ABSTRACT
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.
Subject(s)
Bone Marrow Cells , Hematopoietic Stem Cell Transplantation , Animals , Cell Separation/methods , Coloring Agents , Female , Flow Cytometry/methods , Graft Survival , Hematopoietic Stem Cells/cytology , Male , Mice , Mice, Inbred Strains , Spleen/cytologyABSTRACT
We have previously reported that two or more different subpopulations of bone marrow stem cells exist in mice as determined by cycling status of day-8 and day-14 CFU-S in long term bromodeoxyuridine (BrdU) infusion studies. In the present report, comparisons between cycling of stem cell subpopulations in old and young mice show that, while the general patterns persist, there are some statistically significant differences between corresponding time points of early and late CFU-S cycling patterns in young and old BDF1 mice. In both populations of CFU-S there exist cells which do not enter cycle over a five week period. The method which we have employed allowed the cycling measurements to be made in unstimulated steady-state bone marrow cell populations, since no cell death is caused in vivo.
Subject(s)
Aging/physiology , Hematopoietic Stem Cells/physiology , Animals , Bromodeoxyuridine/pharmacology , Cell Cycle , Male , Mice , Mice, Inbred StrainsABSTRACT
Serial transplantation and competitive repopulation were used to evaluate any loss of self-replicative capacity of bone marrow stem cells in a mouse model with increased and persistent hemopoietic demands. Congenic marrows from old control and from young and old mice with hereditary spherocytic anemia (sphha/sphha) were serially transplanted at 35-day intervals into normal irradiated recipients. Old anemic marrow failed or reverted to recipient karyotype at a mean of 3.5 transplants, and young anemic marrow reverted at a mean of 4.0 transplants, whereas controls did so at a mean of 5.0 transplants. In a competitive assay in which a mixture of anemic and control marrow was transplanted, the anemic marrow persisted to 10 months following transplantation; anemic marrow repopulation was greater if anemic marrow sex matched with the host. It is possible that lifelong stress of severe anemia decreases stem cell reserve in the anemic sphha/sphha mouse marrow. However, marginal differences in serial transplantation number and the maintenance of anemic marrow in a competition assay would suggest that marrow stem cells, under prolonged stress, are capable of exhibiting good repopulating and self-replicating abilities.
Subject(s)
Bone Marrow Transplantation , Cell Cycle , Hematopoietic Stem Cell Transplantation , Spherocytosis, Hereditary/blood , Aging , Animals , Bone Marrow/pathology , Bone Marrow/radiation effects , Cell Cycle/radiation effects , Chronic Disease , Colony-Forming Units Assay , Disease Models, Animal , Erythrocyte Count , Female , Hematopoietic Stem Cells/pathology , Hematopoietic Stem Cells/radiation effects , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Pancytopenia/blood , Pancytopenia/pathology , Radiation Chimera , Spherocytosis, Hereditary/pathology , Spherocytosis, Hereditary/physiopathology , Spleen/pathologyABSTRACT
It was found in a long-term bromodeoxyuridine (BrdU) infusion study that two or more different subpopulations of bone marrow stem cells exist in mice. One of these subpopulations appears to be noncycling and forms approximately 10% of eight-day CFU-S. Another one, a subpopulation of slowly cycling bone marrow cells, is represented as 14-day CFU-S. The 14-day CFU-S have a regular increment in the percentage of the subpopulation entering the cycle over time, with a cell generation half-time of 21 days. The cycling status in these experiments was ascertained by in vivo continuous long-term BrdU infusion. An improved method is presented for long-term BrdU infusion with UV killing of cycled cells.
