Regulation of apoptosis during endocrine development

Funding Type: 
SEED Grant
Grant Number: 
RS1-00477
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Widespread clinical application of cell-based therapies for type 1 diabetes is limited by a serious shortage of islet tissue for engraftment. Therefore, alternative approaches to beta (β) cell generation are required. Presently, much of the promise of regenerative and restorative medicine lies in stem cells. Understanding the molecular events that drive the genesis of functional insulin-producing cells from undifferentiated stem cells is critical to devise strategies that will help restore and/or maintain β cell mass. One avenue of research directed towards this goal is the in vitro expansion of human embryonic stem cells (hESC). Realizing this potential requires a better understanding of the mechanisms that occur as hESCs differentiate into endoderm and subsequently into pancreatic lineage. Embryonic stem cells face several key developmental decisions in the pathway to forming a mature organ. In pancreatic development, the earliest critical steps are gastrulation and subsequent formation of definitive endoderm. Recently, a method to generate definitive endoderm was described (1); however the process is associated with massive cell loss that occurs by the induction of apoptosis, or programmed cell death. The net result of the differentiation protocol is a small population of endodermal cells that limits the experimental and applicable use of these cells. In the proposed studies, we have assembled a multi-disciplined team to explore the mechanisms of apoptosis during differentiation. We will specifically examine the role of the chemokine/chemokine receptor SDF-1/CXCR4 in this process. The appearance of the CXCR4 receptor is a marker for endodermal development. The receptor plays a plays a critical role initiating cell survival or cell death pathways. We hypothesize that inadequate levels of the CXCR4 ligand, SDF-1α, during endodermal development initiates apoptosis and we will test whether the addition of recombinant SDF-1 protects the cells from death. Additionally, we will study the mechanism by which apoptosis is induced during differentiation. We have identified a novel protein kinase A interacting protein, AKIP, that we hypothesize plays a critical role in initiating apoptotic program. Taken together, these studies will help elucidate the molecular mechanisms associated with hESC differentiation and survival. 1. K. A. D'Amour et al., Nat Biotechnol 23, 1534 (Dec, 2005).
Statement of Benefit to California: 
Although Type 1 diabetes can be managed with daily injections of insulin, no cure for this disease exists. Islet transplants offer hope for many with type 1 diabetes, however, a lack of sufficient tissue for transplantation has restricted the number of patients who can receive therapy. Furthermore, a recent report found that more than 75% of islet transplant patients required insulin treatment within 2 years following the transplant. This underscores the need for a renewable and sustainable source for insulin-producing beta (β) cells. To address this problem, we have initiated studies on the mechanisms of islet growth and function in the hopes of generating a renewable cell-based system for transplant by directing differentiation of human embryonic stem cells (hESC) or pancreatic progenitor cells into insulin producing β cells. Before hESC can be routinely used as therapeutic agents, we must unravel the complicated molecular processes underlying differentiation. A critcal first step in differentiation is the transition of hESC from a pluripotent state to a cell of endodermal lineage. An effective protocol for endodermal differentiation was recently developed, however during differentiation many of the cells initiated a cell suicide program, termed apoptosis. The work proposed here will elucidate the molecular mechanisms that drive endodermal apoptosis. Identification of the key enzymes and proteins in this process will ultimately result in our ability to control differentiation, which will in turn generate larger pools of islet precursor cells. The phenomenon of apoptosis is not unique to endodermal formation. Massive apoptosis has been documented in many differentiation protocols. Therefore, this study has broad implications for all aspects of hESC research. By answering a fundamental question about how hESCs survive during the differentiation process, the state and citizens of California will benefit and its citizens because more laboratories will have greater access to a larger population of cells to study various diseases.
Progress Report: 
  • Epigenetic mechanisms play pivotal roles in cell fate determination during development. Long non-coding RNAs (ncRNAs) are in involved in epigenetic gene expression by recruiting epigenetic regulators to target genes. We are dissecting the role of ncRNAs in the stemcellness and differentiation of human embryonic stem cells (hESC). We have used biochemical and molecular approaches to identify ncRNAs, which associate with epigenetic regulators in hESC and are involved in the regulation of the expression of homeotic (HOX) genes. HOX genes encode for key regulators of cell differentiation in Arthropods and Chordates. We have identified 32 ncRNAs, which are transcribed during hESC differentiation. The detailed dissection of the role of two of the identified ncRNAs in HOX gene expression has resulted in novel insights into the role of ncRNAs in hESC differentiation. The ncRNA Mistral plays an important role in ectoderm development, one of the three germ layers that give rise to all cells, tissues and organs. Inactivation of Mistral ncRNA during hESC development prevents ectoderm development, indicating that Mistral is a key regulator of ectoderm development. The second ncRNA Scirocco is involved in epigenetic activation of HOX genes, controlling mesoderm development.
  • The obtained results support a model in which ncRNAs regulate important steps during hESC differentiation. Our results lay a foundation for the application of ncRNAs in hESC differentiation and the development of diagnostic and therapeutic assays to detect and manipulate the differentiation of hESC. The obtained results open novel areas in the filed of stem cell research by providing tools and assays that actively control the differentiation of hESC. These assays represent valuable additions to the efforts aimed at the active control hESC differentiation in order to obtain desired cell types.
  • The establishment and maintenance of mitotically and meitotically stable -epigenetic- gene expression patterns is paramount for cell proliferation and differentiation. Long non-coding RNAs (ncRNAs) have emerged as important regulators of epigenetic gene expression, in particular epigenetic gene silencing. NcRNAs have been associated with imprinting, gene dosage compensation, gene silencing and metastasis. NcRNAs silence gene expression by recruiting epigenetic repressors of the Polycomb group family to target genes. However, the functional importance of ncRNAs in stem cell biology remains unknown.
  • This project is aimed at the dissection of the role and function of ncRNAs in stem cell differentiation. During the funding period, we have focused on the functional characterization of one ncRNA. The ncRNA is transcribed in differentiating embryonic stem cells (ESCs) and facilitates activation of Hox genes. Hox genes are key regulators of cell differentiation and establish the developmental fate of cells. Destruction of Mistral through RNAi attenuates the differentiation of ESCs. Collectively, our results uncover a role for the ncRNAin epigenetic activation of gene expression and stem cell differentiation and identify ncRNAs as important regulators of stem cell differentiation. The obtained results establish a foundation for the development of novel tools and assays that actively control the differentiation of stem cells and direct the differentiation of stem cells into a desired cell type.

© 2013 California Institute for Regenerative Medicine