The clinical potential of human embryonic stem cells (hESC) for transplantation will be realized only when we can develop methods to control the process of tissue differentiation far more efficiently than is currently the case. From over 40 years of experience with adult stem cells, it is recognized that the growth of transplanted bone marrow is generated from the hematopoietic (“blood-forming”) stem and progenitor cells present in the graft. Mature, differentiated cells that accompany the stem cells disappear rapidly after transplantation as they lack the ability to self renew. It is thus essential when designing clinical approaches that use tissue derived from hESC, to specifically target the production of stem and progenitors that will survive, proliferate and differentiate after transplantation. This proposal addresses three fundamental questions for the entire hESC field 1.Do hESC differentiate through the same pathways that exist in adult tissues, 2.How do the conditions in which hESC are initially derived from blastocysts affect their subsequent potential for generating tissue specific stem and progenitor cells, and 3.How can hESC differentiation be regulated to provide large numbers of tissue specific stem and progenitor cells able to engraft and differentiate long term? Studies of hematopoiesis in mice have provided the conceptual basis for the entire field of stem cell biology. However, fundamental biological and technical differences exist in both hematopoietic and embryonic stem cell biology between the murine and human species. Our group has chosen over the past 15 years to focus on the study of human hematopoietic stem cells and lymphoid (immune system) progenitors, more recently bringing these concepts and tools to study hematopoietic differentiation from hESC. In brief, our aims in this proposal are: 1. To understand the pathways along which the blood and immune system are generated from hESC, 2. To assess if the methods by which hESC are derived affect their capacity for hematopoiesis, and 3. To develop the means to expand hematopoietic stem cells derived from hESC. I believe that there are two broad reasons why these studies are important. First, as a pediatric bone marrow transplant physician, I am keenly aware that profound clinical problems remain for my patients. Matched stem cells from healthy donors are often unavailable and poor recovery of the immune system after transplantation results in an unacceptably high incidence of death and illness from infection. Second, as a stem cell biologist I recognize that the well established tools that can be applied specifically to hematopoietic development from hESC are uniquely able to answer some of the most fundamental questions about how hESC generate tissues and how we can best control the process. With these answers we will be able to tailor our approaches for differentiation to all tissue types and move the intriguing biology of hESC more rapidly and safely to the clinic.
The unique combination of pluripotentiality and unlimited capacity for proliferation have provoked hope that hESC will one day provide an inexhaustible source of tissue for transplantation and regeneration. Diseases that might be treated from such tissues affect millions of Californians and their families. However, the clinical potential of hESC for regenerative medicine will be realized only when the process of tissue specific differentiation is significantly more efficient and controlled than is currently the case.
This research proposal has two broad goals. The first is to explore some of the fundamental biologic questions about how individual human embryonic stem cells (hESC) are recruited into a specific pathway of tissue differentiation. Our approach to these questions will be to use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation and offers a range of unique technical tools with which to study these questions rigorously. However, the fundamental concepts formed from these studies will have broad applicability to other types of tissues. By understanding these processes, the development of methods to translate hESC into production of other tissues such as islets, neural cells and cardiac muscle to the clinic will be more successful.
The second goal is to develop approaches to efficiently produce elements of the blood and immune system from hESC for use in transplantation of a variety of diseases. Hematopoietic Stem Cell Transplantation (HSCT, aka Bone Marrow Transplantation) is the most mature example of the clinical application of stem cells, representing a life saving procedure for leukemia, lymphoma, and many other types of blood and immune system diseases. Nonetheless, profound clinical problems remain for the HSCT field particularly in the allogeneic setting. These problems include lack of suitable, matched bone marrow donors for many patients and poor recovery of the immune system after transplantation leading to death and illness from infection in an unacceptably large number of patients. The possibility of producing large numbers of compatible hematopoietic stem and progenitor cells suitable for clinical transplantation presents an opportunity to fundamentally change clinical practice in the HSCT field.
All scientific findings and technical tools developed in this proposal will be made available to researchers throughout California, under the guidelines from the California Institute of Regenerative Medicine.
