Characterization of the Human Hematopoietic Niche During Ontogeny.

Abstract 3626

Poster Board III-562

Studies thus far have shown that there are at least two anatomically and physiologically distinct hematopoietic niches within the bone marrow (BM); the osteoblastic and the vascular. The former is thought to provide the appropriate support for quiescent hematopoietic stem cells (HSC), while the latter allows/induces expansion of the HSC pool. However, it is not entirely known which subpopulations of HSC interact with each niche, and what physiological role each niche type plays in the regulation of stem cell hierarchy and function. We hypothesized that the developmental process whereby the fetal marrow acquires the ability to support hematopoiesis can be used as a model for understanding both the interactions that occur between HSC and the cells comprising the marrow niches, and the role of these niche elements in the initiation/maintenance of hematopoiesis. To this end, human fetal bones between 10- 22 weeks of gestation (gw) were analyzed by flow cytometry and confocal microscopy to identify and characterize stromal/vascular/osteoblastic and hematopoietic elements. Since the development of the vascular bed has been reported to occur in humans between 9-10.5gw, we started the analysis of the human fetal bones at this time point (n=3). CD44 was the only marker widely expressed at this time point, suggesting that these bone rudiments were in an earlier (cartilaginous) stage of development. At 12gw (n=5), 47±4% of the cells isolated from the long bones were CD34+, but less than 1% of these cells were positive for CD45. Further characterization of the CD45⁻CD34⁺ population showed that 74±5.4% of these cells were CD106+; 65±7.2% were CD102+; 10±0.5% were CD31+; and 6.7±2.1 % KDR+. These data suggest that prior to the onset of hematopoiesis, a significant percentage of the cells in the BM are part of the vascular niche, and have an endothelial cell phenotype. Furthermore, the existence of a population of cells with a CD34⁺CD31⁺KDR⁺CD45⁻ phenotype may indicate that, during this stage of development, a hemangioblast-like cell exists in the emergent BM. In addition, the existence of a large mesenchymal/stromal cell population was also found, with 21±2.9% of the cells expressing Stro-1+. CD44 was still widely expressed (55±7.8%) at this time point. Of particular note is the discovery of a cell population in which CD44+ cells co-expressed CD34 in the absence of CD45 (8±1.8%), and another in which CD34+ cells were co-expressing Stro-1 (9±1 %), suggesting the possibility that a common ancestor may exist for these cells. At 16gw (n=6) the overall percentage of CD34+ cells decreased to 15±3.1%, and the majority (72±7%) of the cells harvested from the long bones expressed CD45, showing the change to a predominantly hematopoietic marrow. Furthermore, 12±4.2% of the cells had a CD45⁺CD34⁺ phenotype consistent with that of HSC. At this time point, only 2.38±1% of the cells were CD34+CD106+; 3.5±1.5% were CD34+CD102+; and 1.34±0.8% were CD34+CD31+. In addition, the CD44+CD45- population decreased to only 20±1.2%. At 22gw (n=6), a similar flow cytometric profile was found except that the CD45+CD34+ population had decreased to 6±1.3%. Since phenotypic analysis showed a decrease in the percentage of BM stromal cell precursors from 12 to 22gw, we performed CFU-F assays and found that the frequency of CFU-F in 12gw BM was 0.34-0.45%, the highest found at any time point. At 16gw, the frequency of CFU-F was 0.23-0.25%, and at 22gw, it had decreased to 0.1-0.16%. Nevertheless, even this low level at 22gw was still higher than that of adult BM (0.001-0.02%). Thus, the results from CFU-F assays correlate well with the flow cytometry/confocal microscopy data showing that there is a decrease in mesenchymal/stromal cell precursor frequency/potential as the BM niches mature. To investigate the ability of the stromal layers of different gestational ages to interact with/retain HSC, adhesion assays (n=3) were performed with cord blood (CB) and adult BM-derived CD34+ cells. These studies showed that CB CD34+ cells adhered at similar levels to all of the fetal stromal layers, while adult BM-derived CD34+ cells only adhered efficiently to the 22gw stromal layers. Further studies addressing the cellular interactions that occur within each of the niches at the phenotypic, functional, and transcriptional levels are currently underway.