The ideally engineered bone should have similar structural and functional properties to the native tissue. Although structural integrity is critical for functional bone regeneration, we know less about modulating the structural properties of the engineered bone elicited by bone morphogenetic protein (BMP) than efficacy and safety. Erythropoietin (Epo), a primary erythropoietic hormone, has been used to augment blood transfusion in orthopedic surgery. However, the effects of Epo on bone regeneration are not well known. Here, we determined the role of Epo in BMP2-induced bone regeneration using a cranial defect model. Epo administration improved the quality of BMP2-induced bone and more closely resembled natural cranial bone with a higher bone volume (BV) fraction and lower marrow fraction when compared with BMP2 treatment alone. Epo increased red blood cells (RBCs) in peripheral blood and also increased hematopoietic and mesenchymal stem cell (MSC) populations in bone marrow. Consistent with our previous work, Epo increased osteoclastogenesis both in vitro and in vivo. Results from a metatarsal organ culture assay suggested that Epo-promoted osteoclastogenesis contributed to angiogenesis because angiogenesis was blunted when osteoclastogenesis was blocked by alendronate (ALN) or osteoprotegerin (OPG). Earlier calcification of BMP2-induced temporary chondroid tissue was observed in the Epo+BMP group compared to BMP2 alone. We conclude that Epo significantly enhanced the outcomes of BMP2-induced cranial bone regeneration in part through its actions on osteoclastogenesis and angiogenesis.
Blood and bone are dynamic tissues that are continuously renewed through out life. Early observations based upon the proximity of bone and hematopoietic progenitor populations in marrow suggested that interactions between skeletal and hematopoietic elements are likely to be crucial in the development and function of each system. As a result of these morphologic observations, several groups have demonstrated that the osteoblasts play an important role in hematopoiesis by serving as a specific local microenvironment, or “niche”, for hematopoietic stem cells (HSC). Significant new developments in this area of active investigation have emerged since our last examination of this area in 2005. Here we discuss these new insights into the function and morphology of the HSC niche with a particular focus on cells of the osteoblastic lineage.
The mechanisms that regulate hematopoietic stem cell (HSC) dormancy and self-renewal are well established and are largely dependent on signals emanating from the HSC niche. Recently, we found that prostate cancer (PCa) cells target the HSC niche in mouse bone marrow (BM) during metastasis. Little is known, however, as to how the HSC niche may regulate dormancy in cancer cells. In this study, we investigated the effects of TANK binding kinase 1 (TBK1) on PCa dormancy in the BM niche. We found that binding with niche osteoblasts induces the expression of TBK1 in PCa cells PC3 and C4-2B. Interestingly, TBK1 interacts with mammalian target of rapamycin (mTOR) and inhibits its function. Rapamycin, an mTOR inhibitor, induces cell cycle arrest of PCa cells and enhances chemotherapeutic resistance of PCa cells. As a result, the knockdown of TBK1 decreases PCa stem-like cells and drug resistance in vitro and in vivo. Taken together, these results strongly indicate that TBK1 plays an important role in the dormancy and drug resistance of PCa.
Tumors recruit mesenchymal stem cells (MSCs) to facilitate healing, which induces their conversion into cancer-associated fibroblasts that facilitate metastasis. However, this process is poorly understood on the molecular level. Here we show that the CXCR6 ligand CXCL16 facilitates MSC or Very Small Embryonic-Like (VSEL) cells recruitment into prostate tumors. CXCR6 signaling stimulates the conversion of MSCs into cancer-associated fibroblasts, which secrete stromal-derived factor-1, also known as CXCL12. CXCL12 expressed by cancer-associated fibroblasts then binds to CXCR4 on tumor cells and induces an epithelial to mesenchymal transition, which ultimately promotes metastasis to secondary tumor sites. Our results provide the molecular basis for MSC recruitment into tumors and how this process leads to tumor metastasis.
“Cancer” is a disease that can spread to the other organs over time. The prognosis of cancer patients with metastasis is generally poor. Accordingly, there is an urgent need to establish a greater understanding of metastatic processes. It is highly likely that cancer stem cells (CSCs) are the key cells that mediate metastases, even while the cellular origin of CSCs remains unknown. Growing evidence has also revealed that the microenvironment has profound effects on the regulation of CSCs. Recently, it has been shown that bone metastatic cancer cells target the microenvironment or ‘niche’ which houses hematopoietic stem cells (HSCs). The major function of the HSC niche is to maintain ‘stemness’ of HSCs. These findings suggest that by targeting the HSC niche metastatic cells parasitize the very foundation of hematopoiesis to maintain their stemness. These observations suggest that there will be a need to target the HSC niche to provide effective therapies to eradicate metastatic CSCs.
