Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells

Akutsu, Hidenori; Machida, Masakazu; Kanzaki, Seiichi; Sugawara, Tohru; Ohkura, Takashi; Nakamura, Naoko; Yamazaki-Inoue, Mayu; Miura, Takumi; Vemuri, Mohan C.; Rao, Mahendra S.; Miyado, Kenji; Umezawa, Akihiro

doi:10.1016/j.reth.2014.12.004

Cited by 45 publications

(46 citation statements)

References 32 publications

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“…SEES-3 hESCs (Akutsu et al, 2015) were obtained from the Center for Regenerative Medicine, National Research Institute for Child Health and Development, Japan. H9 hESCs were obtained from WiCell Research Institute, USA.…”

Section: Methodsmentioning

confidence: 99%

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells

et al. 2016

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Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings.

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Section: Methodsmentioning

confidence: 99%

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells

et al. 2016

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“…SEES" ESCs were routinely cultured onto a feeder layer of freshly plated gamma-irradiated mouse embryonic fibroblasts (MEFs), isolated from ICR embryos at 12.5 gestations and passages 2 times before irradiation (30 Gy), in the ESC culture media [15,16]. The ESC media…”

Section: Culture Of Escs Ipscs and Hepatocytesmentioning

confidence: 99%

“…In this study, we propagated hepatocytes or hepatocyte-like cells derived from human PSC, i.e. ESCs and iPSCs, and enriched zone I hepatocytes with ammonia [15,16]. Ammonia-selected cells proliferated in vitro at least up to 30 population doublings for more than 190 days.…”

Section: Introductionmentioning

confidence: 99%

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Tsuneishi

Saku

Miyata

et al. 2020

Preprint

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Zone I and zone III hepatocytes metabolize ammonia through urea cycle and drug by cytochrome P450, respectively. Ammonia has a cytotoxic effect, and can therefore be used as a selection agent for enrichment of hepatocytes. Besides, isolated hepatocytes from livers can be propagated ex vivo under appropriate condition. However, it has not been investigated so far whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed the ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human pluripotent stem cells (PSCs) that are embryonic stem cells (ESCs) and induced pluripotent stem cells. Hepatocyte-like cells after exposure to ammonia highly expressed the CPS1 gene that metabolizes ammonia to carbamoyl phosphate. The resistance to cytotoxicity or cell death by ammonia is probably attributed to the metabolic activity of ammonia in the cells. In addition to the ammonia metabolism-related genes, ammonia-selected PSC-derived hepatocytes increased expression of the CYP3A4 gene, one of the cytochrome P450 genes, that is mainly expressed in zone III hepatocytes. Ammonia-selected hepatocyte-like cells derived from both ESCs and iPSCs can be propagated in vitro up to 30 population doublings for more than 190 days without affecting expression of the liver-associated genes, implying that the ammoniaselected cells have immortality or equivalent life span on the appropriate feeder cells like ESCs and iPSCs. The long-term cultivation of ammonia-selected hepatocyte-like cells resulted in the increased expression of hepatocyte-associated genes such as the CPS1 and CYP3A4 genes. The ammonia selection method to enrich a hepatocyte population was also applicable to immortalized cells from the liver. Ammonia treatment in combination with in vitro propagation will be used to obtain large amounts of hepatocytes or hepatocyte-like cells for pharmacology, toxicology and regenerative medicine.

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“…Medium changes were carried out twice a week thereafter. Edom22-iPSCs (Edom22iPS#S31) (9) and human embryonic stem cells (SEES-5, SEES-6 and SEES-7) were used as controls for Gln-iPSCs (12).…”

Section: Human Cellsmentioning

confidence: 99%

“…For EB formation, iPSC colonies were dissociated into single cells with accutase (Thermo Scientific, MA, USA) and then passaged into the low cell-adhesion 96 well plate dishes at a density of 10,000 cells/well in the iPSC medium without bFGF, and supplemented with ROCK inhibitor (12). After confirming EB formation on day 7, the EBs were harvested and passage to dishes coated with Basement Membrane Matrix (354234, BD Biosciences).…”

Section: Ipsc Culturementioning

confidence: 99%

Gorlin syndrome-induced pluripotent stem cells form medulloblastoma with loss of heterozygosity in PTCH1

Ikemoto

Miyashita

Nasu

et al. 2019

Preprint

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Gorlin syndrome is a rare autosomal dominant hereditary disease with high incidence of tumors such as basal cell carcinoma and medulloblastoma. Disease-specific induced pluripotent stem cells (iPSCs) have now been used as a model to analyze disease pathogenesis as well as an animal model. In this study, we generated iPSCs derived from fibroblasts of four patients with Gorlin syndrome (Gln-iPSCs) with a heterozygous mutation of the PTCH1 gene. Gln-iPSCs from the four patients developed medulloblastoma in 100% (four out of four), a manifestation of Gorlin syndrome, in the teratomas after implantation into immunodeficient mice, but none (0/584) of the other iPSC-teratomas. One of the medulloblastomas had loss of heterozygosity in the PTCH1 gene while benign teratoma, i.e. non-medulloblastoma part, did not, indicating a close clinical correlation between tumorigenesis in Gorlin syndrome patients and Gln-iPSCs.

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Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells

Cited by 45 publications

References 32 publications

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Gorlin syndrome-induced pluripotent stem cells form medulloblastoma with loss of heterozygosity in PTCH1

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