A role for polyamine regulators in ESC self-renewal

Embryonic stem cells (ESCs) depend on extensive regulatory networks to coordinate their self-renewal and differentiation. The polyamine pathway regulator AMD1 was recently implicated in ESC self-renewal and directed differentiation of ESCs to neural precursor cells (NPCs). The polyamines spermine and spermidine are essential for a wide range of biological processes, and their levels are tightly regulated. Here, we review the polyamine pathway and discuss how it can impact polyamine levels, cellular methylation and hypusinated EIF5A levels. We discuss how it could feed into regulation of ESC self-renewal and directed differentiation. We show that in addition to AMD1, a second rate-limiting enzyme in the polyamine pathway, ODC1, can also promote ESC self-renewal, and that both Amd1 and Odc1 can partially substitute for Myc during cellular reprogramming. We propose that both Amd1 and Odc1 are essential regulators of ESCs and function to ensure high polyamine levels to promote ESC self-renewal.     

Identification and isolation of embryonic stem cells in reproductive endocrinology: theoretical protocols for conservation of human embryos derived from <Emphasis Type="Italic">in vitro</Emphasis> fertilization

Background  

Embryonic stem cells (ESC) are pluripotent cells obtained from the inner cell mass (ICM) of blastocysts derived from in vitro culture associated with reproductive endocrinology therapy. Human ESCs are regarded as highly significant since they retain the capacity to differentiate into any of approximately 200 unique cell types. Human ESC research is controversial because to acquire such cells, the ICM of human blastocysts must be manipulated in a way that renders embryos nonviable and unsuitable for transfer in utero. Techniques to yield competent ESCs with conservation of source blastocysts would satisfy many objections against ESC research, but at present such approaches remain largely untested.     

Identification of novel genes and networks governing hematopoietic stem cell development

Hematopoietic stem cells (HSCs) are capable of giving rise to all blood cell lineages throughout adulthood, and the generation of engraftable HSCs from human pluripotent stem cells is a major goal for regenerative medicine. Here, we describe a functional genome‐wide RNAi screen to identify genes required for the differentiation of embryonic stem cell (ESC) into hematopoietic stem/progenitor cells (HSPCs) in vitro. We report the discovery of novel genes important for the endothelial‐to‐hematopoietic transition and subsequently for HSPC specification. High‐throughput sequencing and bioinformatic analyses identified twelve groups of genes, including a set of 351 novel genes required for HSPC specification. As in vivo proof of concept, four of these genes, Ap2a1, Mettl22, Lrsam1, and Hal, are selected for validation, confirmed to be essential for HSPC development in zebrafish and for maintenance of human HSCs. Taken together, our results not only identify a number of novel regulatory genes and pathways essential for HSPC development but also serve as valuable resource for directed differentiation of therapy grade HSPCs using human pluripotent stem cells.     

A novel feeder-free system for human embryonic stem cells and characterization of their sublines with autogenic and allogenic cultivation

We developed a feeder-free system for human embryonic stem cells (ESCs) based on extracellular matrix protein (ECM) as the substrate. ECM was synthesized by mesenchymal stem cells (SC5-MSC) derived from an original ESC line, SC5. The ECM proteins fibronectin and laminin facilitate ESC growth in the feeder-free system. An important component of this system is a conditioned medium from SC5-MSC cells. Two ESC sublines were obtained: SC5-FF cells were cultured in an autogenic, and SC7-FF in an allogenic, feeder-free system. SC5-FF and SC7-FF underwent more than 300 and 115 population doublings, respectively, and retain a normal diploid karyotype. Histochemical and immunofluorescence assays showed that both sublines express undifferentiated ESC markers—alkaline phosphatase, Oct-4, SSEA-4, and TRA-1-81—as well as multidrug resistance transporter ABCG2. PCR assay revealed that undifferentiated SC5-FF cells, like the original SC5 line, maintained on feeder cells express OCT4 and NANOG genes common for somatic cells and DPPA3/STELLA and DAZL genes common for germ line cells. Expression of these genes was gradually diminished during differentiation of embryoid bodies, whereas expression of genes specific for early differentiated cells increased: GATA4, AFP (extraembryonic and embryonic endoderm), PAX6 (neuroectoderm), and BRY (mesoderm). ESC properties (karyotype structure, average time of population doubling, undifferentiated cell number in population) of the SC5 and SC7 and SC5-FF and SC7-FF sublines derived from original ESCs were not altered. It shows that the feeder-free systems, which are more stable than any feeder systems, maintain key ESC properties and may be recommended for fundamental, biomedical, and pharmacological studies performed with human ESCs.     

Epigenetic Signatures Associated with Different Levels of Differentiation Potential in Human Stem Cells

Background

The therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles.

Methodology/Principal Findings

to address this issue, we have investigated the levels of epigenetic regulation in well characterized populations of pluripotent embryonic stem cells (ESC) and multipotent adult stem cells (ASC) at the trancriptome, methylome, histone modification and microRNA levels. Differences in gene expression profiles allowed classification of stem cells into three separate populations including ESC, multipotent adult progenitor cells (MAPC) and mesenchymal stromal cells (MSC). The analysis of the PcG repressive marks, histone modifications and gene promoter methylation of differentiation and pluripotency genes demonstrated that stem cell populations with a wider differentiation potential (ESC and MAPC) showed stronger representation of epigenetic repressive marks in differentiation genes and that this epigenetic signature was progressively lost with restriction of stem cell potential. Our analysis of microRNA established specific microRNA signatures suggesting specific microRNAs involved in regulation of pluripotent and differentiation genes.

