CD4dull+ hematopoietic progenitor cells in murine bone marrow

CD4+ cells comprise approximately 3% to 6% of murine bone marrow (BM) cells. The majority are CD4dull+, but there are two distinct sub populations: CD4 brightly positive Gr-1- cells (CD4hiGr-1-) and CD4+ Gr- 1+ cells (CD4loGr-1lo). CD4hiGr-1- cells are considered to be mature T cells by cell surface antigen expression and morphology. CD4loGr-1lo cells, which comprise approximately 0.6% of the BM cells, express small amount of B220 and Thy1 antigens. Interestingly, colony-forming units (CFU)-spleen and CFU-C are not enriched in this population. However, when injected into lethally irradiated mice, CD4loGr-1lo cells were shown to differentiate into T-cell, B-cell, and myelo-monocyte lineages when assayed 26 weeks after transplantation. Furthermore, donor-derived CD4loGr-1lo cells were present in the recipients' BM at least 16 weeks after transplantation. These observations suggest that murine CD4loGr- 1lo cells in BM have self-renewal capability and retain the ability to differentiate into at least three lineages in long-term hematopoiesis.     

Culture of hematopoietic stem cells purified from murine bone marrow.

The results of the Y-chromosome in situ hybridization experiments, the MRA assessment, and the long-term production of CFU-GM in vitro indicate that our protocol to sort low density WGA+, 15/1.1-, Rh123 dull cells enriches about 200-fold for PHSC. Assays for spleen colony formation (CFU-S) and radioprotection (30-day survival) were shown to be unspecific for PHSC, and, therefore, we lack a quantitative PHSC assay. The absolute number of PHSC in the bone marrow is not known any more, the purity of our sorted population likewise is unknown. Long-term repopulating cells (PHSC) could be separated from short-term repopulating ones by using Rh123 staining. The short-term repopulating cells (Rh123 bright) provided sufficient offspring to protect lethally irradiated mice until endogenous PHSC could reconstitute hematopoiesis. These cells are therefore of interest for bone marrow transplantation, because they provide radioprotection without long-term repopulation and graft-versus-host disease. For gene therapy these cells are of limited use, and PHSC with extensive replication are needed. The PHSC were not cultured successfully. Less than 15% of the sorted Rh123 dull cells responded in semisolid or liquid cultures in the presence of growth factors. Proliferation without differentiation was not observed. This may indicate that the right growth factor has not been found yet. On the other hand, about 30% of the cells responded in stromal layers of long-term bone marrow cultures and prolonged CFU-GM production and cobblestone area formation were observed there, suggesting that cell-cell contact and adherence molecules play a regulatory role in PHSC replication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of engraftable hematopoietic stem cells in murine long-term bone marrow cultures

OBJECTIVE: Long-term bone marrow cultures (LTBMC) are a potential source of hematopoietic stem cells (HSC) for transplantation. Previous reports indicate that feeding LTBMCs induces hematopoietic progenitor cycling, and other studies link HSC cycle phase with engraftability. Our study was initiated to further characterize LTBMC engraftability and determine if a cycle phase-related engraftment defect affects HSC from LTBMCs. MATERIALS AND METHODS: Competitive repopulation of lethally irradiated BALB/c females was used to examine engraftability of LTBMCs under "fed" or "unfed" conditions at 3 to 5 weeks culture. Tritiated thymidine suicide was used to determine the cycle status of HPP-CFC and CFU-S from LTBMCs. RESULTS: Total cell number in LTBMCs decreases from input. Quantitatively, both fed and unfed 3-, 4-, or 5-week cultures compete strongly with fresh marrow for 2 and 8 weeks, but not 6 months, after transplantation. Short-term engraftable HSCs expand between 3 and 5 weeks of culture. Clonal assays indicate no peak in S-phase of CFU-S at 24 and 48 hours after feeding, and fluctuation in both content and cycle status of HPP-CFC after feeding. CONCLUSIONS: Our LTBMCs engraft in all conditions, and the level of engraftment capability does not correlate with cell-cycle phase of CFU-S or HPP-CFC, or with time from feeding. Although the total cell number decreases from input, the proportion of short- and intermediate-term engrafting HSC in whole LTBMCs approximates that of fresh marrow and expands from 3 to 5 weeks in culture, whereas long-term engraftable HSCs are decreased in culture.     