Cancer stem cells; Bone marrow niche; Disseminated tumor cells; and Tumor heterogeneity
The receptor tyrosine kinase Axl is over-expressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our lab indicates that Axl and its ligand GAS6 may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer (PCa) cell lines PC3 and DU145 and has negligible levels of expression in a non-metastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial to mesenchymal transition in PCa cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were down-regulated. Moreover, CoCl2, a hypoxia mimicking agent, prevented GAS6 mediated down-regulation of Axl in these cell lines. Immunochemical staining of human PCa tissue microarrays demonstrated that Axl, GAS6 and Hif1-α (indicator of hypoxia) were all co-expressed in PCa and in bone metastases, compared to normal tissues. Together, our studies indicate that Axl plays a crucial role in PCa metastasis, and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression maintained leading to enhanced signaling.
Bone Metastasis; prostate cancer; EMT; Hypoxia; tumor microenvironment; GAS6; Axl
Disseminated tumor cells (DTCs) are believed to lie dormant in the marrow before they can be activated to form metastases. How DTCs become dormant in the marrow and how dormant DTCs escape dormancy remains unclear. Recent work has shown that prostate cancer (PCa) cell lines express the growth-arrest specific 6 (GAS6) receptors Axl, Tyro3, and Mer, and become growth arrested in response to GAS6. We therefore hypothesized that GAS6 signaling regulates the proliferative activity of DTCs in the marrow. To explore this possibility, in vivo studies were performed where it was observed that when Tyro3 expression levels exceed Axl expression, the PCa cells exhibit rapid growth. When when Axl levels predominate, PCa cells remain largely quiescent. These findings suggest that a balance between the expression of Axl and Tyro3 is associated with a molecular switch between a dormant and a proliferative phenotype in PCa metastases.
Leadership is vital in all professions and organizations. Our purpose was to determine where in dental schools leadership is taught, and to what degree it is emphasized so that we could establish a base line from which to generate recommendations for best practices. Therefore we surveyed all US Deans of Academic Affairs in Dental Schools to determine where in the curriculum leadership is taught and emphasized. Our results showed that leadership training is delivered in many different parts of the curriculum, and at various levels. Generally, respondents indicated that leadership education is delivered either in the setting of practice management, community outreach or in public health settings. In some cases, specific training programs are dedicated specifically to leadership development. Thus several models for leadership development were identified showing design and flexibility to address regional and national needs. In the future it would be of value to assess the effectiveness of the different models and whether single or multiple pathways for leadership training are most beneficial.
A subpopulation of men that appear cured of prostate cancer (PCa) develop bone metastases many years after prostatectomy. This observation indicates that PCa cells were present outside of the prostate at the time of prostatectomy and remained dormant. Several lines of evidence indicate that there are disseminated tumor cells (DTCs) in the bone marrow at the time of prostatectomy. DTCs parasitize the bone microenvironment, where they derive support and impact the microenvironment itself. These DTCs appear to be a heterogeneous population of PCa cells; however, some of them appear to have some aspects of a cancer stem cell (CSC) phenotype as they can develop into clinically detectable metastases. The concept of CSC is controversial; however, several markers of CSC have been identified for PCa, which may represent cells of either basal or luminal origin. These DTCs have now been shown to compete for the hematopoietic stem cell niche in bone, where they may be placed in a dormant state. Interaction with a variety of host factors, including cytokine and cells, may impact the metastatic development and progression, including the dormant state. For example, myeloid cells have been shown to impact both the premetastatic niche and established tumors. Understanding the concepts of how PCa successfully parasitizes the bone microenvironment is paramount toward identifying therapeutic candidates to prevent or diminish PCa bone metastases.
