Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-Hu mice

Abstract

Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte- macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.

Separation of G‐CSF‐mobilized PBSC transplants by counterflow centrifugal elutriation: modest enrichment of CD34+ cells but no loss of primitive haemopoietic progenitors

“…The CD34 þ cells which co-separated with lymphocytes were virtually devoid of CFU-GM. However, in contrast to what has been found for human bone marrow cells (Wagner et al, 1995), these small CD34 þ cells were not enriched with CD34 þ /CD38 ¹ cells. Since nearly all CD34 þ cells were CD38 þ we also analysed the CD38 lo population, which also contains many cells capable of long-term haemopoietic activity in vitro (Breems et al, 1996;Reems & Torok Storb, 1995), although these may be of a less immature state of differentiation (Hao et al, 1995;Prosper et al, 1996).…”

“…The difference between our findings on the distribution of primitive stem cells from PBSC grafts in CCE separations and those reported for murine and human bone marrow (Jones et al, 1990;Wagner et al, 1995) and normal human peripheral blood cells (Herbein et al, 1994) may result from an effect of the mobilization regime on stem cells. Elutriation profiles of murine HPP-CFC have been found to shift to fractions of larger cells when mice were pretreated with 5-fluorouracil and bone marrow was regenerating (Schwartz et al, 1986) and similar findings have been reported for elutriation profiles of human CD34 þ cells mobilized with G-CSF as compared to CD34 þ cells from normal blood (Grimm et al, 1995).…”

“…Elutriation profiles of murine HPP-CFC have been found to shift to fractions of larger cells when mice were pretreated with 5-fluorouracil and bone marrow was regenerating (Schwartz et al, 1986) and similar findings have been reported for elutriation profiles of human CD34 þ cells mobilized with G-CSF as compared to CD34 þ cells from normal blood (Grimm et al, 1995). However, it should be also realized that in the study of Wagner et al (1995) with human bone marrow, progenitors capable of long-term haemopoietic activity in vitro were not exclusively recovered in the fractions with small cells but were also present in the fractions with large cells. Moreover, in other studies with murine bone marrow (Orlic & Bodine, 1992;Orlic et al, 1993) the observation of Jones et al (1990) that only the elutriation fraction with the smallest cells was capable of long-term haemopoietic reconstitution upon transplantation, was not confirmed.…”

“…In another study (Yoder et al, 1993) a distinct subpopulation of murine highly proliferative potential colony-forming cells (HPP-CFC), which required multiple haemopoietic growth factors for maximal proliferation, was isolated in elutriation fractions containing small cells. In separations of human bone marrow by CCE, the fraction with the smallest cells was found to contain progenitor cells capable of long-term haemopoietic activity in vitro, but no CFU-GM (Wagner et al, 1995). Moreover, although early engraftment in patients transplanted with bone marrow grafts from which T cells had been depleted by CCE was not impaired (de Witte et al, 1986;Wagner et al, 1988;Schattenberg et al, 1990), higher rates of late graft failure and mixed chimaerism, compared to patients receiving unseparated bone marrow, have been observed (Wagner et al, 1992;Schattenberg et al, 1989).…”

From stem cells to lymphocytes; biology and transplantation

“…A very different method to enrich qtiiescent HSCs is by size fractionation of bone marrow cells (37)(38)(39)(40)(41). Recently, mouse HSCs were purified by selecting small cells expressing high levels of aldehyde dehydrogenase (ALDH) activity (42,43).…”