Jon S. Odorico - US grants

A comprehensive knowledge of the genetic programs and embryonic inductive events regulating human pancreas development would advance our understanding of congenital pancreatic malformations and aid in developing rational treatment strategies for diabetes mellitus. Significant insights to the mechanisms of pancreatic organogenesis have been gained through recent experiments in mice and chickens. However, critical differences in early developmental pathways between these species and humans places the direct applicability of these data to human development in question. Furthermore, functional studies in human development are restricted by the ethical implications of directly manipulating human embryos. Consequently, a more "human" model based on nonhuman primates would have broad application for studying human development. Recently, embryonic stem (ES) cell lines from nonhuman primates (rhesus macaque and common marmoset) have been derived and their potential for differentiation into derivatives of all three embryonic germ layers (ectoderm, mesoderm, and endoderm) in vivo has been characterized. Their capacity for lineage-restricted differentiation in vitro, however, has not been thoroughly tested to date. in contrast, neural, hematopoietic and muscle lineage differentiation of mouse ES cells in vitro has been extensively studied. Recently, the ability of mouse ES cells to undergo elements of endoderm and pancreatic differentiation in culture has also been demonstrated. Therefore, we will test the hypothesis that pancreatic endocrine and/or exocrine lineage specification can be recapitulated in rhesus ES cells induced to differentiate. The specific aims of this proposal are: l) To determine the expression pattern of endoderm related genes as well as pancreatic exocrine/endocrine-specific genes in rhesus ES cells differentiating in vitro, and 2) To determine the potential of rhesus ES cells for pancreatic lineage differentiation in ES cell derived tumors in immunocompromised mice. A combination of RT-PCR analysis, Northern hybridization, ribonuclease protection assay, in situ hybridization, and immunofluorescence studies will be used to characterize the pattern of pancreatic gene expression. These studies will provide a basis for the refinement of an in vitro primate model of human pancreatic development. Such a model system would have important implications for investigating the mechanisms controlling pancreatic differentiation and for developing new cell-based strategies for treating diabetes.

artificial immunosuppression; human therapy evaluation; immunosuppressive; diabetes mellitus therapy; insulin dependent diabetes mellitus; pancreatic islet transplantation; pancreatic islet function; blood glucose; insulin; hypoglycemic agents; patient oriented research; human subject; clinical research;

stem cell transplantation; embryonic stem cell; pancreatic islets; cell differentiation; Macaca mulatta; animal colony;

DESCRIPTION (provided by applicant): Embryonic stem cells (ESCs) are envisioned as a source of transplantable pancreatic islets to alleviate the donor organ shortage. Despite many recent advances, protocols to differentiate ESCs to homogeneous pools of glucose-responsive insulin-secreting beta cells have yet to be developed. It is widely accepted that the production of these populations will first require generation of definitive endoderm. Thus, the successful derivation and stable propagation of endoderm-restricted stem cell lines would be a significant step forward in the development of pancreas, liver, and lung cells for cell replacement therapies and for improving our understanding of how these tissues are normally formed from this germ layer. We have developed a simple protocol, using magnetic-activated cell sorting (MACS), to isolate endoderm-committed cells from heterogeneous cultures of differentiated murine ESCs on the basis of epithelial cell adhesion molecule (EpCAM) expression. The protocol includes negative selection to remove undifferentiated ESCs and visceral endoderm cells, and positive selection to retain cells that express EpCAM. When sorted cells are placed sub-cutaneously into immunodeficient mice, small EpCAM+PDX1+ nodules highly reminiscent of embryonic pancreatic epithelium develop. Cells from these nodules can be extensively propagated in vitro and have the characteristics expected of an endodermal stem cell (EndSC) or foregut- restricted stem cell (FGSC) population, depending on the duration of growth in vivo. The EndSCs are OCT4+ and express some early endoderm-restricted genes, whereas FGSCs are OCT4- and have high expression of Sox17, Foxa2, and PDX1. EndSCs and FGSCs transplanted back into animals recapitulate the original nodule phenotype, demonstrating their stability in vitro. We hypothesize that only the OCT4+EpCAM+SSEA1- cells recovered from the MACS separation give rise to the in vivo-derived endodermal cell lines. Here, we will directly test this hypothesis by FACS-sorting OCT4+EpCAM+SSEA1- cells using an OCT4GFP ESC line. Cell lines derived from the FACS-sorted population will be compared to the existing MACS-derived EndSCs and FGSCs. We will also study the ability of EndSCs and FGSCs to differentiate in vitro to specific endoderm-derived cell types, including beta cells. To promote differentiation, we will culture cells in an embryonic pancreas environment, expose cells to relevant growth factor signals, and culture cells according to recently published protocols. These studies will contribute not only to improved differentiation efficiency of ESCs to definitive endoderm and pancreatic lineages, but also to a better understanding of germ layer segregation and early embryonic cell fate decisions. PUBLIC HEALTH RELEVANCE: Our preliminary studies have led to the development of cell lines from differentiated mouse embryonic stem cells (ESCs) that appear to be foregut endoderm-committed. These cell lines are non-tumorigenic and grow well in culture, and here we propose to elucidate the cell of origin of these endodermal cell lines and explore their ability to differentiate into pancreatic, liver, and lung cells in vitro. This knowledge will help in establishing protocols for the isolation and differentiation of human endoderm-committed cells for the production of functional cells to be used in transplantation therapies.

