Improving microenvironments to promote hematopoietic stem cell development from human embryonic stem cells
Statement of Benefit to California:
SYNOPSIS: This proposal aims to overcome deficiencies in the generation of functional hematopoietic stem cells (HSCs) derived from hESCs by providing a more physiological milieu for HSCs. Novel stromal cell lines will be developed from early human placentas and fetal livers. These lines may be modified to knockdown differentiating signals (M-CSF) and overexpress Wnt3a. Cultures of hESCs will be monitored for different lineages and for the capacity to undergo terminal differentiation to erythroid, myeloid and lymphoid lineages. Placental villous explants are also able to support hESC-derived early hematopoietic precursors and they will be explored as a natural niche. Transplantation of these HSCs into B,T and NK-deficient mice will allow in vivo exploration either directly or through the use of human implants of bone, fetal liver/thymus and placental villi. Engraftment will be monitored by optical bioluminescence from transfected luciferase containing constructs, by CFU and FACS assays and by secondary transplants. INNOVATION AND SIGNIFICANCE: The most significant aspect of this SEED proposal is the extensive nature of the exploration of the stromal cell lines for their capacity to provide a useful environment for hESCs to differentiate in. Imaginative improvements of the proposal include the use of human implants of bone, fetal liver/thymus and placental villi to facilitate adaptation to the immunodeficient murine environment. Transfection of the hHSCs with GFP and luciferase-containing constructs will allow the monitoring for engraftment by optical imaging of in vivo bioluminescense without sacrificing animals. The investigator proposes to build on the observation that the placenta harbors HSC to develop conditions for deriving HSC from hESC in vitro. The proposal to derive human stromal cell lines from placenta and fetal liver and to engineer them with lentiviral vectors to decrease or increase specific gene expression represents a novel approach which is worthy of testing. The problem to be addressed is important and the significance is contingent on success of the proposed experimental approach. There is little doubt that the investigator will generate descriptive data but ultimate success, namely the ability to generate HSC in large number for potential therapeutic applications is very uncertain. STRENGTHS: The PI is a young investigator obviously committed to a future in ESC: several publications on early hematopoietic differentiation of mESCs and has six funded projects in the same or connected general areas. The PI has access to a list of impressive collaborators as well as access to many hESC lines as potential targets for study of variations on HSC-generation including many grown without xenogeneic cells or proteins. Furthermore, the technical protocols described appear to be of very high quality. Overall, this is a novel approach by a well-trained and enthusiastic investigator who has experience with much of the methodology. WEAKNESSES: A potentially worrisome aspect is the large number of studies that the PI is committed to already (nominally 17% of time on this one). The PI will depend partly on help from several part-time or unpaid assistants and trainees (2 graduate student trainees, one of them only half time on this project, one clinical fellow [no salary] and 2 research associates [one 70% and one 15%]) DISCUSSION: This is an ambitious, productive, young PI who is committed to a large number of studies. The novel approach builds on the previous work describing the ability of the placenta to support hESC-derived hematopoietic precursors, and looking at the terminal differentiation of lymphoid lineages is also new. The work plan is largely descriptive, but this is exploratory research at the early stages. Current funding is largely start-up and reviewers would like CIRM to give this person a chance to attack the proposed problem. Proposal is for descriptive, exploratory research that is appropriate for this type of RFA.