Blood formation from human ES cells

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
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.
Progress Report: 
  • Our CIRM SEED grant proposal was to study the pathways of programmed cell death (cell suicide) in human embryonic stem cells. This is a critical area for several reasons: for example, when we transplant stem cells, we need to know how to keep them from dying so that they can be functional. On the other hand, we also need to know how to induce programmed cell death in stem cells, since it is becoming more and more clear that cancers may be propagated by stem cell populations. For these and many other reasons, it is important to know what pathways of programmed cell death are available to stem cells.
  • There are at least five major forms of programmed cell death: apoptosis (the best described pathway), autophagic cell death, PARP-mediated cell death, paraptosis, and calcium-mediated programmed cell death. Each of these programmed cell death pathways is activated by different stimuli and stresses. In the proposed research, we aimed to determine which of the five major forms of programmed cell death occur in human embryonic stem cells (hESCsP). Furthermore, we evaluated how the repertoire of PCD pathways changes when hESCs differentiate into neurons.
  • We first compiled a list of 322 genes whose activity contributes to these various forms of programmed cell death. Of these 322 genes, 311 were found to be represented on the assay system we used. 153 of these genes were measured with a very high detection confidence (0.95 or greater). We performed a special analysis (unsupervised two-way hierarchical cluster analysis) of these genes and represented the expression profiles in a heat-map. Within this group of genes, we chose to focus our attention first on Bcl-2 family members (both pro-apoptotic and anti-apoptotic) because we found transcripts of these gene families to be some of the most differentially expressed within the 43 samples analyzed. We also focused on this gene family because it is a critical family for the control of programmed cell death.
  • We then quantified all members of the Bcl-2 family amongst hESCs and differentiated cells, working under the hypothesis that overly abundant Bcl-2 family member transcripts in hESCs would point toward apoptotic and/or anti-apoptotic signaling cascades that are especially active in hESCs. We were encouraged when we found that the expression of some Bcl-2 family member genes changed dramatically (some up and others down) when hESCs were differentiated to other cell types.
  • We found that apoptosis is readily activated in hESCs, and, surprisingly, that a subset of p53-induced Bcl-2 family genes (e.g., Noxa and Puma) is highly constitutively expressed in hESCs (in comparison to multiple non-stem-cell primary cells). Whereas the pro-apoptotic genes Noxa and Puma are typically expressed only in response to DNA damage and p53 activity, hESCs constitutively express high levels of Noxa and Puma. This finding suggests that embryonic stem cells might be hyper-sensitive to sources of DNA damage like ultraviolet rays and X-irradiation, compared to other cell types, and furthermore, that p53-independent mechanisms of death induced by DNA damage might be operative in hESCs. However, not all p53-induced genes are up-regulated in these cells, since p21 is not up-regulated. These findings raise the important possibility that cultured hESCs may undergo DNA damage despite appropriate culture conditions, which would be a critical issue for hESC growth for transplantation. Another possibility is that p53, the “guardian of the genome”, is indeed protecting hESCs from DNA damage, in part by having a low threshold to activate programmed cell death, but without activating senescence (since p21 was not found to be up-regulated). Thus p53 may, in hESCs, mediate hypersensitivity to DNA damage, as a mechanism to keep the genomes of hESCs “pristine” for long-term functionality. We are performing follow-up studies to determine the mechanism and implications of the striking constitutive up-regulation of this subset of p53 target genes.
  • We are grateful to CIRM for supporting this SEED grant, especially since it has allowed us to identify novel aspects of programmed cell death and the underlying molecules, and to identify a potentially important novel aspect of human embryonic stem cells that may prove to be important in the consideration of transplantation of these cells and their differentiated derivatives.

© 2013 California Institute for Regenerative Medicine