ISSN:
0006-3592
Keywords:
hematopoietic stem cells
;
quiescence
;
self-renewal
;
multilineage potential
;
single cell culture
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
We have developed methods for detailed characterization of the proliferation kinetics and lineage potential of single human hematopoietic progenitor cells in an in vitro culture system. Fetal bone marrow CD34hi/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 factor (LIF) on a murine stroma cell monolayer. Individual wells were scored for growth between 1 and 10 weeks of culture and analyzed by flow 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 well of approximately 20,000 progeny. Confluent wells were harvested and 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 (CD33+). 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 cells, myeloid cells, and primitive progenitors (CD34+/lin-) which persisted through two expansion cycles. We estimate that more than 36 million cells can be produced from a single cell under these culture conditions. A very small percentage of the CD34hi/lin- population (about 1%) was responsible for the majority of subsequent cell production. Our estimate of stem cell content in fetal bone marrow, defined by self-renewal as well as both B-lymphoid and myeloid differentiation from one cell, is approximately 1/13,000. This assay system provides direct in vitro measurements of the expected characteristics of hematopoietic stem cells (high proliferation potential, multilineage potential, self-renewal, and quiescence), and is therefore well suited to assessment of stem cell activity within various cell populations. © 1996 John Wiley & Sons, Inc.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
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