Transplantation of hematopoietic stem cells obtained by a combined dye
method fractionation of murine bone marrow
I McAlister, NS Wolf, ME Pietrzyk, PS Rabinovitch, G Priestley and B Jaeger
Department of Pathology, University of Washington, Seattle 98195.
Hematopoietic stem cells were purified from murine bone marrow cells (BMC).
Their characteristic density, size, internal complexity, Hoechst 33342 dye
uptake, and wheat germ agglutinin (WGA) affinity were used to distinguish
them from other cells in the bone marrow. BMC suspensions were centrifuged
over Ficoll Lymphocyte Separation Media (Organon Teknika, Durham, NC;
density 1.077 to 1.08). The lower-density cells were drawn off, stained
with Hoechst and labeled with biotinylated WGA bound to streptavidin
conjugated to phycoerythrin (WGA-B*A-PE) or with WGA conjugated to Texas
Red. These cells were then analyzed and sorted by an Ortho Cytofluorograph
50-H cell sorter. The cells exhibiting medium to high forward light
scatter, low to medium right angle light scatter, low Hoechst intensity,
and high WGA affinity were selected. Sorted BMC (SBMC) were stained with
Romanowsky-type stains for morphologic assay, and were assayed in lethally
irradiated (LI) mice for their ability to produce colony-forming units in
the spleen (CFU-S) and for their ability to produce survival. The spleen
seeding factor for day 8 CFU-S upon retransplantation of the isolated cells
was 0.1. The isolated cells were found to have consistent morphology, were
enriched up to 135-fold as indicated by day 8 CFU-S assay, 195-fold as
indicated by day 14 CFU-S assay, and 150 sorter-selected BMC were able to
produce long-term survival in LI mice with retention of donor karyotype.
When recipients of this first transplantation were themselves used as BMC
donors, their number of day 8 and day 12 CFU-S were found to be reduced.
However, 3 X 10(5) of their BMC provided 100% survival among secondary
recipients. When the previously SBMC were competed after one
transplantation against fresh nonsorted BMC in a mixed donor transplant,
they showed the decline in hematopoietic potency normally seen in
previously transplanted BMC. We conclude that the use of combinations of
vital dyes for fluorescence-activated cell sorting (FACS) selection of
survival-promoting murine hematopoietic stem cells provides results
comparable with those produced by antibody- selected FACS and has the
advantage of a method directly transferable to human BMC.
Volume 75,
Issue 6,
pp. 1240-1246,
03/15/1990
Copyright © 1990 by The American Society of Hematology
