Funding opportunities

The role of hyaluronan in regulation of differentiation of hESCs in hematopoietic lineage

Funding Type: 
New Faculty I
Grant Number: 
RN1-00546
Funds requested: 
$2 456 202
Funding Recommendations: 
Not recommended
Grant approved: 
No
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. Since in cell culture, hESC lines are capable of developing into a variety of tissues, including blood cells, and because they can be expanded indefinitely, hESC may serve as an alternative source of transplantable HSCs. While it has been possible to differentiate hESC 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 hESCs require further development. In vivo, the fate of HSC is determined by the local cellular environment, 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 HA is required for differentiation of human ESCs 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 hESC in culture. We will study the cellular and molecular mechanisms mediating the effects of HA on differentiation of hESCs 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 hESCs 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 hESCs and clinically accepted HSCs. We will use this information to guide our further experiments, which will involve testing the regenerative potential of the hESC-derived HSCs in laboratory animals. Overall, the research project will determine whether HSC derived from hESC 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 pluripotent human embryonic stem cells (hESCs) 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 hESC-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) may improve differentiation of hESCs toward HSCs. Thus, the short-term benefits for Californians are 1) translation of our knowledge on adult HSCs biology into the hESCs field; 2) obtaining new theoretical knowledge on generation of HSCs from hESCs; and 3) determination of whether hESC-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 hESC-derived HSCs in non-human primates, hence approaching a “low hanging fruit”.
Review Summary: 
SYNOPSIS: This is an application for support to test the hypothesis that hyaluronic acid (HA) plays a determinative role in differentiation of human embryonic stem cells (hESCs) into hematopoietic lines. Preliminary data of the PI suggests that HA addition improves HSC generation. Based on this, she proposes to study the nature of the effect, including the signaling pathways involved. Discovery of the effects of varying sizes of HA polymers on undifferentiated hESCs and mesodermal derivatives is included in the research plan, along with testing the ability of hESC-derived hematopoietic stem cells to engraft into bone marrow of immune-suppressed mice. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The overall significance of this work relates to the possibility of obtaining true hematopoietic stem cells (HSCs) from hESCs, which would improve the prospects for bone marrow transplantation and other regenerative medicine approaches. Additionally, understanding the influence of abundant extracellular matrix components in cell migration, signaling and differentiation is a fundamentally important problem in hESC differentiation. The proposed work, if successful, would extend our knowledge about hematopoietic differentiation of hESCs, on cellular and mechanistic levels (Aims 1 and 2). This knowledge can be applied to the reconstitution of hematopoiesis (small animal model in Aim 3, possible extensions in future). The experimental design is developed around the hypothesis that HA is required for hematopoietic differentiation from hESCs, and the goal to establish culture conditions for the derivation of hematopoietic cells suitable for in vivo use. The studies described are clear, rational and have some innovative qualities in the overall multilevel approach and experimental methods. A discussion, however, of potential pitfalls and a plan if HA does not successfully stimulate a mesodermal fate or hematopoietic differentiation of a high percentage of hESCs is lacking. The preliminary data demonstrate only modest effects on in vitro hemaotpoietic colony assays. These effects could be either direct or indirect, but an effect at the HSC level is questionable. Much of the rest of the application rests on the likelihood that HA is a critical component. If it is not, the proposal rapidly falls and the entire project will have to be revised. One reviewer felt that even if HA is important, it is likely only one of many components of the niche for HSCs. This is a major concern, and an increased risk for the project as a whole. Three specific aims are proposed: Aim 1 will determine whether HA stimulates a mesodermal fate for stem cells on the road to hematopoiesis, or whether HA can only act on cells already mesodermal in character; and whether or not levels of HA influence cellular response. Aim 2 includes a series of experiments to distinguish effects of low molecular weight HA versus high molecular weight HA, which receptors are being influenced, and what downstream effectors are involved. This aim also includes determining the effect of blocking endogenous HA production by inhibitors of HA synthase, the enzyme that lengthens the molecule. Aim 3 includes functional studies of HSCs derived from hESCs by exposure to HA. The investigators will attempt to engraft in vitro derived hematopocytes into bone marrow of immune-suppressed mice. They will also expose HA-differentiated stem cells to a flow chamber invented by the PI that mimics physiological shear stresses within blood vessels. The team of investigators appears capable, roles are clear, and interactions are well described. Overall, the application addresses a significant area of study and presents a logical plan of research by a qualified investigator with clear institutional commitment. A very significant weakness, however, is the dependence of the entire proposal on Aim1. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI received MD (pediatrics) and PhD training in Russia and then moved to the La Jolla Institute for Molecular Medicine in 1999,and was appointed an Associate Member at the Torrey Pines in 2007. Her PhD in Clinical Immunology from the Russian Academy of Science focused on the biology of hematopoietic stem cells. She has continued her commitment to understanding hematopoiesis. She has considerable experience in the hematopoietic assays needed for her studies and has formed a strong team of investigators that collectively has the knowledge and skills necessary for successful completion of research. The PI has a good publication record germane to the proposed research (30 publications, all of which in the last 10 years), although for the most part her publications are in second to third tier journals. She is 14 years post-PhD and in her 6th year as an independent investigator. She has an NIHR01 grant on nicotine and hematopoiesis. The PI developed a strong and specific career development plan around the anticipated CIRM award, with specifically stated goals and milestones. A detailed timetable for the studies and her career goals for the next five years are included. She maintains strong ties with her mentor at the Burnham Institute, and is enthusiastic and committed to her own teaching and mentoring responsibilities. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: There is a strong letter of support for stem cell research from the Chief Operating Officer of the Torrey Pines Institue for Molecular Studies. The insitutional letter of support states that it will match 50% of the funds of this application, if awarded. It appears that Dr. Khaldoyanidi is the first faculty member recruited, although they have become part of the San Diego stem cell consortium. The institution has a good track record in supporting young faculty. The PI is getting a lot of support and mentoring, along with the growing responsibilities in research and leadership, and the institution seems commited to support her carreer development. Facilities and equipment are available for this project. DISCUSSION: Reviewers felt that the research proposal is weak and largely dependent on the success of the first research aim. If the aim fails there is no project. The preliminary data presented did not provide confidence to the reviewers of success in this aim. Despite this, one reviewer commented that useful information could result from the proposed experiments. The reviewers felt the candidate was clearly an expert in the field of hematopoiesis, but they did not believe the applicant to be strong scientifically when compared to other applicants. The host institution is developing a stem cell science program and is very supportive of the candidate.
Conflicts: 

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