Comparison of gene expression in CD34+ cells from bone marrow and G-CSF-mobilized peripheral blood by high-density oligonucleotide array analysis

Abstract The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.

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Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Blood ◽

10.1182/blood.v106.11.5195.5195 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5195-5195

Author(s):

Lulu Lu ◽

Yongping Song ◽

Baogen Ma ◽

Xiongpeng Zhu ◽

Xudong Wei ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Cord Blood ◽

Peripheral Blood ◽

Lymphocyte Subsets ◽

Color Flow ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Mobilized Peripheral Blood

Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P <0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

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Apoptosis-Independent Activation of Caspase-3 in CD34–Positive (CD34+) Mobilized Cells.

Blood ◽

10.1182/blood.v112.11.2305.2305 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2305-2305

Author(s):

Karine Augeul-Meunier ◽

Carine Crampé ◽

Philippe Farce ◽

Christiane Mounier ◽

Denis Guyotat ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Caspase 3 ◽

Normal Bone Marrow ◽

Hematopoietic Stem ◽

Normal Bone ◽

Peripheral Blood Cd34 ◽

Cd34 Cells ◽

Significant Difference ◽

Mobilized Peripheral Blood

Abstract G-CSF mobilized peripheral blood CD34+ cells are now the preferred and major source of hematopoietic stem and progenitor cells harvested for both autologous and allogeneic transplantation. Several mechanisms, like SDF-1/CXCR4 interactions or degradation of adhesion molecules by proteolytic environnement, are involved in the mobilization process. However this phenomenon is still partially understood. Gene expression analysis has identified an overexpression of the caspase-3 gene in CD34+ mobilized cells, compared to CD34+ from normal bone marrow. Caspase-3 is the main effector of the terminal phase of apoptosis. However recent studies have provided evidence of its implication in non apoptotic cellular processes, such as differentiation, migration and cytoskeleton modelling. We evaluated by multicolour flow cytometry the expression of activated caspase-3 in G-CSF mobilized CD34+/CD45+ cells from blood (n=16), and from apheresis products (n=10). CD34+/CD45+ cells from normal bone marrow (n=4) served as control. Caspase-3 activity on fluorescent substrate (PhiPhiLux method) and apoptosis (Annexin V assay) were also evaluated. Finally we analysed the expression of anti apoptotic proteins Bcl-2, Bcl-Xl, and of Heat Shock Proteins HSP27, HSP70 and HSP90 in the same cell population. There was no significant difference for apoptosis between mobilized and bone marrow CD34+ cells (26% versus 33% apoptotic cells). Activated caspase-3 levels were significantly higher in mobilized CD34+ cells (mean fluorescence intensity 3.64 fold higher). This was consistent with cleavage of caspase-3 substrate observed in mobilized cells, but not in bone marrow CD34+ cells. An increased expression of HSP90 (of which caspase-3 is a client protein) was observed in peripheral CD34+ cells, but there was no variation of BCl-2 and Bcl-Xl expression. Our results show an activation of caspase-3 in the mobilized peripheral blood CD34+ cells, which appears to be independent of apoptosis induction. The role of this activation and possible control by HSPs warrants further analysis to establish its relationship with mobilization mechanisms.

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