The murine long-term multi-lineage renewal marrow stem cell is a cycling cell.

Prevailing wisdom holds that hematopoietic stem cells (HSCs) are predominantly quiescent. Although HSC cycle status has long been the subject of scrutiny, virtually all marrow stem cell research has been based on studies of highly purified HSCs. Here we explored the cell cycle status of marrow stem cells in un-separated whole bone marrow (WBM). We show that a large number of long-term multi-lineage engraftable stem cells within WBM are in S/G2/M phase. Using bromodeoxyuridine, we show rapid transit through the cell cycle of a previously defined relatively dormant purified stem cell, the long-term HSC (LT-HSC; Lineage(-)/c-kit(+)/Sca-1(+)/Flk-2(-)). Actively cycling marrow stem cells have continually changing phenotype with cell cycle transit, likely rendering them difficult to purify to homogeneity. Indeed, as WBM contains actively cycling stem cells, and highly purified stem cells engraft predominantly while quiescent, it follows that the population of cycling marrow stem cells within WBM are lost during purification. Our studies indicate that both the discarded lineage-positive and lineage-negative marrow cells in a stem cell separation contain cycling stem cells. We propose that future work should encompass this larger population of cycling stem cells that is poorly represented in current studies solely focused on purified stem cell populations.

The stem cell continuum: a new model of stem cell regulation.

Most models of hematopoiesis have been hierarchical in nature. This is based on a large volume of correlative data. Recent work has indicated that, at least at the stem/progenitor level, hematopoiesis may, in fact, be a continuum of transcriptional opportunity. The most primitive hematopoietic stem cells are either continually cycling at a slow rate or entering and exiting cell cycle. Associated with this cycle passage are changes in functional phenotype including reversible alterations in engraftment, adhesion protein expression, cytokine receptor expression, homing to marrow, and progenitor cell numbers. Global gene expression, as measured in one point in cycle, is also markedly altered. The differentiation potential of the marrow as it transits cell cycle in response to a set differentiation stimulus also shows marked variations. This cycle-related plasticity has been clearly established for hematopoiesis. It also holds for the ability of murine marrow stem cells to home to lung and to convert to pulmonary cells. These data indicate that bone marrow stem cells can probably not be defined as discrete entities but must rather be studied on a population basis. They also indicate that mathematical modeling will become progressively more important in this field.

p53 expression and environmental tobacco smoke exposure in feline oral squamous cell carcinoma.

The purpose of this study was to determine the prevalence of p53 overexpression in feline oral squamous cell carcinomas (SCC) and to determine, if any, the association between p53 overexpression and lifestyle factors and environmental exposures, including exposure to environmental tobacco smoke (ETS). Questionnaires concerning exposure to ETS and other environmental factors were sent to owners of cats presenting to the Harrington Oncology Program with a diagnosis of oral SCC between 1991 and 2000. Additionally, 23 formalin-fixed biopsy samples from these cats, with information regarding ETS, were evaluated immunohistochemically for p53 expression using the CM-1 clone and the avidin-biotin-horseradish peroxidase method. Of the 23 samples evaluated, 15 (65%) showed positive nuclear staining for the CM-1 clone. Tumor biopsy samples from cats exposed to any ETS were 4.5 times more likely to overexpress p53 than were tumors from unexposed cats (P = 0.19). Among cats with any ETS exposure, those with 5 years or longer of exposure were 7.0 times more likely to overexpress p53 (P = 0.38). Longhaired cats (P = 0.18) and female cats (P = 0.35) were also more likely to show p53 expression in their tumors. These results provide additional support for a relationship between oral SCC development and exposure to household ETS and may implicate p53 as a potential site for carcinogen-related mutation in this tumor.

Tissue injury in marrow transdifferentiation.

