Novel Mechanism in Self-Renewal/Differentiation of Human Embryonic Stem Cells

Funding Type: 
Basic Biology II
Grant Number: 
Award Value: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 

The most prominent feature of the stem cell is its pluripotent capacity to differentiate into various types of cells. The importance of the orchestrated interplay between molecular regulators has been demonstrated in the maintenance of self-renewing pluripotent property or the initiation of differentiation. Advance in the generation of the induced pluripotent stem cells (iPSCs) have been benefitted by our knowledge on the molecular regulation in stem cell renewal/differentiation. Furthermore, the practical use of stem cells for regenerative medicine will be possible through our understanding on the mechanism underlying distinct differentiation process.

Recent progress in stem cell biology has unveiled some important features of molecular and cellular regulations in stem cell pluripotency and differentiation, but it remains largely elusive. The proposed study is based on our recent published findings that demonstrate the significance of the cell cycle regulatory molecule in embryonic stem cell self-renewal and differentiation. Our published data strongly supports that CDK2AP1 (CDK2 associating protein 1) is a competency factor in mouse embryonic stem cell (mESC) differentiation. Even though the difference in molecular regulation between mouse and human has been documented, it is also accepted that they share common molecular mechanism in the maintenance of self-renewal/differentiation. Especially the importance of the role of OCT3/4 in stem cell maintenance and pluripotency has been well documented in both models. This study is focused on the molecular and cellular mechanism and is an innovative research in a sense that the proposed study will unveil a novel mechanism in stem cell regulation.

Specific Aims proposed in this application will significantly advance our understanding in hESC biology towards (1) the epigenetic control of OCT3/4 and its functional contribution to the stem cell pluripotency, (2) the role of cell cycle regulatory mechanism in stem cell self-renewal/differentiation decision, and (3) the potential utilization of molecular regulatory mechanism for future regenerative therapeutics.

Statement of Benefit to California: 

The medicine today is facing two equally pressing issues in the treatment of patients- providing a life-saving quality treatment in the mean time at an affordable cost to every patient. California has the highest healthcare costs of any state in the nation – more than $110 billion per year. In that sense, the regenerative medicine offers an initiative for the future of medicine. The regenerative medicine is the ultimate goal of the future medicine in treatment of patients suffering from both genetic and non-genetic disorder. The benefit of stem cell research is almost unlimited in developing breakthrough cures and treatment for debilitating diseases and injuries, including diabetes, cancer, heart disease, Alzheimer’s, Multiple Sclerosis, HIV/AIDS, Parkinson’s, ALS, osteoporosis and spinal cord injuries. Unfortunately, our current knowledge on stem cell biology is far behind what we need to know in order to make the stem cell therapy available to the patients in the clinics. Only way to improve our practical knowledge and move the field forward is to devote our efforts and resources toward better understanding of the biology and mechanism, which will ultimately lead to the practical translation of our basic knowledge from the laboratory to the treatment of patients in the clinics. In the long run, stem cell therapies may cut California’s skyrocketing healthcare expenditures by reducing the need for expensive, long-term supportive care, which will be unavoidably pressing issues to the citizens of California in the very near future.

Progress Report: 

Specific Aims proposed in this application are designed to significantly advance our understanding in hESC biology towards (1) the epigenetic control of OCT3/4 and its functional contribution to the stem cell pluripotency, (2) the role of cell cycle regulatory mechanism in stem cell self-renewal/differentiation decision, and (3) the potential utilization of molecular regulatory mechanism for future regenerative therapeutics. There are three main specific aims proposed.

Specific Aim 1: Significance of CDK2AP1-mediated molecular regulation in hESC self-renewal/differentiation.

Specific Aim 2: Systems biology analysis to identify key molecular pathways in CDK2AP1-mediated hESC regulation.

Specific Aim 3: Identification of small molecule activator for OCT3/4.
These aims are based on our preliminary study using mouse embryonic stem cells.

The main goal of this proposal is to further translate what we have found with Cdk2ap1 knockout mESC model to human embryonic stem cell biology. Acknowledging the difference between mouse model and human model, we have performed the proposed study in parallel by using our established mESC model and also generating and testing hESC model.

