Distinct hematopoietic progenitor compartments are delineated by the expression of aldehyde dehydrogenase and CD34

Abstract

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSC(lo)) and high expression of aldehyde dehydrogenase (ALDH(br)). Many SSC(lo) ALDH(br) cells coexpress CD34; however, other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets, purified ALDH(br) CD34+, ALDH(neg) CD34+, and ALDH(br) CD34(neg) UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDH(br) CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison, ALDH(neg) CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDH(br) CD34(neg) cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development.

Figure 1.

ALDH^br CD34⁺, ALDH^neg CD34⁺, and ALDH^br CD34^neg cells derived from UCB. UCB cells were labeled with BAAA, as described in “Materials and methods.” (A) Background fluorescence was established in the presence of DEAB. (B,E) SSC^lo ALDH^neg and SSC^lo ALDH^br cell fractions were defined. (C-D) Both cell fractions contained CD34⁺ cells, although the SSC^lo ALDH^br fraction was highly enriched for CD34⁺ cells, to include CD34⁺ CD38^neg cells (n = 8) (D). In most developmental studies, cells expressing lineage-specific antigens were excluded (n = 25). (F) CD34⁺ lin^neg cells were isolated from the SSC^lo ALDH^neg. (G) CD34⁺ lin^neg and CD34^neg lin^neg cells were isolated from SSC^lo ALDH^br UCB (H-M). To ensure purity, each cell fraction was sorted twice. Representative reanalyses depict twice-purified lin^neg SSC^lo ALDH^neg CD34⁺ cells (H,K), lin^neg SSC^lo ALDH^br CD34^neg cells (I,L), and lin^neg SSC^lo ALDH^br CD34⁺ cells (J,M).

Figure 2.

Human hematopoietic engraftment to NOD/SCID bone marrow. NOD/SCID mice received 1000 or 3000 transplanted SSC^lo ALDH^br CD34⁺ cells each. Comparisons were drawn to mice that received transplanted SSC^lo ALDH^neg CD34⁺ or SSC^lo ALDH^br CD34^neg cells (3000 or 10 000 cells per transplant). Transplantation controls included mice that underwent transplantation with 10 000 or 30 000 lin^neg CD34⁺ cells. (A) Human hematopoietic engraftment was assessed by the presence of CD45⁺ ly5^neg (murine CD45^neg) cells in NOD/SCID bone marrow. (B) To confirm engraftment, some marrows were enriched for human cells by depletion of lineage-committed murine cells using density-dependent negative selection. Percentages indicate the % CD45⁺ cells. (C) IgG indicates immunoglobulin G. Human hematopoietic engraftment was determined for (D) B-lymphoid, (E) myeloid, and (F) CD34⁺ progenitor cells. (A-F) Analyses performed at 20 to 21 weeks after transplantation of mice that underwent transplantation with 3000 SSC^lo ALDH^br CD34⁺ cells (A-B) or with 3000 lin^neg SSC^lo ALDH^br CD34⁺ cells (C-F). The percentage of long-term human hematopoietic engraftment for each cell fraction is depicted in panel G (as labeled; 18 to 21 weeks after transplantation; n = 6 UCB). The percentage of short-term human hematopoietic engraftment for each cell fraction is depicted in panel H (as labeled; 6 to 7 weeks after transplantation; n = 3 UCB). Each data point represents the percentage of human hematopoietic engraftment within a single mouse.

Figure 3.

NK development in secondary LTC assays. Secondary NK cultures were established from primary LTCs that had been originally initiated with 50 to 500 cells per culture (n = 6). (A,C) Secondary cultures were analyzed for their content of CD56⁺ NK cells and CD13⁺ myeloid cells. (B,D) To represent the entire data set, the relative percentages of myeloid and lymphoid progeny were plotted for each culture, where each data point represents the average of duplicate cultures. (A-B) High percentages of CD56⁺ cells (more than 50%) were present in secondary cultures derived from primary LTCs originally established with lin^neg SSC^lo ALDH^br CD34⁺ cells. The single exception was from primary cultures that had been initiated with 50 cells/well. (C-D) In contrast, of the cultures established with lin^neg SSC^lo ALDH^neg CD34⁺ cells, 2 secondary cultures gave rise to predominantly CD13⁺ myeloid progeny, and 1 secondary culture had no evident growth.

Figure 4.

Cell development in short-term culture assays. ALDH^neg CD34⁺, ALDH^br CD34⁺, and ALDH^br CD34^neg cells were purified from lin^neg SSC^lo UCB, as depicted in Figure 1. Two hundred to 1000 purified cells were cultured on STO fibroblasts in the presence of IL-3, IL-7, and IL-15 (n = 10). Cultures initiated with ALDH^neg CD34⁺ cells contained higher percentages of CD56⁺ lymphoid progeny than did the ALDH^br CD34⁺ cells (compare panels A and B; see Table 4). Similarly, the ALDH^neg CD34⁺ cell fraction yielded lower percentages of CD13⁺ myeloid progeny (compare panels D and E). Only 2 cultures initiated with ALDH^br CD34^neg cells had growth significant enough to evaluate lineage development, and these gave rise to CD56⁺ progeny (C,F). The CD56⁺ progeny of ALDH^neg CD34⁺ (G-I) and ALDH^br CD34⁺ cells (data not shown) expressed other antigens consistent with NK cells. (J) Relative output of lymphoid cells was higher in cultures initiated with ALDH^neg CD34⁺ cells when compared with paired cultures initiated with ALDH^br CD34⁺ cells. (K) Conversely, the relative cell output of myeloid cells was higher in cultures initiated with ALDH^br CD34⁺ cells when compared with paired cultures initiated with ALDH^neg CD34⁺ cells. Estimations for relative cell output are described in “Materials and methods.” (J-K) Each data point represents an average from duplicate cultures.

Figure 5.

Expression of ALDH and CD34 during early hematopoietic development. Our studies suggested that ALDH^br CD34⁺ cells contained high frequencies of primitive multilineage progenitors, as monitored in NOD/SCID repopulation assays and in LTC. CD34⁺ cells contained short-term progenitors for myeloid and lymphoid compartments; however, the expression of ALDH was maintained most strongly by myeloid progenitors. In contrast, ALDH^neg CD34⁺ cells enriched short-term lymphoid progenitors, suggesting that ALDH may have a diminishing role during early lymphopoiesis. Finally, a population of ALDH^br CD34^neg cells was not efficiently supported within any of the developmental assays used. Based on recent reports, this cell may represent a progenitor for the CD34⁺ compartment.