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Characterization of guinea pig megakaryocyte subpopulations at different
phases of maturation prepared with a Celsep separation system
PK Schick, BP Schick and K Williams-Gartner
Cardeza Foundation for Hematologic Research, Jefferson Medical College,
Philadelphia, PA 19107.
We introduce a new method for preparing subpopulations of guinea pig
megakaryocytes (MK). MK, partially purified by a density gradient, were
separated according to size by sedimentation, starting as a monolayer, in
an albumin gradient at unit gravity. Twenty-two fractions were collected.
Cells were cytocentrifuged, ploidy was assessed by microdensitometry, and
small MK were identified with anti-von Willebrand factor (vWF)
immunoglobulin. Immaturity was assessed by uptake of 3H thymidine and
synthesis of proteoglycans from 35S sulfate. About 88% of cells in
fractions 2 through 18 were MK, of which 90% were viable. Fractions
containing the largest cells were composed of 98% stage III and IV MK;
fractions with the smallest cells contained up to 80% stage I and II MK.
Six MK classes were isolated: immature cells, both stage I and II cells, at
either the 8N, 16N or 32N ploidy class; mature cells, both stage III and IV
cells, at either the 8N, 16N or 32N ploidy class. The fractions were pooled
into three groups: (a) 8% of MK in group 1, fractions 2 through 11, were
immature, and group 1 was composed of 92% of 16N and 32N mature classes;
(b) 29% of MK in group 2, fractions 12 through 15, were immature, and group
2 was composed of 52% 16N mature, 24% 16N immature, and 13% 8N mature
classes; 67% of MK in group 3, fractions 16 through 18, were immature, and
group 3 contained 51% 8N immature, 14% 16N immature, and 18% mature 16N
classes. The mean protein content of the three groups was 1.251, 0.624, and
0.284 mg/10(6) MK, respectively. Nine percent of cells in group 3 but no
cells in group 1 took up large amounts of 3H thymidine. The synthesis of
high-molecular-weight (high-mol-wt) proteoglycans in group 3 and synthesis
of lower mol wt proteoglycans in groups 1 and 2 provided further evidence
for differences in MK maturity. Thus, the method can isolate MK
subpopulations that are viable and can be used to investigate the
biochemical characteristics of MK at different phases of maturation.
Volume 73,
Issue 7,
pp. 1801-1808,
05/15/1989
Copyright © 1989 by The American Society of Hematology
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