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-

Transforming growth factor-beta1 (TGF-beta1) has been described as an efficient growth inhibitor that maintains the CD34(+) hematopoietic progenitor cells in quiescence. The concept of high proliferative potential-quiescent cells or HPP-Q cells has been introduced as a working model to study the effect of TGF-beta1 in maintaining the reversible quiescence of the more primitive hematopoietic stem cell compartment. HPP-Q cells are primitive quiescent stem/progenitor cells on which TGF-beta1 has downmodulated the cytokine receptors. These cells can be released from quiescence by neutralization of autocrine or endogenous TGF-beta1 with a TGF-beta1 blocking antibody or a TGF-beta1 antisense oligonucleotide. In nonhematopoietic systems, TGF-beta1 cooperates with the cyclin-dependent kinase inhibitor, p21(cip1), to induce cell cycle arrest. We therefore analyzed whether endogenous TGF-beta1 controls the expression of the p21(cip1) in the CD34(+) undifferentiated cells using a sensitive in situ hybridization method. We observed that addition of anti-TGF-beta1 is followed by a rapid decrease in the level of p21(cip1) mRNA whereas TGF-beta1 enhances p21(cip1) mRNA expression concurrently with an inhibitory effect on progenitor cell proliferation. These results suggest the involvement of p21(cip1) in the cell cycle control of early human hematopoietic quiescent stem/progenitors and not only in the differentiation of more mature myeloid cells as previously described. The modulation of p21(cip1) observed in response to TGF-beta1 allows us to further precise the working model of high proliferative potential-quiescent cells.

It has been previously demonstrated that prestimulation with cytokines could improve gene transfer in hematopoietic progenitors. However, we have shown that no combination of cytokines so far tested is able to release rapidly in vitro the stem cell compartment from quiescence unless an autocrine transforming growth factor-beta 1 (TGF-beta 1) is blocked by specific oligonucleotide antisense or antiserum (Hatzfeld et al., 1991, J. Exp. Med., 174, 925). We now report that a 10-hr cytokine prestimulation of SBA-CD34high human umbilical cord blood progenitors increases retrovirally mediated transfer of the nls-lacZ reporter gene from 1% to 23.8% and addition of anti-TGF-beta serum doubles this increase (47.3%). Interestingly, the effect of anti-TGF-beta preincubation on gene transfer is most effective on the most immature progenitors, which develop into high proliferative potential mixed colonies with 1-2 x 10(5) cells. Anti-TGF-beta serum pretreatment increases gene transfer in these early colony-forming units granulocyte-erythroid-megakaryocyte-macrophage (CFU-GEMM) from 54.1% to 93.3%. It augments significantly the stability of gene expression in all subpopulations of mixed colonies. Colonies obtained after pretreatment with anti-TGF-beta serum are larger and the expression of the stably integrated recombinant provirus does not reduce their size. This prestimulation method provides a substantial improvement for gene transfer efficiency within the quiescent stem cell compartment that is responsible for long-term engraftment.

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.