Lineage-Committed Pancreatic Progenitors and Stem Cells

Abstract Background Vitamin B3 (nicotinamide) plays important roles in metabolism as well as in SIRT and PARP pathways. It is also recently reported as a novel kinase inhibitor with multiple targets. Nicotinamide promotes pancreatic cell differentiation from human embryonic stem cells (hESCs). However, its molecular mechanism is still unclear. In order to understand the molecular mechanism involved in pancreatic cell fate determination, we analyzed the downstream pathways of nicotinamide in the derivation of NKX6.1+ pancreatic progenitors from hESCs. Methods We applied downstream modulators of nicotinamide during the induction from posterior foregut to pancreatic progenitors, including niacin, PARP inhibitor, SIRT inhibitor, CK1 inhibitor and ROCK inhibitor. The impact of those treatments was evaluated by quantitative real-time PCR, flow cytometry and immunostaining of pancreatic markers. Furthermore, CK1 isoforms were knocked down to validate CK1 function in the induction of pancreatic progenitors. Finally, RNA-seq was used to demonstrate pancreatic induction on the transcriptomic level. Results First, we demonstrated that nicotinamide promoted pancreatic progenitor differentiation in chemically defined conditions, but it did not act through either niacin-associated metabolism or the inhibition of PARP and SIRT pathways. In contrast, nicotinamide modulated differentiation through CK1 and ROCK inhibition. We demonstrated that CK1 inhibitors promoted the generation of PDX1/NKX6.1 double-positive pancreatic progenitor cells. shRNA knockdown revealed that the inhibition of CK1α and CK1ε promoted pancreatic progenitor differentiation. We then showed that nicotinamide also improved pancreatic progenitor differentiation through ROCK inhibition. Finally, RNA-seq data showed that CK1 and ROCK inhibition led to pancreatic gene expression, similar to nicotinamide treatment. Conclusions In this report, we revealed that nicotinamide promotes generation of pancreatic progenitors from hESCs through CK1 and ROCK inhibition. Furthermore, we discovered the novel role of CK1 in pancreatic cell fate determination.

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Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

10.1101/2021.02.02.429390 ◽

2021 ◽

Author(s):

Rabea Dettmer ◽

Isabell Niwolik ◽

Ilir Mehmeti ◽

Anne Jörns ◽

Ortwin Naujok

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Beta Cells ◽

Pluripotent Stem Cells ◽

Reporter Genes ◽

Human Pluripotent Stem Cells ◽

Endogenous Gene ◽

Pancreatic Progenitors ◽

Insulin Producing Cells ◽

Endogenous Genes

AbstractDifferentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. The SOX9 gene plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter genes into the SOX9 locus and a P2A-mCherry reporter gene into the INS locus mediated by CRISPR/CAS9-technology. The knock-ins enable co-expression of the endogenous genes and reporter genes, report the endogenous gene expression and enable the purification of pancreatic progenitors and insulin-producing cells using FACS or MACS. Using these cell lines we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells.

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Modulating cell state to enhance suspension expansion of human pluripotent stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714099115 ◽

2018 ◽

Vol 115 (25) ◽

pp. 6369-6374 ◽

Cited By ~ 12

Author(s):

Yonatan Y. Lipsitz ◽

Curtis Woodford ◽

Ting Yin ◽

Jacob H. Hanna ◽

Peter W. Zandstra

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Large Scale ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

The Body ◽

Altered Expression ◽

Pancreatic Progenitors ◽

Erk Inhibition

The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.

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Pancreatic Progenitors and Organoids as a Prerequisite to Model Pancreatic Diseases and Cancer

Stem Cells International ◽

10.1155/2019/9301382 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Meike Hohwieler ◽

Martin Müller ◽

Pierre-Olivier Frappart ◽

Sandra Heller

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Embryonic Stem ◽

Cell Types ◽

Genetically Engineered ◽

Hereditary Diseases ◽

Pancreatic Diseases ◽

Pancreatic Progenitors

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their unique capacity to stepwise differentiate towards any particular cell type in an adult organism. Pluripotent stem cells provide a beneficial platform to model hereditary diseases and even cancer development. While the incidence of pancreatic diseases such as diabetes and pancreatitis is increasing, the understanding of the underlying pathogenesis of particular diseases remains limited. Only a few recent publications have contributed to the characterization of human pancreatic development in the fetal stage. Hence, most knowledge of pancreatic specification is based on murine embryology. Optimizing and understanding current in vitro protocols for pancreatic differentiation of ESCs and iPSCs constitutes a prerequisite to generate functional pancreatic cells for better disease modeling and drug discovery. Moreover, human pancreatic organoids derived from pluripotent stem cells, organ-restricted stem cells, and tumor samples provide a powerful technology to model carcinogenesis and hereditary diseases independent of genetically engineered mouse models. Herein, we summarize recent advances in directed differentiation of pancreatic organoids comprising endocrine cell types. Beyond that, we illustrate up-and-coming applications for organoid-based platforms.

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Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0365 ◽

2015 ◽

Vol 370 (1680) ◽

pp. 20140365 ◽

Cited By ~ 19

Author(s):

Maria Rostovskaya ◽

Nicholas Bredenkamp ◽

Austin Smith

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Human Pluripotent Stem Cells ◽

Type I ◽

Pancreatic Progenitors ◽

Stem Cell Lines

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

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