cancer stem cell; monocytes; prostate cancer; hematopoietic stem cell; microenvironment
One of the most life-threatening complications of prostate cancer is skeletal metastasis. In order to develop treatment for metastasis, it is important to understand its molecular mechanisms. Our work in this field has drawn parallels between hematopoietic stem cell and prostate cancer homing to the marrow. Our recent work demonstrated that annexin II expressed by osteoblasts and endothelial cells plays a critical role in niche selection. In this study, we demonstrate that annexin II and its receptor play a crucial role in establishing metastasis of prostate cancer. Prostate cancer cell lines migrate toward annexin II and the adhesion of prostate cancer to osteoblasts and endothelial cells was inhibited by annexin II. By blocking annexin II or its receptor in animal models, short-term and long-term localization of prostate cancers are limited. Annexin II may also facilitate the growth of prostate cancer in vitro and in vivo by the MAPK pathway. These data strongly suggest annexin II and its receptor axis plays a central role in prostate cancer metastasis, and that prostate cancer utilize the hematopoietic stem cell homing mechanisms to gain access to the niche.
Annexin II; Annexin II receptor; prostate cancer; metastasis; niche
'Cancer' is a disease that can spread to the other organs over time. The prognosis of cancer patients with metastasis is generally poor. Accordingly, there is an urgent need to establish a greater understanding of metastatic processes. It is highly likely that cancer stem cells (CSCs) are the key cells that mediate metastases, even while the cellular origin of CSCs remains unknown. Growing evidence has also revealed that the microenvironment has profound effects on the regulation of CSCs. Recently, it has been shown that bone metastatic cancer cells target the microenvironment or 'niche', which houses hematopoietic stem cells (HSCs). The major function of the HSC niche is to maintain 'stemness' of HSCs. These findings suggest that by targeting the HSC niche, metastatic cells parasitize the very foundation of hematopoiesis to maintain their stemness. These observations suggest that there will be a need to target the HSC niche to provide effective therapies to eradicate metastatic CSCs.
The hematopoietic stem cell (HSC) niche in the bone marrow has been studied extensively over the past few decades, yet the bone marrow microenvironment that supports the growth of metastatic prostate cancer (PCa) has only been recently considered to be a specialized “niche” as well. New evidence supports the fact that disseminated tumor cells (DTCs) of PCa actually target the HSC niche, displace the occupant HSCs, and take up residence in the pre-existing niche space. This review describes some of the evidence and mechanisms by which DTCs act as molecular parasites of the HSC niche. Furthermore, the interactions between DTCs, HSCs, and the niche may provide new targets for niche-directed therapy, as well as insight into the perplexing clinical manifestations of metastatic PCa disease.
Dormancy; Hematopoietic Stem Cell; Osteoblasts; Prostate Cancer; Metastasis; Niche
Although the hematopoietic stem cell (HSC) niche has been an active area of study, the concept of the bone marrow microenvironment (BMM) harboring a niche for solid metastatic tumor cells has only recently been considered. The HSC niche and microenvironment that is thought to constitute the solid tumor niche share many of the same structural and functional components, suggesting the possibility that the HSC and tumor niche are one in the same. The osteoblast is a critical component for each of these niches, and is important for regulating cellular processes such homing and migration, growth and survival, and quiescence and dormancy. Current understanding of the HSC niche may provide more insight to better defining the solid tumor niche. As role of the niche in regulating these processes is better understood, new insights to the role of the BMM in metastatic disease may be gained, and provide more potential targets for therapy.
niche; bone marrow microenvironment; hematopoietic stem cell; metastasis; disseminated tumor cell
Hematopoietic stem cells (HSC) are maintained in a tightly regulated bone microenvironment constituted by a rich milieu of cells. Bone cells such as osteoblasts are associated with niche maintenance as regulators of the endosteal microenvironment. Bone remodeling also plays a role in HSC mobilization although it is poorly defined. The effects of zoledronic acid (ZA), a potent bisphosphonate that inhibits bone resorption, were investigated on bone marrow cell populations focusing on HSCs, and the endosteal and vascular niches in bone. ZA treatment significantly increased bone volume and HSCs in both young and adult mice (4 week and 4 month old, respectively). ZA increased vessel numbers with no overall change in vascular volume in bones of young and had no effect on vasculature in adult mice. Since both young and adult mice had increased HSCs and bone mass with differing vasculature responses, this suggests that ZA indirectly supports HSCs via the osteoblastic niche and not the vascular niche. Additionally, gene expression in Lin- cells demonstrated increased expression of self-renewal-related genes Bmi1 and Ink4a suggesting a role of ZA in the modulation of cell commitment and differentiation toward a long-term self-renewing cell. Genes that support the osteoblastic niche, BMP2 and BMP6 were also augmented in ZA treated mice. In conclusion, ZA-induced HSC expansion occurs independent of the vascular niche via indirect modulation of the osteoblastic niche.