Primitive hematopoietic cells in murine bone marrow express the CD34 antigen

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin- /10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.     

The bone marrow aspirate of healthy subjects

Bone marrow aspirates were obtained from the right or left posterior superior iliac spine of 50 healthy volunteers, 30 men and 20 women. Reference ranges were derived for each cell type and for the myeloid : erythroid (M : E) ratio. The M : E ratio and the percentage of neutrophils were significantly higher in the women and the erythroid component significantly lower. All 28 evaluable men and 11/17 evaluable women had storage iron present in more than trace amounts. The percentage of erythroblasts with detectable iron granules varied very widely, from 3% to 69% in those with more than a trace of storage iron. Minor dyserythropoietic features were present in a high percentage of subjects and 19/50 subjects had one or two dysplastic megakaryocytes. Granulocytic dysplasia was not detected.     

Functional amelioration of murine galactosialidosis by genetically modified bone marrow hematopoietic progenitor cells

Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disorder galactosialidosis (GS). PPCA(-/-) mice display a disease course similar to that of severe human GS, resulting in nephropathy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overexpressing the corrective enzyme in either erythroid cells or monocytes/macrophages has proven effective for the improvement of the phenotype, and encouraged the use of genetically modified BM cells for ex vivo gene therapy of GS. Here, we established stable donor hematopoiesis in PPCA(-/-) mice that received hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based, bicistronic retroviral vector overexpressing PPCA and the green fluorescent protein (GFP) marker. We observed complete correction of the disease phenotype in the systemic organs up to 10 months after transplantation. PPCA(+) BM-derived cells were detected in all tissues, with the highest expression in liver, spleen, BM, thymus, and lung. In addition, a lysosomal immunostaining was seen in nonhematopoietic cells, indicating efficient uptake of the corrective protein by these cells and cross-correction. Expression in the brain occurred throughout the parenchyma but was mainly localized on perivascular areas. However, PPCA expression in the central nervous system was apparently sufficient to delay the onset of Purkinje cell degeneration and to correct the ataxia. The long-term expression and internalization of the PPCA by cells of systemic organs and the clear improvement of the neurologic phenotype support the use of this approach for the treatment of GS in humans. (Blood. 2002;99:3169-3178)     

Cell cycle status of murine megakaryocyte and granulocyte-macrophage colony-forming cells in bone marrow and spleen.

The cell cycle status of megakaryocyte colony-forming cells (Meg-CFC) and granulocyte-macrophage colony-forming cells (GM-CFC) from the spleen and bone marrow of C57BL mice was evaluated by determining the effects of hydroxyurea (OHU) or cytosine arabinoside (Ara-C), both in vivo and in vitro, upon colony-forming cells (CFC). The concentrations of cells in culture (2 x 10(6) to 4 x 10(6)/ml for spleen and 0.25 x 10(5) to 1.0 x 10(5)/ml for bone marrow) did not alter cell cycle status of either Meg-CFC or GM-CFC. Determination of cell cycle status following in vivo administration of OHU indicated that 25.2% of Meg-CFC and 28.1% of GM-CFC in the spleen, and 26.0% of Meg-CFC and 29.5% of GM-CFC in the bone marrow, were in cycle. In vitro incubation of CFC with OHU showed that in the spleen 25.1% of Meg-CFC and 24.2% of GM-CFC were engaged in DNA synthesis, whereas in bone marrow 28.5% of Meg-CFC and 29.2% of GM-CFC were synthesizing DNA. Incubation with Ara-C, in vitro, gave similar results, with 26.0% of Meg-CFC and 26.2% of GM-CFC in the spleen, and 27.1% of Meg-CFC and 31.4% of GM-CFC in the bone marrow, in cycle. In summary, significant differences were not observed between the cell cycle status of Meg-CFC and GM-CFC, whether derived from spleen or bone marrow. In vitro and in vivo measurements (with OHU) and in vitro measurements with two cytotoxic drugs (OHU versus Ara-C) also provided similar results. The data suggest that the regulation of DNA synthesis in both Meg-CFC and GM-CFC in the murine spleen and bone marrow is similar.     

