The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development

Regulation of stemness in humans: the role of Hypoxia Inducible Factors and Tie2

Thesis (Ph.D.)--University of Washington, 2017Pluripotent stem cells hold great promise for the future of regenerative medicine. Though many studies have examined the genetic determinants of pluripotency, the role of metabolism in stemness acquisition and maintenance remains an active area of investigation. Due to its role in the acquisition of different metabolic states, hypoxia, via action of the Hypoxia Inducible Factors (HIFs), is also suspected to regulate pluripotency. Indeed this low oxygen level is a key feature of a variety of stem cell niches. We thus decided to study the roles of HIFs in the acquisition and maintenance of pluripotency. First, we tested the role of HIFs in stemness acquisition by reprogramming human fibroblasts into induced pluripotent stem cells (iPSCs). We found that even though both HIF1α and HIF2α are needed for iPSC formation, HIF2α overexpression towards the later stages of reprogramming inhibits colony formation through TRAIL activation. Then, in the human embryonic stem cell paradigm we showed that HIF1α is required for the transition from the naïve to the primed hESC, two separate pluripotent states characterized by distinct metabolic profiles. Together these results underline the crucial role of hypoxia and HIFs in the regulation of stemness and metabolism. Another area in which metabolism can act to regulate pluripotency is through the regulation of epigenetics. Previous studies showed that energy metabolism and the availability of certain metabolites can influence stem cells and their stemness capacities. The Polycomb repressive complex 2 (PRC2) has methyltransferase activity on histones, primarily adding the repressive trimethylation mark on histone 3 lysine 27 (H3K27me3). We investigated the role of JARID2, a key protein of this complex, in the maintenance of pluripotency of hESCs. Interestingly, the loss of JARID2 in naïve hESC (2iL-I-F) induces a concomitant loss of H3K27me3 and pluripotency markers. A variety of solid tumors contain a subpopulation of cancer stem cells (CSCs), cancer cells characterized by their ability to self-renew, initiate tumors and lead to metastasis. CSCs share several features with normal adult stem cells in terms of metabolic profile. Clarifying the role of metabolism in stemness acquisition could have interesting applications for cancer stem cells. Interestingly, both primed stem cells and cancer stem cells share a similar glycolytic metabolism. Adult stem cells in the model organism Drosophila melanogaster have been recently used as a model to study cancer stem cells. In vivo studies allow for considerations of the interactions between stem cells and the stem cell niche. These interactions have been shown to be critical in the resistance of stem cells to apoptosis following exposure to ionizing radiation. Germline stem cells are able to survive IR damage through Pvf-1 mediated Tie-2 receptor activation. The human homologs Angiopoietin1 (Ang1) and Tie2 are therefore interesting targets for inhibitors and activators in human stem cells. A combination of in silico designed scaffolds with the binding domain of Ang1, F-domain was used to activate the Tie2 receptor

Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal

To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein

The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development

Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal

<p>To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology–directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.</p

The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development