ALBERT

Description: Because the microenvironment that supports hematopoietic stem cell (HSC) proliferation and differentiation is not fully understood, we adapted a heterotopic bone formation model as a new approach for studying the HSC microenvironment in vivo. Endogenous HSCs homed to tissue-engineered ossicles and individually sorted HSCs from ossicles were able to reconstitute lethally irradiated mice. To further explore this model as a system to study the stem cell niche, ossicles were established with or without anabolic parathyroid hormone (PTH) treatment during the 4-week course of bone development. Histology and micro–computed tomography showed higher bone area-to-total area ratios, thicker cortical bone and trabecular bone, significantly higher bone mineral density and bone volume fraction in PTH-treated groups than in controls. By an in vivo competitive long-term reconstitution assay, HSC frequency in the ossicle marrow was 3 times greater in PTH groups than in controls. When whole bone marrow cells were directly injected into the ossicles after lethal irradiation, the PTH-treated groups showed an enhanced reconstitution rate compared with controls. These findings suggest the residence of HSCs in heterotopic bone marrow and support the future use of this ossicle model in elucidating the composition and regulation of the HSC niche.

Description: Events localized to endosteal surfaces are critical for the maintenance of hematopoetic stem cells (HSCs). Here we explored whether HSCs themselves regulate their microenvironment directly by regulating cytokine expression by osteoblasts (OBs) in response to physiologic demands, or influence the developmental pattern of mesenchymal lineages and thereby indirectly modulate cytokine expression. To test the these possibilities, marrow was isolated from mice 48h after stressing the animals with a single acute bleed (removing 30% of the calculated blood volume by jugular vein venipucture) and in a second control group of non-stressed (puncture only) animals. The Sca-1(+) hematopoietic cells were co-cultured with confluent murine bone marrow stromal (BMSC) and calvarial digested OBs. The presence of HSCs stimulated the basal production of IL-6, SDF-1 and osteoclacin by OBs and BMSCs as determined by ELISA. Co-cultures of HSCs derived from the stressed group produced more IL-6, SDF-1 and osteocalcin relative to the non-stressed group. Newborn dermal fibroblasts did not respond in a similar fashion. To determine if HSCs influence the developmental pattern of the marrow, HSCs derived from stressed and non-stressed animals were separated from either murine OBs or BMSCs using TranswellR membranes and the ability of target cells to differentiate along the osteoblastic lineage was evaluated. A significant proportion of the colonies established from calvarial-derived OB cultures were able to mineralize their extracellular matrix relative whole BMSC population (CFU-OB). In the presence of HSCs, the proportion of non-mineralized (CFU-F) and mineralized colonies from the OB populations significantly increased. Further enhancement of both colony types were induced by the HSCs derived from stressed and non-stressed animals. Adherent cells derived from mixed BMSCs also responded to the presence of HSCs by increasing the generation of CFU-F and OBs relative to the No HSC groups. HSCs derived from stressed vs. the non-stressed groups of animals were better able to induce CFU-OB differentiation. Microarray of HSCs derived from stressed vs. the non-stressed groups, and QRT-PCR of highly purified HSCs (CD150(+), CD48(−), CD41(−), Sca-1(+), cKit(+)) suggested that BMP-2 and BMP-6 were responsible for the activities. Antibody neutralization studies confirmed these observations that BMP-2 and BMP-6 derived from the HSCs themselves alters the developmental pattern of the marrow microenvironment. In conclusion, cross-talk between HSCs-OBs is essential for the development of both cellular populations. These studies demonstrate that at least 2 mechanisms whereby HSCs might set up a paracrine loop with OBs to establish the HSC niche; (i) HSCs directly regulate cytokine expression by OBs in response to physiologic demands or, (ii) HSCs may influence the developmental pattern of mesenchymal lineages and thereby indirectly modulate cytokine expression in the marrow.

Description: We previously developed an in vivo prospective assay for identification of non-cultured cells with MSC potential. Using this assay we identified a population of cells that were slowly cycling and of low density that were capable of multilineage differentiation both in vitro and in vivo (Z. Wang et al, Stem Cells. 2006 24(6):1573). Further characterization of these cells suggested that they resemble a homogenous population of rare Lin−/Sca-1+/CD45− cells that have the morphology and express several markers of undifferentiated embryonic-like stem cells. In vitro the Lin−/Sca-1+/CD45− cells may differentiate into cells from all three germ-layers (M. Kucia et al, Leukemia. 2007 21(2):297). To determine the in vivo fate of this population, we transplanted 500 or 5,000 Lin−/Sca-1+/CD45− cells from a GFP mouse into SCID mice in each group (n=3) immediately after cell sorting to evaluate tissue generation in vivo. At 4 weeks the regenerative potential of these populations was evaluated by micro-CT and histology, and cells were tracked by gross examination of the harvested tissues by fluorescent microscopy. The results showed that a large number of GFP+ cells are located in the implants, indicating that the transplanted cells maintain the ability to contribute to the generation of new tissue. Bone-like tissue was observed in the Lin−/Sca-1+/CD45− group with as low as 500-cells/implant, while 5,000 Lin−/Sca-1+/CD45− cells generated significantly larger mineralized tissue volume, which was confirmed by micro-CT. Lin−/Sca-1+/CD45+ cell only implantation did not form any mineralized tissue, however, while mixed with 2x106 whole bone morrow cells, positive mineralized tissue occurred. Whole bone marrow mixture also improve bone formation in Lin−/Sca-1+/CD45− cell implants compared the actual bone volumes measured by micro-CT. This study demonstrates that non-cultured BM-derived Lin−/Sca-1+/CD45− cells exhibit the capacity to form bone in vivo with as low as 500 cells/implant. Whole bone marrow mixtures can enhance the bone formation, presumably through the interaction of other populations cells. Based on these findings, it is proposed that non-cultured BM-derived Lin−/Sca-1+/CD45− cells are enriched osteogenic cells that can be applied to bone regeneration in vivo.

