IN-VITRO CHARACTERIZATION OF FETAL HEMATOPOIETIC STEM-CELLS - RANGE AND KINETICS OF CELL PRODUCTION FROM INDIVIDUAL STEM-CELLS

We have developed methods for detailed characterization of the prolife ration kinetics and lineage potential of single human hematopoietic pr ogenitor cells in an in vitro culture system. Fetal bone marrow CD34(h l)/lin(-) cells were cultured at one cell per well in the presence of c-kit ligand (KL), interleukin (IL)-3, IL-6, and leukemia inhibitory f actor (LIF) on a murine stroma cell monolayer. Individual wells were s cored for growth between 1 and 10 weeks of culture and analyzed by flo w cytometry for lineage composition. A wide variation in time (1 to 8 weeks) was observed before initial cell division, even in the presence of cytokines promoting cell division in primitive progenitors. Eleven percent of the plated cells eventually produced a confluent culture w ell of approximately 20,000 progeny. Confluent wells were harvested an d individually analyzed by flow cytometry for cell surface phenotype. Forty-eight percent of confluent wells contained primitive progenitors (CD34(+)lin(-)), 16% contained B-lymphoid cells (CD19(+) or CD10(+)), and 100% contained cells committed to the myelo-erythroid lineage (CD 33(+)). CD34(+)/lin(-) cells from confluent wells were replated at one cell per well in secondary culture and the analysis repeated. One of 216 original single cells plated produced populations of B-lymphoid ce lls, myeloid cells, and primitive progenitors (CD34(+)/lin(-)) which p ersisted through two expansion cycles. We estimate that more than 36 m illion cells can be produced from a single cell under these culture co nditions. A very small percentage of the CD34(hl)/lin(-) population (a bout 1%) was responsible for the majority of subsequent cell productio n. Our estimate of stem cell content in fetal bone marrow, defined by self-renewal as well as both B-lymphoid and myeloid differentiation fr om one cell, is approximately 1/13,000. This assay system provides dir ect in vitro measurements of the expected characteristics of hematopoi etic stem cells (high proliferation potential, multilineage potential, self-renewal, and quiescence), and is therefore well suited to assess ment of stem cell activity within various cell populations. (C) 1996 J ohn Wiley & Sons, Inc.