HEMATOPOIETIC STEM CELLS; NICHE; BONE VASCULATURE; BLOOD VESSELS; BISPHOSPHONATE; ZOLEDRONIC ACID
Despite significant improvement in therapy, the prognosis of cancer with bone metastasis is generally poor. Therefore, there is a great need for new therapeutic approaches for metastatic disease. It has been appreciated that tumor cells metastasize to bone using similar mechanisms of hematopoietic stem cell (HSC) homing to bone marrow (e.g. CXCL12/CXCR4). It was recently found that prostate cancer (PCa) cells target the bone marrow microenvironment for HSCs, or HSC niche, during metastasis. Importantly, these disseminated PCa cells can be mobilized out of the niche using HSC mobilizing agents. These findings suggest that bone marrow HSC niche is a potential therapeutic target for metastatic disease. Therefore, the hypothesis worth considering is that agents that can disrupt the interactions between tumor cells and the HSC niche may prove efficacious when used in conjunction with standard chemotherapeutic agents. Although further understanding of the tumor-niche interactions is needed, the concept of targeting the niche in conjunction with chemotherapy could open up new windows to eradicate incurable metastatic diseases.
Prostate cancer (PCa) metastases and hematopoietic stem cells (HSCs) frequently home to the bone marrow where they compete to occupy the same HSC niche. We have also shown that under conditions of hematopoietic stress, HSCs secrete the bone morphogenetic proteins (BMPs)-2 and BMP-6 that drives osteoblastic differentiation from mesenchymal precursors. Because it is not known, we examined if metastatic PCa cells can alter regulation of normal bone formation by HSCs and hematopoietic progenitor cells (HPCs). HSC/HPCs isolated from mice bearing non-metastatic and metastatic tumor cells were isolated and their ability to influence osteoblastic and osteoclastic differentiation was evaluated. When the animals were inoculated with the LNCaP C4-2B cell line which produces mixed osteoblastic and osteolytic lesions in bone, HPCs but not HSCs were able to induced stromal cells to differentiate down an osteoblastic phenotype. Part of the mechanism responsible for this activity was the production of BMP-2. On the other hand, when the animals were implanted with PC3 cells that exhibits predominantly osteolytic lesions in bone, HSCs derived from these animals were capable of directly differentiating into tartrate-resistant acid phosphatase (TRAP) positive osteoclasts through an interleukin-6 (IL-6) mediated pathway. These studies for the first time identify HSC/HPCs as novel targets for future therapy involved in the bone abnormalities of PCa.
Hematopoietic stem cells; osteoblast; osteoclast; differentiation; disseminated tumor cells
The role of erythropoietin (Epo) and Epo/Epo receptor (EpoR) signaling pathways for production of red blood cells are well established. However, little is known about Epo/EpoR signaling in non-hematopoietic cells. Recently, we demonstrated that Epo activates JAK/STAT signaling in hematopoietic stem cells (HSCs), leading to the production of bone morphogenetic protein 2 (BMP2) and bone formation and that Epo also directly activate mesenchymal cells to form osteoblasts in vitro. In this study, we investigated the effects of mTOR signaling on Epo-mediated osteoblastogenesis and osteoclastogenesis. We found that mTOR inhibition by rapamycin blocks Epo-dependent and -independent osteoblastic phenotypes in human bone marrow stromal cells (hBMSCs) and ST2 cells, respectively. Furthermore, we found that rapamycin inhibits Epo-dependent and -independent osteoclastogenesis in mouse bone marrow mononuclear cells and Raw264.7 cells. Finally, we demonstrated that Epo increases NFATc1 expression and decreases cathepsin K expression in an mTOR-independent manner, resulting in an increase of osteoclast numbers and a decrease in resorption activity. Taken together, these results strongly indicate that mTOR signaling plays an important role in Epo-mediated bone homeostasis.
