Our bodies are made of about 2 billions of different cell types that have their own functions. Diseases like diabetes are largely caused by a breakdown in cell function or by cell death. The major issue of diabetes is an inability to control the level of glucose (sugar) in the blood. Blood glucose levels are normally controlled by insulin produced by islet cells of pancreas. In people with Type I diabetes islet cells are being destroyed by autoimmune system, which is mainly occurred in children. A lifetime of diabetes results in severe and debilitating consequences including kidney failure, adult blindness, nerve damage and cardiovascular disease (leading to limb amputations, heart attack and stroke). The majority of current constructive techniques rely on supply of donor tissues for replacement; however, the major hurdle is finding enough number of human islets to be transplanted into all the people who is waiting on the list for this treatment. The supply of islets donated from cadaver will never be balanced with demand. There is currently no available cure for diabetes that would prevent the occurrence of these consequences. This reality has driven researchers and clinicians to find alternate strategies to cure diabetes. An ideal pancreas, which reproduces the physiological response of the normal pancreas to the glucose changing, would drastically reduces the occurrence of secondary illness and improve the quality of live of diabetic people. Embryonic stem cells (ESC) can make all cell types existing in human body, however at the moment they can not be used for clinical application because the risk of tumor formation and the lack of knowledge for efficient differentiation in insulin-producing cells. We propose to create a novel human cellular system that will address these unmet need, especially for cell therapy. Our goal is to prune all bad characteristics of ESC by selecting a new population of cells from ESC, which can not only maintain to grow indefinitely but also produce enough number of clinical grade cells to make up the destroyed insulin-secreting cells. Our new cells will be called pruned-ESC. In order to demonstrate that insulin producing cells from our pruned-ESC be safely used, we will combine gene and cell therapies for curing Type I diabetes. We will insert an active master gene into pruned-ESC, which is crucial for normal development of pancreas, particularly for insulin-producing cells. Pruned-ESC with a master gene will be given to Type I diabetic mice to evaluate the feasibility of our system for clinical application. We believe that our combined gene and cell technology along with pruned-ESC will allow us to investigate that reasons of Type I diabetes and offer an evidence that is Type I diabetes can be curable in the near future.
Statement of Benefit to California:
Diabetes mellitus (DM) is estimated to affect approximately 18.2 million people in the US alone, and more than 150 million people worldwide. California’s ethnically diverse population is disproportionately affected by diabetes. The overall prevalence of diabetes among California adults is increasing. A study by UCLA Center for Health Policy Research, comparing the years 2001 and 2003, showed that in 2003 nearly 1.7 million California adults age 18 and over (6.6%) has been diagnostic with diabetes in 2003, up from 1.5 million (6.2%) in 2001. By the year 2020, the prevalence of diabetes in California is expected to exceed four million people. Without normal glucose homeostasis over the long term, complications involving the eyes, kidneys, nerves and cardiovascular system are common, which reduce quality of life and significantly increase morbidity, mortality, and cost. For this reason new therapeutic solutions for diabetes should be a priority for California. Stem cell research is among the hottest fields in today’s medical research because these cells have the potential to replace cells that are dysfunctional or lost. The use of stem cells for curing disease and ending disabilities may change the medical treatment in this century. However, there are some major roadblocks that need to be resolved before these cells are implemented in ordinary medical care. Reliable methods for isolation and expansion of stem cells need to be established, efficient differentiation protocols need to be developed, and stem cell plasticity causing tumorigenicity needs to be controlled. This proposal aims to address all of these roadblocks. We will use a novel approach to select a new stem cell population from human embryonic stem cells, which retain differentiation capability but lose propensity for teratoma formation. Our overall goal is to evaluate the potential use of this novel stem cell system for the treatment of a devastating, yet incurable, disease, diabetes mellitus. If we succeed this project our developed system may become the basic of new treatment solutions for diabetes, giving the California a clear competitive advantage over other states in area of stem cell research.
