To explore the use of stem/progenitor cells from peripheral blood (PB) for allogeneic transplantation, we have studied the mobilization of progenitor cells in normal donors by growth factors. Normal subjects were administered either granulocyte-macrophage colony-stimulating factor (GM-CSF) at 10 micrograms/kg/d, or G-CSF at 10 micrograms/kg/d, or a combination of G- and GM-CSF at 5 micrograms/kg/d each, administered subcutaneously for 4 days, followed by leukapheresis on day 5. Mononuclear cells expressing CD34 (CD34+ cells) were selectively enriched by affinity labeling using Dynal paramagnetic microspheres (Baxter Isolex; Baxter Healthcare Corp, Santa Ana, CA). The baseline CD34+ cells in peripheral blood before mobilization was 0.07% +/- 0.05% (1.6 +/- 0.7/microL; n = 18). On the fifth day after stimulation (24 hours after the fourth dose), the CD34+ cells were 0.99% +/- 0.40% (61 +/- 14/microL) for the 8 subjects treated with G-CSF, 0.25% +/- 0.25% (3 +/- 3/microL, both P < .01 v G-CSF) for the 5 subjects administered GM-CSF, and for the 5 subjects treated with G- and GM-CSF, 0.65% +/- 0.28% (41 +/- 18/microL, P < .5 v GM-CSF). Parallel to this increase in CD34+ cells, clonogenic assays showed a corresponding increase in CFU- GM and BFU-E. The total number of CD34+ cells collected from the G-CSF group during a 3-hour apheresis was 119 +/- 65 x 10(6) and was not significantly different from that collected from the group treated with G- and GM-CSF (101 +/- 35 x 10(6) cells), but both were greater than that from the group treated with GM-CSF (12.6 +/- 6.1 x 10(6); P < .01 for both comparisons). Analysis of the CD34+ subsets showed that a significantly higher percentage of cells with the CD34+/CD38- phenotype is found after mobilization with G- and GM-CSF. In the G-CSF group, immunomagnetic selection of CD34+ cells permitted the enrichment of the CD34+ cells in the apheresis product to 81% +/- 11%, with a 48% +/- 12% yield and to a purity of 77% +/- 21% with a 51% +/- 15% recovery in the G- and GM-CSF group. T cells were depleted from a mean of 4.5 +/- 2.0 x 10(9) to 4.3 +/- 5.2 x 10(6) after selection, representing 99.9% depletion. We conclude that it is feasible to collect sufficient numbers of PB progenitor cells from normal donors with one to two leukapheresis procedures for allogeneic transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38-cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38-subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38-cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.

Multilineage differentiation of human fetal bone marrow CD34+ cell subsets was examined using a single-cell liquid culture assay. Four CD34+ cell populations, ie, (1) CD38-, HLA-DR+, (2) CD38-, HLA-DR-, (3) CD38+, HLA-DR-, and (4) CD38+, HLA-DR+ cells, were sorted as single cells into 96-well flat-bottom culture plates containing long-term culture medium supplemented with interleukin-3, interleukin-6, stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor, erythropoietin, basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1). Single CD34+, CD38-, HLA-DR+ cells had the highest replating efficiency as well as the highest replating efficiency. The cellular composition of the single-cell progeny was studied by morphologic and/or flow cytometric examination. Only the progeny of single CD34+ cells that lacked CD38 could give rise to each of the hematopoietic cell lineages. The expansion of the progeny of single CD34+, CD38-, HLA-DR+ cells was examined in more detail and showed three clearly distinguishable growth patterns: 28% (SD, +/- 10%; n = 14) of the single cells formed cell clusters/colonies; 9% (SD, +/- 4%; n = 14) formed dispersed cells; and 11% (SD, +/- 6%; n = 14) gave rise to a mixture of cell clusters and dispersed cells. The dispersed cell growth pattern was reduced when SCF or bFGF and IGF-1 was absent in the growth factor cocktail. The replating ability of the dispersed cells was considerably larger than that of cells with other growth patterns, in that 76% of the cells that gave rise to dispersed cells and 54% of the cells that gave rise to dispersed cells as well as cell clusters gave rise to a second generation, but only 7% of the cells that gave rise to cell clusters gave rise to a second generation. The second generation of cells continued to produce third and fourth generations after repetitive replating, except for the replated cells from cell clusters. In contrast with the first-generation progeny, SCF did not have an influence on the replating ability of the cells. Only in the progeny of single CD34+, CD38-, HLA-DR+ cells that gave rise to dispersed cells was each of the hematopoietic cell lineages found, ie, B lymphocytes, neutrophils, monocytes, macrophages, osteoclasts, basophils/mast cells, eosinophils, erythrocytes, megakaryocytes, and platelets.

Using multidimensional flow cytometry we have defined and quantified the human T-cell differentiation pathway, focusing on those events occurring among the most immature thymocytes and putative bone marrow (BM) T-precursors. Early thymocytes were found to express the CD34 antigen and consisted of a mean 1.2% of cells within human pediatric (n = 9) and 2.0% in fetal thymi (n = 4). All CD34+ thymocytes were atypical blast by morphology, expressed intracytoplasmatic, but not cell surface, CD3, and were cell surface CD2+, CD5+, CD7+, CD38+, CD45+, CD45RA+, CD49d+, and LECAM-1(Leu8)high. CD34high thymocytes lacked surface expression of CD4 and CD8, but as CD34 expression diminished there was a coordinate increase in CD4 levels, followed by the appearance of CD8. The expression of CD1 and CD10 also increased concomitant with the loss of CD34, whereas expression of LECAM-1 diminished with CD34 downregulation. The differential expression of these antigens on early thymocytes (as well as the number of thymocytes displaying these patterns) was highly reproducible among the nine pediatric and four fetal specimens examined, suggesting a precise, stereotyped regulation of early differentiation events. Cell populations with antigen expression patterns suggestive of pluripotent stem cell (CD34high, CD38-), or non-T-lineage committed stem cells (CD34+, CD33+ or CD34+, CD19+) were not identified in either fetal or pediatric thymi (sensitivity = 1/10(4)). The presence of cells with the antigenic profile of the earliest CD34+ thymocytes was explored in human BM. Putative BM T-cell precursors with the appropriate phenotype (CD34+, CD7+, CD5+, CD2+, LECAM-1high) were readily identified in fetal specimens (constituting +/- 2% of the CD34+ population), but could not be reliably detected in adults. In contrast with thymi, only 13% of these cells expressed cytoplasmatic CD3, suggesting the presence of the immediate precursor of the putative prothymocyte population. This was further supported by the detection of CD34bright, CD7+, CD2-, CD5-, LECAM-1moderate cells in fetal specimens. Our results document the flow of cell surface differentiation during T-lymphopoiesis and suggest that T-lineage features are first acquired in the BM. The ability to reproducibly identify and isolate T-cell precursor populations of precisely defined maturational stage in marrow and thymus by multiparameter flow cytometry will facilitate characterization of the molecular events controlling T-lineage differentiation.