Recent findings indicate that adult BM contains cells that can differentiate into mature, nonhematopoietic cells of multiple tissues including cells of the kidney, lung, liver, skin and GI tract and fibers of heart and skeletal muscle. Recently the number of these observations has substantially increased, but there is a lack of information on the mechanistic issues in stem cell plasticity. In three different models for skin, liver and skeletal muscle plasticity, we have shown that following transplantation of the marrow cells from green fluorescent protein (GFP) transgenic mice, high levels of conversion of marrow cells can be identified. Injury to the tissue was the single most important factor for this phenomenon since the incidence of marrow to other tissue conversions significantly increased after tissue injury was implemented. Our studies also demonstrate the effect of radiation on the extent of marrow conversion.

Tolerance induction by costimulator blockade in 100 cGy treated hosts with varying degrees of genetic disparity.

Long-term multilineage allochimerism can be obtained in H2-mismatched B6.SJL to BALB/c transplants with host irradiation of 100 cGy, donor spleen cell pre-exposure and costimulator blockade with anti-CD40 ligand (CD40L) antibody. We evaluated this allochimerism approach in murine marrow transplants with different degrees of major histocompatibility complexe (MHC) mismatching; these include: (1) H2-mismatched transplant H2Kk to H2Kb, (2) full haplo-identical transplant H2Kbd to H2Kbk, (3) a partial haplo-identical transplant H2Kd to H2Kbd and (4) an MHC class II mismatch. Levels of chimerism increased up to 12 weeks and then stayed relatively stable up to 1 year after transplant. At 18 weeks post-transplant, the H2-mismatched, haplo-identical, partial haplo-identical and class II-mismatch transplants evidenced 17.9+/-4.4, 40.7+/-0.9, 25.1+/-4.19 and 33.7+/-3.5% donor chimerism, respectively. Dropping the anti-CD40 antibody treatment and spleen cells or changing the schedule of antibody to one injection, in haplo-identical or full-mismatched transplants resulted in no donor-derived chimerism. On the other hand, these still resulted in minor chimerism in class II-mismatched transplants. Lineage analysis of peripheral blood at 6 and 12 months post-transplant demonstrated a significant shift toward increased chimeric lymphocytes and decreased chimeric granulocytes in the full H2 as compared with haplo-identical or class II transplants. Transplantation with anti-CD40L antibody eliminated both graft-versus-leukemia and graft-versus-host disease (GVHD) and delayed lymphocyte infusion did not rescue animals from fatal leukemia. In conclusion, under the conditions of our tolerization regimen, a haplo transplant gives higher engraftment levels than a full H2 mismatch, and despite lower engraftment levels, a class II-mismatched transplant can be successfully accomplished with only 100 cGy and no CD40L blockade.

Influence of timing of administration of 5-fluorouracil to donors on bone marrow engraftment in nonmyeloablated hosts.

We evaluated the engraftment and the cell cycle status of marrow cells at various times after 5-fluorouracil (5-FU) administration. 5-FU (150 mg/kg) was given to donor male BALB/c mice at 1, 2, 6, or 12 days prior to marrow harvest. The donor cells were then assessed in host nonmyeloablated female mice. Bone marrow engraftment of marrow treated with 5-FU was evaluated and compared to marrow treated with diluent (phosphate-buffered saline) at 3 and 10 weeks after marrow infusion. Our data show a rapid induction of an engraftment defect 1 day after 5-FU, persistence of this defect through day 6, and a recovery by day 12. Experiments using hydroxyurea (which selectively kills cells in the S phase) to determine the cell cycle status indicated that cells that engrafted in post-5-FU marrow were noncycling at days 1, 2, and 12 but cycling at day 6. Post-5-FU bone marrow was also analyzed in vitro by colony assays and its cycling status determined by 3H-thymidine suicide assay. High-proliferative-potential colony-forming cells (HPP-CFCs) and low-proliferative-potential colony-forming cells (LPP-CFCs) decreased rapidly 1 day after 5-FU, with a nadir observed at day 6 for HPP-CFCs and day 2 for LPP-CFCs. By day 12, LPP-CFCs showed a total recovery, but HPP-CFCs were still defective. Significant numbers of HPP-CFCs were cycling, mostly at days 6 and 8 after 5-FU, whereas LPP-CFCs appeared quiescent except at day 2. These results emphasize the importance of timing if post-5-FU marrow is used for gene therapy or marrow transplantation.

