat. Commun.7:11247 doi: 10.1038/ncomms11247 (2016)
Cell Type	Adipocyte	Adipocyte (white adipose, [WAP])	Adipocyte (WAP)	Adipocyte
Species tested in	human	Human, mouse	porcine	human
Isolation step	Liposuction from hip, stromal vascular fraction (SVF) separated from adipose tissue Zuk et al., 2001; CD31- cells isolated after magnetic bead separation; single cell isolation;	PromoCell (GmBH) and ADSC (StemPro; Invitrogen) are derived from s.c. fat of a 63-year-old Caucasian female and of a 22-year-old female, respectively. They were confirmed to be >95% CD44+ and >95% CD31−/CD45−. Similar results of iPS efficiencies and feeder independence were obtained with both of the hADS cells. hWP (PromoCell) is derived from the s.c. fat of a 38-year-old Caucasian female. Cells were cultured in MesenPRO RS medium (for hADS) or DMEM plus 10% hiFBS (for hWP) and used within five passages	Subcutaneous adipose tissue was obtained from the 28-day-old Danish Landrace female piglets, and enzymatically dissociated in Dulbecco’s modified Eagle medium/F12 (DMEM/F12) with 0.09% collagenase type I and 10% fetal bovine serum (FBS, Life Technologies, USA). After incubating in 37°C for 90 min, the dissociation was terminated by washing medium (DMEM/F12 with 10% FBS), followed by centrifugation for 5 min (3206g). Pellet was resuspended with washing medium, and sequentially filtered through 250 mm, 80 mm and 25 mm nylon mesh to remove the tissue debris. Being washed for three times, cells were suspended with DMEM/F12 containing 10% FBS, 50 mg/ml vitamin C and 10 ng/ml basic fibroblast growth factor (bFGF, Pepro Tech, USA),	Method based on Heng et al.: mRNA transfection-based, feeder-free, induced pluripotent stem cells derived from adipose tissue of a 50-year-old patient * First mRNA-based IPSC reprogramming of adipose tissue-derived MSCs. * First IPSC of cells from an “old” patient. * First IPSC reprogramming which is completely feeder-free. * First IPSC reprogramming which uses chemically defined culture media. (no viral vectors needed; chemical differentiation). * 4 of 12 clones retain normal chromosomes to 30 passages
Immortilization step (genes needed)	Not needed	Not needed	Not needed	Used hTERT to immortalize their vanillic acid inducible lineage-control system
Reprogramming step (genes needed)		Reprogramming of human adipose cells was carried out essentially as described (4, 12). hWP (5,000/cm2) or hADS cells (3,000/cm2) were plated in six-well plates. The cells were infected with the combination of human reprogramming retroviruses (c-Myc, Klf4, Oct4, or Sox2 in pMXs; Addgene) that had been produced in 293T cells cotransfected with gag/pol and VSV-G as described above. EGFP retrovirus was included at 1/40 volume as internal controls for transduction efficiencies. One well from each group was saved for counting cell numbers. On day5, cells were passed onto 10-cm dishes covered with feeder MEFsor onto 6-cm dishes without MEFs. Cells were cultured in DMEM/F12 plus 20% KSR supplemented with β-mercaptoe-thanol (0.1%), NEAA (1×), Glutamax (1%), and 10 ng/mL FGF2 (“cDF12” media). Medium was changed every day. On days 18–28, individual colonies were picked and cultured feeder-free in defined mTeSR1 medium on plates coated with matrigel, which was prepared according to the previously described formula (13, 14), and changed daily. Dispase was used to passage cells. For feeder-free and XF induction of hiPS cells, hADS cells were plated either in StemPro XF-MSC SFM with CELLstart coating (Invitrogen) or in DMEM plus 2% human serum. Retrovirus containing four factors and EGFP was pro-duced in “XF-cDF12” media containing XF-KSR (Invitrogen).	inducible lentiviralus carrying reprogramming factors (Oct4, Sox2, c-Myc and Klf4	Reprogramming induced by addition of vannilic acid. Requires stable transfection of multiple vectors:   1. The constitutively expressed vanillic acid-sensitive olfactory G protein-coupled receptor MOR9-1 (pCI-MOR9-1; PhCMV-MOR9-1-pA and pCK53[reporter gene to check expression];) 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).   2. induces transcription factor VanA1 (pSP1, PCRE-VanA1-pA   3. Neurogenin 3 (required for the development of all endocrine cells in the pancreas) (pSP12 (pASV40-Ngn3cm←P3VanO2mFT-miR30Pdx1g-shRNA-pASV40) for endocrine specification)   4. pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40). Results in the expression of the homeobox factor Pdx1 and the factor MafA, which results in the production of the final beta-like cell.
Reprogramming step (media conditions needed)	cultures were incubated with DMEM/F12 containing 10% FBS, 0.5 M 1-methyl-3 isobutylxanthine, 1 M dexamethasone, 10 g/ml insulin (Novo Nordisk, Copenhagen, Denmark) and 100 M indomethacin (Dumex-Alpharma, Copenhagen, Denmark) for 3 wk (makes mesodermal lineage; muscle, bone, neural).	xeno-free medium NutriStem (Stemgent), up to 56 days. Because matrigel is of the mouse tumor origin, plates used for this condition were coated with a humanized defined substrate, CELLstart (ThermoFisher, Invitrogen (NOTE: spontaneous differentiation not reproducible however can use factors or media from other papers)	Lif-2i medium was added into the plated for another 2 days, which consisted of DMEM/F12, 15% KSR, GlutaMAXTM-1 (Life Technologies, USA), 0.1 mM 3-mercapto- ethanol, N2 (Life Technologies, USA), B-27 (Life Technologies, USA), PD0325901 (Selleck, USA), CHIR99021 (Selleck, USA), mouse Lif (Millipore, USA) and 4 tg/ml DOX. Finally, iPSCs colonies were picked and maintained in Lif-2i medium for the following culture. For passaging, they were dissociated using TrypLE (ThermoFisher, Life Technologies, USA) every 2 to 3 days.	Induced system only requires presence of vanillic acid to produce beta like cells which secrete insulin. Does not produce alpha (glucagon) or delta (somatostatin) secreting cells   However, this system is Concentration-dependent on vanillic acid MEANING depending on low, medium, or high exposure to vanillic acid (the inducer of the system) will result in different cell types:   [VA conc]   Cell type 0                     hIPSC   Medium     endocrine Progenitor     High               Beta Cell
Cell Survival time (days)	·       CD31(-) Proliferative for 8 months ·       12 of 68 clones could produce 1 million cells and differentiate into adipogenic, osteogenic, and neurogenic cells	·       Not tested ·       Can form all germ layers but function not tested, only markers ·       No exogenous factors required to support iPsc; ·       Generate iPsc efficiently from Lin(-) population	Not tested	Inducible system gives tight control of differentiation process and can be modified to produce other cell types   Excellent GLUCOSE Control of insulin production   No data on cell survival of precursor stem cells but differentiated beta cells are functional for at least 2 weeks   “ 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.”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The Inducible System for generating Beta-like cells FromA programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

