Macrophage inflammatory protein 1 alpha, interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro.

Abstract

Factors that induce proliferation of the human hematopoietic stem cell are ill-defined. Primitive hematopoietic progenitors can be maintained and differentiate in stroma-dependent, long-term bone marrow cultures (LTBMC), originally described by Dexter et al. (Dexter, T. M., L. H. Coutinho, E. Spooncer, C. M. Heyworth, C. P. Daniel, R. Schiro, J. Chang, and T. D. Allen. 1990. Molecular Control of Haemopoiesis). However, 70-80% of primitive progenitors capable of reinitiating secondary stromal cultures (LTBMC-initiating cells [IC]) are lost over a period of 5 wk in such cultures. We have recently described a novel "stroma-noncontact" culture system, in which hematopoietic progenitors are separated from the stromal layer by a 0.4-micron microporous filter membrane. Primitive progenitors in such cultures can not only differentiate into committed progenitors, but are also maintained to a greater extent than in "Dexter" cultures. However, still only 50% of the originally seeded LTBMC-IC are recovered at week 5. Since maintenance of primitive progenitors may depend not only on growth-promoting factors but also on factors that inhibit differentiation and/or proliferation, we evaluated the effect of macrophage inflammatory protein 1 alpha (MIP-1 alpha) or "stem cell inhibitor" in combination with the growth-inducing factor interleukin 3 (IL-3) on the recovery of LTBMC-IC from stroma-noncontact cultures. We demonstrate that addition of MIP-1 alpha alone to stroma-noncontact cultures does not change the number of LTBMC-IC present after 8 wk, indicating that this factor may not directly inhibit or stimulate proliferation of primitive progenitors. Addition of the growth stimulatory cytokine, IL-3, alone results in exhaustion of LTBMC-IC after 8 wk of culture, possibly as a result of their terminal differentiation. However, LTBMC-IC can be maintained for at least 8 wk when grown in stroma-noncontact cultures supplemented with both MIP-1 alpha plus IL-3. This effect depends on soluble (ill-defined) stromal factors, and results from a direct interaction of these cytokines with the progenitor population or its progeny, but not the stroma.

Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34+DR− cells and of some hemopoietic subsets from CD34+ and CD34+DR+ cells

“…28 This molecule has also been shown to support the maintenance of LTC-IC from BM CD34 + DR − cells. 29 These experiments combined MIP-1␣ with IL-11 and SCF 28 or with IL-3 and conditioned medium. 29 We assessed whether this beneficial effect of MIP-1␣ held true when combined with a cocktail of positive growth factors such as IL-3, IL-6, IL-11 and SCF.…”

True

“…3 Addition of IL-3 and MIP-1␣ increases this to 100%. 7 Likewise, primitive lymphoid progenitors are maintained in M2-10B4 non-contact cultures supplemented with MIP-1␣ and IL-3. 8 Although other cell lines, such as NIH3T3 and FHS-173-We, support LTC-IC in contact cultures equally well as M2-10B4 feeders, these two feeders poorly support LTC-IC in non-contact conditions.…”

“…[1][2][3][4][5] We and others have shown that primitive LTC-IC and primitive lymphoid progenitors can be maintained in stroma-based cultures even without contact with the stromal feeders. [6][7][8][9][10][11] Up to 50% of LTC-IC are maintained without exogenous cytokines in transwells above primary human bone marrow stroma or the murine fibroblast line M2-10B4, for 8 weeks. 3 Addition of IL-3 and MIP-1␣ increases this to 100%.…”

NOD/SCID repopulating cells but not LTC-IC are enriched in human CD34+ cells expressing the CCR1 chemokine receptor

“…[11][12][13][14][15][16][17] Evidence for an effect of MIP-1␣ on LTC-IC was provided by Verfaillie who showed prolonged survival of LTC-IC in cultures containing MIP-1␣ IL-3 and stromal cell supernatant or heparan sulphate. [18][19][20] IL-3 and MIP-1␣ were also required for survival of primitive human haemopoietic cells with myeloid and lymphoid differentiation potential in vitro. 18,21 Furthermore, both mRNA and protein for MIP-1␣ have been detected in the adherent stromal layers of long-term bone marrow cultures where the LTC-IC reside.…”

“…[18][19][20] IL-3 and MIP-1␣ were also required for survival of primitive human haemopoietic cells with myeloid and lymphoid differentiation potential in vitro. 18,21 Furthermore, both mRNA and protein for MIP-1␣ have been detected in the adherent stromal layers of long-term bone marrow cultures where the LTC-IC reside. 22,23 As the action of MIP-1␣ on stem and progenitor cells is the result of direct interaction with the target cells, the responsive cells must bear the appropriate receptors for the chemokine.…”