Host marrow stem cell potential and engraftability at varying times after low-dose whole-body irradiation.

High levels of chimerism in syngeneic BALB/c transplants were reported when hosts were exposed to 1 Gy (100 cGy) whole body irradiation (WBI) and infused with 40 x 10(6) marrow cells. The recovery of host stem cells and alterations of enhanced host engraftability at varying times after 1 Gy WBI have now been evaluated in this study. Male BALB/c marrow (40 x 10(6) cells) was infused into female BALB/c hosts immediately or at 6, 12, and 24 weeks after 1 Gy WBI of host female BALB/c mice; engraftment percentages 8 weeks after cell injection at week 0, 6, 12, or 24 were 68% +/- 12%, 45% +/- 15%, 51% +/- 12%, or 20% +/- 8%, respectively. Eight-week engraftment levels in nonirradiated hosts average 7.7%. Conversely, engraftable stem cells measured at 8 weeks postengraftment in 1 Gy--exposed hosts were reduced to 8.6% +/- 3% of nonirradiated mice at time 0, 35% +/- 12% 6 weeks later, 49% +/- 10% at 3 months, and 21% +/- 7% at 6 months. Engraftment was still increased and stem cell decreased 1 year after 1 Gy. Furthermore, the primary cells transplanted into 1 Gy hosts can be serially transplanted, and the predominant effect of 1 Gy is directly on engrafting stem cells and not through accessory cells. These data show that transplantation in 1 Gy mice may be delayed until recovery of hematopoiesis, suggesting strategies in allogeneic transplantation to avoid the adverse effects of cytokine storm. The incomplete recovery of engraftable stem cells out to 12 months indicates that stem cell expansion, especially in patients previously treated with radiomimetic drugs, may not be feasible. (Blood. 2001;98:1246-1251)

The fleet feet of haematopoietic stem cells: rapid motility, interaction and proteopodia.

Haematopoietic stem cells (HSCs) have been extensively characterized regarding in vivo engraftment, surface epitopes and genetic regulation. However, little is known about the homing of these rare cells, and their intrinsic motility and membrane deformation capacity. We used high-speed optical-sectioning microscopy and inverted fluorescent videomicroscopy to study highly purified murine lineage-negative, rhodamine-low, Hoechst-low HSCs over time under various in vitro conditions. We discovered extremely rapid motility, directed migration to stromal cells and marked membrane modulation. High resolution images with three-dimensional reconstruction showed the general presence of microspikes. Further, pseudopodia (proteopodia) were observed that were induced by stromal-derived factor-1 and steel factor. Proteopodia were directed towards and were quenched by stromal cells, at times bridged HSCs, and could rapidly retract or detach from cells. Proteopodia were also observed in vivo with homed HSCs in frozen sections of murine spleen, lung and heart. This is the first demonstration that HSCs are both fast and highly malleable in phenotype.

Adhesion receptor expression by hematopoietic cell lines and murine progenitors: modulation by cytokines and cell cycle status.

Hematopoietic progenitor cells are incubated with cytokine combinations for in vitro expansion of stem cells and to enhance retrovirus-mediated gene transfer. Optimization of the engraftment of these treated cells would be critical to the success of stem cell transplantation or gene therapy. Previous studies demonstrated that a 48-hour incubation of donor BALB/c bone marrow with a mixture of four cytokines (IL-3, IL-6, IL-11, and SCF), resulted in expansion of primitive progenitor/stem cells but a loss of long-term engraftment in nonmyeloablated or myeloablated recipients. We have established the expression pattern for a number of adhesion receptors by normal hematopoietic progenitors and cell lines and the modulation in expression induced by cytokines or cell cycle progression to ascertain the molecular basis for such defective engraftment. Northern blot analysis demonstrated that the cytokine combination of IL-3, IL-6, IL-11, and SCF dramatically down-regulated alpha 4 integrin receptor expression in HL-60 cells. Synchronized FDC-P1 cells exhibited modulation of alpha 4 expression through cell cycle progression, both by quantitative RT-PCR and flow cytometry. Normal murine bone marrow lineage-depleted, Sca+ cells expressed a number of adhesion receptors, including alpha L, alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, beta 1, L-selectin, CD44, and PECAM as assessed by flow cytometry, immunofluorescence, and RT-PCR. There was modulation of the expression of several of these receptors after incubation in the four cytokines for 24 and/or 48 hours: the proportion of cells expressing alpha L, alpha 5, alpha 6, and PECAM increased, whereas the proportion of cells expressing alpha 4 and beta 1 decreased, after cytokine incubation. There was a demonstrable concomitant decline in adhesion of these cells to fibronectin after the cytokine incubation, a finding that correlates with the decrease in expression of alpha 4. These changes in adhesion receptor expression and function with cytokines and during cell cycle transit may be critical to stem cell homing and engraftment after transplantation, as multiple receptors could be involved in the process of rolling, attachment to endothelium, endothelial transmigration, and migration within the marrow space.

Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice.

Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.

Immunofluorescence characterization of key extracellular matrix proteins in murine bone marrow in situ.

The mechanism of hemopoietic stem cell homing to the bone marrow involves molecular interactions that mediate the recognition and interaction of these cells with the marrow microenvironment, including the extracellular matrix. On selective binding, this environment, in combination with soluble cytokines, regulates stem cell proliferation and differentiation. Using immunofluorescence labeling, we analyzed the location of the prominent extracellular matrix proteins fibronectin, collagen Types I, III, and IV, and laminin in sections of murine femoral bone marrow. Collagen Types I, IV, and fibronectin were localized to the endosteum, the region of the femoral microenvironment for which homing stem cells have a high affinity. The results further demonstrated a strong spatial association of collagen Type IV and laminin with the bone marrow vessels, including arterioles, veins, and sinuses. Fibronectin was distributed throughout the central marrow region, and all the proteins analyzed except collagen Type III were present in the bone, although at different levels. Fibronectin, collagen Types III and IV, and laminin were also present in the periosteum. The distinct locations of particular extracellular matrix proteins support the notion that they may play an important mechanistic role in the homing of engrafting cells.

Synchronized cell-cycle induction of engrafting long-term repopulating stem cells.

We have recently defined the window for marrow stem cell homing into nonablated hosts as the first 24 hours posttransplant. Within this homing window, donor cells rapidly cleared from the peripheral blood and lungs and plateaued in the marrow. We have now assessed the cell-cycle status of the engrafting cells capable of contributing to long-term hematopoiesis using administration of hydroxyurea (HU), a chemotherapy agent with S-phase cell-cycle specificity. HU was given at very short periods following a male bone marrow transplant (0, 3, 6, 12, and 15 hours) into female nonablated hosts, and donor cell engraftment was analyzed after 6 weeks. The data show that quickly after transplant (12 hours), greater than half of the engrafting cells capable of contributing long-term to all levels of the hematopoietic hierarchy are in S-phase. Analysis after 6 weeks included whole bone marrow, peripheral blood, primitive cells with high proliferative potential, and mature lineage-restricted marrow cells. These donor cells appear to be naturally synchronized. When HU was administered at any of the other time points, there was little evidence of cell death 6 weeks postengraftment.

Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse model.

Increasingly, allogeneic and even more often autologous bone marrow transplants are being done to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide an opportune means of delivering genes in transfected, engrafting stem cells. However, despite its widespread clinical use and promising gene therapy applications, relatively little is known about the mechanisms of engraftment in marrow transplant recipients. This is especially so in the nonablated recipient setting. Our data show that purified lineage negative rhodamine 123/Hoechst 33342 dull transplanted hematopoietic stem cells engraft into the marrow of nonablated syngeneic recipients. These cells have multilineage potential, and maintain a distinct subpopulation with "stem cell" characteristics. The data also suggests a spatial localization of stem cell "niches" to the endosteal surface, with all donor cells having a high spatial affinity to this area. However, the level of stem cell engraftment observed following a transplant of "stem cells" was significantly lower than that expected following a transplant of the same number of unseparated marrow cells from which the purified cells were derived, suggesting the existence of a "nonstem cell facilitator population," which is required in a nonablated syngeneic transplant setting.