SYNOPSIS: The proposed research will attempt to derive novel hESC populations that are partially differentiated, multipotent, nontumorigenic, homogeneous, indefinitely expandable in the absence of feeder cells, and with the potential to be mass produced at clinical grade for transplantation. Aim 1 will develop a cell-culture strategy to select a stem cell population that is more developmentally restricted and then to define conditions to expand these cells indefinitely while maintaining homogeneity that can be applied to mass production. Aim 2 will modify these optimized hESCs by the forced constitutive expression of Pdx1 and assess the growth, gene expression pattern and beta-cell characteristics of these cells in culture and after in vivo engraftment in the NOD-scid diabetic mouse model. INNOVATION AND SIGNIFICANCE: The generation of unlimited nontumorigenic human embryonic stem cells of clinical quality with broad developmental potential is the principle goal of stem cell technology. The twelve human ES cell lines derived by the CHA Regenerative Medicine Institute could be a valuable new resource. The innovative aspect of this proposal is the possibility that the CHA hESC lines can be grown in a simple medium without a feeder layer for 20 passages without losing developmental potential. STRENGTHS: Strong preliminary results suggest that the human ESCs trained to grow on human feeder cells may provide a valuable source of cells that could be optimized for growth in the absence of feeder cells to provide clinical grade cells for transplantation. The investigators have the training and support for the proposed experiments. An impressive group of advisors has been assembled. Dr. KY Cha has extensive experience with human embryo culture and directed the derivation of 12 hESC lines at CHARMI Korea. Dr Warburton is an established investigator with expertise in endoderm development, particularly lung organogenesis. Dr. DeFilippo, an expert in tissue regeneration using animal models, will consult on animal experimentation. Members of the CHARMI Scientific Advisory Board will contribute expertise in human ESC differentiation (K-S Kim, McLean Hospital & Harvard Medical School) and human ESC biology and culture (HM Chung, Director CHA Stem Cell Insitiute, Korea). All have strong publication records in their respective fields. WEAKNESSES: Although the CHA hESC lines have been characterized thoroughly in-house, it appears that none of the lines have been described in the literature, including the hes11 and hes12 lines, which because they have been trained to grow on human feeder layers, will be the focus of this study. It is unclear which, if any, of the CHA lines have been conditioned without feeder cells altogether, a major claim in this proposal. Therefore, although these cell lines appear promising, sufficient information is not currently available to judge their potential or their suitability for the proposed studies. Specific goals for Aim 1 are described: to induce partial differentiation in order to obtain a novel multipotent hESC population without potential for teratoma formation; to expand homogeneous ESC cultures indefinitely, to develop methods for mass production of clinical grade ESCs. However, experimental plans designed to attain these goals are generally lacking. For example, it is unclear how partial differentiation will be induced, what direction in terms of embryonic development this differentiation is intended, what the nature of the culturing protocol to direct this differentiation will be, how the characterization of the stem cells from amniotic fluid will help guide the hESC cultures, and what explicit criteria or markers will be used to judge whether the state of differentiation has been attained. In addition, evidence suggests that any fully differentiated or committed cell is not tumorigenic. Thus, why would anyone want to compromise the pluripotency and growth of hESCs by 'pruning' or partially differentiating them for no benefit? Aim 2 will force expression of Pdx1 in the CHA hESC lines to induce beta-cell differentiation in vitro and in vivo. The purpose of this genetic modification is unclear. Success with this experiment cannot contribute in a meaningful way to an understanding of the inherent potential of the hESC cells to acquire beta-cell properties and engrafting potential without genetic manipulations. It is an artificial means of inducing pancreatic development that will be irrelevant to any final human protocol. Moreover, Pdx1 expression might be expected to induce ductal and acinar development as well, although this will not be examined. There is NO evidence that forced overexpression of Pdx1 drives the differentiation of hESCs to become insulin producing beta cells. On the contrary, it appears as though Pdx1 directly activates the transcription of certain genes such as insulin, without causing a change in cellular differentiation. Engraftment at organ sites other than pancreas will not be investigated, although this may an important consideration. Additionally, claims of accomplishments without documentation, the lack of organization in the application, presence of extraneous information and the consistent lack of specific experimental plans and criteria to judge outcomes gives little confidence for successful completion of goals. DISCUSSION: There was no further discussion following the reviewers' comments.