osteoblasts; HSCs; osteoclasts; erythropoietin; mTOR; rapamycin
Cross-talk between hematopoietic stem cells (HSCs) and the cells comprising the niche is critical for maintaining stem cell activities. Yet little evidence supports the concept that HSCs regulate development of the niche. Here the ability of HSCs to directly regulate endosteal development was examined. Marrow was isolated 48h after ‘stressing’ mice with a single acute bleed or from control non-stressed animals. ‘Stressed’ and ‘non-stressed’ HSCs were co-cultured with bone marrow stromal cells to map mesenchymal fate. The data suggest that HSCs are able to guide mesenchymal differentiation towards the osteoblastic lineage under basal conditions. HSCs isolated from animals subjected to an acute stress were significantly better at inducing osteoblastic differentiation in vitro and in vivo than from control animals. Importantly, HSC-derived BMP-2 and BMP-6 were responsible for these activities. Furthermore, significant differences in the ability of HSCs to generate a BMP response following stress were noted in aged and in osteoporotic animals. Together these data suggest a coupling between HSC functions and bone turnover as in aging and in osteoporosis. For the first time, these results demonstrate that HSCs do not rest passively in their niche. Instead, they directly participate in bone formation and niche activities.
HSCs; Niche; Osteoblasts; MSCs; Endosteal
For metastasis to occur, tumor cells must first detach from their tissue of origin. This requires altering both the tissue of origin and the cancer cell. Once detached, cancer cells in circulation must also acquire survival mechanisms. Although many may successfully disseminate, variation exists in the efficiency with which circulating tumor cells home to and invade the bone marrow as metastastic seeds. Disseminated tumor cells that do successfully invade the marrow are secured by cell–cell and cell–extracellular matrix adhesion. However, establishing a foothold in the marrow is not sufficient for disseminated tumor cells to create metastases. A significant latent phase must be overcome by either rescuing cellular proliferation or attenuating micrometastatic mass dormancy programs. Finally, growing metastases fuel osteolysis, osteoblastogenesis and T-cell differentiation, creating a variety of tumor phenotypes. Each step in the metastatic cascade is rich in biological targets and mechanistic pathways.
anoikis; autophagy; circulating tumor cells; disseminated tumor cells; dormancy; metastasis; osteoblastogenesis; osteolysis; survival
Bone is the preferred metastasis site of advanced prostate cancer (PCa). Using an in vivo murine model of human PCa cell metastasis to bone, we noted that the majority of animals that develop skeletal metastasis have either spinal lesions or lesions in the bones of the hindlimb. Much less frequently, lesions develop in the bones of the forelimb. We therefore speculated whether the environment of the forelimb bones is not permissive for the growth of PCa. Consequently, data on tumor prevalence were normalized to account for the number of PCa cells arriving after intravascular injection, marrow cellularity, and number of hematopoietic stem cell niches. None of these factors were able to account for the observed differences in tumor prevalence. An analysis of differential gene and protein levels identified that growth arrest specific-6 (GAS6) levels were significantly greater in the forelimb versus hindlimb bone marrow. When murine RM1 cells were implanted into subcutaneous spaces in immune competent animals, tumor growth in the GAS6-/- animals was greater than in GAS6+/+ wild-type animals. In an osseous environment, the human PC3 cell line grew significantly better in vertebral body transplants (vossicles) derived from GAS6-/- animals than in vossicles derived from GAS6+/+ animals. Together, these data suggest that the differences in tumor prevalence after intravascular inoculation are a useful model to study the molecular basis of tumor dormancy. Importantly, these data suggest that therapeutic manipulation of GAS6 levels may prove useful as a therapy for metastatic disease.
The hematopoietic stem cell (HSC) niche in the bone marrow has been studied extensively over the past few decades, yet the bone marrow microenvironment that supports the growth of metastatic prostate cancer (PCa) has only been recently considered to be a specialized ‘niche' as well. New evidence supports the fact that disseminated tumor cells (DTCs) of PCa actually target the HSC niche, displace the occupant HSCs and take up residence in the pre-existing niche space. This review describes some of the evidence and mechanisms by which DTCs act as molecular parasites of the HSC niche. Furthermore, the interactions between DTCs, HSCs and the niche may provide new targets for niche-directed therapy, as well as insight into the perplexing clinical manifestations of metastatic PCa disease.
