EXTENSIVE PROLIFERATIVE CAPACITY OF SINGLE ISOLATED CD34(-BLOOD CELLSIN SUSPENSION-CULTURE(++) HUMAN CORD)

Nonadherent, low-density T-lymphocyte-depleted (NALT(-)) CD34(+++) cel ls from normal human cord blood were assessed in suspension culture fo r the effects of recombinant cytokines on their proliferation, differe ntiation, and generation of myeloid progenitor cells. In this cell pop ulation, 82% of cells expressed c-kit protein as assessed by in situ h ybridization, and their cloning efficiency was 85% when cells were pla ted at low cell numbers with combinations of growth factors. CD34(+++) cells were sorted as 1, 5, or 10 cell(s) per well and also at 5000 ce lls per dish to initiate stromal-free suspension cultures in the prese nce of steel factor (SLF), interleukin (IL)-1(alpha), and 1L-3. Forty- eight percent of the wells started with a single CD34(+++) cell were p ositive for growth after 14 days, and the wells contained greater than 5 x 10(3) cells by 21-28 days. Progenitors were assayed weekly with c ultures initiated with 1 or 5000 cells. While the fold expansion of nu cleated cells was greater in cultures initiated with one cell per well (>5000 compared to 791-fold expansion for 5000 cells), the fold expan sion of progenitors was greater than 5000 cells were used to initiate cultures. Under optimal conditions, there was, respectively, a 160-, 1 64-, and 57-fold output of high proliferative potential colony-forming cells, granulocyte-macrophage colony-forming units, and erythroid bur st-forming units/granulocyte erythroid macrophage megakaryocyte colony -forming units within 1-3 weeks for cultures initiated with 5000 CD34( +++) cells compared with respective fold increases of 29, 16, and 1, f or single-initiated cultures. These results demonstrate the expansion capacity of single CD34(+++) cord blood cells and demonstrate that fac tors in addition to SLF, IL-1(alpha), and IL-3 are necessary for optim al expansion of progenitors from single isolated CD34(+++) cells.