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

Catherine Verfaillie, Karin Blakolmer, Philip B Mc Glave

Research output: Contribution to journalArticle

217 Scopus citations

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 longterm 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 shortterm 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 ≤ 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 Lin34+DR cells yielded significantly greater numbers of CFC than LTBMC initiated with plated Lin 34+DR+ cells (p ≤ 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 ≤ 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 ≤ 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. In contrast, LTBMC initiated with either plated or panned Lin34+DR cells generated high numbers of CFC for a minimum of 12 wk. These studies present the first evidence that further purification of 34+/DR- cells using an additional immunomagnetic depletion of committed myeloid and lymphoid progenitors results in a Lin 34+DR population that is significantly enriched (424- fold) for primitive progenitors capable ofinitiating and sustaining growth of committed myeloid progenitors in LTBMC for at least 12 wk. These studies also provide the first evidence that primitive progenitors capable of adhering avidly to irradiated bone marrow-derived stroma when panned for 2h are present exclusively in the Lin 34+DR population. In contrast, Lin 34+DR cells, which are committed clonogenic precursors, do not exhibit the ability to adhere to irradiated stroma. Further study of these cell populations will allow detailed analysis of interactions between primitive hematopoiesic stem cells and the bone marrow microenvironment.

Original languageEnglish (US)
Pages (from-to)509-520
Number of pages12
JournalJournal of Experimental Medicine
Volume172
Issue number2
DOIs
StatePublished - Aug 1 1990

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