Conclusions/Significance

Our study leads us to propose a model where the level of epigenetic regulation, as a combination of DNA methylation and histone modification marks, at differentiation genes defines degrees of differentiation potential from progenitor and multipotent stem cells to pluripotent stem cells.     

Induced pluripotent stem cells

Induced pluripotent stem cells (iPS) result from a reprogramming of somatic cells via transduction with viral vectors expressing the Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28 genes, which are essential for the establishment and maintenance of the pluripotent state. In properties, iPS are almost fully similar to embryonic stem cells (ESC). To date, iPS have been obtained from various differentiated cells of mice and humans. Along with ESC, iPSs are highly promising for research and medicine.     

WNTing embryonic stem cells

Embryonic stem cells (ESCs) - undifferentiated cells originating from preimplantation stage embryos - have prolonged self-renewal capacity and are pluripotent. Activation of the canonical Wnt pathway is implicated in maintenance of and exit from the pluripotent state. Recent findings demonstrate that the essential mediator of canonical Wnt signaling, β-catenin, is dispensable for ESC maintenance; however, its activation inhibits differentiation through derepression of T cell factor 3 (Tcf3)-bound genes. Wnt agonists are useful in deriving ESCs from recalcitrant mouse strains and the rat and in nuclear reprogramming of somatic stem cells. We discuss recent advances in our understanding of the role of canonical Wnt signaling in the regulation of ESC self-renewal and how its manipulation can improve pluripotent ESC derivation and maintenance.     

Evaluation of unrestricted somatic stem cells as a feeder layer to support undifferentiated embryonic stem cells

The use of unrestricted somatic stem cells (USSCs) holds great promise for future clinical applications. Conventionally, mouse embryonic fibroblasts (MEFs) or other animal‐based feeder layers are used to support embryonic stem cell (ESC) growth; the use of such feeder cells increases the risk of retroviral and other pathogenic infection in clinical trials. Implementation of a human‐based feeder layer, such as hUSSCs that are isolated from human sources, lowers such risks. Isolated cord blood USSCs derived from various donors were used as a novel, supportive feeder layer for growth of C4mES cells (Royan C4 ESCs). Complete cellular characterization using immunocytochemical and flow cytometric methods were performed on murine ESCs (mESCs) and hUSSCs. mESCs cultured on hUSSCs showed similar cellular morphology and presented the same cell markers of undifferentiated mESC as would have been observed in mESCs grown on MEFs. Our data revealed these cells had negative expression of Stat3, Sox2, and Fgf4 genes while showing positive expression for Pou5f1, Nanog, Rex1, Brachyury, Lif, Lifr, Tert, B2m, and Bmp4 genes. Moreover, mESCs cultured on hUSSCs exhibited proven differentiation potential to germ cell layers showing normal karyotype. The major advantage of hUSSCs is their ability to be continuously cultured for at least 50 passages. We have also found that hUSSCs have the potential to provide ESC support from the early moments of isolation. Further study of hUSSC as a novel human feeder layer may lead to their incorporation into clinical methods, making them a vital part of the application of human ESCs in clinical cell therapy. Mol. Reprod. Dev. 79: 709–718, 2012. © 2012 Wiley Periodicals, Inc.     

Cryopreservation and quality control of mouse embryonic feeder cells

Stem cell research is a highly promising and rapidly progressing field inside regenerative medicine. Embryonic stem cells (ESCs), reprogrammed “induced pluripotent” cells (iPS), or lately protein induced pluripotent cells (piPS) share one inevitable factor: mouse embryonic feeder cells (MEFs), which are commonly used for ESC long term culture procedures and colony regeneration. These MEFs originate from different mouse strains, are inactivated by different methods and are differently cryopreserved. Incomprehensibly, there are to date no established quality control parameters for MEFs to insure consistency of ESC experiments and culture. Hence, in this work, we developed a bench-top quality control for embryonic feeder cells.According to our findings, MEFs should be inactivated by irradiation (30 Gy) and cryopreserved with optimal 10% DMSO at 1 K/min freezing velocity. Thawed cells should be free of mycoplasma and should have above 85 ± 13.1% viability. Values for the metabolic activity should be above 150 ± 10.5% and for the combined gene expression of selected marker genes above 225 ± 43.8% compared to non-irradiated, cryopreserved controls. Cells matching these criteria can be utilized for at least 12 days for ESC culture without detaching from the culture dish or disruption of the cell layer.     

LncKdm2b controls self‐renewal of embryonic stem cells via activating expression of transcription factor Zbtb3

Divergent long noncoding RNAs (lncRNAs) represent a major lncRNA biotype in mouse and human genomes. The biological and molecular functions of the divergent lncRNAs remain largely unknown. Here, we show that lncKdm2b, a divergent lncRNA for Kdm2b gene, is conserved among five mammalian species and highly expressed in embryonic stem cells (ESCs) and early embryos. LncKdm2b knockout impairs ESC self‐renewal and causes early embryonic lethality. LncKdm2b can activate Zbtb3 by promoting the assembly and ATPase activity of Snf2‐related CREBBP activator protein (SRCAP) complex in trans. Zbtb3 potentiates the ESC self‐renewal in a Nanog‐dependent manner. Finally, Zbtb3 deficiency impairs the ESC self‐renewal and early embryonic development. Therefore, our findings reveal that lncRNAs may represent an additional layer of the regulation of ESC self‐renewal and early embryogenesis.