A novel 37-Kd adhesive membrane protein from cloned murine bone marrow stromal cells and cloned murine hematopoietic progenitor cells

The adhesion of hematopoietic progenitor cells to bone marrow stromal cells is critical to hematopoiesis and involves multiple effector molecules. Stromal cell molecules that participate in this interaction were sought by analyzing the detergent-soluble membrane proteins of GBI/6 stromal cells that could be adsorbed by intact FDCP-1 progenitor cells. A single-chain protein from GBI/6 cells having an apparent molecular weight of 37 Kd was selectively adsorbed by FDCP-1 cells. This protein, designated p37, could be surface-radiolabeled and thus appeared to be exposed on the cell membrane. An apparently identical 37- Kd protein was expressed by three stromal cell lines, by Swiss 3T3 fibroblastic cells, and by FDCP-1 and FDCP-2 progenitor cells. p37 was selectively adsorbed from membrane lysates by a variety of murine hematopoietic cells, including erythrocytes, but not by human erythrocytes. Binding of p37 to cells was calcium-dependent, and was not affected by inhibitors of the hematopoietic homing receptor or the cell-binding or heparin-binding functions of fibronectin. It is proposed that p37 may be a novel adhesive molecule expressed on the surface of a variety of hematopoietic cells that could participate in both homotypic and heterotypic interactions of stromal and progenitor cells.     

Cytological characterization of murine bone marrow and spleen hematopoietic compartments for improved assessment of toxicity in preclinical gene marking models

Gene therapy has proven its potential to cure diseases of the hematopoietic system, but potential adverse reactions related to insertional mutagenesis by integrating gene vectors and chromosomal instability in long-lived repopulating cells have emerged as a major limitation. Preclinical gene therapy in murine models is a powerful model for assessment of gene marking efficiency and adverse reactions. However, changes in the hematologic composition after transplantation with retrovirally modified hematopoietic stem cells have not been well investigated in large cohorts of animals by systematic cytological analyses. In the present study, cytological analyses of bone marrow and spleen were performed in a large cohort (n?=?58) of C57BL/6J mice over an extended observation period after gene marking. Interestingly, we observed hematological malignancies in four out of 30 animals transplanted with dLNGFR (truncated form of the human p75 low-affinity nerve growth factor receptor) and tCD34 modified stem/progenitor cells. Our data demonstrate that cytological analysis provides important information for diagnosis of hematological disorders and thus should be included in preclinical studies and performed in each investigated animal. Together with histological analysis, flow cytometric analysis, and other analyses, the quality and predictive value of preclinical gene therapy studies will be improved.     

