Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro

Abstract

To develop a purification strategy for isolating the most primitive hematopoietic stem cells present in normal human marrow we have combined cell separation techniques with an assay for cells that initiate sustained hematopoiesis in vitro in the presence of irradiated human marrow adherent cells. These “feeders” were established by subculturing 2- to 6-week-old primary long-term marrow culture adherent layers at a density of 3 x 10(4) irradiated cells per square centimeter. Test “long-term culture (LTC)-initiating cells” were plated on top of the feeders and the cocultures then maintained as standard long-term marrow cultures with half-media changes and removal of half of the nonadherent cells each week. The total number of myeloid, erythroid, and multilineage clonogenic progenitors present after 5 weeks was used to provide a quantitative assessment of the number of LTC-initiating cells originally added. Using this assay, the density, light scatter, and two cell surface antigen properties of LTC- initiating cells have been defined and compared with cells capable of directly forming colonies in methylcellulose. While the majority of the clonogenic cells were found in the high forward light scatter (FLS) “blast” window, LTC-initiating cells had significantly lower FLS properties and in this respect were more similar to lymphocytes. LTC- initiating cells also expressed less HLA-DR antigen than clonogenic cells. The majority of LTC-initiating cells were found in the top 2% of the CD34 (My10) fluorescence profile, whereas clonogenic cells were found throughout the top 5% of the CD34 fluorescence profile. By combining low FLS, low orthogonal light scatter (OLS), low HLA-DR expression, and high CD34 expression, a population could be obtained that was enriched for LTC-initiating cells approximately 800-fold over unseparated marrow. This population contains only 0.06% of the marrow cells and 2% of the total clonogenic cells, but retains 50% to 60% of the LTC-initiating cells present in the original marrow. The ability to purify these two populations independently shows that the LTC and clonogenic assays identify distinct, although not necessarily nonoverlapping cell types in human marrow. Since clonogenic cells are derived from LTC-initiating cells, the LTC assay clearly detects a more primitive population. The availability of a simple approach that allows the purification of such cells by three orders of magnitude in high yield should be useful for the investigation of early events in hematopoiesis as well as for the definitive isolation of human hematopoietic stem cells with long-term in vivo repopulating potential.

Interleukin 6 receptor expression by human cord blood‐ or peripheral blood‐derived primitive haematopoietic progenitors implies acquisition of different functional properties

“…Long-term bone marrow culture (LTBMC). Ten thousand sorted CD34 1 IL-6R 1 or IL-6R 2 cells were plated on allogeneic irradiated bone marrow (BM) stromal layers cultured in stromal medium [a-medium, 10% FCS, 10% horse serum (Bio Whittaker, Walkersville, MD, USA), 5 Â 10 25 mol/l 2-ME and 10 26 mol/l hydrocortisone (Japan Upjohn, Tokyo, Japan)], according to the reported method with minor modifications (Sutherland et al, 1989;Gunji et al, 1993;Sakabe et al, 1998).…”

“…The significance of differences between mean values was determined using the two-tailed Student's t-test. The frequency of LTC-ICs in the initial cell population was calculated by Poisson statistics, as reported elsewhere (Taswell, 1981;Sutherland et al, 1989).…”

“…Moreover, telomerase expression by subpopulations of CB-and PB-derived CD34 1 cells was investigated using a sensitive polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) (Ohyashiki et al, 1996a, b). Then the pattern of telomerase expression in each subpopulation was compared with the distribution of LTC-ICs and ELTC-ICs, which represent primitive haematopoietic progenitor cells in vitro (Sutherland et al, 1989;Hao et al, 1996), in relation to the IL-6R expression by these primitive stem/progenitor cells. Our results suggested that telomerase activity was differently expressed or upregulated by CB-and PB-derived CD34 1 IL-6R 1 or IL-6R 2 cells in response to cytokine stimulation and that IL-6R expression was differently regulated between these two populations of stem cells.…”

Detection and clinical significance of human acute myeloid leukaemia progenitors capable of long‐term proliferation in vitro

“…Normal haematopoiesis occurs as the result of the ordered proliferation and differentiation of a hierarchy of progenitors, which have been categorized by their functional potential as measured by in vitro and in vivo assays (Lord & Spooncer, 1986;Andrews et al, 1989;Sutherland et al, 1989a;Jones et al, 1990;Larochelle et al, 1996). In acute myeloid leukaemia (AML), only a subset of leukaemic cells are capable of proliferation in methylcellulose (Moore et al, 1973;Minden et al, 1979), suggesting that AML blasts are heterogeneous in their proliferative potential, at least in vitro.…”

“…These studies have suggested that such cells exist in AML, and that they are phenotypically distinguishable from the majority of blasts and AML colony-forming cells (Lapidot et al, 1994;Sutherland et al, 1996;Ailles et al, 1997;Blair et al, 1997Blair et al, , 1998Bonnet & Dick, 1997;Blair & Sutherland, 2000). As longterm (5±8 week) growth in in vitro assays is important to detect the most primitive normal progenitors, (Sutherland et al, 1989a(Sutherland et al, , 1990Hao et al, 1996), we reasoned that longterm in vitro assays may also detect primitive AML progenitors. Initially, we evaluated 2±8 week production of colony-forming units (CFU) from growth factor containing suspension cultures (SC) in the absence of stroma as a quantitative assay for the more primitive AML progenitors.…”