 

 

 

(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 ofPdx1 (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_cmexpression ramps up, it initiates a positive-feedback loop by inducing the genomic counterparts Pdx1_gand 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_g expression 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.

 

 

Supplemental Movies showing control of expression of each component of the inducible system in real time.

Movies

1. Supplementary Movie 1 (1,838 KB)

Time-lapse fluorescence microscopy of hMSC-TERT cotransfected with the lineage control network vectors pCI-MOR9-1 (PhCMV-MOR9-1-pA), pSP1 (PCRE-VanA1-pA), pSP12 (pA-Ngn3cm←P3VanO2mFTmiR30Pdx1g-shRNA-pA) and pSP24 (PCREm-EYFP-pA; visual plug-in for pSP17) and grown for 59h in the presence of medium (2μM) vanillic acid concentration.

2. Supplementary Movie 2 (4,394 KB)

Time-lapse fluorescence microscopy of hMSC-TERT cotransfected with the lineagecontrol network vectors pCI-MOR9-1 (PhCMV-MOR9-1-pA), pSP1 (PCRE-VanA1-pA), pSP12 (pA-Ngn3cm←P3VanO2mFTmiR30Pdx1g-shRNA-pA) and pSP24 (PCREm-EYFP-pA; visual plug-in for pSP17) and grown for 48h in the presence of high (400μM) vanillic acid concentration.

 

Figure 5: Characterization of glucose-sensitive insulin-secreting beta-like cells programmed by the synthetic lineage-control network. (a) Quantitative analysis of lineage-controlled beta-like cells co-stained for VanA₁ and either insulin (C-peptide), glucagon or somatostatin. The cells staining positive for VanA₁ are containing the lineage-control network. Data are the means±s.d. (n=3). (b,c) Human pancreatic islets and beta-like cells produced by programming hIPSC-derived pancreatic progenitor cells using the synthetic lineage-control network or the growth-factor/chemical-based differentiation technique were exposed to low (2.8 mM), medium (10 mM), high (20 mM) glucose as well as high glucose and potassium chloride (30 mM) before intracellular (b) and secreted (c) insulin (C-peptide) levels were profiled using ELISA. Data are the means±s.d. of duplicate experiments (n=6). Statistics by Student’s t-test; *P<0.05, **P<0.005, ****P<0.0001; ns, not significant. (d) Beta-like cells produced by programming hIPSC-derived pancreatic progenitor cells using the synthetic lineage-control network or the growth-factor/chemical-based differentiation technique and cultivated for 4 weeks were exposed to low (2.8 mM), medium (10 mM), high (20 mM) glucose as well as high glucose and potassium chloride (30 mM) before secreted insulin (C-peptide) levels were profiled using ELISA. Data are the means±s.d. of duplicate experiments (n=6). Statistics by Student’s t-test; **P<0.005; ns, not significant. (e) Transmission-electron micrographs of human pancreatic islets and lineage-controlled beta-like cells. Scale bars, 1 μm (human pancreatic islets) and 2 μm (beta-like cells differentiated by the synthetic lineage-control network).