SYNOPSIS: The applicant will investigate the process of tissue-specific differentiation from human embryonic stem cells (hESCs) with the eventual aim of making it more efficient and controlled than is currently the case. Three basic questions will be addressed: 1) To what extent are differentiation pathways of postnatal tissue recapitulated during hES cell differentiation? 2) How do techniques used for hES cell derivation, propagation, and differentiation affect the tissue-specific stem and progenitor cell generation? 3) How can hES cell differentiation be regulated, especially in the direction of hematopoietic stem cells that are capable of regeneration after transplantation? To this end, the investigators propose to define the clonogenic stages of differentiation of lymphohematopoietic cells derived from hESCs, determine their intrinsic lymphohematopoietic potential, and develop approaches to regulate expansion of engraftable hematopoietic stem cells and lymphoid progenitors.
IMPACT AND SIGNIFICANCE: Whether hematopoietic stem cells derived from hESCs will one day provide an inexhaustible source of tissue for transplantation remains speculative. At this point, attempts to engraft such cells in immune deficient mice have been largely unsuccessful except for some low level engraftment when cells were infused directly into the marrow cavity. This could be either the result of homing defects in cells derived from hESCs or, alternatively, low frequency of true hematopoietic cells. The applicants' work will address this by applying immunophenotypic functional molecular and transplantation assays that were developed to study cord blood and marrow populations. Their planned efforts to develop a quantitative assay to measure hematopoietic potential and clonal diversity as well as work on culture conditions to facilitate recruitment of hESCs to the hematopoietic pathway will increase our understanding of how to most efficiently propagate such cells. The same can be said for the other specific aims, which are focused on determining intrinsic lymphohematopoietic potential of hESC lines derived and propagated in different conditions and developing an understanding of how to regulate the expansion of engraftable hematopoietic stem cells. If successful, the project will allow the discrimination of discrete stages in the development of mature blood cells from hESCs, which is not currently possible, and will shed light on potential differences in the hematopoietic potential of different hESC lines. In addition, the applicant proposes use of a novel reagent for subsequent expansion of hematopoietic precursor cells generated from hESCs.
The success of this work would represent a major therapeutic advance, enabling a new paradigm of genetic medicine for inherited disorders. To do this, one has to learn how to direct hESCs to a preferentially, and hopefully, uniquely hematopoietic fate. In addition, it could allow for large scale production of hematopoietic stem cells for cancer therapy. Achieving this goal requires not just creating hematopoietic stem cells from hESCs, but doing so in large number. These are two extremely important targets for hESC research.
QUALITY OF THE RESEARCH PLAN: The research design and methods are well developed in the applicant's laboratory and are based on 15 years of work in identifying, manipulating, and transplanting rare multipotent and lymphoid-restricted clonogenic populations from human umbilical cord blood and bone marrow. The applicants have experience with lentiviral marking that will allow them to track the clonal origin of hematopoietic lineages produced from hESCs. They have experience with clonal analyses as well as culture conditions required for these studies. Their most ambitious aim is to develop approaches to regulate expansion of engraftable hematopoietic stem cells. Their experience with expansion of cells from cord blood will be helpful. The dimerizer technique proposed for that purpose is elegant and based on solid work carried out by others. The time line seems realistic.
In general, the plan addresses an important and interesting area, but in several areas the experimental design is too vague to evaluate (particularly regarding the assays for establishing purity of sorted cell populations in Aim 1.1, the cytokine testing in Aim 1.3, and the discrimination of "intrinsic" and "extrinsic" factors in Aim 2), fails to consider potential difficulties in interpretation, or does not provide adequate alternatives if the proposed approaches are unsuccessful. In addition, relevant preliminary data is lacking, particularly regarding the proposed in vivo assays. While nice data is provided showing engraftment of human bone marrow cells in NOD/SCID mice, since much of the proposal relies on engraftment of hESC-derived cells, data demonstrating feasibilty of this technique seems essential.
There are concerns as well with the Competitive Limiting Dilution Assay proposed in Aim 2. If the hESCs are marked in the fully undifferentiated state, and then the same integrant shows up in the B and myeloid lineages, this could reflect the existence of a multipotent hESC-derived hematopoietic stem cells, but also could result from self-renewal of the marked hESCs prior to differentiation, each of which subsequently generated 1 lymphoid and 1 myeloid progentor.
Finally, regarding Aim 3, if hESCs are being investigated as a renewable, expandable source of hematopoietic stem cells, then the choice to focus on expansion of hESC-derived hematopoietic stem cells seems to require some additional justification, especially as hematopoietic stem cells (from any source!) have been notoriously difficult to expand ex vivo.
