A reliable, reproducible, clinically safe and cost effective technology for generating therapeutically relevant quantities of red blood cells from human embryonic stem cells (hESC) has potential to have a profound clinical significance on the blood supply in the clinics. This is a proposal to develop such a bioreactor with optimal conditions for production of erythrocytes from hESCs based on our recent lab-scale culture conditions. Production of large quantities of mature red blood cells from human ES cells represents, in principle, a limitless source for erythrocyte transfusions. Some patient populations, such as Sickle cel Disease and Thalassemia require frequent blood transfusions and allosensitization to minor (non ABO/RhD) antigens is quite frequent (5 to 35%). In particular , allosensitization is of special concerns in the treatment of sickle cell accuse of significant disparities in the prevalence of variety of non ABO/RhD blood antigens between the donor pool (typically white) and the patient population (typically of African descent). Generation of ES-derived ORh-negtive blood products on a large scale will allow to avoid these problems and create banks of safe blood products, independent of donors, for transfusion medicine needs. This will be especially useful for rare red blood-cell types with limited donor availability. In addition, with possible design of improved universal donor erythrocytes with a limited antigenic repertoire through genetic manipulation will have tremendous clinical significance. In the clinical settings, hES cell–derived erythrocytes would offer a number of superiorities over packed red blood cells (PRBC) currently used in clinical practice. These cells will: 1) have a greatly reduced risk of infection, 2) be a cohort of young cells that will have longer in vivo lifespan, excellent oxygen transport function and improved intravascular survival, 3) be always available, 4) be type O, Rh(D) negative and of a phenotype selected to minimize the risk of a hemolytic reactions, 5) be convenient to use. The generation of universal blood donor-banks will require automated system of production and strategies to increase efficacy of this process, which is the goal of the proposed work.
Statement of Benefit to California:
There has recent alarms on chronic shortage of safe blood and blood products and recommendations on new strategies to prevent transmission of HIV and other blood-borne pathogens, such as collecting blood only form donors at the lowest infectious risk (reports of Fifty-Eighth World Health Assembly in May of 2005). It is also proposed to introduce legislation to eliminate paid blood donation “expect in limited circumstances of medical necessity”. Production of large quantities of mature red blood cells from human ES cells represents, in principle, a limitless source for erythrocyte transfusions. Some patient populations, such as Sickle cel Disease and Thalassemia require frequent blood transfusions and allosensitization to minor (non ABO/RhD) antigens is quite frequent (5 to 35%). In particular , allosensitization is of special concerns in the treatment of sickle cell accuse of significant disparities in the prevalence of variety of non ABO/RhD blood antigens between the donor pool (typically white) and the patient population (typically of African descent). Generation of ES cell-derived ORh-negtive blood products on a large scale will allow to avoid these problems and create banks of safe blood products, independent of donors, for transfusion medicine needs. The generation of universal blood donor-banks will require automated system of production and strategies to increase efficacy of this process. This is a proposal to develop such a bioreactor with optimal conditions for production of erythrocytes from hESCs based on recent success achieved in lab-scale culture conditions. If successful, this would provide tremendous advantages for the State-of –California: unlimited supply of safe blood, ability to produce blood with specific, low antigenic phenotypes for transfusions in patients with b-thalassemia and Sickle Cell Anemia. Since Bay Area, specifically Oakland has a very large Sickle Cell and Thalassemic population, the economic and health advantages will be enormous.
