The central goal of this proposal is to investigate the cellular and molecular events during the early stages of primitive stem cell commitment to the hematopoietic lineages. We have studied murine totipotent embryonal stem (ES) cells in methyl-cellulose culture and observed the emergence of erythroid cells and macrophages in clonally expanding ES colonies. We have also isolated two murine cDNA clones from the library of a murine multipotential hematopoietic cell line (FDCP-Mix) which cross hybridize with two human hematopoietic specific genes, vav and HS-1, whose functions are unknown.
Our specific aims are to determine when vav and HS-1 are expressed in the early stages of hematopoietic stem cell differentiation and how perturbation of their expression affect hematopoiesis. Therefore, we propose: 1) to define the optimal growth conditions for ES cell derived hematopoietic cells using various cytokines including the recently identified stem cell factor, SCF-1; 2) to characterize and sequence the murine cDNA clones we have isolated, determine their homology to human clones and evaluate by RNA analysis the specificity-of their expression in murine hematopoietic cells; 3) to generate polyclonal antibodies to vav and HS-1 using fusion proteins and use the antibodies to further characterize their protein by SDS polyacrylamide gel analysis of hematopoietic cells and employ immunocytochemical techniques to localize and establish the kinetics of protein expression in ES cell derived hematopoietic cells and 4) to use ,anti-sense RNA vectors and anti-sense oligonucleotides to block the expression of these proteins in ES cells in order to understand their function in hematopoietic differentiation. These experiments should 1) ascertain the usefulness of such an in vitro model system for future studies in hematopoiesis, 2) shed light on the function and significance of hematopoietic specific genes, 3) contribute to the investigations of pathological changes in hematopoietic cells and 4) be relevant to our understanding of the ,fundamental process of cellular differentiation.
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| Adra, C N; Lelias, J M; Kobayashi, H et al. (1994) Cloning of the cDNA for a hematopoietic cell-specific protein related to CD20 and the beta subunit of the high-affinity IgE receptor: evidence for a family of proteins with four membrane-spanning regions. Proc Natl Acad Sci U S A 91:10178-82 |
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| Lelias, J M; Adra, C N; Wulf, G M et al. (1993) cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the rho GTP-binding proteins. Proc Natl Acad Sci U S A 90:1479-83 |
| Wulf, G M; Adra, C N; Lim, B (1993) Inhibition of hematopoietic development from embryonic stem cells by antisense vav RNA. EMBO J 12:5065-74 |
