Using optimal culture conditions in which the transforming growth factor 11 (TGF-j1) inhibitory loop has been interrupted by antiuense TGF-131 oligonudeotides or antf-TGF-1 serum, we have compared the proliferative capacities and the abUities ofthe CD34+ CD38-ceil populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38-fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38-cells from a typical umbilical cord blood sample produce equivalent numbers of colonyformdngunits (CFU)-gnulocyte/erythrocyte/macrophage/ megakaryocyt, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult tansplantation. In addition, the colonies resulting from the umbical cord blood samples were signiflcantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparatons, as esimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is pardaly compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbiical cord blood is a suitable source of cells for adult transplantation.Small amounts of near-term or neonatal mouse blood has been demonstrated to have the potential to completely reconstitute the hematopoietic system of adult mice (1). This finding has prompted extensive analysis of human umbilical cord blood as a potential source for cells for hematopoietic reconstitution in man (1-4). Various nonmalignant (3, 5) and malignant (7,8) hematopoietic diseases and malignant nonhematopoietic diseases (9, 10) have been successfully treated in children by using umbilical cord blood transplantation. Umbilical cord blood is easily obtainable and therefore could be used to develop banks of HLA-typed cells for general use in adult transplantation ifthe engraftment potential were high enough. Here we report methods for evaluating the relative potentials for engraftment and their use in comparing umbilical cord blood and bone marrow as cellular sources for transplantation.In the past, engraftability of bone marrow samples has largely been analyzed through estimation of the content of colony-forming unit (CFU)-anulocyte/macrophage (GM) (11,13 Purfication of CD34+ Cells. Mononuclear cells were obtained as described (20,23). Hematopoietic progenitors expressing the CD34 antigen were purified using the Applied Immune Sciences (Santa Clara, CA) procedure [soybean agglutinin (SBA) and CD34 CELLector flasks] (20, 24). These cells were then recovered, concentrated, and used for fluorescence-activated cell sorting (FACS), phenotypic anal-

Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting- gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.

We have previously shown that early human CD34high hematopoietic progenitors are maintained quiescent in part through autocrine transforming growth factor-beta 1 (TGF-beta 1). We also demonstrated that, in the presence of interleukin-3, interleukin-6, granulocyte colony-stimulating factor, and erythropoietin, TGF-beta 1 antisense oligonucleotides or anti-TGF-beta serum have an additive effect with KIT ligand (Steel factor [SF]), which suggests that they control different pathways of regulation in these conditions. This finding also suggests that autocrine TGF-beta 1 might suppress c-kit expression in primitive human hematopoietic progenitors. We have now distinguished two subpopulations of CD34high cells. One subpopulation expresses a c- kit mRNA that can be downmodulated by exogenous TGF-beta 1 within 6 hours. Another subpopulation of early CD34high cells expresses a low or undetectable level of c-kit mRNA, but its expression can be upmodulated within 6 hours by anti-TGF-beta. These effects disappear 48 hours after induction and cannot be maintained longer than 72 hours, even if TGF- beta 1 or anti-TGF-beta serum are added every day. Similar kinetics, although delayed, are observed with KIT protein expression. On the contrary, no specific effect of TGF-beta 1 was observed on c-fms, GAPDH, and transferrin receptor gene expression in these early progenitors. These results clarify the complex interaction between TGF- beta 1 and SF in normal early hematopoietic progenitors. SF does not switch off the TGF-beta 1 inhibitory pathway. Autocrine TGF-beta 1 appears to maintain these cells in a quiescent state, suppressing cell division by downmodulating the receptor of SF, a key cytokine costimulator of early progenitors.