Funding opportunities

Hematopoietic differentiation of human pluripotent stem cells

Funding Type: 
New Faculty II
Grant Number: 
Funds requested: 
$2 456 202
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Transplantation of hematopoietic stem cells (HSCs) derived from blood or bone marrow is a frequently used procedure for treatment of blood disorders including cancers and immunodeficiencies due to chemotherapy. However, restoration of blood cell production does not always work, because of 1) incompatibility of donor and host cells; and 2) the limited quantities of HSC that can be obtained from adult donors or umbilical cord blood. Human pluripotent stem cells (hPSCs), such as embryonic (hESCs) and induced pluripotent stem cells (iPSCs), can give rise to all tissues in a body including blood cells. Since hPSCs are capable of developing into blood cells in cell culture, and because they can be expanded indefinitely, hPSC may serve as an alternative source of transplantable HSCs. While it has been possible to differentiate hPSC into cells that resemble HSCs, when they were tested in animal models, they had limited ability for long-term reconstitution of blood cells. Thus, current protocols for generation of hHSCs from hPSCs require further development. In vivo, the fate of HSC is determined by the local cellular microenvironment, which is termed the “niche”. We have demonstrated that a complex sugar called hyaluronan (HA) regulates the fate of adult HSCs in the niche. We have also found that endogenous HA is required for differentiation of human hESCs into HSCs in culture. Thus, in this grant application we propose to test the hypothesis that HA is an important factor for production of HSCs from hPSC in culture. We will study the cellular and molecular mechanisms mediating the effects of HA on differentiation of hPSCs into HSCs. This will allow us to develop new culture techniques for the generation of transplantable HSCs. We will further compare the HSCs derived from hPSCs with clinically accepted HSC preparations. We will use a variety of methods for this comparison, including in vitro hematopoietic assays and a 3-dimensional flow chamber device that we developed. This novel device will allow us to study the interactions between HSCs and the vasculature under conditions of physiological shear stress. Through these experiments we will discover the similarities and differences between HSCs derived in a culture dish from hPSCs and clinically accepted HSCs. We will use this information to guide our further experiments, which will involve testing the regenerative potential of the hPSC-derived HSCs in laboratory animals. Overall, the research project will determine whether HSC derived from hPSC can be used as an alternative source for the HSCs that are currently so valuable for treatment of human blood disorders.
Statement of Benefit to California: 
The concept of stem cell based therapy implies that damaged tissues can be repaired by tissue-specific stem cells that generate mature functionally active progeny. Among the variety of tissue-specific multipotent stem cells, only hematopoietic stem cells (HSCs) have been routinely used in clinical practice for over than 30 years. In currently used protocols, HSCs are isolated either from adult sources (bone marrow or mobilized peripheral blood) or neonatal (umbilical cord blood) and used to regenerate hematopoiesis (the production of mature blood cells) in patients with genetic disorders of hematopoietic system and in cancer patients. Despite of the obvious success of the HSC transplantation approach, there are obstacles for the effective HSC-based therapy: due to a shortage of HLA-matched donors and the technical limits for in vitro expansion of HSCs only one third of patients receive the required HSC transplantation. Thus, alternative sources for HSCs are needed, and the human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPSC), theoretically might be a good source. Since the overall concept of using HSC for therapy has been proven, adult or neonatal HSCs can serve as an excellent reference to define whether the hPSC-derived HSCs are useful and safe, thus representing “a low hanging fruit” project. Current knowledge on generation of HSCs from hESCs suggests that the properties of hESC-derived HSCs, including multipotency, regenerative capacity and survival, depend on culture conditions used to differentiate hESCs. Based on our knowledge of the adult hematopoietic regulatory microenvironment (i.e. the niche), which determines the fate of HSCs, and new findings generated in our laboratory, we propose to test the hypothesis that the complex sugar called hyaluronan (HA) is required for differentiation of hESCs toward HSCs. Thus, the short-term benefits for Californians are 1) translation of our knowledge on adult HSCs biology into the hPSCs field; 2) obtaining new theoretical knowledge on generation of HSCs from hPSCs; and 3) determination of whether hPSC-derived HSCs resemble adult and neonatal HSCs qualities. If proven successful by in vivo and in vitro homing and engraftment studies, the next ultimate step will be evaluation of efficacy and safety of the hPSC-derived HSCs in non-human primates, hence approaching a “low hanging fruit”.
Review Summary: 
The Principal investigator (PI) for this application proposes to investigate the role of hyaluron (HA)-mediated pathways in regulating the developmental shifts that allow generation of hematopoietic stem cells (HSC) from hPSC (human pluripotent stem cells) including hESC (human embryonic stem cells) and iPSC (induced pluripotent stem cells). Published studies have shown HA is an important regulatory element for hematopoiesis in adult bone marrow, and new preliminary data in this proposal suggest HA may be important for hPSC differentiation to HSC. This proposal will initially focus on characterizing the molecular mechanisms mediating these HA effects using specific inhibitors and knockdown approaches. The cellular target and developmental stage where HA is required will be analyzed, with the stated goal of defining if HA manipulation can enhance HSC generation from pluripotent cells. Final studies will evaluate and compare engraftment and hematopoietic reconstitution abilities of in vitro hPSC-derived HSC and HSC in current clinical use in an in vivo model and in a novel 3-dimensional flow chamber model of human vasculature. Reviewers agree that this is a well-written research plan that outlines a logical and well-justified series of experiments. The main goal is to generate engraftable blood-forming stem cells from pluripotent cell lines. This would be a significant accomplishment. The reviewers expressed some concerns which impact on the feasibility of achieving meaningful results. The studies to define the molecular mechanisms induced by HA are straightforward. However, the reviewers noted that it appears that these studies will be performed on a mixed population of differentiating cells, potentially including cell types that are irrelevant for the hematopoiesis-specific phenotype making it very difficult to distinguish direct from indirect HA affects. Similarly, they pointed out that optimal evaluation of stem cell biology and definition of cellular targets requires clonal or at least “purified” cell subsets. This was not been discussed in the proposal and resulted in reduced overall enthusiasm for the proposal. The reviewers further noted that it was not clear from the proposal that manipulation of HA or the corresponding signaling pathways will result in significantly greater numbers of in vitro derived HSC. The preliminary data included in the proposal is suggestive of a role for HA in HSC derivation from hPSC, however, it was unclear how specific was the observed effect. Reviewers concurred that a novel aspect of this proposal is the use of a 3-D flow chamber to compare the ability of derived HSC with adult HSC to interact with a model of the human vasculature under conditions of physiological shear (e.g. homing). That is an area that is not typically evaluated in stem cell biology, but may be critical for therapeutic delivery. The PI received his/her training from institutions inside and outside of USA. S/he has a long history of studying CD44, HA and hematopoiesis. She/he has a good publication record with 5 papers in the last 3 years and currently holds 2 grants, one from NIH (a R41) and one from the University of California Tobacco-Related Disease Research program. There is a good career development plan including continued interest and participation in commercial development activity. The mentors and institutional support are appropriate and supportive. The institution does not have much of an established track record in stem cell biology, however the broader research community in the area clearly makes this one of the leading areas for stem cell research.

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