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.

Hematopoietic Stem Cells: Uncomfortable Considerations.

PURPOSE: This report defines new concepts of hematopoietic stem cell biology. RECENT FINDINGS: We have utilized 3 different approaches which show that long-term repopulating hematopoietic stem cells are actively cycling and always changing phenotype. In addition this is reversible. This indicates that the stem cell cannot be purified by current epitope selection approaches. The vast bulk of hematopoietic stem cells are discarded in different populations when stem cells are purified to lineage negative c-kit positive and Sca-1 positive cells. Studies to define the hematopoietic niche have been largely carried out on these irrelevant purified cells and thus are not definitive. Studies have indicated the presence of baseline stem cells which function during the normal lifetime of mice. Baseline hematopoiesis appears to be run by thousands of relatively short lived clones with limited differentiation capacity. Thus there appear to be two basic hematopoietic stem cell modes; emergency and baseline.

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.

Mesenchymal stromal cell-derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells.

Mesenchymal stromal cells (MSCs) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 h to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 h post irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% dimethyl sulfoxide at -80 °C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells.

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.

The marrow cell continuum: stochastic determinism.

Traditional models of hematopoiesis have been hierarchical in nature. Over the past 10 years, we have developed data indicating that hematopoiesis is regulated in a continuum with deterministic and stochastic components. We have shown that the most primitive stem cells, as represented by lineage negative rhodamine(low) Hoechst(low) murine marrow cells are continuously or intermittently cycling as determined by in vivo BrdU labeling. When marrow stem cells are induced to transit cell cycle by in vitro exposure to cytokines, either IL-3, IL-6, IL-11, and steel factor or thrombopoietin, FLT3 ligand, and steel factor, they progress through cycle in a highly synchronized fashion. We have determined that when the stem cells progress through a cytokine stimulated cell cycle the homing, engraftment, adhesion protein, global gene expression, and hematopoietic differentiation phenotypes all change in a reversible fashion. This has led to the continuum model, in which, with cycle transit, chromatin is continually changing altering open transcription areas and providing a continually changing landscape of transcriptional opportunity. More recently, we have extended the changing differentiation profiles to differentiation into lung cells and found that non-hematopoietic differentiation also shows cycle related reversibly modulation. These observations all together support a continuum model of stem cell regulation in which the phenotype of the marrow stem cells is continually and reversibly changing over time.

The stem cell continuum: considerations on the heterogeneity and plasticity of marrow stem cells.

Traditional models of hematopoiesis have been hierarchical. Recent evidence showing that marrow stem cells are a cycling population and that the hematopoietic phenotype of these cells reversibly changes with cycle transit have suggested a continuum model of stem cell regulators. Studies on marrow cell conversion to lung cells have extended this continuum to cycle-related differentiation into nonhematopoietic stem cells. We postulate that stem cells transiting cell cycle continually change their chromatin structure, thus providing different windows of transcriptional opportunity and a continually changing phenotype. Final outcomes with this continuum model would be determined by the specific chromatin state of the cell and the presence of specific differentiation inducers.

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.

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.

The marrow stem cell: the continuum.

The marrow hematopoietic stem cell is currently being redefined as to all aspects of its phenotype and its total differentiation capacity. This redefinition now includes its plasticity as to production of nonhematopoietic and hematopoietic cell types, the determinants of its in vivo engraftment potential and its expression of stem cell functional characteristics.

Marrow stem cell potential within a continuum.

On the basis of our studies of the fluctuation of the hematopoietic stem cell phenotype with cell cycle trnsit, we hypothesize that the ability of marrow stem cells to convert to nonhematopoietic cells will also vary at different points in the cell cycle. The new biology of stem cells has an impact on many fields including developmental biology and stem cell biology and the clinical potential is enormous.

Lymphohematopoietic stem cell engraftment.

Traditional dogma has stated that space needs to be opened by cytoxic myeloablative therapy in order for marrow stem cells to engraft. Recent work in murine transplant models, however, indicates that engraftment is determined by the ratio of donor to host stem cells, i.e., stem cell competition. One hundred centigray whole body irradiation is stem cell toxic and nonmyelotoxic, thus allowing for higher donor chimerism in a murine syngeneic transplant setting. This nontoxic stem cell transplantation can be applied to allogeneic transplant with the addition of a tolerizing step; in this case presensitization with donor spleen cells and administration of CD40 ligand antibody to block costimulation. The stem cells that engraft in the nonmyeloablated are in G0, but are rapidly induced (by 12 hours) to enter the S phase after in vivo engraftment. Exposure of murine marrow to cytokines (IL-3, IL-6, IL-11 and steel factor) expands progenitor clones, induces stem cells into cell cycle, and causes a fluctuating engraftment phenotype tied to phase of cell cycle. These data indicate that the concepts of stem cell competition and fluctuation of stem cell phenotype with cell cycle transit should underlie any new stem cell engraftment strategy.

Stem cell engraftment strategies.

The donor stem cell phenotype and host microenvironment determine the outcome of a stem cell transplant. In a series of transplant studies in syngeneic male to female or congenic Ly5.1/Ly5.2 models in which hosts have received no or minimal irradiation (100 cGy), evidence overwhelmingly supports the concept that syngeneic engraftment is determined by stem cell competition. These approaches can be extended to H-2 mismatched allogeneic mouse combination when antigen pre-exposure and CD40-CD40 ligand antibody blockage are employed. A human trial in patients with resistant neoplasia infusing pheresed blood with 10(8) CD3 cells/kg showed that tumor responses and complete chimerism occur with very low levels of CD34+ cells/kg and that the extent of previous treatment is a critical factor in determining chimerism. A major feature of transplants is the phenotype of the donor stem cell. This phenotype shows dramatic reversible plasticity involving differentiation, adhesion protein expression, and engraftment with cytokine-induced cell-cycle transit. Homing is probably also plastic. Marked fluctuations in engraftment capacity are also seen at different points in marrow circadian rhythm.

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.

Chiaroscuro hematopoietic stem cell.

These observations suggest several immediate clinical strategies. In gene therapy, approaches could be targeted to obtain cycling of hematopoietic stem cells and gene-carrying retrovirus vector integration followed by engraftment at an appropriate time interval which favors engraftment. The same type of approach can be utilized for stem cell expansion approaches. Alternatively marrow or peripheral stem cell engraftment can be obtained with minimal to no toxicity in allochimeric strategies in such diseases as sickle cell anemia or thalassemia. A similar approach could be useful in obtaining cell engraftment with minimal toxicity in therapies employing cellular immune (T-cell and NK-cell) attack against cancer. These areas of clinical application are outline in Table 3.

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.

Fluorescence activated cell sorting: a window on the stem cell.

Fluorescence activated cell sorting (FACS) in the field of stem cell biology has become an indispensable tool for defining and separating rare cell populations with a high degree of purity. Steady progress has been made in this regard, but the intrinsic lability of the stem cell phenotype presents a different challenge and there are many technical caveats. FACS remains, however, the technology of choice for reporting and characterizing rare cell populations such as stem 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.