Funding opportunities

Cell-Cell Interactions Promote Differentiation of Human Embryonic Stem Cells to Insulin-Secreting Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00379
Funds requested: 
$487 500
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
One million people in the United States have insulin dependent diabetes - a disease that elevates blood glucose and may result in kidney failure, blindness and amputation. Transplantation of insulin-producing beta cells can establish normal blood sugar levels without the need for insulin injections but multiple doses of cells are required and diabetes returns within 2-3 years in most islet transplant patients. This failure with time is thought to be primarily from “exhaustion” and other insults to an inadequate number of engrafted beta cells. If an abundant source of beta cells was available, long-term success would likely improve. Human embryonic stem cells are a promising source of beta cells for transplantation. A specific cell line of “pluripotential” stem cells differentiates into forerunners of beta cells. The forerunner cells do not make insulin but they can be identified and, when transplanted together with embryonic pancreas or blood vessel cells, will transform into insulin-producing cells. Prior studies have accomplished this transformation only after transplantation in rodents - in vivo - where the insulin-producing cells are not accessible for easy study or harvest for transplantation. We plan to isolate and grow human forerunner cells in the laboratory then mix them in a Petri dish with appropriate cells to coax them into becoming insulin-secreting cells. Once we have an ongoing colony of transformed insulin-producing cells, we will transplant them into diabetic mice (a strain that does not reject human tissue) to assess their ability to reverse insulin-dependent diabetes. These human embryonic stem cell investigations will deepen our understanding of stem cell biology and, potentially, lead to successful long-term treatment of insulin dependent diabetes.
Statement of Benefit to California: 
One million people in the United States have insulin dependent diabetes - a disease that elevates blood glucose and may result in kidney failure, blindness and amputation. Transplantation of insulin-producing beta cells can establish normal blood sugar levels without the need for insulin injections but multiple doses of cells are required and diabetes returns within 2-3 years in most islet transplant patients. This failure with time is thought to be primarily from “exhaustion” and other insults to an inadequate number of engrafted beta cells. If an abundant source of beta cells was available, long-term success would likely improve. Human embryonic stem cells are a promising source of beta cells for transplantation. A specific cell line of “pluripotential” stem cells differentiates into forerunners of beta cells. The forerunner cells do not make insulin but they can be identified and, when transplanted together with embryonic pancreas or blood vessel cells, will transform into insulin-producing cells. Prior studies have accomplished this transformation only after transplantation in rodents - in vivo - where the insulin-producing cells are not accessible for easy study or harvest for transplantation. We plan to isolate and grow human forerunner cells in the laboratory then mix them in a Petri dish with appropriate cells to coax them into becoming insulin-secreting cells. Once we have an ongoing colony of transformed insulin-producing cells, we will transplant them into diabetic mice (a strain that does not reject human tissue) to assess their ability to reverse insulin-dependent diabetes. These human embryonic stem cell investigations will deepen our understanding of stem cell biology and, potentially, lead to successful long-term treatment of insulin dependent diabetes. The proposed research will improve our understanding of stem biology; specifically, how stem cells become insulin-secreting cells. This work will enhance California's standing as a leader in cutting-edge stem cell research, a position that will translate into economic gains through the stimulation of biotechnology investment and scientific endeavor. For citizens of California with insulin dependent diabetes this work could ultimately led to an effective treatment through the transplantation of insulin-secreting cells or, perhaps, the regeneration of patients' own damaged beta cells.