Description: The mechanisms of bone and blood formation have traditionally been viewed as distinct, unrelated processes, but compelling evidence suggests that they are intertwined. Based on observations that hematopoietic precursors reside close to endosteal surfaces, it was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). Preceding these observations are studies demonstrating that the disruption or perturbation of normal osteoblastic function has a profound and central role in defining the operational structure of the HSC niche. These observations provide a glimpse of the dimensions and ramifications of HSC-osteoblast interactions. Although more research is required to secure a broader grasp of the molecular mechanisms that govern blood and bone biology, the central role for osteoblasts in hematopoietic stem cell regulation is reviewed herein from the perspectives of (1) historical context; (2) the role of the osteoblast in supporting stem cell survival, proliferation, and maintenance; (3) the participation, if any, of osteoblasts in the creation of a stem cell niche; (4) the molecules that mediate HSC-osteoblast interactions; (5) the role of osteoblasts in stem cell transplantation; and (6) possible future directions for investigation.

Description: Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin−Sca-1+Rag-2− BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Δ-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.

Description: Based on anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, we have begun an analysis of osteoblast/hematopoietic cell interactions. We explore here the functional interdependence between these two cell types from the standpoint of de novo cytokine secretion. We determined that, over a 96-hour period, CD34+ bone marrow cells had no significant effect on osteoblast secretion of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-β1 , but in some experiments minor increases in leukemia inhibitory factor levels were observed. However, when CD34+ bone marrow cells were cocultured in direct contact with osteoblasts, a 222% ± 55% (range, 153% to 288%) augmentation in interleukin-6 (IL-6) synthesis was observed. The accumulation of IL-6 protein was most rapid during the initial 24-hour period, accounting for nearly 55% of the total IL-6 produced by osteoblasts in the absence of blood cells and 77% of the total in the presence of the CD34+ cells. Cell-to-cell contact does not appear to be required for this activity, as determined by coculturing the two cell types separated by porous micromembranes. The identity of the soluble activity produced by the CD34+ cells remains unknown, but is not likely due to IL-1β or tumor necrosis factor-α, as determined with neutralizing antibodies. To our knowledge, these data represent the first demonstration that early hematopoietic cells induce the production of molecules required for the function of normal bone marrow microenvironments, in this case through the induction of hematopoietic cytokine (IL-6) secretion by osteoblasts.

Description: Metastasis is the most deadly aspect of many solid tissue cancers. The majority of metastases utilize pathways established to regulate hematopoietic stem cell (HSC) homing to gain access to the marrow. Whether solid tumors target the niche to establish footholds in the marrow is unknown. Using prostate caner (PCa) as a model, we determined if PCa and HSC compete for the HSC niche. To determine if disseminated PCa competes for the niche and prevents HSC engraftment, sham-operated or tumor-implanted NOD/SCID mice (Ly5.1) were transplanted with marrow cells from Ly5.2 donor animals. To preserve the niche, the transplanted animals received no preconditioning. At 16 weeks, greater HSC engraftment was observed in sham-operated group than in the tumor-bearing group as reflected by mature HSC progeny in the peripheral blood (0.7 ± 0.4% vs. 0.2 ± 0.2%, p = 0.022). Multiphoton microscopy revealed that PCa and SLAM receptor isolated HSCs co-localize at the endosteal bone surfaces in vivo and in vitro, further suggesting that PCa compete for the niche. Since osteoblasts (OBs) are vital components of the niche, it was determined if alterations in OB numbers also regulates metastasis. Here, vertebrae from control or conditional osteoblast knockout (Col2.3Δ-TK) mice were transplanted into SCID animals. Following treatment with ganciclovir which ablated the osteoblasts, metastasis to these tissues was evaluated. Fewer disseminated PCa were recovered from the Col2.3Δ-TK transplants than vehicle treated transplants (41.6 ± 23.7% vs. 100.0 ± 69.9%, p = 0.009). If metastases target the niche, then we should be able to mobilize metastases using HSC mobilizing regimes. Here, PCa tumors were established and removed. Later G-CSF was administered, and PCa numbers were determined in the blood. More PCa cells were seen in the blood after G-CSF treatments vs. the vehicle group (616.9 ± 463.0 cells vs. 143.7 ± 159.1 cells, p = 0.025). Part of the mechanism responsible for the mobilization of PCa was an increase in the production of MMP-2 and MMP-9 in the marrow. Finally, we evaluated if soluble factors produced by PCa alter HSCs activities. Animals were treated with either control or PCa conditioned medium for 3 days, and then HSC were harvested. Alterations of many mRNA transcripts involved in HSC self renewal were observed in animals exposed to the PCa conditioned medium. Our findings indicate that PCa and HSCs compete for the osteoblastic HSC niche, and that PCa utilize the same mechanisms as HSCs to gain access and egress from the niche. Taken together, these data provide the first evidence that the HSC niche serves as pre-metastatic niche for solid tumors and plays a central role in bone metastases.