Proliferation Of Mesenchymal Progenitor Cells Research Articles

IL-17RA Signaling in Prx1+ Mesenchymal Cells Influences Fracture Healing in Mice.

Fracture healing is a complex series of events that requires a local inflammatory reaction to initiate the reparative process. This inflammatory reaction is important for stimulating the migration and proliferation of mesenchymal progenitor cells from the periosteum and surrounding tissues to form the cartilaginous and bony calluses. The proinflammatory cytokine interleukin (IL)-17 family has gained attention for its potential regenerative effects; however, the requirement of IL-17 signaling within mesenchymal progenitor cells for normal secondary fracture healing remains unknown. The conditional knockout of IL-17 receptor a (Il17ra) in mesenchymal progenitor cells was achieved by crossing Il17raF/F mice with Prx1-cre mice to generate Prx1-cre; Il17raF/F mice. At 3 months of age, mice underwent experimental unilateral mid-diaphyseal femoral fractures and healing was assessed by micro-computed tomography (µCT) and histomorphometric analyses. The effects of IL-17RA signaling on the osteogenic differentiation of fracture-activated periosteal cells was investigated in vitro. Examination of the intact skeleton revealed that the conditional knockout of Il17ra decreased the femoral cortical porosity but did not affect any femoral trabecular microarchitectural indices. After unilateral femoral fractures, Il17ra conditional knockout impacted the cartilage and bone composition of the fracture callus that was most evident early in the healing process (day 7 and 14 post-fracture). Furthermore, the in vitro treatment of fracture-activated periosteal cells with IL-17A inhibited osteogenesis. This study suggests that IL-17RA signaling within Prx1+ mesenchymal progenitor cells can influence the early stages of endochondral ossification during fracture healing.

Tumor-derived mesenchymal progenitor cell-related genes in the regulation of breast cancer proliferation.

Breast cancer (BC) is one of the most common malignancies worldwide, and its development is affected in various ways by the tumor microenvironment (TME). Tumor-derived mesenchymal progenitor cells (MPCs), as the most important components of the TME, participate in the proliferation and metastasis of BC in several ways. In this study, we aimed to characterize the genes associated with tumor-derived MPCs and determine their effects on BC cells. Tumor-derived MPCs and normal breast tissue-derived mesenchymal stem cells (MSCs) were isolated from tissues specimens of patients with BC. We conducted culture and passage, phenotype identification, proliferation and migration detection, inflammatory factor release detection, and other experiments on isolated MPCs from tumors and MSCs from normal breast tissues. Three paired tumor-derived MPCs and normal breast tissue-derived MSCs were then subjected to transcriptome analysis to determine the expression profiles of the relevant genes, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to further confirm gene expression. Subsequently, the overexpression plasmids were transfected into tumor-derived MPCs, and the expression of various inflammatory factors of tumor-derived MPCs and their proliferation were characterized with a cell viability test reagent (Cell Counting Kit 8). Subsequently, the transfected tumor-derived MPCs were cocultured with BC cells using a conditioned medium coculture method to clarify the role of tumor-derived MSCs in BC. Tumor-derived MPCs expressed stem cell characteristics including CD105, CD90, and CD73 and exhibited adipogenic and osteogenic differentiation in vitro. The proliferation of tumor-derived MPCs was significantly lower than that of normal breast tissue-derived MSCs, and the invasive metastatic ability was comparable; however, MPCs were found to release inflammatory factors such as interleukin 6 (IL-6) and transforming growth factor β (TGF-β). Transcriptome analysis showed that stomatin (STOM), collagen and calcium binding EGF domains 1 (CCBE1), and laminin subunit alpha 5 (LAMA5) were significantly upregulated in tumor-derived MPCs. Among them, STOM was highly expressed in tumor-derived MPCs, which mediated the slow proliferation of MPCs and promoted the proliferation of BC cells. STOM, CCBE1, and LAMA5 were highly expressed in tumor-derived MPCs, with STOM being found to retard the proliferation of MPCs but promote the proliferation of BC cells. There findings present new possibilities in targeted microenvironmental therapy for BC.

Age-associated callus senescent cells produce TGF-β1 that inhibits fracture healing in aged mice.

