For many therapeutic reasons it is important to have available large numbers of blood cells. However, it is difficult to generate large numbers of specialized blood cells that have the ability to neutralize autoimmunity and response to tumor cell growth. In this study we would develop a technique that would allow the production of large numbers of different types of blood cells from human embryonic stem cells. For example, a subset of white blood cells, called dendrititc cells, is currently manipulated in the laboratory in a manner that allows them to attack cancer cells. The same cells also are altered in the laboratory to counter-act the development of autoimmune diseases. A problem with these experiments is that it is difficult to isolate large numbers of these cells, since they are relatively rare. With the technology that is described in this grant application we would be able to generate large numbers of such cells in the laboratory using as a starting point, human embryonic stem cells.
In this study we would develop an approach that would allow the production of large numbers of different types of blood cells from human embryonic stem cells. For example, a subset of white blood cells, called dendrititc cells, is currently manipulated in the laboratory in a manner that allows them to attack cancer cells. The same cells also are altered in the laboratory to counter-act the development of autoimmune diseases. A problem with these experiments is that it is difficult to isolate large numbers of these cells, since they are relatively rare. With the technology that is described in this grant application we would be able to generate large numbers of such cells in the laboratory using as a starting point, human embryonic stem cells. The approach is novel and straightforward and could be applied immediately once it has been established.
SYNOPSIS: The goal of the proposed research is to produce from hESCs long term, expandable cultures of hematopoietic progenitor cells that can be differentiated in vitro to T-lineage cells for gene replacement strategies. Based on previous success with mouse hematopoietic precursor cells, the first aim will generate large numbers of human hematopoietic progenitor cells by depressing levels of bHLH E-proteins, key transcriptional inducers that antagonize cell cycle progression and promote development. To avoid genetic manipulation of hESCs, E-protein function will be inhibited reversibly with Id1 linked to a cell-penetrating peptide; Id1 is a natural antagonist of E-protein activity that forms a heterodimer with E-proteins that is incapable of binding DNA. Aim 2 will attempt to identify chemicals from a large, nonproprietary, synthetic compound library that can mimic the ability of the Notch ligand DL-1 to induce T-lineage differentiation of the cultured human hematopoietic progenitors from Aim 1.
SIGNIFICANCE AND INNOVATION: The generation of long term, multipotential human hematopoietic progenitor cell lines from hESC, if successful, would create cells that could be used to readily generate specific hematopoietic cell types, such as lymphocytes or dendritic cells, on a large scale for cell based therapeutic approaches. Currently it is not possible to significantly expand human hematopoietic stem cells (hHSC) or multipotent hematopoietic progenitors, and the ability to prepare sufficient numbers of differentiated cells from hESC is a critical technical hurdle for cell replacement therapy. Solutions to this problem, as exemplified in this application, have great significance. This proposal presents a rational, innovative and well-designed program to derive large-scale cultures of hematopoietic progenitor cells from hESCs with strong potential to be useful for replacement therapies and is likely to be successful. The efficacy of this approach with mouse stem cells and known parallels with the biology of human hematopoietic progenitor cells indicate that it should be feasible with human cells as well.
The strategy is based on the well-known role of E-proteins to push hematopoietic progenitors toward differentiation and away from self-renewal and pluripotency. The use of a proven E-protein inhibitor capable of penetrating cell membranes to modulate E2A activity in human hematopoietic progenitors is a highly innovative advance that should provide the means to induce the expansion of hematopoietic progenitor cells without resorting to genetic manipulations. The added advantage of simple reversibility by removing the inhibitor from the culture medium facilitates the necessary next step of directed differentiation that would make them suitable for therapeutic use.
