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