Review Summary: 
SYNOPSIS: The goal is to develop co-culture strategies to induce hESC differentiation into insulin-secreting beta-like cells. Since attempts to derive functional insulin-secreting cells from human ES cells have had limited success, this research will test if other tissue sources, known to favor endocrine development in vivo, can assist development in vitro. Goal 1 is to follow the Semb protocol to prepare partially differentiated hESCs that co-express Pdx1 and Isl1 markers and bear an integrated insulin-promoter/GFP reporter. The enriched population will be co-cultured with 4 different human cell preparations to induce further differentiation monitored by the activation of the INS-eGFP reporter. The cells for co-culture are nestin+ cells from cultured islets; duct cells discarded from islet isolation; endothelial cells from umbilical vessels and from dermal microvessels. Goal 2 will examine the properties of the insulin-expressing cells and their ability to regulate glucose in NOD SCID mice. SIGNIFICANCE AND INNOVATION: In the long term, human embryonic stem cells will very likely become a viable source of insulin-secreting cells for transplantation therapy of diabetics. The technology to produce functional beta-like cells from human ES cells, however, is currently inadequate, in part because the identity and biochemical nature of critical growth factors and morphogens are unknown. This remains true in spite of the report since the submission of this application on the production in vitro of insulin-producing endodermal cells from human ES cells in the absence of co-culturing with inducing cells. The level of insulin production and the extent of differentiation were low and the ability of these cells or any future versions derived from them to control blood glucose is uncertain. Therefore, it is prudent to continue investigation of alternative schemes that previous research suggests might be effective. The use of several potential cellular sources of inducers is a novel and worthwhile idea to pursue. Another reviewer stated that this is an ambitious grant focused on differentiating pancreatic islets from one line of human embryonic stem cells. The premise underlying the proposal is that the various non-islet cells that normally provide trophic support and differentiation signals to islets must be present in co-culture in order for HES cells to differentiate into islets. Consequently there is a lot of primary cell work involved in the proposal and the specific aims are (1) To transfect SA002 HES cells with an Insulin-eGPF insulin promoter reporter (2) To differentiate HES cells into pancreatic progenitor cells that express Pds1, Foxa2 and Isl1. (3) To culture human endothelial and pancreatic cells (ductal cells and nestin-positive populations) (4) To co-culture the differentiated pancreatic progenitors with HUVEN/HMEC or HPCD/HNPC using a layered ECM strategy. (5) To characterize the insulin-secreting phenotype of the resultant fully differentiated cells (6) To implant the differentiated cells under the kidney capsule of NOD mice and follow their function in vivo. STRENGTHS: The PI is an accomplished pancreas-transplant surgeon with expertise in the use of fetal pancreas for transplantation and long-standing interest in the clinical application of basic science advances to the treatment of type I diabetes. He has assembled a collaborative team with broad expertise in embryonic stem cells, endothelial cells (Dr. Talavera), isolated human islets (Dr. Kandeel), and the use of transcription factors to force human ES cell differentiation toward a pancreatic phenotype (Dr. Benvenisty). WEAKNESSES: The experiments require a wide variety of specialized cellular reagents and considerable protocol-specific expertise, not all of which is available to the applicants or their collaborators. The protocols that use antibodies to identify partially differentiated endocrine cells and nestin-positive cells that then will be picked manually for further culturing are not feasible due to the effects of immunological procedures, including fixation, on viable cells. It is unlikely that the partially differentiated Pdx1/Isl1 co-positive cells will have sufficient replicating potential to be propagated to the extent that they can be used to derive clonal lines, and then the further matured insulin+ cells expanded as needed for subsequent experiments, and in the long-term transplantation. Moreover, many of the developmental potential attributed to some of the tissue and cell preparations are controversial, and so the theoretical basis for the projected benefits of some co-culture combinations is not secure. From the cellular complexity of the co-culturing protocols, the uncertainty of induction efficiency, and the difficulty of coaxing differentiated cells to replicate, it is difficult to foresee a manageable plan to scale up production for the numbers of purified, differentiated cells needed for effective transplantation. The PI is an experienced transplant surgeon with a broad outlook on pancreatic islet transplantation but he has not been particularly active in the lab (by CV and PubMed). The problem he picked has been a hard one for stem cell biologists, and has stymied very experienced stem cell researchers. The methods are very complex and dependent on each other. Thus at the end, if there is not success, it will be very difficult to trouble shoot the entire protocol. Sufficient checks at various steps are not presented. Some of the basic issues in the protocols are confusing. Is the plan to establish a permanent line in Aim 1? If so, then the strategy for transfection should be discussed and some basic issues of efficiency of transfection of HES cells acknowledged. Also if this is the foundation on which much of the other work is based, maybe a lentiviral infection would be better? At least some acknowledgement of the pros and cons of the methodology is deserved. The PI has access to pancreatic ductal tissue through the donor network and will have considerable assistance from an experienced pancreas cell culture expert at the City of Hope. Nonetheless, the pancreata that are not transplanted are likely to be of lesser quality than those designated for research, and so there may be difficulty gaining sufficient, good quality cells for use in the experiments. Some background data would have helped to strengthen the case here. PDMS is really toxic to cells, and the fibronectin coating is likely not to be sufficient for HES cells or their differentiated progeny to stick to it. This is only one of several technical hurdles that will likely hurt this effort. It is confusing to read that the differentiated cells will be identified using antibodies and then subsequently hand passaged. How is that possible? Would a FACS strategy help—has anyone sorted off these markers? Or are cells of similar morphologies going to be taken from other plates than those fixed and stained? Details of the co-cultures are also missing. Are the ratios of various cells to others important? Is cell-cell contact important or can conditioned media be used? On page 5, why is clonal assay needed at 7c? In general there are too many technically difficult and risky procedures interfaced to make the reviewer optimistic that the plan will succeed. DISCUSSION: This proposal is from a very experienced clinician with great promise who is proposing to tackle a complex laboratory research problem that would challenge even seasoned laboratory investigators.
Conflicts: 

© 2013 California Institute for Regenerative Medicine