Functional, phenotypic and molecular characterization of cytokine low-responding circulating CD34+ haemopoietic progenitors.
Circulating CD34+ cell populations characterized by a low rate (up to five) or high rate (more than five) of cell divisions were isolated from 8 d cultures in the presence of stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), Flt3 ligand and Peg-rHu megakaryocyte growth and development factor (Peg-rHuMGDF) using the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometric cell sorting. Phenotypic characterization of cells which had experienced up to five divisions (CFDA-SEbright) showed a similar surface antigen expression to starting, freshly isolated CD34+ cells. Conversely, cells which experienced more than five divisions (CFDA-SEdim) showed a differentiating behaviour, down-regulating CD34 antigen and acquiring differentiation markers. CFDA-SEbright cells were significantly enriched in CD105 (endoglin) positive precursors as compared to both freshly isolated CD34+ and CFDA-SEdim cells. Functional analysis indicated that CFDA-SEbright had a 3-fold and 10-fold greater cumulative cloning efficiency as compared to freshly isolated CD34+ cells and CFDA-SEdim cells, respectively. CFDA-SEbright cells retained the vast majority of LTC-IC and showed a LTC-IC frequency 2.8-fold higher than that found in freshly isolated CD34+ cells. RT-PCR and Western blot analyses showed significantly higher bcl-2 RNA and protein levels in CFDA-SEbright cells as compared to freshly isolated CD34+ and CFDA-SEdim cells. This study indicates that cytokine low-responding circulating CD34+ cells (CFDA-SEbright cells) represent a functionally, phenotypically and molecularly distinct multipotent progenitor population with biological properties associated with primitive precursors.
Pierelli
L
L
Cattedra di Ematologia, Istituto di Ostetricia e Ginecologia, Universita' Cattolica del Sacro Cuore, Roma, Italy.
Scambia
G
G
Fattorossi
A
A
Bonanno
G
G
Battaglia
A
A
Rumi
C
C
Marone
M
M
Mozzetti
S
S
Rutella
S
S
Menichella
G
G
Romeo
V
V
Mancuso
S
S
Leone
G
G
D015703:Antigens, CD; D018952:Antigens, CD34 / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology*; D000071063:Endoglin; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000145:classification* / Q000187:drug effects / Q000473:pathology; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D010051:Ovarian Neoplasms / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011956:Receptors, Cell Surface; D013388:Succinimides; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D015703:Antigens, CD; D018952:Antigens, CD34; D016207:Cytokines; C064206:ENG protein, human; D000071063:Endoglin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D013388:Succinimides; D019010:Vascular Cell Adhesion Molecule-1