Blood formation from human ES cells
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.
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.