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 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 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 Expression of miRNAs from the Imprinted DLK1/DIO3 Locus Signals the Osteogenic Potential of Human Pluripotent Stem Cells

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1523 ◽  
Author(s):  
Laetitia Barrault ◽  
Jacqueline Gide ◽  
Tingting Qing ◽  
Lea Lesueur ◽  
Jorg Tost ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Osteogenic Differentiation ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Osteogenic Potential ◽  
Human Pluripotent Stem Cell ◽  
Stem Cell Lines

Substantial variations in differentiation properties have been reported among human pluripotent cell lines (hPSC), which could affect their utility and clinical safety. We characterized the variable osteogenic capacity observed between different human pluripotent stem cell lines. By focusing on the miRNA expression profile, we demonstrated that the osteogenic differentiation propensity of human pluripotent stem cell lines could be associated with the methylation status and the expression of miRNAs from the imprinted DLK1/DIO3 locus. More specifically, quantitative analysis of the expression of six different miRNAs of that locus prospectively identified human embryonic stem cells and human-induced pluripotent stem cells with differential osteogenic differentiation capacities. At the molecular and functional levels, we showed that these miRNAs modulated the expression of the activin receptor type 2B and the downstream signal transduction, which impacted osteogenesis. In conclusion, miRNAs of the imprinted DLK1/DIO3 locus appear to have both a predictive value and a functional impact in determining the osteogenic fate of human pluripotent stem cells.

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 Clonal derivation of white and brown adipocyte progenitor cell lines from human pluripotent stem cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Michael D. West ◽  
Ching-Fang Chang ◽  
Dana Larocca ◽  
Jie Li ◽  
Jianjie Jiang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Progenitor Cell ◽  
Human Pluripotent Stem Cells ◽  
Brown Adipocyte

Inhibition of Chronic Myelogenous Leukemia Stem Cells with Novel Wnt Antagonists.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 238-238 ◽  
Author(s):  
Edward Kavalerchik ◽  
Jason Gotlib ◽  
Ifat Geron ◽  
Annelie Abrahamsson ◽  
Wolfgang Wrasidlo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myelogenous Leukemia ◽  
Reporter Gene ◽  
Myelogenous Leukemia ◽  
Beta Catenin ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Wnt Inhibitors

Abstract Introduction A growing proportion of chronic myelogenous leukemia (CML) patients show evidence of disease progression. Recent research suggests that leukemia stem cells (LSC) that share phenotypic characteristics with granulocyte-macrophage progenitors (GMP) are involved in CML progression. These LSC have aberrantly gained self-renewal capacity as a result of enhanced Wnt/beta-catenin signaling. We assayed the capacity of novel Wnt/beta-catenin antagonists to inhibit CML LSC. Methods To assay the efficacy of a novel Wnt inhibitor, MC-001, HEK293 cells were transfected with a Wnt-dependent reporter gene and expression plasmid for Dsh. After 16h, the cells were treated for 24 h with MCC-001, a novel marine sponge derived inhibitor, at varying concentrations and the reporter gene activity was measured. All cells were also transfected with a b-gal reporter gene to control for transfection efficiency. To assess the effects of MCC-001 and other Wnt inhibitors on Wnt/beta-catenin induced self-renewal, hematopoietic stem cells (HSC), GMP and lineage positive cells from normal (n=8) and advanced phase CML (n=8) peripheral blood and marrow (n=8) were clone sorted with the aid of a FACS Aria into methocult media (Stem Cell Technologies) with or without Wnt inhibitors including recombinant Dkk1, lentiviral axin or MCC-001. On day 10, individual colonies were plucked and replated in new methylcellulose and the replating efficiency determined at day 10. To establish an in vivo CML LSC model, HSC, GMP and lineage positive cells were transduced with a lentiviral luciferase GFP for 48 hours and transplanted intrahepatically into newborn immunocompromised mice (RAG2−/−gamma−/−) mice that facilitate high levels of human hematopoietic progenitor engraftment. Results The HEK293 beta-catenin reporter assay revealed that the MC-001 IC50 was 2.1 microM. In comparative Wnt inhibitor replating assays (n=8), recombinant Dkk1 did not inhibit CML HSC (n=8) while lentiviral axin and MCC-001 (at 2 and 10 microM) inhibited both CML HSC and CML GMP at doses that spared normal HSC replating (Figure 1). Transplantation of CML HSC, GMP and lineage positive cells into RAG2−/−gamma−/− mice demonstrated that only CML GMP provided serial transplantation potential and thus, were enriched for the LSC population (Figure 2). Conclusions Selective Wnt/beta-catenin inhibition with a marine sponge derived beta-catenin antagonist, MCC-001, blocks in vitro replating capacity of CML LSC at doses that spare normal HSC. Current experiments focus on in vivo inhibition of LSC self-renewal with novel Wnt inhibitors in a robust CML LSC bioluminescent imaging model (Figure 2). Figure 1. Chronic Myelogenous Leukemia Stem Cell Inhibition with MCC-001: A novel β-catenin Inhibitor Figure 1. Chronic Myelogenous Leukemia Stem Cell Inhibition with MCC-001: A novel β-catenin Inhibitor Figure 2. Bioluminescent Chronic Myelogenous Leukemia Stem Cell Transplantation Model. Figure 2. Bioluminescent Chronic Myelogenous Leukemia Stem Cell Transplantation Model.

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.

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