Growth Factor Induction of Cytosolic Protein Tyrosine Kinase Activity in Human Haemopoietic Progenitor Cells Isolated by Flow Cytometry

Abstract

We employed a highly sensitive method to assay protein tyrosine kinase activity in extracts of subpopulations of CD34+ bone marrow progenitor cells isolated by fluorescence activated cell sorting in an attempt to better define how growth-factor induction of enzymatic activity relates to progenitor cell maturation. FACS analysis confirmed that, under the conditions employed, essentially all of the CD34+ cells in adult human marrow that lacked the CD38 antigen were devoid of the myeloid maturation marker CD33 as well as the lineage antigens: CD10, 13, 14, 15, 16, 19, 71 and glycophorin A. A variable portion (50-90%) of these CD34+, CD38- progenitor cells expressed HLA-DR. CD34+, CD38- cells that did not express HLA-DR were found to lack detectable levels of either membrane or cytosolic tyrosine kinase activity. HLA-DR+ progenitor cells that lacked CD38 possessed elevated levels of cytosolic tyrosine kinase activity but only low levels of plasma membrane activity. In contrast, CD34+ cells that expressed CD38 (and HLA-DR) possessed high levels of membrane-associated tyrosine kinase activity. A cocktail of haemopoietic growth factors that included IL-3, IL-6 and stem cell factor effectively induced tyrosine kinase activity in CD34+, CD38-, HLA-DR- progenitor cells. Growth factor induction of tyrosine kinase activity in these cells was not inhibited by actinomycin D or cyclohexamide. Most of the tyrosine kinase activity induced by these growth factors was recovered from the cytosolic fraction of disrupted cells. Thus, induction of cytosolic tyrosine kinase activity is an early event in the response of uncommitted haemopoietic cells to haemopoietic growth factors. Subsequent activation of membrane tyrosine kinases may initiate key transduction processes as these cells begin to differentiate.

Life and Death of Cardiac Stem Cells

“…This can only be obtained by fixation of cells to make them permeable for the detection of intracellular components. 42,43 Similar problems exist with immunolabeling. With either protocol, cell viability is lost, precluding further study.…”

Lineage Commitment of HLA-DR/CD38-Defined Progenitor Cell Subpopulations in Bone Marrow and Mobilized Peripheral Blood Assessed by Four-Color Immunofluorescence

“…Studies of the growth development. Nevertheless, many studies of the funccharacteristics (12)(13)(14)(15)(16)(17)(18), transplantation potential (19)(20), tional potential of hematopoietic stem cells are underreplating efficiency (21)(22)(23)(24)(25), and biochemical character-taken with CD34 cells selected on the basis of the degree istics (26) of phenotypically defined progenitor cell subof expression of either CD38 or HLA-DR. populations have facilitated definition of early pathways In previous work, three-color flow cytometric analyof maturation and differentiation. These studies indicate sis has been used to assess the expression of various antithat the earliest progenitor cells in adult human bone margens associated with lineage commitment on hematopoirow lack the CD38 differentiation antigen and stain dimly etic cells expressing various levels of CD38…”

“…(1,4,11,(27)(28)(29)(30)(31)(32)(33). In addition, several groups of investigators have comparatively assessed the biochemical, functional, and proliferative characteristics of HLA-DRdim and HLA-DRbright hematopoietic cells (26,34,35). Rustin et al directly compared the growth characteristics of CD34+/CD38-and CD34+/HLA-DR-cells isolated from adult bone marrow and found substantial quantitative differences between the two subpopulations with regard to the content of primitive progenitors (32).…”

Cardiac Stem Cells and Mechanisms of Myocardial Regeneration

“…FACS analysis is extremely helpful in separating stem cells in various categories according to the expression of one or more surface epitopes (101, 146, 481), but the distinction between undifferentiated and early committed cells necessitates caution. Sophisticated analyses of nuclear and cytoplasmic proteins can be performed by FACS, but they require fixation of the cells to make them permeable and amenable to the detection of intracellular components (174,192,255). However, the viability of the cells is lost precluding any subsequent in vivo or in vitro study.…”