STRENGTHS: A major strength of the proposal is the experience of the principal investigator (PI), who has a solid track record with in vitro work involving hematopoietic stem cells. This includes work on gene transfer owing to close collaboration with Dr. Kohn, a co-investigator on this application. The preliminary studies include a demonstration that hematopoietic stem cells (not hESC-derived) can be dynamically imaged in immunodeficient mice. Studies also show enhanced engraftment of stem cells after dimerization of a constitutively expressed signaling domain of the cytokine receptor MPL and treatment with a diffusable synthetic ligand (AP 20187); again, cells were not yet hESC-derived. The work is logically planned and is likely to provide important basic information on lymphoid hematopoietic progenitors generated from hESCs and their potential transplantability in NOD/SCID mice.
The major weakness is the lack of an in vivo readout of long-term functionality of the hESC-derived hematopoietic stem cells. Also the experimental plan is vague at crucial points and is over-ambitious, e.g. identifying each step in the lymphomyeloid differentiation pathway, which is high risk with respect to success. There is a lack of clear criteria for establishing purity of isolated populations. More preliminary data would have been useful, particularly regarding in vivo hES transplantation. As noted above, there is also the potential for alternative interpretations of data that thus may not allow clear conclusions
There is a question of relevance of the fusion-protein mediated expansion of hESC-derived hematopoietic stem cells. The field is at such a state of infancy that the applicant will have to begin at the very beginning. Most of the proposal is very basic culture work (aim 1) and assay development and validation (aim 2).
The lineage direction and expansion experiments in Aim 3 are long shots, which might or might not work. They assume that the same signal transduction pathways activated by Tpo and Flt-3l in hematopoietic stem cells will turn on in hESCs, with the same effects. Maybe, but maybe not.
The pathways targeted in aim 3 are fairly limited. Given the experience with others, e.g. HOX gene pathways, the focus on cytokine pathways seems too narrow.
DISCUSSION: Directed differentiation is an important aspect of hESC biology to pursue, and blood cells are important to study. hESC-derived hematopoietic stem cells have great potential as an inexhausible source of cells for transplantion. The results could be relevant to transplantation as well as to in-vitro work. The PI plans to address the issue of efficiently generating engraftable human hematopoietic stem cells by addressing phenotype, function and transplantability. Specifically, this proposal will address phenotyping, quantitative assay for clonogenic potential, and work on culture conditions which reviewers think is important. One reviewer felt that the proposed plan is realistic; the most ambitious aim is to develop approaches to regulate expansion of engraftable human hematopoietic stem cells. Another reviewer felt that this is an overly ambitious proposal that is beyond the scope of a 4-yr project. Even if it works, the mouse may not be comparable to humans. For example, humans make as many blood cells in one day as the mouse makes in a lifetime. The main issues to work on are the culture and propagation conditions and the PI has 15 years experience with culture conditions, cell marking, etc. The PI's experience from cord blood will also be helpful.
A third reviewer felt that the clonal analysis with respect to marking is good, but the assay proposed may not give the clonal readouts (marks) in the pluripotent state. One doesn't know if the "mark" represents the proliferation state or whether the cells have already differentiated into hematopoietic stem cells (thus you have two lineages).
In summary, the applicant is a strength and has established good collaborations with Dr. D. Kohn who will contribute to vector design for clonal marking. A weakness is the lack of long-term readout for the function of human hematopoietic stem cells simply because this hasn't worked in mouse. Another problem is there is no quantitiative technique to identify progenitors of each lineage - they are grown as a clump. Specific aims 1 & 2 are exactly where the field needs to start; results obtained would be valuable for all. Aim 3 is somewhat narrow but still a good place to start. Aim 2 however is very ambitious; seems beyond scope of a 4 year grant. The applicant has not described how progenitors will be functionally analyzed. While one reviewer was happy to see an attempt to do clonal analysis with viral marking, (s)he did not agree that the assay will give a clonal readout. This is because the investigators are infecting hESC and it is not clear that a marked cell will duplicate before it differentiates to hematopoietic stem cells. Hence they did not see this approach as a conclusive measure of clonality.
PROGRAMMATIC REVIEW: There was strong enthusiasm for the very strong track record and qualifications of this applicant, coupled with the quality of the team. One reviewer noted that this proposal introduces precise assays - an important consideration in the ESC field. Another reviewer reiterating the concerns about whether the proposed approaches would show clonality. The Working Group voted to recommend to the ICOC for funding based on the outstanding record of the applicant and that it brings a precise quantitative assay that could be generally useful with many tissues.