Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice

Abstract

Increasing use of purified or cultured human hematopoietic cells as transplants has revealed an urgent need for better methods to predict the speed and durability of their engraftment potential. We now show that NOD/SCID-beta2 microglobulin-null (NOD/SCID-beta2m-/-) mice are sequentially engrafted by two distinct and previously unrecognized populations of transplantable human short-term repopulating hematopoietic cells (STRCs), neither of which efficiently engraft NOD/SCID mice. One is predominantly CD34+CD38+ and is myeloid-restricted; the other is predominantly CD34+CD38- and has broader lymphomyeloid differentiation potential. In contrast, the long-term repopulating human cells that generate lymphoid and myeloid progeny in NOD/SCID mice engraft and self-renew in NOD/SCID-beta2m-/- mice equally efficiently. In short-term expansion cultures of adult bone marrow cells, myeloid-restricted STRCs were preferentially amplified (greater than tenfold) and, interestingly, both types of STRC were found to be selectively elevated in mobilized peripheral blood harvests. These results suggest an enhanced sensitivity of STRCs to natural killer cell-mediated rejection. They also provide new in vivo assays for different types of human STRC that may help to predict the engraftment potential of clinical transplants and facilitate future investigation of early stages of human hematopoietic stem cell differentiation.

Figure 1

Different engraftment kinetics of human cells in NOD/SCID-β2m^–/– and NOD/SCID mice. Groups of recipients were sacrificed 3, 6, and 13 weeks after transplantation, and the types and numbers of human cells present in the BM were determined by FACS analysis. (a) Total human CD45/71⁺ cells in NOD/SCID-β2m^–/– mice (filled symbols, 13–14 mice/point) and NOD/SCID mice (open symbols, 15–16 mice/point) were calculated from data pooled from two independent experiments. (b) Absolute numbers of cells belonging to different human lineages present at different times. (c) Relative distribution of different types of human cells present after different periods in NOD/SCID-β2m^–/– (filled bars) and NOD/SCID mice (open bars, same experiments as in b). Data for the 3-week NOD/SCID mice are not shown in this panel because of the low numbers of human cells present in these mice at this early time point. (d) Representative FACS profile of cells harvested from the BM of a NOD/SCID-β2m^–/– mouse 3 weeks after transplantation of 2.5 × 10⁵ human lin^– BM cells. Note the high number of human erythroid (glycophorin A⁺) and megakaryocytic (CD41⁺) cells. Values for all figure parts are the mean ± SEM. ^ASignificant difference, P < 0.05.

Figure 2

Phenotype of STRC-M and STRC-ML and their expansion in short-term culture. CD34⁺CD38⁺ cells (filled symbols) in adult BM show an initially greater ability than CD34⁺CD38^– cells (open symbols) to engraft NOD/SCID-β2m^–/– mice for 3 weeks (P < 0.01), but CD34⁺CD38^– cells have an equivalent ability to produce this activity in 5-day expansion cultures (P > 0.1). In contrast, CD34⁺CD38⁺ cells contribute much less than the CD34⁺CD38^– cells to the 8-week engraftment of NOD/SCID-β2m^–/– mice (P < 0.01) and show a parallel decline in this activity after 5 days in culture. Each symbol corresponds to the level of engraftment seen in an individual mouse originally injected with the yield of CD38⁺ or CD38^– cells obtained from a starting equivalent of 10⁵ CD34⁺ cells either directly (pre-culture) or after 5 days of culture with FL, SF, IL-3, IL-6, and G-CSF (post-culture). Different symbols show the results of two separate experiments. Bar, mean of each group. ^ASignificant differences, P < 0.05.

Figure 3

The distribution of 6-week repopulating activity in NOD/SCID-β2m^–/– mice between the G₀/G₁ and S/G₂/M fractions of 5-day human CB cell-expansion cultures is indistinguishable from that measured for total cells or other progenitors. CD34⁺ CB cells were cultured for 5 days in serum-free medium supplemented with SF, FL, IL-3, IL-6, and G-CSF. G₀/G₁ and S/G₂/M cells were then isolated after DNA staining with Hoechst 33342. Approximately half of the fractionated cells were transplanted in NOD/SCID-β2m^–/– mice immediately after their isolation. The other half of each fraction was first cultured for an additional 16 hours before being transplanted. (a) The proportion of engrafted mice (and the average level of engraftment) for the two populations compared (G₀/G₁ and S/G₂/M). (b) The corresponding data for total cells, CD34⁺ cells, CFC, and LTC-IC in the G₀/G₁ (open bars) and S/G₂/M (filled bars) fractions from the same experiments. All values shown are the mean ± SEM of data pooled from three experiments.

Figure 4

Model indicating a hierarchy of transplantable human hematopoietic cells with distinct biological properties. LTRCs include CD34^–CD38^– cells (22, 26) as well as cells expressing CD34 but not CD38 (3, 21). The engraftment ability of LTRCs is restricted to the G₀/G₁ phases of the cell cycle (30) and are the only human cells that efficiently engraft NOD/SCID mice. STRC-ML are CD34⁺CD38^–, and their ability to engraft NOD/SCID-β2m^–/– mice is not cell cycle–restricted. Most freshly isolated STRC-M express both CD34 and CD38.