ABSTRACT Diabetes and its complications still claim the lives of millions of people despite continuing advances in insulin delivery technology primarily because insulin fails to achieve perfect glycemic control. On the other hand, beta cell replacement therapies including vascularized pancreas and isolated islet transplantation are able to fully restore normoglycemia, achieve insulin-independence and can delay end-organ complications. However, these latter therapies suffer from two key limitations, the shortage of organs and the need for life- long immunosuppression to prevent allograft rejection. Furthermore, the intrahepatic portal vein islet transplantation site used in humans is far from ideal and many islets are lost after implantation. An ideal beta cell replacement therapy strives towards both generating an abundant supply of functional beta cells and identifying a minimally invasive, well-vascularized, retrievable site for transplantation that is clinically applicable. After years of research it is now well established that human pluripotent stem cells (hPSCs) can be directed to differentiate into highly enriched physiological functional islet-like clusters (ILCs) in vitro that are capable of curing diabetes in mice. The extracellular matrix (ECM) is a critical component of the cellular niche that helps maintain cellular differentiation and provides tissue-specific signals to guide the fate and behavior of cells. Recent progress in the decellularization of organs has spurred great interest in using natural matrix for regenerative medical applications; yet, few studies have focused on the pancreas in general and the human pancreas to date has not been effectively decellularized and studied. Appreciating the importance of tissue-specific ECM, we have established effective techniques for the decellularization and delipidization of human pancreas tissue to produce several types of natural matrix constructs, including intact 3D matrix, molded sponge scaffolds and a spontaneous gelling hydrogel (hP-ECM). With the challenges of identifying a clinically applicable transplant site that provides for immediate and sufficient oxygen and nutrient delivery, we believe there is compelling rationale to take advantage of the proven proangiogenic and anti-inflammatory properties of ECs and MSCs. Thus, transplanting ILCs with hPSC-derived endothelial cells (ECs) and hPSC-derived mesenchymal stromal cells (MSCs), each providing essential properties, combined with hP-ECM into a prevascularized deviceless retrievable subcutaneous site might provide a more optimal transplant platform. Now, based on this innovative technology we aim to obtain a better understanding of the composition and function of natural hP-ECM in the context of hPSC differentiation to beta cells. The immediate objectives are to characterize human pancreatic extracellular matrix and to use this natural matrix in combination with stem cell-derived ? cells, ECs and MSCs to reconstruct endocrine tissue capable of glucose- stimulated insulin-secretion after transplantation to mice. Our specific aims are to: 1) Comprehensively characterize the human pancreatic and islet ECM proteome, or matrixome, and compare the matrixome of different developmental ages using advanced quantitative mass spectrometry methods in collaboration with Dr. Linjun Li, 2) Construct a hP-ECM - cellular composite tissue graft combining hPSC-ILCs with ECs +/- MSCs and test its function in an immunodeficient murine diabetes model. Ultimately, we envision a bioengineered composite endocrine organ as a highly innovative regenerative medicine strategy for producing potentially autologous insulin-producing tissue for transplantation. These basic enabling studies are the first steps towards developing an effective, minimally invasive transplant platform that is available for all patients with diabetes.