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L3MBTL1 deficiency directs the differentiation of human embryonic stem cells toward trophectoderm.

ABSTRACT: Human embryonic stem cells (hESCs) can be used to study the early events in human development and, hopefully, to understand how to differentiate human pluripotent cells for clinical use. To define how L3MBTL1, a chromatin-associated polycomb group protein with transcriptional repressive activities, regulates early events in embryonic cell differentiation, we created hESC lines that constitutively express shRNAs directed against L3MBTL1. The L3MBTL1 knockdown (KD) hESCs maintained normal morphology, proliferation, cell cycle kinetics, cell surface markers, and karyotype after 40 passages. However, under conditions that promote spontaneous differentiation, the L3MBTL1 KD cells differentiated into a relatively homogeneous population of large, flat trophoblast-like cells, unlike the multilineage differentiation seen with the control cells. The differentiated L3MBTL1 KD cells expressed numerous trophoblast markers and secreted placental hormones. Although the L3MBTL1 KD cells could be induced to differentiate into various embryonic lineages, they adopted an exclusive trophoblast fate during spontaneous differentiation. Our data demonstrate that depletion of L3MBTL1 does not affect hESC self-renewal, rather it enhances differentiation toward extra-embryonic trophoblast tissues.

SUBMITTER: Hoya-Arias R

PROVIDER: S-EPMC3202892 | biostudies-other | 2011 Nov

REPOSITORIES: biostudies-other

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L3MBTL1 deficiency directs the differentiation of human embryonic stem cells toward trophectoderm.

Hoya-Arias Ruben R Tomishima Mark M Perna Fabiana F Voza Francesca F Nimer Stephen D SD

Stem cells and development 20110403 11

Human embryonic stem cells (hESCs) can be used to study the early events in human development and, hopefully, to understand how to differentiate human pluripotent cells for clinical use. To define how L3MBTL1, a chromatin-associated polycomb group protein with transcriptional repressive activities, regulates early events in embryonic cell differentiation, we created hESC lines that constitutively express shRNAs directed against L3MBTL1. The L3MBTL1 knockdown (KD) hESCs maintained normal morpholo ...[more]

PMID: 21341991

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Project description:ObjectiveIn vitro production of a definitive endoderm (DE) is an important issue in stem cell-related differentiation studies and it can assist with the production of more efficient endoderm derivatives for therapeutic applications. Despite tremendous progress in DE differentiation of human embryonic stem cells (hESCs), researchers have yet to discover universal, efficient and cost-effective protocols.Materials and methodsIn this experimental study, we have treated hESCs with 200 nM of Stauprimide (Spd) for one day followed by activin A (50 ng/ml; A50) for the next three days (Spd-A50). In the positive control group, hESCs were treated with Wnt3a (25 ng/ml) and activin A (100 ng/ml) for the first day followed by activin A for the next three days (100 ng/ml; W/A100-A100).ResultsGene expression analysis showed up regulation of DE-specific marker genes (SOX17, FOXA2 and CXCR4) comparable to that observed in the positive control group. Expression of the other lineage specific markers did not significantly change (p<0.05). We also obtained the same gene expression results using another hESC line. The use of higher concentrations of Spd (400 and 800 nM) in the Spd-A50 protocol caused an increase in the expression SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration of activin A (25 ng/ml) was not able to up regulate the DE-specific marker genes. Then, A50 was replaced by inducers of definitive endoderm; IDE1/2 (IDE1 and IDE2), two previously reported small molecule (SM) inducers of DE, in our protocol (Spd-IDE1/2). This replacement resulted in the up regulation of visceral endoderm (VE) marker (SOX7) but not DE-specific markers. Therefore, while the Spd-A50 protocol led to DE production, we have shown that IDE1/2 could not fully replace activin A in DE induction of hESCs.ConclusionThese findings can assist with the design of more efficient chemically-defined protocols for DE induction of hESCs and lead to a better understanding of the different signaling networks that are involved in DE differentiation of hESCs.

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L3MBTL1 deficiency directs the differentiation of human embryonic stem cells toward trophectoderm.

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L3MBTL1 deficiency directs the differentiation of human embryonic stem cells toward trophectoderm.

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