SYNOPSIS: Production of mature erythrocytes from SC at clinically useful rates from adult hematopoietic SC has been reported. The PI proposes to develop large scale culture bioreactors for for this purpose, controlLing all critical aspects of the problem. He will assess the morphology, viability, relevant marker and gene expression patterns and numerical efficiency of the process to generate a mathematical model to relate reactor variables to experimental observations and optimize design and conditions. The work will be performed in collaboration with Dr. Carrier, who will supply hES cell lines grown without serum or feeder cells and who will assist with culture conditions and additives. Dr. Nakamura, who recently described efficient techniques for enucleating erythrocites in culture, will also collaborate and will actually visit the PI's Lab during the grant period. Dr. Srivastava, an Assistant Project Scientist, will apparently perform the culture work. SIGNIFICANCE AND INNOVATION: As described, this project will attempt to enable a scale-up of methods for culturing hSC in an attempt to generate clinical quantities of RBCs of clinical quality. The advantages of a practical system would be presumably enhanced by starting from hESC grown without exposure to xenogeneic serum or feeder cells (available from Dr. Carrier). The innovative aspect of this proposal, other than the use of O, Rh-negative hESC, is the attention to the bioengineering aspects of intermediate or large scale culture of these cells. This attention may yield general principles to support similar culture methods for these and other eventual uses. If hRBC of appropriate quality could be raised in large enough amounts at reasonable cost, the results of these studies could change medical practice in a major way. The harvesting of blood for transfusion has become increasingly difficult and expensive as regulatory efforts to reduce the risk of infectious disease transmission become more and more sophisticated. Using universal donors for RBC and possibly nuclear transfer from recipients for marrow replacement are mentioned and would be certainly facilitated by success in these experiments. The significance of this proposal relates to the potential use of hES cells as a source of either hematopoietic stem cells (HSCs) or red blood cells (RBCs) for clinical use in either bone marrow transplantation or replacement of red blood cells. The innovation relates to the experience of the investigators in development of bioreactor systems for large scale cell growth. The novel concept would be to develop an O-Rhneg source for blood products. STRENGTHS: - The PI is an expert in the physical and mathematical aspects of flows through capillary channels in solids and other types of vessels. This should enable an elegant study of design characteristics for a bioreactor. The CV does not list references on bioreactor design per se. - The collaborators, Drs. Carrier (20% effort) and Nakamura (90 and 75% on years 1 and 2), are well known experts in the use of stem cells for studies of differentiation. Dr Carrier will provide hES cell lines of presumably clinical grade and Dr. Nakamura will help with the cultures, particularly to obtain the terminal differentiation and enucleation of erythrocytic precursors. - The large time commitment of Dr. Nakamura is a particular strength. Despite his full time commitment to the RIKEN BioResource Center in Tsukuba, he will spend essentially full time in this project the first year and 75% of it in the second, as visitor to UCSD. - Dr. Srivastava appears to have experience with stem cells. - The proposal describes generating EBs from hES cells in a scale-up mode, isolating CD34+ cells for erythroid cell growth, and then enucleation. WEAKNESSES: - The required expertise in the tissue culture aspects of this proposal lie with the senior collaborators. Dr. Srivastava is a junior colleague who is described as having several years of handling stem cell studies. He is an author of several papers with Dr. Carrier, including one on the migration into the brain by cells descended from transplanted ESC. Presumably he will do the hands-on work for the cultures, assisting Dr. Nakamura, with a 50% effort. - The PI's own expertise is in the fluid dynamic aspects of the reactor and may well be secondary to the contribution of the collaborators. There is little specificity in the proposed work about how he will carry out the declared objectives in practice. - There is no description of concern regarding critical issues in practical implementation: clinical suitability of the RBCs produced and cost. Yet these aspects are of utmost importance to the success of the proposal. - Although mentioned in general terms, there is no development of the idea of generating embryonic hematopoietic stem cell lines for transplantation. Yet this may be the most effective way to restore bone marrow from hESC possibly matching the recipient's genotype completely. - This proposal shows little regard for potential difficulties in hematopoietic cell differentiation from hES cells. One might question the value of the proposed goal, that is production of red cells from hES cells for clinical use. There is no mention of the scale-up that would actually be required to provide a source for administration. There are substantial differences in the ease of in vitro differentiation of human and mouse Es cells. Indeed, there is still uncertainty as to how adult type red cells can be produced from hES cells. The feasibility of the entire venture is under question. Alternatively, the focus might be placed on generation of HSCs from hES cells, as fewer cells would likely be needed for reconstitution of hosts and these HSCs would generate all cell types. The proposal is also not well written in many places. RECOMMENDED CHANGES: The proposal should be more focused on the work of the PI in developing a practical biorreactor (or bioreactors) for the declared purpose. An admittedly secondary issue at this time, cost, will eventually determine whether hESC derived RBCs will be used. It should be part of the description and discussion, given the sharp focus on the practical objective. The PI needs to consider and estimate the level of scaleup required. The authors need to examine how many red cells that might be able to produce on small scale and whether these cells have the properties of adult type red cells in terms of hemoglobin composition and rheological properties. DISCUSSION: There was no further discussion following the reviewers' comments.