Cellular senescence plays an important role in human diseases, including osteoporosis and osteoarthritis. Senescent cells (SCs) produce the senescence-associated secretory phenotype to affect the function of neighboring cells and SCs themselves. Delayed fracture healing is common in the elderly and is accompanied by reduced mesenchymal progenitor cells (MPCs). However, the contribution of cellular senescence to fracture healing in the aged has not to our knowledge been studied. Here, we used C57BL/6J 4-month-old young and 20-month-old aged mice and demonstrated a rapid increase in SCs in the fracture callus of aged mice. The senolytic drugs dasatinib plus quercetin enhanced fracture healing in aged mice. Aged callus SCs inhibited the growth and proliferation of callus-derived MPCs (CaMPCs) and expressed high levels of TGF-β1. TGF-β–neutralizing Ab prevented the inhibitory effects of aged callus SCs on CaMPCs and promoted fracture healing in aged mice, which was associated with increased CaMPCs and proliferating cells. Thus, fracture triggered a significant cellular senescence in the callus cells of aged mice, which inhibited MPCs by expressing TGF-β1. Short-term administration of dasatinib plus quercetin depleted callus SCs and accelerated fracture healing in aged mice. Senolytic drugs represent a promising therapy, while TGF-β1 signaling is a molecular mechanism for fractures in the elderly via SCs.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-β signaling

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-β signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-β signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-β signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

Transcriptome analysis of tumor-derived mesenchymal progenitor cells shows that CHST15 is a fibrosis regulator of retroperitoneal liposarcoma.

BackgroundRetroperitoneal liposarcoma (RPLS) is a rare, biologically heterogeneous tumor with distinct clinical characteristics, such as frequent local recurrence, repeated relapse, and rare distant metastasis. No effective targeted therapy is available for RPLS. Here, we aim to determine the pathological functions and therapeutic potential of carbohydrate sulfotransferase 15 (CHST15) in RPLS.MethodsTumor-derived mesenchymal progenitor cells (MPCs) and normal adipose derived mesenchymal stem cells (MSCs) were obtained from patients with RPLS. MPCs and MSCs were isolated and characterized based on surface markers, proliferation, and differentiation using flow cytometry and molecular staining. Transcriptome analysis was performed to decipher expression profile of differentiation-related genes in 3 paired MSCs and MPCs. Further confirmation of genes were performed using quantitative real-time polymerase chain reaction (qRT-PCR). Plasmids overexpressing CHST15 were transfected into adipose MSCs to examine fibrosis-related gene expression at mRNA level by real-time PCR.ResultsThe tumor stromal-derived MPCs expressed CD105, CD73, and CD90, and exhibited osteogenic and adipogenic differentiation potential in vitro. The proliferation of tumor-derived MPCs was significantly lower than that of normal adipose-derived MSCs (P<0.001). Transcriptome analysis revealed upregulation of IL-7R, ALPL, PKNOX2, and CHST15 in tumor-derived MPCs. CHST15 was highly expressed in tumor-derived MPCs (P<0.001). CHST15 mediated fibrosis-related FGF2 gene expression in MSCs (P<0.05) and MPCs (P<0.001).ConclusionsCHST15 is upregulated in tumor-derived MPCs and regulates fibrosis in RPLS. This provides clues for development of novel therapeutic strategies by targeting CHST15-induced MPC activation in RPLS.

Characterization of plasma fibronectin for migration, proliferation, and differentiation on human articular chondrocytes.

Plasma fibronectin (pFN) plays a crucial role in wound healing by binding to integrins and inducing cell migration. It is known to induce the migration and proliferation of mesenchymal progenitor cells in vitro, which play a key role during microfracture in cartilage repair. Endogenous chondrocytes from the native cartilage of the defect rim might aid in cartilage repair. In this study, the effect of pFN on proliferation, migration, and differentiation was tested on human articular chondrocytes. Results showed that treatment with pFN increased the migration of chondrocytes in a range of 1-30μg/ml as tested with no effect on proliferation. TGFβ3-induced chondrogenesis was not affected by pFN. Especially, gene expression of matrix metalloproteinases was not increased by pFN. Plasma FN fragmentation due to storage conditions could be excluded by SDS-PAGE. Moreover, bioactivity of pFN did not alter during storage at 4°C and 40°C for up to 14days. Taken together, pFN induces the migration but not proliferation of human articular chondrocytes with no inhibitory effect on chondrogenic differentiation. Additionally, no loss of activity or fragmentation of pFN was observed after lyophilization and storage, making pFN an interesting bioactive factor for chondrocyte recruitment.