Multipotential hematopoietic stem cells in the bone marrow

It has been generally held that human hematopoietic stem cells are lineage-negative CD34⁺ CD38^?. However, murine hematopoietic stem cells were reported to be CD34^?. We have characterized the surface phenotypes of murine hematopoietic stem cells by using a murine transplantation model. Our studies revealed that the majority of the stem cells in normal adult mice are CD34^? while a minority (15%–20%) being CD34⁺. Our studies also revealed that stem cells that are activated by injection of 5-fluorouracil in vivo, exposure to cytokines in vitro, or mobilization by G-CSF are CD34⁺ and that CD34 expression is reversible. It has been reported that fetal murine hematopoietic stem cells are CD34⁺. Our studies revealed that stem cells of juvenile mice are CD34+ and that the developmental change from CD34⁺ to CD34^? state takes place between 7 and 10 weeks of age. In adult mice, expression of CD38 by steady-state and activated stem cells was completely reciprocal of CD34 expression. Activated stem cells and the minority population of the stem cells in the normal mice are CD34⁺ CD38^?. In contrast, the majority of stem cells in normal adult mice are CD34- CD38+. Recently, we studied CD38 expression by stem cells of neonatal and juvenile mice. Stem cells of newborn mice are CD38^?. About half of the stem cells of 5-week-old mice are CD38⁺. Finally, our studies indicated that some of the CD34⁺ stem cells in the bone marrow of normal adult mice express lineage markers such as Mac-1 and CD4. These studies in a murine model clearly documented that expression of both CD34 and CD38 by stem cells is under developmental control and may be subject to changes induced by activation of the stem cells. In order to test whether or not these principles apply to human stem cells we tested surface phenotypes of human stem cells using two xenotransplantation techniques. Studies based on human/sheep xenograft model indicated that a significant portion of adult human long-term engrafting cells are CD34^?. Similar to mouse stem cells expression of CD34 by human stem cells was reversible. Studies based on our newborn NOD/SCID/β-microglobulin^null mice indicated that human cord blood stem cells are CD34⁺ CD38^?. These results appear to support the validity of studies of murine stem cells to provide insight into human stem cells.     

Non-side-population hematopoietic stem cells in mouse bone marrow

Most hematopoietic stem cells (HSCs) are assumed to reside in the so-called side population (SP) in adult mouse bone marrow (BM). We report the coexistence of non-SP HSCs that do not significantly differ from SP HSCs in numbers, capacities, and cell-cycle states. When stained with Hoechst 33342 dye, the CD34(-/low) c-Kit(+)Sca-1(+)lineage marker(-) (CD34(-)KSL) cell population, highly enriched in mouse HSCs, was almost equally divided into the SP and the main population (MP) that represents non-SP cells. Competitive repopulation assays with single or 30 SP- or MP-CD34(-)KSL cells found similar degrees of repopulating activity and frequencies of repopulating cells for these populations. Secondary transplantation detected self-renewal capacity in both populations. SP analysis of BM cells from primary recipient mice suggested that the SP and MP phenotypes are interconvertible. Cell-cycle analyses revealed that CD34(-)KSL cells were in a quiescent state and showed uniform cell-cycle kinetics, regardless of whether they were in the SP or MP. Bcrp-1 expression was similarly detected in SP- and MP-CD34(-)KSL cells, suggesting that the SP phenotype is regulated not only by Bcrp-1, but also by other factors. The SP phenotype does not specify all HSCs; its identity with stem cell function thus is unlikely.     

Senescence of hematopoietic stem cells and bone marrow failure

Functional failure in hematopoietic stem cells (HSCs) may bring fatal consequences because HSCs are the ultimate source of mature blood cells, which need continuous replenishment. One potential cause of HSC dysfunction is senescence, in which HSCs and progenitor cells enter a state of proliferative arrest. HSC senescence is genetically regulated and one particular regulator is the telomerase gene. Mutations in the telomerase gene complex have been found in patients with bone marrow failure syndromes. During a normal lifetime, HSC clones function over the long term and may not show any functional loss under normal circumstances. However, pathologic environments may limit HSC proliferation, accelerate HSC turnover, and shorten the functional life of HSCs, leading to HSC clonal exhaustion and senescence.     

Fractionation of mouse bone marrow by adherence separates primitive hematopoietic stem cells from in vitro colony-forming cells and spleen colony-forming cells.

