Abstract
It is well established that in adults, long-term repopulating hematopoietic stem cells (HSC) are mitotically quiescent cells that reside in specialized bone marrow (BM) niches that maintain the dormancy of HSC. Our laboratory demonstrated that the engraftment potential of human HSC (CD34+ cells) from BM and mobilized peripheral blood (MPB) is restricted to cells in the G0 phase of cell cycle but that in the case of umbilical cord blood (UCB) -derived CD34+ cells, cell cycle status is not a determining factor in the ability of these cells to engraft and sustain hematopoiesis. We used this distinct in vivo behavior of CD34+ cells from these tissues to identify genes associated with the engraftment potential of human HSC. CD34+ cells from BM, MPB, and UCB were fractionated into G0 and G1 phases of cell cycle and subjected in parallel to microarray and proteomic analyses. A total of 484 target genes were identified to be associated with engraftment potential of HSC. System biology modeling indicated that the top four signaling pathways associated with these genes are Integrin signaling, p53 signaling, cytotoxic T lymphocyte-mediated apoptosis, and Myc mediated apoptosis signaling. Our data suggest that a continuum of functions of hematopoietic cells directly associated with cell cycle progression may play a major role in governing the engraftment potential of stem cells. While proteomic analysis identified a total of 646 proteins in analyzed samples, a very limited overlap between genomic and proteomic data was observed. These data provide a new insight into the genetic control of engraftment of human HSC from distinct tissues and suggest that mitotic quiescence may not be the requisite characteristic of engrafting stem cells, but instead may be the physiologic status conducive to the expression of genetic elements favoring engraftment.
Highlights
Life-long maintenance of the hematopoietic system is sustained by highly specialized hematopoietic stem cells (HSC) [1,2,3]
To identify genes differentially expressed between quiescent (G0) and cycling (G1) cells, we examined the global mRNA expression profiles of sorted and qRT-PCR verified G0 and G1 cells from bone marrow (BM), mobilized peripheral blood (MPB), and umbilical cord blood (UCB)
In case of MPB, 1705 genes were differentially expressed; 840 genes were upregulated in G0 cells and 865 genes were upregulated in G1 cells
Summary
Life-long maintenance of the hematopoietic system is sustained by highly specialized hematopoietic stem cells (HSC) [1,2,3]. When human mobilized peripheral blood (MPB) and BM CD34+ cells in G0 or G1 phase of cell cycle were examined, only those in G0 were capable of in-vivo long-term multilineage engraftment [4] Both mitotically quiescent as well as cycling HSC from umbilical cord blood (UCB), fetal liver and human fetal bone marrow [5,6] retained their ability to engraft in NOD/SCID mice. These studies established that in adult tissues, a hierarchical order of hematopoietic potential can be assembled based on the mitotic status of HSC whereby only cells in G0 engraft. The availability of six groups of human CD34+ cells from three distinct tissues with previously established functional capabilities allowed us to carefully investigate the genetic control of pathways implicated in engraftment and to examine the degree of homogeneity or heterogeneity between functionally similar (all G0 groups of cells and G1 cells from UCB) but phenotypically different (G0 and G1 cells from UCB) groups of cells in the absence of the impact of cell cycle regulatory genes
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