STRENGHTS: A particular strength of this proposal is the accomplishments of Dr. Murre and his colleagues that are directly relevant to this proposed research. Dr. Murre is a longstanding expert in the biology of bHLH factors and has applied highly innovative approaches toward the analysis of mouse hematopoietic progenitors and their development in culture. He and his colleagues are one of a very few groups with experience with all facets of this project, save the technology of high throughput screening of the synthetic compound library. For this he has recruited Dr. James Rush of the Genomics Institute of the Novartis Research Foundation. Screening for synthetic compounds that can induce differentiation of human hematopoietic progenitors along specific developmental pathways is a key strength of this proposal.
The loss of E2A activity in murine hematopoietic progenitors has been shown to generate long term cells with erythroid, myeloid and lymphoid developmental potentials. The first Aim builds upon this observation, and the proven effectiveness of the proposed experimental strategies in other settings. For example, previous work by the applicant showed that forced expression of Id proteins in human hematopoietic precursor cells blocked B cell development; therefore, E-proteins are likely to play the same regulatory roles in human hematopoiesis as they have been shown to do during murine hematopoiesis. Consequently, inhibition of E2A by Id1 also can be expected to promote the growth of human hematopoietic progenitors without compromising their pluripotency. The use of Id-(Arg)n fusion to test the reversible inhibition of E2A activity is a another scientific strength.
In addition, recombinant Id3 fused to a cell-penetrating peptide derived from HIV TAT was shown to effectively enter primary pro-B cells, reduce the binding of E2A to DNA, and affect target gene expression. For this proposal, a synthetic peptide with increased ability to cross biological membranes will be used in place of the TAT peptide to direct Id1 uptake by the hematopoietic progenitor cells. A variety of other protocols required for the success of the proposed studies are in hand, such as the production of hematopoietic progenitor cells, their isolation by FACS, growth of the cells in culture and induction to T-lineage fate. A collaboration with Dr. Karl Willert, who heads an experienced human ES cell facility at UCSD, will provide further expertise for dealing with human ESCs.
WEAKNESSES: There are a few weaknesses in this application that are minor and affect the enthusiasm for this project very little. First, the use of a cell-penetrating peptide to reversibly alter behavior of hESCs is innovative, though it is an unproven strategy in this specific context for the expansion of human hematopoietic progenitors. Second, in Aim 2 the collaboration established with the GNF, with its expertise and track record of successes, greatly enhances the likelihood of identifying inducing compounds from the technically demanding high throughput screen of the chemical library proposed. Nonetheless, success with this approach first and foremost requires a robust luciferase reporter cell line with proven effectiveness in the demanding setting of a large scale screen. While the preTa promoter has been useful in other settings, it remains to be seen whether it will be effective in this screen with these mouse and human hematopoietic progenitor lines (e.g., it is not clear whether sufficient numbers of hematopoietic progenitors bearing the preTa-luciferase reporter can be acquired for the massive screening). Third, although the use of HUES-1 cells, which are not NIH-approved, is used as justification for CIRM funding, the suitability of this cell line for these studies is not discussed, and the use of NIH-authorized lines is not considered. Thus the force of this argument is uncertain. Finally, discovery of synthetic compounds that can induce hematopoietic differentiation needs to be more strongly articulated as cytokines can be used for generating specific lineages.
DISCUSSION: Reviewers were very enthusiastic about the strong science in this proposal which has a good chance of success. The applicant has directly relevant experience in the murine system, and the approach of depressing bHLH E-proteins to get hHSC has been shown to be feasible in other systems. The greatest strength of this proposal is the ability to use Id1 as opposed to genetic manipulation to modulate E-protein function. This is an inventive strategy, and the natural antagonistic effects of Id1 have already been demonstrated in mouse. Another key strength of proposal is that it could result in long term cultures of multipotent progenitor hHSC that could be used to further derive specific lineages. In Aim 2, the use of a synthetic chemical library to find compounds that can mimic effects of the Notch pathway on T-cell differentiation is a valid and useful approach. Reviewers commented that in terms of screening the compound library, new compounds for T-cell specific differentiation aren't necessarily needed because cytokines that can do this already exist. Other reviewers noted that the compound screen may be important for generating key progenitor lineages. The other weaknesses of the proposal are modest given the importance of the work.