Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells

Abstract

Investigations linking human megakaryocyte development and cell biology have been hindered by an inability to obtain large, relatively pure megakaryocyte cell preparations from in vitro stem cell cultures. We report here that such preparations can be generated from liquid cultures of normal human peripheral blood mononuclear cells stimulated by a serum source of megakaryocyte colony stimulating activity (Meg- CSA, the 0% to 60% ammonium sulfate protein fraction of aplastic canine serum). Adherent-depleted peripheral blood mononuclear cells are suspended at 5 x 10(5) to 10(6) cells/mL in supplemented liquid culture medium, platelet-poor human plasma 20% (vol/vol) and 1 to 2 mg/mL serum Meg-CSA protein. After 12 to 14 days of incubation, megakaryocytes constitute 3.0 +/- 2.9% (mean +/- SD, n = 8) of the unseparated cultured cell population. Megakaryocytes can be enriched by counterflow centrifugal elutriation to a purity of 58 +/- 14% (+/- SD) with a recovery of 13 +/- 7% and a viability of 67 +/- 19%. This algorithm results in the average isolation of approximately 3 x 10(5) enriched megakaryocytes from a 100-mL starting volume of peripheral blood. Cultured megakaryocytes exhibit normal light and ultrastructural morphology by Wright-Giemsa staining and electron microscopic analysis. After a 12-day culture interval, enriched megakaryocyte preparations exhibit morphologic stage distributions that are similar to normal human marrow. Stage distributions move rightward with culture duration indicating partial synchrony of megakaryocyte maturation. On cytospin preparations, megakaryocyte diameter averages 30.2 +/- 1.5 microns and increases with maturation stage. Flow cytometric analyses demonstrate the expression of platelet glycoproteins (GP) Ib and IIb/IIIa by the cultured megakaryocytes. The modal ploidy of the enriched cells at day 12 of culture is 16N and most remaining megakaryocytes are 8N or 32N. Liquid culture of serum Meg-CSA-stimulated human peripheral blood mononuclear cells represents a valuable investigative tool that should permit studies of human megakaryocyte biology that have not been possible in the past.

Enrichment and characterization of peripheral blood‐derived megakaryocyte progenitors that mature in short‐term liquid culture

“…Megakaryocyte culture media consisted of IMDM (GIBCO, Grand Island, NY) supplemented with 1% Glutamine Pen-strep (Irvine Scientific, Santa Ana, CA). Preliminary experiments indicated that 20-30% normal, heparinized, platelet-poor, human AB plasma was required for megakaryocyte development, in agreement with other reports [16][17][18]. By including the following components of a serum-free culture system, the optimal concentration of human plasma could be reduced to 10%: 2-mercaptoethanol ( lo4 M), pyruvic acid (1 10 mg/ml), cholesterol (7.8 mg/ml), adenosine, guanine, cytidine, uridine, thymidine, 2-deoxycytosine, 2-deoxyadenosine, 2-deoxyguanosine (10 mg/ml each, Sigma); human recombinant insulin (10 mg/ml), human transferrin (300 mg/ml), soybean lipids (1%, Boehringer Mannheim, Indianapolis, IN); human recombinant basic fibroblast growth factor ( 2 ng/ml, Genzyme, Cambridge, MA); human recombinant epidermal growth factor (15 ng/ml), platelet-derived growth factor (10 ng/ml, Amgen, Inc., Thousand Oaks, CA).…”

“…Cells fractionated by CCE were plated at 1.25 and 2.5 x lOs/ml in triplicate. The culture consisted of 15% citrated, platelet-poor, human AB plasma in Iscove's modified Dulbecco's medium (IMDM) with additives as described by Mazur et al [16] and incubated 12 days at 3 7 T , 5% C 0 2 in air. Plates were subsequently fixed with methanol:acetone, stained with antibodies to GPIb and GPIIb/IIIa (Biodesign, Kennebunkport, ME) and a secondary goat anti-mouse fluorescent isothiocyanate ([FITC], Southern Biotechnology Assoc., Inc., Birmingham, AL).…”

Recombinant human megakaryocyte growth and development factor (rhumgdf), a ligand for c‐mpl, produces functional human platelets in vitro

“…An average of 4.4 rt 0.3 x lo6 CD34' cells were obtained per unit (n = 25), with an average purity of 90 f 5% (n = 12). Cells were frozen until use [38] then thawed and plated at 5 x 106/ml in megakaryocyte growth medium (meg-GM) [31] with 10% normal human AB plasma (HuAB) [39] and 10 ng/ml purified, mammalian cell-derived, rHuMGDF [ 11. After eight days, cultures were transferred to fresh culture wells and supplemented with 20-50% fresh meg-GM + 10% HuAB. Cultures were monitored for the appearance of proplatelets which occurred one to three days after replating.…”

Ex Vivo Large‐Scale Generation of Human Platelets from Cord Blood CD34 + Cells

“…Cord blood (CB) represents a large and readily available source of HSC. Several investigators [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] have previously made efforts to obtain megakaryocytic progenitors from HSC in bone marrow, peripheral blood, or CB by using a liquid culture system. Furthermore, there have been some clinical trials in which in vitro expanded megakaryocytic progenitors were infused to patients receiving high-dose chemotherapy.…”