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.

294 NAÏVE STATE-LIKE PLURIPOTENT STEM CELL LINES DERIVED FROM PORCINE EMBRYONIC FIBROBLASTS

2013 ◽  
Vol 25 (1) ◽  
pp. 294
Author(s):  
J.-K. Park ◽  
K.-H. Choi ◽  
D.-C. Son ◽  
J.-I. Oh ◽  
C.-K. Lee
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Embryonic Stem ◽  
Embryonic Fibroblasts ◽  
Primed State ◽  
Exogenous Factors ◽  
Stem Cell Lines

A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is the “naïve” state, which is characterised by small, round or dome-shaped colony morphologies, LIF and BMP4 signalling pathways, and 2 active X chromosomes in females; mouse embryonic stem cells (mESC) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSC) or human embryonic stem cells (hESC). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and nodal/activin signalling pathways, and X chromosome inactivation in females. Meanwhile, a few studies have reported that primed pluripotent stem cell lines could be reverted to a naïve pluripotent state using various exogenous factors including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions, and upregulation of Oct3 or klf4. Therefore, the purpose of this study was to investigate whether a LIF-dependent naïve pluripotent stem cell line could be derived from porcine embryonic fibroblasts (PEF) via various previously reported factors. We were able to successfully induce PEF into a naïve state-like pluripotent stem cell line by viral infection using FUW-tetO-hOCT4, FUW-tetO-hSOX2, FUW-tetO-hKlf4, FUW-tetO-hMYC, and FUW-M2rtTA obtained from Addgene and addition of 2i and LIF. These naive state-like pluripotent stem cells display mESC-like morphologies, clonogenicity by trypsin, and expression of Oct4, Sox2, Nanog, and SSEA1 using PCR, immunocytochemistry, and fluorescence-activated cell sorting. All cell lines maintained stemness characteristics and stable morphology for more than 30 passages. In addition, naïve state-like pluripotent stem cells could be induced to differentiate to fibroblast-like cells by withdrawal of doxycycline, lif, and 2i. These differentiated cells could be regenerated into naïve state-like pluripotent stem cells by addition of doxycycline, lif, and 2i. We suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines and needs various exogenous factors, including continuous transgene expression, GSK3β and MEK inhibitors (2i), and LIF to be induced into naïve state-like pluripotent stem cells. This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

Abstract 010: A Separable Upstream Promoter of the Slc8a1 (NCX1) Gene Efficiently Marks Cardiac Cell Populations Derived from Human Pluripotent Stem Cells

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Alejandro Hidalgo-Gonzalez ◽  
Dmitry A Ovchinnikov ◽  
James Hudson ◽  
Justin Cooper-White ◽  
Wolvetang Ernst
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Cardiac Cells ◽  
Protein Quantification ◽  
Egfp Expression ◽  
Cardiac Cell

The sarcolemmal Na+/Ca2+ exchanger SLC8A1(NCX) regulates intracellular Ca+ in cardiomyocytes from early developmental stages. The upstream-most SLC8A1(NCX1) promoter is well conserved amongst the homoeothermic animals and contains putative binding sites for transcription factors of the NKX, GATA, STAT and CDX families. We hypothesized that functional cardiac cells with mature cardiac structural markers will express the sarcolemmal Na+/Ca2+ calcium antiporting channel, important for proper functional contractivity of in vitro differentiated human pluripotent stem cells. Pseudotyped lentiviral particles delivering NCX1cp-EGFP reporter cassette were used to confirm the efficiency and specificity of the reporter in rodent foetal cardiac cell isolates, and to establish stable human pluripotent stem cell lines. Cells were differentiated using a 2D induction protocol, and gene expression analysis and protein quantification carried at day 16. Initial NCX1cp-EGFP expression was observed from day 10-11 of cardiac differentiation. Beating foci were visualized 1-2 day after initial NCXCP-EGFP expression, reporter expression was confined to the grouped and individual beating cells, and highly correlated with the efficiency of spontaneously contractile cell production. At later stages, NCX1cp-EGFP expression correlated with clusters of formed spontaneously contractile units harbouring essentially all cardiomyocytes present in cultures, as evidenced by colocalization of high levels of cardiac troponin T (cTnT) and α-actinin proteins. The EGFP+ sorted fraction of differentiated cultures was found to be highly enriched in both early (ISL1, TBX5) and late (cTnT, MYH6) cardiomyocyte markers when compared to the EGFP- fraction. We conclude that a ~3 kb genomic fragment of the distal cardiac-specific promoter of the SLC8A1(NCX1) containing the upstream-most exon of the gene is sufficient to drive the expression of a lentiviral reporter in both rodent heart-derived primary and human (embryonic and induced) pluripotent stem cell-derived cardiac cells. Isolation of a homogenous and functional cardiomyogenic population represents one of the key objectives for cardiac tissue engineering, and in particular in vitro drug screening applications.

