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Summary:In The remarkable proliferation and cell renewal processes in the human hematopoietic system are believed to be supported by a small population of hematopoietic stem cells. 1,2 A major challenge in stem cell research is the establishment of culture systems that facilitate in vitro maintenance and augmentation of stem cell activity. This is of great impor- tance not only for hematopoietic stem cell transplantation but also for gene therapy. It is also an important step towards cellular and molecular understanding of the regulatory mechanisms that mediate the commitment vs self-renewal decision of stem cells.In the last two decades a number of hematopoietic cytokines have been identified. Studies using recombinant hematopoietic cytokines have revealed that hematopoietic cytokines alone or in combination support the proliferation of hematopoietic progenitors and that it is possible to increase the number of progenitors in culture. 3 Recently, several groups of investigators have reported that combinations of early-acting cytokines support the maintenance or expansion of progenitors and even of transplantable stem cells in both murine 4-7 and human systems. [8][9][10][11] On the other hand, however, the importance of stromal cells in the maintenance and expansion of primitive progenitors including transplantable stem cells have been repeatedly demonstrated. 12-20 A number of stromal cell lines have been reported to support the proliferation of human primitive progenitors. MS-5 is one such cell line, derived from noninfected Dexter-type murine long-term marrow culture, which supports CFU-S maintenance 21 and acts synergistically with human growth factors to stimulate the formation of blast colonies and macroscopic colonies from CD34 + CD38 Ϫ primitive progenitors in short-term methylcellulose assays. 22 MS-5, alone 23 or in combination with SCF and G-CSF, 24,25 also supports the proliferation of primitive lymphohematopoietic progenitors that are capable of differentiation along both myeloerythroid and lymphoid lineages and their differentiation toward the B cell lineage. Furthermore, a recent study has demonstrated that, in the presence of multiple cytokines, MS-5 cells promote a net increase of LTC-IC through self-renewal divisions. 26 In the present study, we demonstrate synergistic effects of MS-5 cells and early-acting hematopoietic growth factors, FL and TPO on the expansion of human cord blood CD34 + cells, CD34 + CD38 Ϫ cells, CFU-C and CAFC. Using an SRC assay we also found that transplantable stem cell activity was slightly augmented or at least maintained in our culture system.

We identified the cell cycle status of CD34+ cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34+ cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34+ cells in PB were cycling. BM CD34+ cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34+cells. In addition, when cycling and dormant BM CD34+cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34+ cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34+ progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment. © 1998 by The American Society of Hematology.

The relationships between transfused cell number of CD34+ cell subpopulations divided by HLA-DR and CD33 antibodies and hematopoietic recovery patterns after peripheral blood progenitor cell transplantation (PBPCT) subsequent to myeloablative chemoradiotherapy were investigated in 14 children with cancer. Both logarithm of transfused CD34+ cell number/10⁶/kg and logarithm of transfused cell number/10⁶/kg of the CD34+HLA-DR+CD33+ sub-population, which is supposed to be myeloid-committed cells, were correlated with myeloid recovery after PBSCT, though they were not correlated with erythroid or platelets recovery. On the other hand, logarithm of transfused cell number/10⁶/kg of CD34+HLA-DR-CD33- subpopulation, which is supposed to be immature progenitor cells, was not correlated with myeloid recovery but correlated with erythroid recovery and platelet recovery. These results suggested that rapid myelopoiesis after PBPCT occurs following transfusion of sufficient numbers of myeloid-committed cells and complete hematopoietic reconstitution occurs after transfusion of sufficient numbers of immature hematopoietic progenitor cells.

To study the cytokine regulation of early stages of human Blymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34؉ CD38