Showing papers by "Huck-Hui Ng published in 2012"

Ncoa3 functions as an essential Esrrb coactivator to sustain embryonic stem cell self-renewal and reprogramming

Abstract: Embryonic stem cell (ESC) pluripotency depends on a well-characterized gene regulatory network centered on Oct4, Sox2, and Nanog. In contrast, little is known about the identity of the key coregulators and the mechanisms by which they may potentiate transcription in ESCs. Alongside core transcription factors, the orphan nuclear receptor Esrrb (estrogen-related receptor β) is vital for the maintenance of ESC identity and furthermore is uniquely associated with the basal transcription machinery. Here, we show that Ncoa3, an essential coactivator, is required to mediate Esrrb function in ESCs. Ncoa3 interacts with Esrrb via its ligand-binding domain and bridges Esrrb to RNA polymerase II complexes. Functionally, Ncoa3 is critical for both the induction and maintenance of pluripotency. Through chromatin immunoprecipitation (ChIP) sequencing and microarray experiments, we further demonstrate that Ncoa3 shares overlapping gene regulatory functions with Esrrb and cooperates genome-wide with the Oct4–Sox2–Nanog circuitry at active enhancers to up-regulate genes involved in self-renewal and pluripotency. We propose an integrated model of transcriptional and coactivator control, mediated by Ncoa3, for the maintenance of ESC self-renewal and somatic cell reprogramming.

A role for polyamine regulators in ESC self-renewal.

Abstract: 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.

Stem cell genome-to-systems biology.

Abstract: Stem cells are capable of extended proliferation and concomitantly differentiating into a plethora of specialized cell types that render them apropos for their usage as a form of regenerative medicine for cell replacement therapies. The molecular processes that underlie the ability for stem cells to self-renew and differentiate have been intriguing, and elucidating the intricacies within the genome is pertinent to enhance our understanding of stem cells. Systems biology is emerging as a crucial field in the study of the sophisticated nature of stem cells, through the adoption of multidisciplinary approaches which couple high-throughput experimental techniques with computational and mathematical analysis. This allows for the determination of the molecular constituents that govern stem cell characteristics and conjointly with functional validations via genetic perturbation and protein location binding analysis necessitate the construction of the complex transcriptional regulatory network. With the elucidation of protein-protein interaction, protein-DNA regulation, microRNA involvement as well as the epigenetic modifications, it is possible to comprehend the defining features of stem cells at the system level.

Computer and Statistical Analysis of Transcription Factor Binding and Chromatin Modifications by ChIP-seq data in Embryonic Stem Cell

Abstract: Advances in high throughput sequencing technology have enabled the identification of transcription factor (TF) binding sites in genome scale. TF binding studies are important for medical applications and stem cell research. Somatic cells can be reprogrammed to a pluripotent state by the combined introduction of factors such as Oct4, Sox2, c-Myc, Klf4. These reprogrammed cells share many characteristics with embryonic stem cells (ESCs) and are known as induced pluripotent stem cells (iPSCs). The signaling requirements for maintenance of human and murine embryonic stem cells (ESCs) differ considerably. Genome wide ChIP-seq TF binding maps in mouse stem cells include Oct4, Sox2, Nanog, Tbx3, Smad2 as well as group of other factors. ChIP-seq allows study of new candidate transcription factors for reprogramming. It was shown that Nr5a2 could replace Oct4 for reprogramming. Epigenetic modifications play important role in regulation of gene expression adding additional complexity to transcription network functioning. We have studied associations between different histone modification using published data together with RNA Pol II sites. We found strong associations between activation marks and TF binding sites and present it qualitatively. To meet issues of statistical analysis of genome ChIP-sequencing maps we developed computer program to filter out noise signals and find significant association between binding site affinity and number of sequence reads. The data provide new insights into the function of chromatin organization and regulation in stem cells.

Investigating the bona fide differentiation capacity of human pluripotent stem cells.

