Abstract

The bone marrow (BM) repopulating potential of hematopoietic stem cells (HSCs) is directly related to the cell cycle status of these cells. In general, only mitotically quiescent HSCs retain the ability to engraft and sustain long-term multilineage reconstitution in conditioned recipients. In a series of studies, our laboratory previously examined the effect of cell cycle status on the engraftment potential of human HSC from three different hematopoietic tissues. Only CD34+ cells in G0 phase of cell cycle (G0CD34+) from adult human BM or mobilized peripheral blood (MPB) engrafted successfully in conditioned NOD/SCID mice whereas those in G1 phase of cell cycle (G1CD34+) failed to do so. In contrast, both G0CD34+ and G1CD34+ cells from cord blood (CB) engrafted effectively. In the present study, we used the distinct in vivo behavior of these groups of adult and neonatal cells as the basis for genotypic and proteomic analyses in which it was possible to align multiple profiles of functional and non-functional HSC and therefore derive a genetic and protein fingerprint that may be associated with Engraftment potential of human stem cells. Human CD34+ cells from BM, MPB, and CB were sorted into G0 and G1 phases of cell cycle and the cell cycle status of each isolated fraction was further confirmed by the expression or lack thereof of Ki67 by qRT-PCR. Agilent Whole Human Genome Oligo Microarrays were used for genotyping (three independent samples from each tissue for a total of 18 groups) and a Linear Mixed Effect Model was used to identify differentially expressed genes, with at least a two-fold increase in expression and false discovery rate <0.05. An LC-MS/MS proteomic analysis of the same 18 groups of cells in addition to 6 others (total of four independent samples from each tissue) was also conducted in parallel. Differential expression of cellular proteins was calculated using a proprietary algorithm. A total of 190 genes were highly expressed in engrafting cells (all three groups of G0CD34+ cells and CB-derived G1CD34+ cells) whereas 1039 genes were highly expressed in non-engrafting cells (BM- and MPB-derived G1CD34+ cells). Out of the 190 differentially regulated genes in engrafting cells, 161 genes have a known function. Of these, 84 are present in the nucleus and 23 are transcription regulators including ARNTL, BCL6B, DMTF1, HES1, HLF, IFI16, and ZNF326. System Biology modeling indicated that the top four signaling pathways associated with these genes are Wnt signaling, PPARα/RXRα activation, Amyloid processing, and IGF1 signaling. Of the 1039 differentially regulated genes in non-engrafting cells, 273 are present in the nucleus and 69 are transcription regulators including CALR, CyclinE1, CEBPB, CIITA, MYC, MAPK1, and NOTCH4. System Biology modeling implicated these genes in multiple signaling pathways with the top four being the antigen presentation pathway, role of BRCA1 in DNA damage response, IL4 signaling, and the G1/S checkpoint regulation. However, proteomic analysis identified a total of 646 proteins that were detected in the lysates of all six groups of cells. Of these, 70 proteins had a significant differential expression with less than 5% false discovery rate between paired groups. The genes of only 9 proteins were differentially expressed in either the engrafting or non-engrafting cells including TPT1 (in the engrafting group) and ALDOA, MPO, TUBB, CALR, ACTB, ACTG1, PRTN3, ANXA1 (in the non-engrafting group). Functional studies aimed at discerning the roles of these proteins in stem cell function are underway. These studies demonstrate that the overlap between genomic and proteomic analysis of the same groups of engrafting and non-engrafting hematopoietic cells is rather limited but that simultaneous analysis with both protocols may identify unique modulators of stem cell function. Furthermore, protein expression analysis may be more useful in identifying pathways, the activation of which results in the loss of stem cell function since these pathways remain inactive in the mitotically and metabolically inactive engrafting cells.

Disclosures: No relevant conflicts of interest to declare.

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