Béatrice Panterne

p21cip1 mRNA is controlled by endogenous transforming growth factor-?1 in quiescent human hematopoietic stem/progenitor cells

Transforming growth factor‐β1 (TGF‐β1) 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‐β1 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‐β1 has downmodulated the cytokine receptors. These cells can be released from quiescence by neutralization of autocrine or endogenous TGF‐β1 with a TGF‐β1 blocking antibody or a TGF‐β1 antisense oligonucleotide. In nonhematopoietic systems, TGF‐β1 cooperates with the cyclin‐dependent kinase inhibitor, p21cip1, to induce cell cycle arrest. We therefore analyzed whether endogenous TGF‐β1 controls the expression of the p21cip1 in the CD34+ undifferentiated cells using a sensitive in situ hybridization method. We observed that addition of anti‐TGF‐β1 is followed by a rapid decrease in the level of p21cip1 mRNA whereas TGF‐β1 enhances p21cip1 mRNA expression concurrently with an inhibitory effect on progenitor cell proliferation. These results suggest the involvement of p21cip1 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 p21cip1 observed in response to TGF‐β1 allows us to further precise the working model of high proliferative potential‐quiescent cells. J. Cell. Physiol. 184:80–85, 2000.

Cryopreservative effect of leupeptin on early human bone marrow progenitors

SummaryLeupeptin, a thiol- and serine-proteinase inhibitor of low molecular weight, quickly enters viable cells. This property has been used to protect cells during thawing against intracellular proteolytic activities released by injured lysosomes. The bone marrow nucleated cells were frozen without rate-controlled freezing devices. Concentrations ranging from 0.1 to 1µM of leupeptin allow to recover 87% of the most immature multipotent bone marrow progenitors which can develop in vitro into large multilineage colonies, instead of 58% recovery without leupeptin. The protective effect of leupeptin is particularly useful to freeze cells difficult to cryopreserve or when freezing-control equipments are not available.

Inhibition of autocrine TGF-ß in CD34+ human progenitor cells reveals their potentiality to engraft adults and improves gene transfer efficiency