For Specific Aim 1 in the Year 1, we have further detailed our knowledge on how CDK2AP1 epigenetically regulates Oct3/4 promoter at the molecular level. We have recently made a novel finding that CDK2AP1 may play a role in the regulation of Oct3/4 in the sequence specific manner along with NuRD complex. This has been also confirmed in hESC model. We have also found that CDK2AP1 may exert its effect through changes in cellular localization and conformation during the differentiation of hESC. We are in the process of further detailing this mechanism to link the cell cycle regulatory role of CDK2AP1 through CDK2/RB and the epigenetic role in stem cell differentiation. We have generated necessary hESC cell lines to further perform the proposed studies. At the end of the Year 2 as planned, we are expecting to submit a manuscript and related to the Aim 1. An NIH RO1 grant will be submitted for the next cycle related to our new findings.

For Specific Aim 2, we have performed and obtained significant amount of data that advance our understanding on the molecular role of CDK2AP1 in stem cell differentiation. We have newly found CDK2AP1 significantly alter DNA methylation of key signaling pathways, especially Wnt pathway that is known to have a significant impact on stem cell maintenance and differentiation. We have detailed molecular axis related to the epigenetic regulation of Wnt pathway by CDK2AP1 and a manuscript is in the preparation. Using the hESC cell lines we have generated, we will perform systems biology approach as proposed in the Year 2. This will enable us to generate significant amount of data related to the epigenetic role of CDK2AP1 in hESC differentiation.

For Specific Aim 3, we have generated hESC sublines with human OCT3/4 promoter-GFP and human CDK2AP1-GFP for chemical library screening. We will generate more deletion mutants of CDK2AP1 promoter-RFP lines and test them before we initiate the works proposed in the Aim 3. We will initiate the proposed Aim 3 near the end of the Year 2.

During the reporting period, we have made significant research progress towards (1) the epigenetic control mediated by cyclin dependent kinase 2-associated protein 1 (CDK2AP1) through Nucleosome Remodeling and Deacetylation (NuRD) complex and its functional contribution to the stem cell pluripotency, (2) Role of Cdk2ap2, a sibling molecule of Cdk2ap1 in stem cell self-renewal, (3) the role of cell cycle regulatory CDK2AP1 in the regulation of OCT4 promoter, and (4) genome wide molecular signaling pathways affected by CDK2AP1 in ESCs. These are briefly summarized as follows.

(1) CDK2AP1 is a novel regulatory factor in NuRD-mediated Wnt signaling in embryonic stem cells (Manuscript submitted). NuRD complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered as a key player in the formation of functional NuRD complex. We show that CDK2AP1 plays a role in self-renewal/differentiation of ESC by engaging MBD3 to the promoter region of Wnt signaling related genes. The occupancy of MBD3 on several Wnt signaling gene promoters was significantly lost in the absence of CDK2AP1, resulting in hyper-activation of Wnt signaling. We propose that the transcriptional modulation of Wnt pathway mediated by MBD3/NuRD requires the presence of essential auxiliary component, such as CDK2AP1, that may aid the association of the complex with the specific focal region of the target promoters.

(2) Cdk2ap2 Is a Novel Regulator for Self-Renewal of Murine Embryonic Stem Cells (In Press. Stem Cells and Development). To understand the function of Cdk2ap2 during early development, we generated mESCs with homozygous disruption of the endogenous Cdk2ap2 locus (Cdk2ap2tr/tr). The Cdk2ap2tr/tr mESCs, when grown in a complete growth medium, showed an early differentiation phenotype characterized by flattened colonies and a distinct intercellular boundary. We also observed downregulation of Nanog and upregulation in markers of mesoderm and endoderm differentiation. Cdk2ap2tr/tr mESCs were able to form embryoid bodies (EBs); however, those EBs were unhealthy and had an increased level of apoptosis. Cdk2ap2 under normal conditions has a biphasic expression, suggesting regulatory roles in early-versus-late stem cell differentiation. These data begin to add to our understanding of how Cdk2ap2 may be involved in the regulation of self-renewal of stem cells during early embryogenesis.

