PhD student, Monash University
Pluripotency is an important feature of human embryonic stem cells and human induced pluripotency stem cells. Recent reports of the exact characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a broad spectrum of naive characteristics. Furthermore, our characterisation identifies KLF4 as a conduit for conversion of primed hPSCs into naive t2iLGöY hPSCs underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.
Abstract: Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.
Pub.: 13 Sep '14, Pinned: 25 Aug '17
Abstract: Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.
Pub.: 19 Jul '16, Pinned: 25 Aug '17
Abstract: The molecular mechanisms and signalling pathways that regulate the in vitro preservation of distinct pluripotent stem cell configurations, and their induction in somatic cells by direct reprogramming, constitute a highly exciting area of research. In this Review, we integrate recent discoveries related to isolating unique naive and primed pluripotent stem cell states with altered functional and molecular characteristics, and from different species. We provide an overview of the pathways underlying pluripotent state transitions and interconversion in vitro and in vivo. We conclude by highlighting unresolved key questions, future directions and potential novel applications of such dynamic pluripotent cell states.
Pub.: 11 Feb '16, Pinned: 25 Aug '17
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