Functional and kinetic characterization of granulocyte colony‐stimulating factor‐primed CD34⁻ human stem cells

Abstract

Summary.We assessed the functional properties and the kinetic status in vitro, and the engraftment potential in vivo of human haematopoietic stem cells according to the expression of CD34 antigen. Lin ) CD34 ) and Lin ) CD34 + cells were isolated from granulocyte colony-stimulating factor-primed peripheral blood (PB) cells of healthy donors. The CD34 ) cell fraction did not contain either clonogenic cells in semisolid culture or long-term culture initiating cells (LTC-IC). However, stroma-dependent liquid cultures and cytokines induced CD34 expression on a minority of stem cells, acquisition of clonogenic capacity and generation of LTC-IC. Significantly higher percentages of quiescent G 0 cells and lower percentages of cycling G 1 cells were found in Lin ) CD34 ) cells when compared with Lin ) CD34 + cells. Kinetic quiescence of Lin ) CD34 ) cells was associated with a significantly higher expression of the negative regulators of the cell cycle, p27 Kip1 and p21 cip1/waf1 . Cytokine-mediated induction of CD34, in vitro, resulted in cycling of stem cells and downregulation of p27. There was a higher rate of human long-term engraftment in immunocompromised non-obese diabetic (NOD)/recombination activating gene 1 null and NOD/severe combined immunodeficient-b 2 microglobulin null mice injected with CD34 + cells. Thus, our study indicated that CD34 expression on human PB stem cells was associated with haematopoietic activity, cell-cycle recruitment and downregulation of p27 Kip1 in vitro and higher engraftment capacity in vivo.

Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia

“…5,15,16 Previous studies suggested that circulating progenitor cells differ from their BM counterparts with respect to cell-cycle characteristics and proliferative response to cytokines, [17][18][19][20] which is also reflected in our finding of differential ERK phosphorylation and may reflect effects mediated by the microenvironment.…”

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

“…[4][5][6]15 However, the CD34 neg fraction suffers from a profound impairment in migration after IV transplantation compared with the CD34 pos fraction. 5,7,8,11,13,[49][50][51][52] Our flow cytometric and western blot data revealed a more pronounced E-selL activity on the CD34 pos subset of the Lin neg CD38 neg fraction than on the CD34 neg subset, suggesting that the enhanced migratory ability of CD34 pos cells is due to its possession of a specific set of functional E-selLs, required for proper migration and homing of HSPCs. In agreement with these results, a previous study illustrated that BM and fetal liver-derived CD34 pos subsets rolled over immobilized E-selectin with higher efficiency than CD34 neg subsets.…”

The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration

“…The expression of human CD45 antigen in the BM and PB of mice that underwent transplantation was confirmed by reverse-transcription-polymerase chain reaction (RT-PCR) and Southern blotting as previously reported. 25 For comparative homing assessment in the BM and PB samples, CD34 ϩ CD45 ϩ cells were further evaluated for their relative content in PKH67-and PKH26-positive cells (ie, cells treated with UTP or mock medium, respectively).…”

“…Genes showing a detection called "A" (absent) in all samples were excluded. 25 The generated lists and, independently, the GCOS-generated absolute analysis data were uploaded onto GeneSpring software version 7.2 (Silicon Genetics, Redwood City, CA). To normalize data, each measurement was divided for the 50th percentile of all signals in that sample.…”