(3) Epigenetic regulation of Oct4 by CDK2AP1 (Presented in the ISSCR 2012, Manuscript in preparation). Cell cycle regulators are gaining a more prominent role, these include molecules such as p27 that have been shown to play a role in cell cycle kinetics to maintain a pluripotent state and regulate specific genes involved in pluripotency. In our studies we have found that CDK2AP1 plays a key role in NuRD-mediated Oct3/4 silencing by epigenetically regulating the Oct4 promoter during differentiation of both mESC and hESC. Detailed analysis of the Oct4 promoter revealed an absence of DNA methylation at the proximal enhancer (PE) region in differentiated Cdk2ap1-/- mESC. In parallel, we have seen an increase in H3K9 acetylation at the same region in Cdk2ap1-/- mESC. We have found CDK2AP1 occupancy at the PE region in mESC as well as hESC embryoid bodies. Furthermore, in ESC we have observed interdependency in CDK2AP1 and MBD3 binding to the OCT4 promoter. In hESC nuclear translocation of CDK2AP1 upon differentiation was distinct from mESC. CDK2AP1 plays a significant role in stem cell differentiation by association with the NuRD complex on specific promoter regions, changing chromatin accessibility and leading to the silencing of the Oct4 promoter during differentiation in ESC.

(4) Global epigenetic signatures and signaling pathways affected by CDK2AP1 in ESCs (Manuscript in preparation). To gain insight into the genome wide molecular effects of CDK2AP1 in ESC differentiation, we have performed systems biology analysis by combining DNA methylation array analysis and gene expression array analysis on both mESC and hESC samples. From DNA methylation (MeDIP-seq on hESC) and gene expression array analysis, we found that there were global methylomic and transcriptional changes due to the deletion of in ESCs. Detailed analyses revealed that the deletion of Cdk2ap1 led to site-specific methylation changes. We have constructed a complete meta-network map of mESCs built from 12 different mouse gene expression arrays to explain how nucleosome remodeling and differentiation are interconnected and linked. In addition, by using our Cdk2ap1 knockout mESC model, we have demonstrated that CDK2AP1 may affect the repertoire and function of miRNA involved in stem cell self-renewal/differentiation. This finding demonstrates that CDK2AP1, either through cell cycle regulatory mechanism or transcriptional control, may affect the function of miRNA in ESC.

Public Summary: During the reporting period of the Yr3 (Aug. 1, 2012- Jul. 31, 2013), we have made significant research progress towards (1) Epigenetic role of CDK2AP1 in ESC maintenance/differentiation by regulation of WNT pathway. This work was an extension of the previous year and completed for a publication. (2) We have performed bioinformatics analysis on our own datasets and publicly available datasets to identify novel molecular factors in self-renewal of hESCs. Findings from this study were submitted to Stem Cells and now in revision for publication. (3) We have performed detailed analysis to define molecular role of CDK2AP1 and MBD3 in mouse and human ESCs, which are briefly summarized as below.

(1) CDK2AP1 is a novel regulatory factor in NuRD-mediated Wnt signaling in embryonic stem cells (Published in JBC 87(49): 41103–41117): In continuation of work from the previous year, we have completed the study and successfully revised the manuscript for publication in JBC during the first quarter of this reporting period. Potential role of MBD3/NuRD in the repression of pluripotency genes in ESC has been demonstrated. However, it is still elusive on the detailed molecular mechanism, such as the necessity of any auxiliary factor priming the complex formation or conferring target specificity. Our study further elucidates the epigenetic role of CDK2AP1 in stem cell maintenance via association with MBD3/NuRD complex and demonstrates the importance of a complimentary molecule to MBD3/NuRD in the regulation of ESC. One area of interest is how NuRD recruitment occurs on the target gene promoters, whether it is focal, broadly distributed or dependent on the pattern of the gene. In this study, we found a novel regulatory factor within the NuRD complex, which may function as a guide in NuRD recruitment to specific WNT genes to regulate stem cell pluripotency.