Abstract: Human pluripotent stem cells (hPSCs) have been perennially paraded as a source of cells for cell replacement therapies because they can (theoretically) give rise to any single cell type within the human body 1. Hence, they can create in vitro a vast number of any human cell type to replace the diseased cell population that a patient might require — this is a salient goal that regenerative medicine aspires to deliver on 2. However, despite the ever-expanding menagerie of therapeutically relevant differentiated lineages being created from hPSCs, usage of these stem cell-derived progeny for regenerative medicine still remains an uncertainty. The question here is whether hPSC lines are faithful to their parental pluripotent antecedents in the embryo from which they were derived. During the enterprise of embryonic development, native pluripotent cells housed within the blastocyst give rise to the entire repertoire of differentiated cell types within the human body that successfully subserve important physiological responsibilities. When put to the test, will hPSCs properly differentiate in vitro to produce physiologically functional differentiated cell types that are equatable to the bona fide differentiated lineages that we actually find in the human body? Or will they produce differentiated progeny that partially resemble the cells that we see in vivo? Concerns have surfaced — for example, hPSC-derived pancreatic β-cells sometimes fail to secrete insulin in response to glucose, unlike authentic pancreatic β-cells found in vivo3 and hPSC-generated hematopoietic stem cells rarely contribute to the hematopoietic systems of recipient animals 4 — which potentially disqualify usage of these cells to treat diabetes or hematological deficiencies. In a recent paper published in Cell Research, Patterson et al. investigate the fidelity of differentiated cell types created from hPSCs — were they made in the likeness of actual bodily cells found in the human embryo? To this end, the authors differentiated hPSCs into cell types representative of the three fetal germ layers from which all of the embryo proper arises — neural progenitors (definitive ectoderm), fibroblasts (mesoderm), and hepatocytes (definitive endoderm) — and then they used microarrays to interrogate whether hPSC-derived cell types were transcriptionally similar to cells from the same lineage taken from actual human fetuses (Figure 1) 5. While pervasive transcriptional congruities were found between fetus-derived and hESC/hiPSC-derived cell types, some striking differences were reported — around ∼10% of assayed genes were differentially expressed between embryo-derived and hPSC-derived cells (Figure 1). Moreover, no matter if hESCs or hiPSCs were specified into neural progenitors, fibroblasts, or hepatocytes, their resultant differentiated offspring continued to express pluripotency-associated genes LIN28A, LIN28B, and DPPA4, amongst others (Figure 1). Rarely if ever did genuine fetus-derived differentiated cells express any of these three pluripotency markers — hPSC-derived cells generally expressed these genes thousands of times higher than their embryonic counterparts. It is possible that some malingering undifferentiated hPSCs persisted throughout the differentiation protocols, consequently contributing to the elevated expression of these pluripotency genes in hPSC-derived lineages. Whatever the reason, enduring expression of pluripotency genes in stem cell-derived progeny is translationally troublesome; for example, one notes that expression of LIN28 in differentiated cells is tumorigenic 6. Might hPSC-derived differentiated cells be prone to oncogenic subversion? If so, this could indeed pose a serious impediment to the exploitation of differentiated derivatives of hPSCs for cell-based therapies in human patients. Figure 1 To assess the clinical fitness of differentiated cell types generated from human pluripotent stem cells (hPSCs; both embryonic stem cells and induced pluripotent stem cells), Patterson et al. systematically differentiated hPSCs into neural progenitors, ... In light of their findings that hPSC-derived cells were similar but not identical to bona fide embryo-derived cells, the authors posited that these discrepancies might be an issue of “developmental maturation” — the immediate differentiated progeny of hESCs and hiPSCs might resemble very early differentiated cell types found in the nascent fetus, not more matured differentiated cell types found at later developmental stages. Indeed, this appeared to be partially the case — while hPSC-derived neural progenitors differentially expressed ∼10% of assayed genes as compared to neural progenitors taken from week 16 human fetal spinal cord, the number of differentially expressed genes was halved when they were compared against spinal cord progenitors sourced from weeks 6.5-8 of fetal development (although a subset of genes remained to be differentially expressed regardless of what week of fetal development was chosen). Based on these findings, Patterson et al. advance that hPSC-derived lineages might resemble the respective cell types taken from human fetuses prior to developmental week 6. The main import to be abstracted from the findings of Patterson et al. is that hPSC-derived differentiated cell types transcriptionally resemble but are not identical to their embryonic counterparts; and also those hPSC-derived lineages continue to aberrantly express select pluripotency genes. The reasons for these transcriptional incongruities are unclear — for example, we have now become cognizant of the fact that hPSCs in vitro differentially express a large number of genes ( > 1 700) as compared to native pluripotent cells found in human blastocysts 7. These precocious differences between hPSCs and authentic embryonic pluripotent cells might have contributed to the differences in gene expression seen in their differentiated endpoints — if there are already transcriptional issues with the hPSCs that we are starting out with altogether in the beginning of these in vitro differentiation regimens. Or perhaps hPSCs are not in themselves defective, but our current differentiation procedures are yet to be fully optimized and do not completely recapitulate developmental cell type specification programs found in the embryo, yielding “incompletely programmed” differentiated cell types that are similar yet different from fetal cells 9. What do these findings portend for the usage of hPSCs for regenerative medicine? Are transcriptional incongruences between hPSC-derived and fetal cells causes for concern? One utilitarian argument to be waged is that it is inconsequential that hPSC-derived progeny are transcriptionally divergent from their in vivo counterparts — as long as they can physiologically perform. This also invokes another challenge. The apparent “developmental naivety" of hPSC-derived cells also remains an important clinical concern — to frame it in the above example of β-cells; adult β-cells secrete insulin in response to glucose, but fetal β-cells do not 3. Clearly, should hPSCs be capable of differentiating into cell types similar to those found in the fetus, procedures must be elaborated to “mature” these cells into adult-like cells with the appropriate physiological functionalities that are appropriate to transplant into diseased adult patients 8. Nevertheless, in any event, this present report by Patterson et al. is timely, beckoning concern in regards to the filial piety of hPSCs and their differentiated offspring. This is the first experimental demonstration that there are clear transcriptional differences between multiple hPSC-derived cell types and their in vivo counterparts. Perhaps further studies delving into their epigenetic differences might yield interesting insights as well. More importantly, whether these cells are still physiologically functional enough to be therapeutically exploited or the differentiation procedures need be improved are the questions now.