In an attempt to understand better the regulation of stem cell function in chimeric immunodeficient mice transplanted with human cells, and the filiation between progenitor cells identified in vitro and in vivo, we assessed the different compartments of hematopoietic progenitors found in the marrow of CB17-severe combined immunodeficiency (SCID) mice (34 mice, 9 experiments) after intravenous injection of 2 to 3 x 10(7) cord blood mononuclear cells. On average 6.3 +/-4 x 10(5) human cells were detected per four long bones 4 to 6 weeks after the transplant predominantly represented by granulomonocytic (CD11b+) and B lymphoid (CD19+) cells. Twenty five percent of these human cells expressed the CD34 antigen, of which 90% coexpressed the CD38 antigen and 50% the CD19 antigen. Functional assessment of progenitor cells (both clonogenic and long-term culture-initiating cells [LTC-IC]) was performed after human CD34+ cells and CD34+/CD38- cells have been sorted from chimeric CB17-SCID marrow 3 to 10 weeks after intravenous (IV) injection of human cells. The frequency of both colony-forming cells and LTC-IC was low (4% and 0.4%, respectively in the CD34+ fraction) when compared with the frequencies of cells with similar function in CD34+ cells from the starting cord blood mononuclear cells (26% +/- 7% and 7.2% +/- 5%, respectively). More surprisingly, the frequency of LTC-IC was also low in the human CD34+ CD38- fraction sorted from chimeric mice. This observation might be partly accounted for by the expansion of the CD34+ CD19+ B-cell precursor compartment. Despite their decreased frequency and absolute numbers, the differentiation capability of these LTC-IC, assessed by their clonogenic progeny output after 5 weeks in coculture with murine stromal cells was intact when compared with that of input LTC-IC. Furthermore the ratio between clonogenic progenitor cells and LTC-IC was similar in severe combined immunodeficiency (SCID) mice studied 4 weeks after transplant and in adult marrow or cord blood suspensions. Results generated in experiments where nonobese diabetic (NOD)-SCID mice were used as recipients indicate a higher level of engraftment but no change in the distribution of clonogenic cells or LTC-IC. These results suggest that the hierarchy of hematopoietic differentiation classically defined in human hematopoietic tissues can be reconstituted in immunodeficient SCID or NOD-SCID mice.

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo- erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood- derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo- erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood- derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

Key Points BM niches differentially support T-ALL. BM niches differentially protect T-ALL cells from chemotherapy.