Transcriptomic analysis of different tissue layers in antler growth Center in Sika Deer (Cervus nippon)

BackgroundWith the unprecedented rapid growth rate (up to 2.75 cm/day), velvet antler is an invaluable model for the identification of potent growth factors and signaling networks for extremely fast growing tissues, mainly cartilage. Antler growth center (AGC) locates in its tip and consists of five tissue layers: reserve mesenchyme (RM), precartilage (PC), transition zone (TZ), cartilage (CA) and mineralized cartilage (MC). The aim of this study was to investigate the transcription dynamics in the AGC using RNA-seq technology.ResultsFive tissue layers in the AGC were collected from three 3-year-old male sika deer using our previously reported sampling method (morphologically distinguishable). After sequencing (15 samples; triplicates/tissue layer), we assembled a reference transcriptome de novo and used RNA-seq to measure gene expression profiles across these five layers. Nine differentially expressed genes (DEGs) were selected from our data and subsequently verified using qRT-PCR. The results showed a high consistency with the RNA-seq results (R2 = 0.80). Nine modules were constructed based on co-expression network analysis, and these modules contained 370 hub genes. These genes were found to be mainly involved in mesenchymal progenitor cell proliferation, chondrogenesis, osteogenesis and angiogenesis. Combination of our own results with the previously published reports, we found that Wnt signaling likely plays a key role not only in stimulating the antler stem cells or their immediate progeny, but also in promoting chondrogenesis and osteogenesis during antler development.ConclusionWe have successfully assembled a reference transcriptome, generated gene expression profiling across the five tissue layers in the AGC, and identified nine co-expressed modules that contain 370 hub genes and genes predorminantly expressed in and highly relevant to each tissue layer. We believe our findings have laid the foundation for the identification of novel genes for rapid proliferation and chondrogenic differentiation of antler cells.

Pentosan polysulfate binds to STRO-1+ mesenchymal progenitor cells, is internalized, and modifies gene expression: a novel approach of pre-programing stem cells for therapeutic application requiring their chondrogenesis

BackgroundThe pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. However, the mechanism(s) of action of PPS in mediating these effects remains unresolved.In the present report we address this issue by investigating the binding and uptake of PPS by MPCs and monitoring gene expression and proteoglycan biosynthesis before and after the cells had been exposed to limited concentrations of PPS and then re-established in culture in the absence of the drug (MPC priming).MethodsImmuno-selected STRO-1+ mesenchymal progenitor stem cells (MPCs) were prepared from human bone marrow aspirates and established in culture. The kinetics of uptake, shedding, and internalization of PPS by MPCs was determined by monitoring the concentration-dependent loss of PPS media concentrations using an enzyme-linked immunosorbent assay (ELISA) and the uptake of fluorescein isothiocyanate (FITC)-labelled PPS by MPCs. The proliferation of MPCs, following pre-incubation and removal of PPS (priming), was assessed using the Wst-8 assay method, and proteoglycan synthesis was determined by the incorporation of 35SO4 into their sulphated glycosaminoglycans. The changes in expression of MPC-related cell surface antigens of non-primed and PPS-primed MPCs from three donors was determined using flow cytometry. RNA sequencing of RNA isolated from non-primed and PPS-primed MPCs from the same donors was undertaken to identify the genes altered by the PPS priming protocol.ResultsThe kinetic studies indicated that, in culture, PPS rapidly binds to MPC surface receptors, followed by internalisation and localization within the nucleus of the cells. Following PPS-priming of MPCs and a further 48 h of culture, both cell proliferation and proteoglycan synthesis were enhanced. Reduced expression of MPC-related cell surface antigen expression was promoted by the PPS priming, and RNA sequencing analysis revealed changes in the expression of 42 genes.ConclusionThis study has shown that priming of MPCs with low concentrations of PPS enhanced chondrogenesis and MPC proliferation by modifying their characteristic basal gene and protein expression. These findings offer a novel approach to re-programming mesenchymal stem cells for clinical indications which require the repair or regeneration of cartilaginous tissues such as in osteoarthritis and degenerative disc disease.

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction.

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/β-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/β-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Pericyte response to contraction mode-specific resistance exercise training in human skeletal muscle.

