Long-term reconstituting hematopoietic stem cells (PHSC) in adult murine bone marrow can be isolated using monoclonal antibodies specific for certain cell-surface molecules and FACS sorting. A polyclonal antibody raised against the murine homologue of the human sialomucin CD34 has been used to show that mCD34 is expressed on murine hematopoietic stem and progenitor cells from both the fetal liver and adult bone marrow compartments. We have used a rat monoclonal antibody to identify a population of CD34+ cells isolated from murine yolk sac to test the hypothesis that PHSC in the yolk sac express CD34 and to determine expression of other PHSC “markers”. C57BL/6J embryos were removed from timed-pregnant dams at d9.0 post-coitus (pc), yolk sacs were dissected, and a single cell suspension was prepared by brief collagenase (0.1%) treatment. Cells were incubated with anti-mCD34 or isotype control antibody, sorted, and CD34+ yolk sac were transplanted into intrapartum busulfan conditioned congenic newborn mice. Recipient congenic animal (n = 11) peripheral blood was analyzed 1 - 6 months post-transplant and high level (≥ 40% all lineages) donor type Gpi-1 was present in B and T lymphocytes, granulocytes, and red blood cells in animals transplanted with d9.0 pc yolk sac cells. Thus CD34+ yolk sac expressing cells include PHSC. In contrast to adult bone marrow PHSC that express MHC class I (H-2K) and Sca-1 but not AA4.1, CD34+ yolk sac PHSC express AA4.1 but not H-2K or Sca-1. Further analysis of stem cell markers is in progress, but the present results indicate that the cell surface phenotype of PHSC varies considerably depending on the developmental stage of murine ontogeny. We speculate that the behavior of PHSC may also vary during murine ontogeny. Understanding how PHSC behavior changes during development is of importance for contemplation of in utero PHSC therapies of human disease.
