Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma

Abstract

We enriched bone marrow cells from 10 normal individuals for primitive hematopoietic progenitors using a two-step technique, and examined resultant primitive progenitors for their in vitro long-term repopulating capacity and their ability to adhere to irradiated stroma. Immunomagnetic depletion of mature myeloid and lymphoid progenitors resulted in a lineage-negative (Lin-) cell population. Subsequent dual-color fluorescence activated sorting of cells with low forward and vertical light scatter properties, expressing CD34 antigen (34+) and either bearing (DR+) or lacking (DR-) the HLA-DR antigen, resulted in the selection of Lin-34+ DR+ and Lin-34+ DR- cell populations. When the Lin-34+ DR+ cell fraction was cultured in a short-term methylcellulose assay, we demonstrated a 61-fold enrichment for colony forming cells (CFC) compared with undepleted bone marrow mononuclear cells. In contrast to the Lin-34+ DR+ cells, direct culture of Lin-34+ DR- cells in short-term methylcellulose generated significantly less CFC (p less than or equal to 0.001). We then compared the capacity of Lin-34+ DR+ and Lin-34+ DR- cells to generate sustained hematopoiesis when plated in long-term bone marrow culture (LTBMC). When LTBMC were initiated with plated Lin-34+ DR+ cells, we recovered high numbers of CFC during the first week, but observed a rapid decline in the number of harvested CFC over the following weeks. No CFC could be recovered after week 7. In contrast, LTBMC initiated with plated Lin-34+ DR- cells yielded significantly greater numbers of CFC than LTBMC initiated with plated Lin-34+ DR+ cells (p less than or equal to 0.001), and this was sustained for at least 12 wk of culture. The Lin-34+ DR+ population was only 6.6-fold enriched for primitive progenitors capable of initiating and sustaining hematopoiesis in LTBMC when compared with undepleted bone marrow mononuclear cells, while the Lin-34+ DR- population was 424-fold enriched for such primitive progenitors (p less than or equal to 0.001). Finally, we examined the capacity of both Lin-34+ DR+ and Lin-34+ DR- populations to adhere to irradiated allogeneic stroma. We used a previously described "panning method" in which cells are plated onto stroma for 2 h, the nonadherent cells removed by extensive washing, and the adherent fraction maintained under conditions favoring LTBMC growth. When stroma was panned with Lin -34+ DR+ cells, 79 +/- 10% of the cells were recovered in the panning effluent. In contrast, when stroma was panned with Lin -34 + DR- cells, significantly fewer (37 +/- 7%) (p less than or equal to 0.001) cells were recovered in the panning effluent. Unlike LTBMC initiated with plated Lin -34 + DR+ cells, virtually no CFC were recovered from LTBMC initiated with panned Lin -34 + DR+ cells.(ABSTRACT TRUNCATED AT 400 WORDS)