Using antisense TGF-s1 oligonucleotides (ODN) or anti-TGF-s serum in single cell and clonal culture of enriched progenitors from normal human bone marrow cells and umbilical cord blood, we have demonstrated that early hematopoietic progenitors produce autocrine TGF-s1. These cells could not be triggered into cycle by optimal concentrations of growth factors available, which included IL-3, IL-6, G-CSF, SL and Epo. Using medium with this cytokine cocktail supplemented with either antisense TGF-s 1 ODN or anti-TGF-s serum, we first estimated the proliferative capacity and ability to generate early progenitors in long-term cultures of bone marrow and umbilical cord blood cells. The results showed that the capacity for long-term engraftment of a 100-200 ml sample of umbilical cord blood is greater than that of the volume of bone marrow typically used to engraft an adult. The release from quiescence of the early progenitor cells by anti-TGF-s serum was also used to improve gene transfer efficiency, since retroviral gene transfer requires a proliferative status of the target cell. High efficiency (95%) of gene transfer was obtained in early colony-forming unit-granulocyte-erythroidmegakaryocyte-macrophage (CFU-GEMM) with a stable gene expression. Development and size of the colonies were not affected by the retrovirally-mediated gene transfer. Among the normal hematopoietic progenitor populations, the earliest pluripotent cells are largely quiescent while a larger proportion of later, lineage-restricted cells are in active phases of the cell cycle (Lajtha and Schofield, 1974). Using antisense TGF-s1 oligonucleotides (ODN) or anti TGF-s serum in single cell and clonal culture of enriched progenitors from normal human bone marrow cells, we have demonstrated that early colony forming cells produce autocrine TGF-61. Indeed, antisense TGF-s ODN or anti TGF-s serum enhance colony formation from early progenitor cells such as CFU-GEMM and early BFU-E: we obtained twice more mixed colonies and erythroid bursts than in the control cultures.These colonies were larger (Hatzfeld et al., 1991). These cells could not be triggered into cycle by optimal concentrations of growth factors available, which included IL-3, IL-6, G-CSF and Epo. We have further shown that, although inclusion of SF increases the frequency of colony formation by early progenitor cells, it still does not reverse the effects of TGF-81 in culture and many cells remain quiescent (Li et al., 1994). We have therefore used cultures with our standard cytokine cocktail supplemented with SF and either antisense TGF-81 ODN or anti-TGF-s serum: 1) to compare the proliferative capacity and ability to generate early progenitors in long-term cultures of bone marrow and umbilical cord blood cells. 2) to improve gene transfer efficiency since retroviral gene transfer requires a proliferative status of the target cell. To evaluate the proliferative capacity of bone marrow and umbilical cord blood progenitors, cells obtained from iliac crest of healthy individuals or from umbilical cord blood with the appropriate consent of the donors were purified as previously described, using the AIS procedure (SBA and CD34 CELLector flasks; Applied Immune Sciences Inc., Santa Clara, CA) (Cardoso et al., 1995). The isolated CD34+ population obtained by this procedure was 95 ± 3% pure. Cells were then cultured with the cocktail of cytokines in the presence of 21mers phosphorothioate TGF-s1 ODN or turkey anti-TGF-s blocking serum, either in liquid cultures for expansion or in clonogenic assays to evaluate the types of progenitors. The CD34+ CD38- population was purified by FACS. When cultured in the presence of IL-3, IL6, GM-CSF, SF, and Epo, colony formation by early progenitors (CFU-GEMM, HPP-CFC, and early BFU-E) in the cord blood CD34+CD38- fraction, but not in the CD34+CD38+ fraction, was significantly (1.5 to 2 fold) enhanced by antisense TGF-B1 ODN. Late progenitors (CFU-E, CFU-M, and CFU-G) in the same preparation were unaffected by the antisense ODN. These results suggest that some subpopulations of CD34+ CD38- cells are under the control of autocrine TGF-s. Cumulative evaluation of the respective number of colonies obtained after more than 35 days of culture leads to the conclusion that, although a typical bone marrow sample prepared for adult bone marrow transplant contains significantly more CD34+ cells than a typical cord blood sample (100-200 ml), the ability to generate early progenitors in primary culture are quite comparable for both samples. This results from the higher frequency of CD34+ CD38- cells in the cord blood preparations and from the higher proliferative potential of these cells in culture. Indeed, the CD34+ CD38- cord blood fraction yielded similar number of CFU-GEMM but almost 3 fold more early BFU-E and 2 fold more CFU-GM than did the corresponding bone marrow cell preparation (Cardoso et al, 1993). We took advantage of releasing the early progenitors from quiescence to improve transfer efficiency since the proliferative status of the target cell is a prerequisite for an efficient transfer. Purified cord blood CD34+ cells were prestimulated in the presence of cytokines and anti TGF-s serum. They were then transfected by cocultured for 40 h on 40% confluent NB16, a packaging cell line which express the E. Coli Bgalactosidase, kindly provided by Drs J.M. Heard and O. Danos (Ferry et al., 1991). Transduced CD34+ cells were then tested in clonogenic assays and colonies were stained with X-Gal as substrate (Strair et al., 1990). Results showed that addition of anti-TGF-s serum increased the transfer efficiency from 24% without anti-TGF-s serum up to 47.3% in the presence of anti-TGF-s serum. Interestingly, the effect of anti-TGF-s is most effective on the most immature progenitors, which develop into large mixed colonies (CFU-GEMM) with l-2xl05 cells. Anti-TGF-s serum pretreatment increases gene transfer in CFU-GEMM from 54 to 95%. It augments significantly the stability of gene expression in subpopulations of mixed colonies and the expression of the stably integrated recombinant provirus does not reduce their size. In conclusion, the elucidation of the mechanisms controlling cell proliferation, especially the role of inhibitors like TGF-s 1, will be ofinterest in clinical applications for transplantation with cell expansion as well as for gene therapy.