Hospitals experience recurrent shortages of blood, especially type O, Rh (D) negative red blood cells (RBC) which are critical for trauma victims and many other patients who undergo major surgery or are being treated for serious illness. Blood Banks depend on the altruism of the public to donate RBC; however the donor pool has become increasingly restricted as new donor exclusions become necessary. The awareness of the trend toward increasing blood shortages motivated us to investigate donor-independent RBC, which will be consistent in quality, essentially infection-free and available on a large scale. With the advent of research on embryonic stem cells, the opportunity exists to develop RBC and eventually to create donor-independent, universal donor (O,Rh-negative) RBC banks. In the clinical setting, human ES cell–derived RBC would be expected to have a number of advantages over packed RBC (PRBC) currently used in clinical practice: 1) they will have a greatly reduced risk of infection, 2) they will be a cohort of young cells of consistent characteristics and quality that will have excellent oxygen transport function and improved intravascular survival, 3) they will be always available, 4) they will be type O, Rh-negative and of a phenotype selected to minimize the risk of a hemolytic reactions due staff or blood bank errors, 5) they will be convenient to use. Human ES cells can be expanded indefinitely in vitro and may eventually be derived by reprogramming of somatic cells or taken from a bank representing major haplotype combinations. This unlimited expansion allows large absolute numbers of erythrocytes to be generated, enabling the continuous replenishment of banked ES-derived RBC samples. This project is based on our proof-of-principle preliminary data, which demonstrate that hematopoietic progenitors can be generated from human ES cells and that strategies can be developed to enhance the efficiency of this process. The work will progress in three stages: 1) development of culture conditions that are serum-free, feeder-lines free; 2) development of Rh-negative ES cells line with normal karyotype and 3) development of strategies to enhance the process of proliferation and differentiation. The characterization of final product, optimization of production and storage conditions will be developed in subsequent proposals. At the conclusion of these studies we will establish 1) novel serum-free, feeder-cell free culture conditions for non-NIH, California-derived human ES cells, 2) specific methods to select donors and generate novel O, Rh-negative, extensively phenotyped ES cells lines for the generation of future, universal donor transfusion products, 3) novel humanES cells line with enhanced proliferative capacity. The long-term objective which will be addressed in future grants is to develop ES-derived erythrocytes for banking and transfusion medicine protocols.
Statement of Benefit to California:
The recent Fifty Eight World Health Assembly in May of 2005, was alarmed by chronic shortage of safe blood and blood products and recommended new strategies to prevent transmission of HIV and other blood-borne pathogens, such as collecting blood only form donors at the lowest infectious risk. It also proposed to introduce legislation to eliminate paid blood donation “expect in limited circumstances of medical necessity”. 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. Dr. Carrier has developed collaborations with Dr. David Smootrich for IVF Clinic in La Jolla. He has generated a bank of 1000 fertilized eggs and has a list of volunteer donors, who want to donate eggs for research. Dr. Smootrich is in the position to provide fertilized eggs that have a favorable phenotype to become universal blood donors, eg ORh-negative (cde/cde), and also negative for Kell, Duffy(a), Kidd(a). According to data from Marion Read, Ph.D. from the New York Blood Center, we will have to screen 1000 embryos to identify such phenotype. We propose to use the currently available embryos to develop reliable, efficient, cost-effective methods to screen and select optimal embryo donors for RBC production for a universal blood bank. The generation of universal blood donor-banks will require automated system of production and strategies to increase efficacy of this process. Dr. Carrier has developed collaboration with the Bioengineering Department at UCSD and experiments are in progress to develop a bioreactor device for this purpose. 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 the Bay Area, specifically Oakland has a very large Sickle Cell and Thalassemic population, the economic and health advantages will be enormous. In addition, the scale up of this project into a practicable method to supply blood for transfusion will generate economic benefits, through the establishment of large scale manufacturing operations, and also by attracting new biotech firms and supporting existing ones.