Fractionation of mouse bone marrow by adherence to tissue culture plastic was used to characterize the adhesive properties of hematopoietic stem (HS) cells capable of long-term reconstitution. The adherent fraction that represents approximately 13% of the total marrow population was virtually devoid of in vitro colony-forming cells and spleen colony-forming cells but did contain approximately 30% of the total HS cells recovered from the procedure. These cells could be detected by both the competitive repopulation assay and by repopulation of W/Wv recipients. In approximately 60% of the recipients from the competitive repopulation experiments, the contribution of the adherent marrow cells was relatively low early (8 to 10 weeks) after transplantation. With time, however, the hematopoietic contribution from these cells increased, reaching a stable level 20 to 30 weeks posttransplantation. In the remaining recipients (40%), the contribution from adherent cells was already significant within 8 to 10 weeks of transplantation and did not change dramatically throughout the course of the experiment. Adherent bone marrow containing significant numbers of HS cells was unable to protect mice from radiation death, indicating that these early cells in the absence of later-stage progenitors are unable to provide this function.     

Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow

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.     

Bone marrow origin of hematopoietic progenitors and stem cells in murine muscle

It has been reported that mononuclear cells harvested from murine skeletal muscle are capable of hematopoietic reconstitution of lethally irradiated mice. First, the nature of the hematopoietic progenitors in the muscle of C57BL/6-Ly-5.1 mice was examined by means of methylcellulose culture. The types and incidences of colonies grown from muscle mononuclear cells were different from those cultured from bone marrow (BM) or peripheral blood mononuclear cells. The next step was to examine the origin of the hematopoietic progenitors and stem cells in the muscle with the use of Ly-5.2 mice that had been made chimeric by transplantation of Ly-5.1 BM cells. The percentages of Ly-5.1 cells cultured from the muscle of the chimeric mice correlated with those cultured from BM, indicating BM origin of hematopoietic progenitors in the muscle. Long-term hematopoietic engrafting cells in the muscle of the chimeric mice were also derived from BM. However, mobilization of progenitors into circulation by granulocyte colony-stimulating factor did not change the population of hematopoietic progenitors in the muscle. It is proposed that hematopoietic progenitors and stem cells in the muscle tissue are of BM origin but their transition from BM to muscle may be a slow process.     

Distribution of marrow repopulating cells between bone marrow and spleen early after transplantation

Whether hematopoietic stem cells (HSCs) home selectively to bone marrow (BM) early after transplantation remains an issue of debate. Better understanding of homing mechanisms may benefit BM transplantation protocols in cases of limited graft cell number or nonmyeloablative conditioning regimens. Using flow cytometry and serial transplantation to stringently identify HSCs, trafficking patterns of long-term engrafting cells were mapped between BM and spleen early after transplantation. Low-density BM cells were tracked in irradiated or nonirradiated mice 1, 3, 6, and 20 hours after transplantation, at which time recipient BM and spleen were analyzed for recovery of primitive donor cells by phenotype and adhesion molecule expression. In addition, phenotypically defined HSC-enriched or HSC-depleted grafts were tracked 20 hours after transplantation in recipient BM and spleen and analyzed for recovery and long-term repopulating potential in mice undergoing serial transplantation. Regardless of irradiation status, recovery of donor Sca-1+ lin- cells was higher at most time points in recipient BM than in spleen, while recovery of total Sca-1+ cells was variable. A significantly higher percentage of BM-homed donor Sca-1+ cells expressed CD43, CD49e, and CD49d 20 hours after transplantation than spleen-homed cells, which contained significantly more non-HSC phenotypes. Furthermore, BM-homed cells were significantly enriched for cells capable of secondary multilineage hematopoiesis in mice undergoing serial transplantation compared with spleen-homed cells. These results support the notion of specific homing of HSCs to BM by 20 hours after transplantation and provide a basis for the enhanced engraftment potential afforded some Sca-1+ lin- cells subfractionated on the basis of adhesion molecule expression.