Immunological study of in vitro maturation of human megakaryocytes

Abstract

Human megakarocyte colonies were grown from the bone marrow in plasma clot or methyl cellulose cultures. Maturation of the megakaryocytic cells was sequentially studied from day 5 to day 16 of culture by fluorescent labelling with a panel of monoclonal and polyclonal antibodies against different platelet glycoproteins (Gp), P1 A1 antigen, factor VIII RAg platelet factor 4 (PF 4), fibrinogen and platelet-derived growth factor (PDGF). Expression of Gp Ib was also studied by immunogold technique at electron microscopy. The first cells identifiable by these antibodies were found at day 5 of culture. They had the size of a lymphocyte. These small megakaryocyte precursors already expressed all the platelet antigens, HLA-DR and transferrin receptors and were devoid of erythroid or myeloid markers. Among the platelet antigens, Gp IIIa was the most sensitive marker for the identification of these precursors. However, double-fluorescent labelling demonstrated that the different platelet markers were coexpressed in a large majority of cells. Interestingly, cytoplasmic markers demonstrated that these small megakaryocyte precursors were themselves heterogenous by morphological criteria. During maturation, expression of Gps, particularly of Gp Ib, increased while the labelling pattern of anti factor VIII RAg and anti PF 4 antibodies switched from diffuse to granular staining. PDGF could also be detected in the megakaryocytes grown in culture.

Serum proteome profiling detects myelodysplastic syndromes and identifies CXC chemokine ligands 4 and 7 as markers for advanced disease

“…CXCL4 is already expressed in maturating megakaryocytes (37). Disturbed maturation of megakaryocytes and ␣-granule defects in platelets are frequent features of MDS (38)(39)(40) and might be reflected by either low CXCL4 expression or a release deficiency in MDS platelets.…”

CD34+/CD41a+ cells best predict platelet recovery after autologous peripheral blood stem cell transplantation

“…Glycoproteins IIb and IIIa (CD41a, CD41b) are reported to be expressed first during megakaryocytic differentiation, while glycoprotein Ib (CD42a, CD42b) appears to be a later marker. 26,[29][30][31] In conclusion, this is the first report to examine the correlation between the numbers of CFU-MK and CD34 + /CD41a + cells infused and the time to platelet recovery, as well as the correlation between the number of CFU-MK and CD34 + /CD41a + cells. The threshold for rapid platelet recovery was 1 ϫ 10 5 /kg CD34 + /CD41a + cells or 1 ϫ 10 5 /kg CFU-MK.…”

Expression of a Surface Antigen (MA6) by Peripheral Blood CD34+Cells Is Correlated with Improved Platelet Engraftment and May Explain Delayed Platelet Engraftment Following Cord Blood Transplantation

“…While clinical data suggest that the time to platelet engraftment is correlated with the dose of CD34 + hematopoietic progenitor cells transplanted [1,2], other measures of hematopoietic progenitors contained within the transplanted tissue have also shown some correlation [3][4][5][6][7][8]. Lineage-committed MK progenitors can be characterized by in vitro colony-forming assays and their generation of burst-and colony-forming units (BFU-MK and CFU-MK, respectively) [9][10][11][12]. However, these in vitro measures of MK progenitors are of limited value.…”