Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice

M Bhatia, JCY Wang, U Kapp… - Proceedings of the …, 1997 - National Acad Sciences
Proceedings of the national academy of Sciences, 1997National Acad Sciences
The purification of primitive human hematopoietic stem cells has been impaired by the
absence of repopulation assays. By using a stringent two-step strategy involving depletion of
lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell
population that is highly enriched for cells capable of multilineage repopulation in nonobese
diabetic/severe combined immunodeficient (NOD/SCID) recipients. These S CID-r
epopulating c ells (SRCs) were exclusively found in a cell fraction that expressed high levels …
The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) recipients. These SCID-repopulating cells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34+ CD38 cells. The highly purified SRC were capable of extensive proliferation in NOD/SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400,000 progeny 6 weeks after the transplant. Detailed flow cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34+ CD38 cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and/or expanding these cells during in vitro culture.
National Acad Sciences