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RS1-00192-1: An Optimal Bioreactor For Production Of Erythrocytes From Human Embryonic Stem Cells

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

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 (provided by applicant)

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 {REDACTED} has a very large Sickle Cell and Thalassemic population, the economic and health advantages will be enormous.

Review

SYNOPSIS OF PROPOSAL: Production of mature blood cells from stem cells at clinically useful rates from adult hematopoietic stem cells has been reported. The PI proposes to develop large scale culture bioreactors for for this purpose, controlling all critical aspects of the problem. He/she will assess several 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 researchers with stem cell expertise.

INNOVATION AND SIGNFICANCE: As described, this project will attempt to enable a scale-up of methods for culturing human stem cells in an attempt to generate clinical quantities of red blood cells of clinical quality. The innovative aspect of this proposal 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 human red blood cells 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.

STRENGTHS OF PROPOSAL:The Principal Investigator (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 charactyeristics for a bioreactor, although curriculum vitae does not list references on bioreactor design per se.

The collaborators are well known experts in the use of stem cells for studies of differentiation, and the large time commitment of one collaborator is a particular strength.

The proposal describes specifics for generating embryoid bodies from hESC in a scale-up mode, which is also considered a strength

WEAKNESSES OF PROPOSAL: The required expertise in the tissue culture aspects of this proposal lie with the senior collaborators,and not with the PI. 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, including clinical suitability of the red blood cells 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, although 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. Furthermore, the proposal is also not well written in many places.

The reviewers suggest 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 red blood cells will be used. It should be part of the description and discussion, given the sharp focus on the practical objective. In doing this, the PI needs to consider and estimate the level of scaleup required and include an examination of how many cells will be needed.

DISCUSSION: There was no further discussion following the reviewers’ comments.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Lansing, Sherry