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

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.

scholarly journals New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells

2021 ◽  
Author(s):  
Rabea Dettmer ◽  
Isabell Niwolik ◽  
Ilir Mehmeti ◽  
Anne Jörns ◽  
Ortwin Naujok
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Beta Cells ◽  
Cell Lines ◽  
Reporter Gene ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Beta Catenin ◽  
Pancreatic Progenitors ◽  
Insulin Producing Cells

AbstractDifferentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. SOX9 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 gene into the SOX9-locus and a P2A-mCherry reporter gene into the INS-locus mediated by CRISPR/CAS9-technology. The knock-ins enabled co-expression of the endogenous and reporter genes and report on the endogenous gene expression. Furthermore, FACS and MACS enabled the purification of pancreatic progenitors and insulin-producing cells. 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. Our new protocol offers an alternative route towards stem cell-derived beta cells, pointing out the importance of Wnt/beta-catenin inhibition and the efficacy of EGF for the development of pancreatic progenitors, as well as the significance of 3D culture for the functionality of the generated beta cells. Graphic Abstract

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

2015 ◽  
Vol 370 (1680) ◽  
pp. 20140365 ◽  
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.

scholarly journals High Oxygen Condition Facilitates the Differentiation of Mouse and Human Pluripotent Stem Cells into Pancreatic Progenitors and Insulin-producing Cells

2014 ◽  
Vol 289 (14) ◽  
pp. 9623-9638 ◽  
Author(s):  
Farzana Hakim ◽  
Taku Kaitsuka ◽  
Jamiruddin Mohd. Raeed ◽  
Fan-Yan Wei ◽  
Nobuaki Shiraki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
High Oxygen ◽  
Pancreatic Progenitors ◽  
Insulin Producing Cells ◽  
Oxygen Condition

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

2018 ◽  
Vol 115 (25) ◽  
pp. 6369-6374 ◽  
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.

scholarly journals Probing the mechanism for hydrogel-based stasis induction in human pluripotent stem cells: is the chemical functionality of the hydrogel important?

2020 ◽  
Vol 11 (1) ◽  
pp. 232-240 ◽  
Author(s):  
M. Sponchioni ◽  
C. T. O'Brien ◽  
C. Borchers ◽  
E. Wang ◽  
M. N. Rivolta ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Block Copolymer ◽  
Embryonic Stem Cell ◽  
Pluripotent Stem Cells ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cells ◽  
Essential Parameter ◽  
Chemical Functionality

It is shown that hydroxyl functionality is required to induce stasis in human embryonic stem cell colonies immersed within wholly synthetic block copolymer worm gels with comparable storage moduli. Thus gel softness does not appear to be an essential parameter for stasis induction.

scholarly journals Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

2019 ◽  
Author(s):  
Koray D. Kaya ◽  
Holly Y. Chen ◽  
Matthew J. Brooks ◽  
Ryan A. Kelley ◽  
Hiroko Shimada ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Retinoic Acid ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Mitochondrial Morphology ◽  
Rod Photoreceptor ◽  
Molecular Staging ◽  
Organoid Cultures

ABSTRACTRetinal organoids generated from human pluripotent stem cells exhibit considerable variability in temporal dynamics of differentiation. To assess the maturity of neural retina in vitro, we performed transcriptome analyses of developing organoids from human embryonic and induced pluripotent stem cell lines. We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrated that addition of 9-cis retinal, instead of widely-used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. Our studies thus provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids, which should facilitate disease modeling and evaluation of therapies in vitro.Summary StatementThree-dimensional organoids derived from human pluripotent stem cells have been extensively applied for investigating organogenesis, modeling diseases and development of therapies. However, substantial variations within organoids pose challenges for comparison among different cultures and studies. We generated transcriptomes of multiple distinct retinal organoids and compared these to human fetal and adult retina gene profiles for molecular staging of differentiation state of the cultures. Our analysis revealed the advantage of using 9-cis retinal, instead of the widely-used all-trans retinoic acid, in facilitating rod photoreceptor differentiation. Thus, a transcriptome-based comparison can provide an objective method to uncover the maturity of organoid cultures across different lines and in various study platforms.

scholarly journals Generation of Pancreatic Progenitors from Human Pluripotent Stem Cells by Small Molecules

2020 ◽  
Author(s):  
Yuqian Jiang ◽  
Chuanxin Chen ◽  
Lauren N. Randolph ◽  
Songtao Ye ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Small Molecules ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Pancreatic Progenitors

scholarly journals Valproic acid Promotes the in Vitro Differentiation of Human Pluripotent Stem Cells Into Spermatogonial Stem Cell-like Cells

2021 ◽  
Author(s):  
Xiaotong Wang ◽  
Mengyuan Qu ◽  
Zili Li ◽  
Yuting Long ◽  
Kai Hong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Pluripotent Stem Cells ◽  
Wnt Signaling Pathway ◽  
Human Pluripotent Stem Cells ◽  
Sequencing Analysis ◽  
Upregulated Genes

Abstract Background: Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on the SSCLC induction. Methods: We examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. The percentage of haploid cells and cells expressed meiotic and spermatid genes were also detected. RNA-sequencing analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols.Results: The combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. High concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-sequencing analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and the expression of genes in Wnt signaling pathway. Conclusions: VPA robustly promotes the differentiation of human pluripotent stem cell lines into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that inactivation of Wnt signaling pathway might be a cause of SSC depletion in azoospermia testes. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.

scholarly journals Human Pluripotent Stem Cells to Model Islet Defects in Diabetes

2021 ◽  
Vol 12 ◽  
Author(s):  
Diego Balboa ◽  
Diepiriye G. Iworima ◽  
Timothy J. Kieffer
Keyword(s):  
Stem Cells ◽  
Beta Cells ◽  
Pluripotent Stem Cells ◽  
Pancreatic Beta Cells ◽  
Molecular Mechanisms ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Pancreatic Endocrine Cells ◽  
Onset Of Diabetes ◽  
And Function

Diabetes mellitus is characterized by elevated levels of blood glucose and is ultimately caused by insufficient insulin production from pancreatic beta cells. Different research models have been utilized to unravel the molecular mechanisms leading to the onset of diabetes. The generation of pancreatic endocrine cells from human pluripotent stem cells constitutes an approach to study genetic defects leading to impaired beta cell development and function. Here, we review the recent progress in generating and characterizing functional stem cell-derived beta cells. We summarize the diabetes disease modeling possibilities that stem cells offer and the challenges that lie ahead to further improve these models.

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