Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability

Abstract

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold, P < 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1(+)lin- adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11, flt3-ligand, and Steel factor. Moreover, the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures, long-term culture-initiating cells increased 7- +/- 2-fold, myeloid colony-forming cells increased 140- +/- 36-fold, and total nucleated cells increased 230- +/- 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1(+)lin- marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension, suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119- CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult.

Figure 1

Proportion of negative mice (mice not showing repopulation of both lymphoid and myeloid compartments) 4–12 months after being transplanted with WGA⁺ Sca-1⁺lin⁻ or CD34⁻Sca-1⁺lin⁻ marrow cell populations (○) or the cells derived from them after 9–10 days incubation in 100 ng/ml IL-11, 100 ng/ml FL, and 50 ng/ml SF (•). The data for the cultured cells are expressed per freshly isolated purified cell equivalents to allow their increased absolute numbers to be seen directly on this graph. Data are pooled from the six experiments described in Table 1 (4–15 recipients per point). The lines shown connect the CRU frequency values determined from the two sets of pooled data by the method of maximum likelihood (26) (at 37% negative mice, see text) to the origin.

Figure 2

Expansion of total cells, CFC, LTC-IC, and CRU in cultures of Sca-1⁺lin⁻ marrow cells stimulated in vitro by 100 ng/ml IL-11, 100 ng/ml FL, and 50 ng/ml SF for 5 (open bars, n = 2 experiments), or 9–10 days (hatched bars, n = 5–7 experiments). The fold increase (±1 SEM) was calculated by dividing the number of progenitors detected in the 5- or 9- to 10-day cultures by the corresponding number of progenitors calculated to be present in the cells used to initiate each culture. (Data include 1 experiment not described in Table 1.)

Figure 3

Levels of engraftment maintained in individual C57BL/6 mice injected with 40 Sca-1⁺lin⁻CD34⁻ cells (day 0) or the cells generated from the equivalent of 30 or 32 of these cells after 5 (day 5) or 10 days (day 10) of culture in IL-11 plus FL and SF, respectively.