SYNOPSIS: The investigator proposes to develop a system for generating O-Rh-negative red cells from human embryonic stem cells. The PI indicates that she has access to 1000 fertilized eggs from which embryonic stem cells of the appropriate genotype can be derived. Her proposal includes 2 specific aims: 1) enhancement of self renewal and proliferative capacity of human ES cells by bcl-2 transfection and 2) establishment of serum-free, feeder-free culture conditions. In collaboration with a company, the PI has designed and developed a hematopoietic-specific microplate biomaterial microarray, which allows screening of "hundreds of cytokine combinations" with or without human mesenchymal stem cells using a type of stromal support. SIGNIFICANCE AND INNOVATION: This investigator has indeed identified a significant problem, namely the limited availability and potential biocontamination of currently utilized blood products. Conceptually, the proposal is innovative in attempting to establish hESC-derived cell lines that produce red blood cells (RBC) with a “universal donor” phenotype in unlimited numbers for transfusion purposes. If the cell line is appropriate to the task of producing RBC in the amounts needed for public health, (some 12 million transfusions in the US per year), this approach would have profound significance on the practice of transfusion medicine and optimal treatment of individuals who are chronically dependent on red cell transfusions. Developing such a cell line is an important achievement but if it is intended for practical use, the efficiency of the process through proliferation, maturation and enucleation must be improved. The PI’s proposal to do this by constitutively expressing an anti-apoptotic gene, bcl-2, is reasonable and important, and may be essential for the practical purpose intended. The study and optimization of the multiple interactions between the factors involved in erythropoiesis in-vitro and the proper level of expression will be a very tedious brute-force event, and the PI appears to have prepared the way by collaborating in the development of an instrument that provides increased efficiency in the search. Otherwise, the search for proper conditions might well be “endless”, as the PI states. In addition to the practical usefulness of obtaining a cell line with all or most of the desired characteristics, this proposal would advance the state-of-the-art of induced differentiation and genetic manipulation of hESCs. STRENGTHS: The PI is a recognized expert in both hematopoiesis and hESC culture, has prior experience in stem cell research with both murine ES and human ES cells, and has apparently already obtained proof of principle for the derivation of erythropoietic cells from EC. The approach will require the PI’s knowledge of hESC behavior in culture and an understanding of the multiple steps of erythrocyte generation, and the PI will be assisted by well-chosen collaborators and advisers who are recognized experts in their respective fields. This team is well suited to accomplish the experimental steps that seem to be required. Although only H1 is mentioned specifically, the PI will have access to many novel lines to be developed by an IVF clinic in La Jolla (letter from Dr. Smotrich), and additional RBC-producing lines might be developed eventually when useful red blood cell phenotypes are uncovered. WEAKNESSES: Although much detail is properly given regarding hESC handling, other important aspects are not mentioned, such as: when will the cell lines be characterized for blood group genotypes? The task of making and caring for these lines until their typing is finished will not be inconsiderable. Typing both parents could limit the numbers but this will not be an inconsiderable task and a strategy is clearly required. The rationale for various experiments is also lacking. For example, while the biomaterial microarray system may be useful, little rationale is provided as to which combinations of cytokines will in fact be screened. Moreover, little justification is provided for the choice of bcl-2 enforced expression to prevent apoptosis of ES cell derivatives (the stated rationale is that its over-expression prevents apoptosis of erythroid cells in the absence of erythropoietin). There is some indication that this group has performed "proof of principle" experiments indicating the feasibility of generating enucleated erythrocytes from ES cells, but such data are not provided in the application. The investigator provides an extensive description of the plans to generate ES cells expressing bcl-2 in Aim1. However, in the Expected Results and Alternative Options, the PI indicates that "in our preliminary experiments including conventional culture conditions containing serum, ES cell clones expressing bcl-2 and non-transfected ES cells lines grew with similar kinetics". Thus it is unclear whether new experiments are proposed or whether they are describing data that already have been obtained. An hESC line with stable expression of the bcl-2 construct will be needed. Has such a line been developed? Dr. Azad’s letter only mentions “transient expression”. The proposal is difficult to read at times because of typos and errors of diverse kinds, including grammatical errors. Finally, the PI does not elaborate on the advantages to the people of California of funding the both groups. DISCUSSION: One reviewer suggested that Dr. Carrier, with the help of Dr. Reed might test all available hESC cell lines for the desired blood group types.