(2) Discovery of novel molecular factors in self-renewal of hESCs (Manuscript in revision: Stem Cells). Our understanding of self-renewal and differentiation capacity of human embryonic stem cells (hESCs) remains elusive on the detailed molecular mechanisms. Molecular markers defining self-renewing pluripotent embryonic stem cells have been identified by relative comparisons between undifferentiated and differentiated cells. Most of previous analyses have been done under a specific differentiation condition that may present significantly different molecular changes over other experimental conditions. Therefore, it is currently unclear if there are true consensus markers that define undifferentiated hESCs. Our current study elucidates the global regulation of stem cell beyond the well-known stem cell factors by combining over 33 microarrays and the latest bioinformatic tools. We examined if there are a set of key genes consistently altered during differentiation of hESCs regardless of differentiation conditions. By comprehensive genome-wide consensus microarray analyses, we have profiled gene expression signatures that are most significantly affected by differentiation in hESCs from our own microarray data sets as well as publically available microarrays. Our finding has unveiled the novel molecular markers that determine self-renewal and form intramodular hubs. Bioinformatics approach for the identification of new molecular markers defining undifferentiated hESCs, interacting partners and interconnectivity analyses may contribute to delineating molecular mechanisms of stem cell self-renewal/differentiation and can be a useful tool to identify molecular factors inducing stemness from different cell types.

(3) Identification of molecular targets that are specifically regulated by CDK2AP1/MBD3 in mESCs and hESCs: To examine the specific molecular effect of CDK2AP1/MBD3 interaction, we restored Cdk2ap1 wt or Mbd3 binding mutant in Ckd2ap1 ko mESCs and performed gene expression microarray analysis (Affymetrix GeneChip Mouse Genome 430 2.0 array). In addition, we have performed genome-wide ChIP-seq analysis to identify specific molecular targets in hESCs that are associated with CDK2AP1, MBD3, Mi-2beta or active histone H3. Analysis is being done to profile changes in molecular marks that are associated with these molecules during differentiation of hESCs. Results will be essential to defining molecular mechanisms in the regulation of hESC self-renewal/differentiation by MBD3/NuRD complex.

Public Summary: We have performed additional analysis during the NCE period (Aug. 1, 2013 – Nov. 30, 2013) to continue and finish what has been attempted during the Yr 3 funding period. The following is the Summary of research findings during the NCE period.

To define molecular signatures that are regulated by CDK2AP1 either in association with MBD3 or independently of MBD3, we have taken advantage of our ESC model system. We have generated mESC lines from Cdk2ap1-/- cells by restoring either the wild type Cdk2ap1 or the MBD3 binding mutant form of Cdk2ap1. We then performed gene expression microarray analysis. During this NCE period, we have completed bioinformatics analysis of data as briefly presented in the progress report. From this approach, we were able to identify significant differences in molecular roles of CDK2AP1 and MBD3. Some of important pathways are regulated collaboratively by CDK2AP1 and MBD3. In addition we found some important cellular functions that are distinctively regulated by either CDK2AP1 or MBD3. This result demonstrates that CDK2AP1 may participate in the regulation of certain genes through interaction with MBD3/NuRD, while it maintains its own molecular role apart from NuRD complex. We will continue to further define the significance of these molecular roles in ESC biology and also their in vivo significance in development.

As a way to define molecular targets that are specifically associated with CDK2AP1/MBD3-NuRD in hESCs, we have performed the genome-wide ChIP-seq analysis with undifferentiated and differentiated hESCs. We have identified molecular marks that CDK2AP1, MBD3, Mi-2beta or active histone H3 associates and examined how those marks change during the differentiation of hESCs into embryoid bodies. This analysis enabled us to profile gene targets that are under the regulation by MBD3/NuRD during the differentiation of hESCs and also targets of CDK2AP1-mediated MBD3/NuRD control. Results from this analysis will set the basis for further analysis towards defining molecular mechanisms underlying the target selection or specificity of epigenetic regulation by MBD3/NuRD in hESCs. As an additional approach to define the role of CDK2AP1/MBD3/NuRD in neural differentiation of hESCs, we have established a neural induction model from hESCs and performed ChIP-seq and gene expression analysis. Currently we are in the process of analyzing the data and plan to continue beyond the funding period. Our ultimate goal of this approach is to define the role of CDK2AP1 in conferring molecular specificity in the spatial or temporal regulation by MBD3/NuRD in ESCs and during development.