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 Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Cell Research ◽  
2021 ◽  
Author(s):  
Minglei Zhi ◽  
Jinying Zhang ◽  
Qianzi Tang ◽  
Dawei Yu ◽  
Shuai Gao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Biological Research ◽  
Marker Genes ◽  
Stem Cell Lines ◽  
Cloned Gene

AbstractPig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

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 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 OP0073 ESTABLISHMENT OF HUMAN INDUCED PLURIPOTENT STEM CELL-LINES (IPSC) FOR IN VITRO MODELLING HAND OSTHEOARTHRITIS

2019 ◽  
Author(s):  
Rocío Castro-Viñuelas ◽  
Clara Sanjurjo-Rodríguez ◽  
María Piñeiro-Ramil ◽  
Tamara Hermida Gómez ◽  
Isaac Fuentes-Boquete ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Stem Cell Lines ◽  
Induced Pluripotent ◽  
In Vitro Modelling

GENERATION OF RABBIT PLURIPOTENT STEM CELL LINES

2012 ◽  
Vol 24 (1) ◽  
pp. 286
Author(s):  
A. Dinnyes ◽  
M. K. Pirity ◽  
E. Gocza ◽  
P. Osteil ◽  
N. Daniel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Pluripotent Cells ◽  
Domestic Species ◽  
Isolation And Characterization ◽  
Induced Pluripotent ◽  
Pluripotency Genes

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in the rabbit. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit full pluripotency in vivo. Moreover, similarly to several domestic species, markers used to characterize the putative ESCs are not fully adequate because studies in domestic species have revealed that they are not specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing rabbit embryo. The status of isolation and characterization of the putative pluripotency genes in rabbit will be discussed. Using rabbit specific pluripotency genes we might be able to reprogram somatic cells and generate induced pluripotent stem cells more efficiently thus overcome some of the challenges towards harnessing the potential of this technology. This study was financed by EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; RabPstem, PERG07-GA-2010-268422; PluriSys, HEALTH-2007-B-223485; AniStem, PIAP-GA-2011-286264), NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; Plurabbit, OMFB-00130-00131/2010 ANR-NKTH/09-GENM-010-01.

Epigenetic regulation in stem cell development, cell fate conversion, and reprogramming

2015 ◽  
Vol 6 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Kazuyuki Ohbo ◽  
Shin-ichi Tomizawa
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Cell Fate ◽  
Cell Lines ◽  
Cell Fate Decisions ◽  
Micro Rnas ◽  
Stem Cell Lines ◽  
Cell Fate Conversion

AbstractStem cells are identified classically by an in vivo transplantation assay plus additional characterization, such as marker analysis, linage-tracing and in vitro/ex vivo differentiation assays. Stem cell lines have been derived, in vitro, from adult tissues, the inner cell mass (ICM), epiblast, and male germ stem cells, providing intriguing insight into stem cell biology, plasticity, heterogeneity, metastable state, and the pivotal point at which stem cells irreversibly differentiate to non-stem cells. During the past decade, strategies for manipulating cell fate have revolutionized our understanding about the basic concept of cell differentiation: stem cell lines can be established by introducing transcription factors, as with the case for iPSCs, revealing some of the molecular interplay of key factors during the course of phenotypic changes. In addition to de-differentiation approaches for establishing stem cells, another method has been developed whereby induced expression of certain transcription factors and/or micro RNAs artificially converts differentiated cells from one committed lineage to another; notably, these cells need not transit through a stem/progenitor state. The molecular cues guiding such cell fate conversion and reprogramming remain largely unknown. As differentiation and de-differentiation are directly linked to epigenetic changes, we overview cell fate decisions, and associated gene and epigenetic regulations.

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