Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation

Abstract

Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.

Quantitation and characterization of human megakaryocyte colony‐forming cells using a standardized serum‐free agarose assay

“…The fact that the surface phenotype of CFC-Mk was found to resemble that of BFU-E more closely than that of CFC-GM is also suggestive of this concept. However, it is also notable that many CFC-Mk were found to be distinct from other types of CFC (erythroid, granulopoietic or multipotent) in their expression of CD41, confirming the observations of Debili et al (1992) and Nichol et al (1994). Thus, isolation of cells with a CD34 þ CD45RA ¹ CD41 þ phenotype allows a high and selective enrichment of a subpopulation of CFC-Mk that constitute approximately half of all the CFC-Mk present in normal human marrow.…”

Ultrastructural analysis of the distribution of the vitronectin receptor (αvβ3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the α‐granule membrane

“…Increases in GP IIb-IIIa content during MK maturation and decreases in membrane antigens such as CD34 and CD4 are already well characterized (Debili et al, 1992;Dolzhanskiy et al, 1996). Notwithstanding, it should be emphasized that our studies do not as yet distinguish between the surface and total pool of VnR in these cells.…”

Endocytosed factor V is trafficked to CD42b+ proplatelet extensions during differentiation of human umbilical cord blood‐derived megakaryocytes

“…On Day 12, a population of CD41 − CD42b − cells capable of factor V endocytosis was also observed, and approximately 12% of the cells that endocytosed factor V also expressed CD34. Expression of CD34 by immature megakaryocytes derived ex vivo from human bone marrow (Debili et al, 1992;Norol et al, 1998) or mobilized blood cells (Debili et al, 2001;Norol et al, 1998) has been described previously. CD41 + CD42b + megakaryocytes transition from a mitotic cell cycle to endomitosis, and CD34 disappears during endomitosis (Cramer Borde & Vainchencker, 2013).…”