Significant variations in differentiation properties between independent mouse ES cell lines cultured under defined conditions

Ward, Christopher M.; Barrow, Katie M.; Stern, Peter L.

doi:10.1016/j.yexcr.2003.10.017

Cited by 45 publications

(34 citation statements)

References 32 publications

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“…ES cells differentiated into EBs by the removal of LIF from the defined medium and then differentiated as a monolayer have been described to exhibit varied expression of marker transcripts characteristic of mesoderm, endoderm and ectoderm lineages (Ward et al, 2004). In agreement with these results, our ES-like cells derived from differentiated EB also expressed markers of differentiation characteristic of the three layers.…”

Section: Discussionsupporting

confidence: 86%

Inadvertent presence of pluripotent cells in monolayers derived from differentiated embryoid bodies

Ramírez¹,

Pericuesta²,

Fernández‐González³

et al. 2007

Int. J. Dev. Biol.

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The therapeutic use of embryonic stem (ES)-derived cells is restricted by a risk of teratoma formation. To test the hypothesis that some cells with pluripotency characteristics remain following the differentiation of embryoid bodies (EB) into monolayer cells, we transformed mouse ES cells using constructs comprised of the mTert promoter coupled to green fluorescent protein. In this manner, EBs could be identified as showing gradually diminishing expression of the fluorescent marker as a consequence of differentiation. After 2 weeks of incubation, however, small groups of fluorescent cells remained in the differentiated monolayer. When these cells were isolated, cultured and expanded under ES cell culture conditions, they recovered their ES cell morphology (herein denoted ES-EB). We found by immunocytochemistry, reverse transcription-PCR and bisulphite analysis that despite the fact that some of these ES-EB lost their capacity to express some pluripotency markers characteristic of ES cells and undergo the epigenetic modification (hypo-methylation) of some retrotransposons (RT), after several passages in ES media, the cell colonies recovered their capacity for both pluripotency marker expression and RT methylation. Furthermore, when assessed for their ability to form chimeras, most ES-EB lines were unable to do so, although they recovered this potential for chimera production after some passages in ES cell media. Our results highlight the need for specific screening of differentiated cells before their therapeutic use and indicate that under adequate culture conditions, cells that loose their potential for expressing key markers of pluripotency can recover this fundamental embryonic stem cell property.

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Section: Discussionsupporting

confidence: 86%

Inadvertent presence of pluripotent cells in monolayers derived from differentiated embryoid bodies

Ramírez¹,

Pericuesta²,

Fernández‐González³

et al. 2007

Int. J. Dev. Biol.

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show abstract

“…LIF actions on the later LC stages are not well understood but may cause decreased steroidogenesis (24) through lowering of cholesterol substrate availability in the mitochondria (25). In most stem cells (e.g., mouse embryonic stem cells), LIF withdrawal is a stimulus for differentiation (26). These data suggest that the LIF signaling pathway is present in the LC lineage, and that it may have a role in stimulating renewal of the identified putative SLCs.…”

Section: Discussionmentioning

confidence: 99%

In search of rat stem Leydig cells: Identification, isolation, and lineage-specific development

Dong

Sottas

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

238

301

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Leydig cells (LCs) are thought to differentiate from spindle-shaped precursor cells that exhibit some aspects of differentiated function, including 3␤-hydroxysteroid dehydrogenase (3␤HSD) activity. The precursor cells ultimately derive from undifferentiated stem LCs (SLCs), which are postulated to be present in testes before the onset of precursor cell differentiation. We searched for cells in the neonatal rat testis with the abilities to: (i) proliferate and expand indefinitely in vitro (self renew); (ii) differentiate (i.e., 3␤HSD and ultimately synthesize testosterone); and (iii) when transplanted into host rat testes, colonize the interstitium and subsequently differentiate in vivo. At 1 week postpartum, spindle-shaped cells were seen in the testicular interstitium that differed from the precursor cells in that they were 3␤HSD-negative, luteinizing hormone (LH) receptor (LHR)-negative, and platelet-derived growth factor receptor ␣ (PDGFR␣)-positive. These cells were purified from the testes of 1-week-old rats. The cells contained proteins known to be involved in LC development, including GATA4, c-kit receptor, and leukemia inhibitory factor receptor. The putative SLCs expanded over the course of 6 months while remaining undifferentiated. When treated in media that contained thyroid hormone, insulin-like growth factor I, and LH, 40% of the putative SLCs came to express 3␤HSD and to synthesize testosterone. When transplanted into host rat testes from which LCs had been eliminated, the putative SLCs colonized the interstitium and subsequently expressed 3␤HSD, demonstrating their ability to differentiate in vivo. We conclude that these cells are likely to be the sought-after SLCs.c-kit ͉ leukemia inhibitory factor ͉ platelet-derived growth factor receptor ␣ ͉ puberty ͉ steroidogenesis L eydig cells (LCs) are the primary source of testosterone in the male, and their differentiation in the testes during puberty is a signature event in the development of the male body plan. It is hypothesized, but far from proven, that LCs first arise from undifferentiated stem cells [stem LCs (SLCs)] (1-3). It has been suggested that, in rats, the putative SLCs are present in the testis at birth, and that by 11 days postpartum, at least some of their progeny express LC-specific genes and thus become committed to the LC lineage (4, 5).The committed cells subsequently undergo phased transitions through progenitor and immature stages and ultimately to terminally differentiated adult LC stage (6). In particular, progenitor LCs (PLCs) form during days 12-28 postpartum (presumably from SLCs). The PLCs proliferate and also exhibit some aspects of differentiated function, including 3␤-hydroxysteroid dehydrogenase (3␤HSD) activity (7). Luteinizing hormone (LH) receptors (LHRs) first appear as the PLCs differentiate, suggesting that SLCs are likely to be independent of LH control (8). The development of the steroidogenic capacity of PLCs requires stimulation by LH (9). The mitotic activity of PLCs gradually is reduced, and the cells enlarge...

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“…Levels of Lif mRNA increased in the testis 24 h after EDS treatment and then returned to basal levels within 3-5 days. In embryonic stem cells LIF withdrawal is a stimulus for differentiation (Ward et al 2004) and it is possible that the transient change in levels of Lif after EDS acts both to enhance stem cell proliferation and then to permit Leydig cell differentiation.…”

Section: Discussionmentioning

confidence: 99%

Leydig cell re-generation and expression of cell signaling molecules in the germ cell-free testis

O’Shaughnessy

Morris²,

Baker³

2008

Reproduction

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Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3b-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17b-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif ), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place. Reproduction (2008) 135 851-858

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Significant variations in differentiation properties between independent mouse ES cell lines cultured under defined conditions

Cited by 45 publications

References 32 publications

Inadvertent presence of pluripotent cells in monolayers derived from differentiated embryoid bodies

Inadvertent presence of pluripotent cells in monolayers derived from differentiated embryoid bodies

In search of rat stem Leydig cells: Identification, isolation, and lineage-specific development

Leydig cell re-generation and expression of cell signaling molecules in the germ cell-free testis

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