dormancy; hematopoietic stem cell; metastasis; niche; osteoblasts; prostate cancer
Since leadership is an essential part of the oral health professions, oral health educators can play an essential role in establishing a culture of leadership and in mentoring students to prepare them for future leadership roles within the profession. However, leadership training for oral health professionals is a relatively new concept and is frequently not found within dental and dental hygiene curricula. The purpose of this article is to propose several models for leadership training that are specific to the oral health professions. The authors hope that providing an overview of leadership programs in academic dental institutions will encourage all U.S. and Canadian dental schools to begin developing a culture that promotes leadership development.
dental education; allied dental education; leadership; leadership training; interprofessional education
Previously we reported that annexin 2 (anxa2) plays an important role in hematopoietic stem cell (HSC) localization to the endosteal/osteoblastic marrow niche. The study explored the role that annexin 2 plays in presenting stromal derived factor-1 (SDF-1 or CXCL12) to HSCs.
Materials and Methods
Competitive long-term bone marrow transplant (CLT-BMT) assays were used to determine if HSC engraftment is altered in annexin 2-deficient animals. Colony-forming cell assays, CXCL12 Elisa, and real-time RT-PCR analyses were employed to determine stem or progenitor cell mobilization by G-CSF. Immunohistochemistry, immunoprecipitation, binding assays, and chemotactic assays were employed to determine if annexin 2 is associated with CXCL12. Degradation assays were also used to determine if annexin 2 and CXCL12 protect each other from proteolytic degradation.
Anxa2−/− animals have fewer HSC in their marrows, and the HSCs in anxa2−/− animals express less CXCR4 and CXCR7 suggesting a cell intrinsic defect. Transplantation studies of wild-type marrow into anxa2−/− animals demonstrated a cell extrinsic defect in the anxa2−/− animals. CXCL12 binds directly to annexin 2, and this interaction facilitates the presentation of CXCL12 to HSCs. Yet the binding of CXCL12 to annexin 2 does not protect CXCL12 from proteolytic cleavage following stem or progenitor cell mobilization by G-CSF.
These results suggest that annexin 2 serves as an anchor for CXCL12 to help in the localization of HSCs to the niche.
Annexin 2; CXCL12; osteoblasts; HSCs; localization; homing; mobilization
We have utilized in vitro and mouse xenograft models to examine the interaction between breast cancer stem cells (CSCs) and bone marrow derived mesenchymal stem cells (MSCs). We demonstrate that both of these cell populations are organized in a cellular hierarchy in which primitive aldehyde dehydrogenase (ALDH) expressing mesenchymal cells regulate breast CSCs through cytokine loops involving IL6 and CXCL7. In NOD/SCID mice, labeled MSCs introduced into the tibia traffic to sites of growing breast tumor xenografts where they accelerate tumor growth by increasing the breast cancer stem cell population. Utilizing immunochemistry, we identified “MSC-CSC niches” in these tumor xenografts as well as in frozen sections from primary human breast cancers. Bone marrow derived mesenchymal stem cell may accelerate human breast tumor growth by generating cytokine networks that regulate the cancer stem cell population.
A prospective in vivo assay was used to identify cells with potential for multiple lineage differentiation. With this assay, it was first determined that the 5-fluorouracil resistant cells capable of osseous tissue formation in vivo also migrated toward stromal derived factor-1 (SDF-1) in vitro. In parallel, an isolation method based on fluorescence-activated cell sorting was employed to identify a very small cell embryonic-like Lin−Sca-1+CD45− cell that with as few as 500 cells was capable of forming bone-like structures in vivo. Differential marrow fractionation studies determined that the majority of the Lin−Sca-1+CD45− cells reside in the subendosteal regions of marrow. To determine whether these cells were capable of differentiating into multiple lineages, stromal cells harvested from Col2.3ΔTK mice were implanted with a gelatin sponge into SCID mice to generate thymidine kinase sensitive ossicles. At 1.5 months, 2,000 green fluorescent protein (GFP)+ Lin−Sca-1+CD45− cells were injected into the ossicles. At harvest, colocalization of GFP-expressing cells with antibodies to the osteoblast-specific marker Runx-2 and the adipocyte marker PPARγ were observed. Based on the ability of the noncultured cells to differentiate into multiple mesenchymal lineages in vivo and the ability to generate osseous tissues at low density, we propose that this population fulfills many of the characteristics of mesenchymal stem cells.