Basic Biology I
Hematopoietic stem cell transplantation provides a life-saving treatment for individuals who have certain genetic abnormalities affecting their hematopoietic system, or for individuals treated for malignant diseases such as leukemia. However, some individuals are unable to receive this life saving treatment due to the inability to find a suitably matched hematopoietic stem cell donor. While umbilical cord blood may alleviate some of those issues, there are still concerns associated with cost and complexity. Previous studies have identified that human embryonic stem cells are able to differentiate into hematopoietic stem cells in the laboratory research setting. While these cells are able to resemble hematopoietic stem cells, very little success has been met regarding the ability of the cells to functionally engraft in the bone marrow using methodologies similar to that used for bone marrow transplantation purposes. Previously we have elucidated some of the key regulators involved in the multiple steps of hematopoietic stem cell engraftment. We aim to use this knowledge to define optimal conditions that allow for the differentiation of human embryonic stem cells into hematopoietic stem cells that are able to function in settings of bone marrow transplantation. This will involve identification of the molecules known to be involved in the multiple coordinated steps of hematopoietic stem cell engraftment and the manipulation of the cells to allow for efficient expression. It is anticipated that these studies will allow for identification of the methods for efficient differentiation of in vivo functional hematopoietic stem cells that can be used in clinical settings of bone marrow transplantation.
Statement of Benefit to California:
While hematopoietic stem cell transplantation provides a life-saving treatment for many individuals, in ethnically diverse states such as California, fewer individuals are unable to find suitable donors than in less ethnically diverse regions due to biases in the unrelated marrow donor registries. Therefore, alternatives need to be developed which are able to eliminate these issues. One such approach for hematopoietic stem cell transplantation is to derive the stem cells from a range of human embryonic stem cells so that all patients will be equally able to find matched donor cells. However, the efficient differentiation protocols to achieve this have yet to be defined. Using our knowledge of the mechanisms of hematopoietic stem cell engraftment, we aim to define methods that allow for the differentiation of functional hematopoietic stem cells. This may ultimately allow for the routine use of this potentially limitless supply of cells in the clinical need of bone marrow transplantation.
The goal of this proposal is the derivation of functional hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs). While many groups have had success differentiating hESCs into HSCs with appropriate in vitro characteristics, no group to date has shown that these cells can functionally engraft in adult bone marrow in vivo. In Aim 1 the applicant proposes to define the optimal culture conditions for differentiation of hESCs to HSCs using cell surface expression analysis and in vitro assays as readouts. In Aim 2 the applicant will analyze the HSCs derived in Aim 1 for their adhesion to extracellular matrix proteins in vitro, their chemotactic response and their homing and lodgment in immunodeficient mice. Finally, in Aim 3, the applicant proposes to demonstrate the ability of hESC-derived HSCs to functionally engraft in the bone marrow microenvironment in immunodeficient mice, including a humanized mouse model to provide a human hematopoietic microenvironment. Reviewers agreed that the potential impact of this proposal is high. The inability to derive functional HSCs from hESCs is a significant bottleneck to the development of pluripotent stem cell-based therapies for diseases of the hematopoietic and immune systems. Reviewers appreciated that the proposal addresses one of the more enigmatic problems in the field, one which many laboratories have been working on for years without success. However, one reviewer felt that this proposal contains nothing conceptually novel that has not been tried before and failed. This reviewer also commented that much of the data obtained would be descriptive and would not address key mechanistic questions. Reviewers raised a number of concerns about the research plan and preliminary data that lead them to question the project’s feasibility. They were particularly concerned about Aim 1 and the fact that Aims 2 and 3 depend on its success. One reviewer noted that the strategies proposed in Aim 1 are not new and thus may not succeed where others have failed. In addition, the endpoints that will be used to assess differentiation of hESCs to HSCs are not established predictors of HSC function. This reviewer acknowledged that the applicant proposes an alternative strategy of small molecule screening but could not evaluate its potential due to a lack of detail. Another reviewer was concerned that Aims 1 and 2 did not include a clearly defined, quantitative measure for evaluating the differentiation optimization process. This reviewer also would have appreciated the inclusion of more relevant preliminary data in the proposal, specifically some indication that the applicant has had success differentiating hESCs to HSCs. The reviewers praised Aim 3 as a well conceived and crucial in vivo experiment but were uncertain of its value given their concerns about the feasibility of Aims 1 and 2. Another reviewer noted that the alternative strategy described in Aim 3, stimulating a particular hematopoietic niche with an agonist, is only valid if indeed this niche has been validated. This reviewer would have liked some discussion of the controversy surrounding that issue. The reviewers praised the applicant as an outstanding young investigator with an excellent publication record. They worried about a lack of experience working with hESCs but noted the significant percent effort contributed by a collaborating expert in hESC culture and manipulation. Reviewers found the applicant’s resources to be more than adequate and the research team highly qualified to carry out the proposed experiments. Overall, while the reviewers appreciated the significance and potential impact of the scientific question addressed in this proposal, they raised significant questions about the feasibility of its research plan.