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. 1997 May 13;94(10):5320-5.
doi: 10.1073/pnas.94.10.5320.

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

M Bhatia  1 , J C Wang, U Kapp, D Bonnet, J E Dick
Affiliations

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

M Bhatia et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Purification of CD34+ CD38−, CD34+ CD38+, and CD34− cell subsets. (A) Schematic diagram depicting the strategy used to obtain highly purified cells. Light density mononuclear cells from whole CB or BM were separated into lineage negative (Lin−) or positive (Lin+) fractions by staining with monoclonal antibodies against lineage antigens, followed by purification on an immunomagnetic column (StemSep). Lin− cells were stained with monoclonal antibodies specific for CD34 and CD38 and sorted on a FACStar Plus cell sorter into CD34+ CD38− and CD34+ CD38+ populations. Lin+ cells were stained with anti-CD34 and sorted for the CD34− population. (B) Expression of CD34 and CD38 on mononuclear cord blood cells analyzed by flow cytometry. The CD34+ cells that also express CD38 make up 0.5% of the cells in the lymphocyte/blast window (R1 in C), whereas 0.1% are CD38 negative. (C) Forward and side scatter plot of Lin− cells showing the live gate (R1) used for flow cytometry. (D) Dot plot showing that Lin− cells are highly enriched for both CD34+ and CD34+ CD38− cells. (E) Histogram of CD34 expression on Lin− cells. R2 represents the flow cytometry sorting gate used to purify CD34+ cells. (F) Histogram of CD38 expression on Lin− cells. R3 and R4 represent the gates used to sort CD38− and CD38+ cells. (G) Histogram of CD34 expression on Lin+ cells. R5 indicates the gate used to select CD34− cells (H and I). Flow cytometric reanalysis of CD34+ CD38+ (H), CD34+ CD38− (I), and CD34− (J) sorted cell fractions. Purities are given as a percentage of the cells in the particular lymphocyte/blast window.
Figure 3
Figure 3
Multilineage differentiation of human CD34+ CD38− cells in NOD/SCID mice. Bone marrow from a highly engrafted mouse transplanted with 5,000 CD34+ CD38− CB cells was stained with various human-specific monoclonal antibodies and analyzed by flow cytometry. (A) Histogram of CD45 (pan-leukocyte marker) expression indicating that 35% of the cells present in the murine bone marrow are human. (B) Analysis of lineage markers was done on cells within gate R1 (CD45+). (C) Cells in R1 were further gated based on forward and side scatter properties into lymphoid (R2), blast (R3) and myeloid (R4) windows. (D) Isotype control for nonspecific IgG staining of PE and FITC fluorescence. (E–I) Expression of the following markers is shown: E, myeloid marker CD33 and monocytic marker CD14 (R3 and R4); F, myeloid marker CD13 and mature granulocyte marker CD15 (R3 and R4); G, pan-B cell markers CD19 and CD20 (R2 and R3); H, CD19 and mature B cell marker sIgM (R2 and R3); I, CD38 and the immature hematopoietic marker CD34 on cells from R2, R3, and R4.
Figure 2
Figure 2
Human SRCs are exclusively CD34+CD38−. (A) Representative Southern blot analysis of individual NOD/SCID mice transplanted with CD34+ CD38−, CD34+ CD38+, and CD34− cells from CB. DNA was extracted from the murine bone marrow 8 weeks after the transplant and hybridized with a human chromosome 17-specific α-satellite probe. (B) Summary of the level of human cell engraftment in the BM of mice transplanted with purified cell fractions from 14 CB (solid circles) and two BM (open circles) samples. Numbers indicate the dose of CD34+ CD38− cells transplanted. Up to 60,000 CD34+ CD38+ cells and 400,000 CD34− cells were transplanted.
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