Skeletal muscle satellite cells (SCs) are important for muscle repair and hypertrophy in response mechanical stimuli. Neuron-glial antigen 2-positive (NG2(+)) and alkaline phosphatase-positive (ALP(+)) pericytes may provide an alternative source of myogenic progenitors and/or secrete paracrine factors to induce Pax7(+) SC proliferation and differentiation. The purpose of this study was to investigate NG2(+) and ALP(+) cell quantity, as well as SC content and activation, in human skeletal muscle following prolonged concentric (Conc) or eccentric (Ecc) resistance training. Male subjects engaged in unilateral resistance training utilizing isolated Ecc or Conc contractions. After 12 wk, muscle biopsies were analyzed for NG2(+) and ALP(+) pericytes, total Pax7(+) SCs, activated SCs (Pax7(+)MyoD(+)), and differentiating myogenic cells (Pax7(-) MyoD(+)). NG2(+) cells localized to CD31(+) vessels and the majority coexpressed ALP. NG2(+) pericyte quantity decreased following both Conc and Ecc training (P < 0.05). ALP(+) pericyte quantity declined following Conc (P < 0.05) but not Ecc training. Conversely, total Pax7(+) SC content was elevated following Conc only (P < 0.001), while Pax7(+)MyoD(+) SC content was increased following Conc and Ecc (P < 0.001). Follow up analyses demonstrated that CD90(+) and platelet-derived growth factor receptor-α (PDGFRα)(+) mononuclear cell proliferation was also increased in response to both Conc and Ecc training (P < 0.01). In summary, resistance training results in a decline in pericyte quantity and an increase in mesenchymal progenitor cell proliferation, and these events likely influence SC pool expansion and increased activation observed posttraining.

Identification of a progenitor cell population destined to form fracture fibrocartilage callus in Dickkopf-related protein 3-green fluorescent protein reporter mice.

Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3-green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.

Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells

To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen invitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P= .00041; Regen, P= .00029; Dr.PRP, P= .00051; PRP-A, P < .0001; and PRP-C, P < .0001) stimulated migration of MPCs. All platelet concentrates but one (Dr.PRP, P= .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P= .027; Regen, P= .0029; PRP-A, P= .00021; and PRP-C, P= .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP preparations did induce such differentiation. These findings suggest differing outcomes with PRP treatment in stem cell-based cartilage repair. Our findings may help to explain the variability of results in studies examining the use of PRP clinically.

Wnt/β-catenin signaling regulates the proliferation and differentiation of mesenchymal progenitor cells through the p53 pathway.

ObjectiveMesenchymal progenitor cells (MPCs) are found in articular cartilage from normal controls and patients with osteoarthritis (OA). Nevertheless, the molecular mechanisms of the proliferation and differentiation of these cells remain unclear. In this study, we aimed to determine the involvement of Wnt/β-catenin signaling in regulating the proliferation and differentiation of MPCs.MethodsMPCs were isolated from the articular cartilage of normal and OA patients. Cells were sorted by immunomagnetic cell separation. Cell proliferation capacity was evaluated using the MTT assay. Toluidine blue staining and immunostaining with anti-collagen II or anti-aggrecan antibodies were used to determine the chondrogenic differentiation capabilities of MPCs. The mRNA and protein expression of target genes were examined by quantitative real-time polymerase chain reaction and Western blotting, respectively. Knock-down of p53 expression was achieved with RNA interference.ResultsMost cells isolated from the normal and OA patients were CD105+ and CD166+ positive (Normal subjects: CD105+/CD166+, 94.6%±1.1%; OA: CD105+/CD166+, 93.5%±1.1%). MPCs derived from OA subjects exhibited decreased differentiation capabilities and enhanced Wnt/β-catenin activity. Inhibition of Wnt/β-catenin signaling promoted proliferation and differentiation, whereas activation of this pathway by treatment with rWnt3a protein decreased the proliferation and differentiation of normal MPCs. Additionally, Wnt/β-catenin signaling positively regulated p53 expression, and silencing of p53 increased proliferation and differentiation of MPCs.ConclusionsWnt/β-catenin regulated the proliferation and differentiation of MPCs through the p53 pathway.

Can thrombin-activated platelet releasate compensate the age-induced decrease in cell proliferation of MSC?

Mesenchymal progenitor cells (MSCs) are promising for cell-based regeneration therapies. In elderly patients a reduced proliferation of MSCs has been described. Platelet-rich plasma (PRP) contains important factors necessary for osteogenic regeneration. The aim of this study was to find out whether the age-induced decrease in cell proliferation can be compensated by the use of supernatant of centrifuged, activated PRP (tPR). MSCs of donors of three age groups (A: young, 14-16 years, B: middle age, 36-46 years, C: older, 74-83 years) were expanded with 20% FCS alone or supplemented with thrombin-activated platelet releasate (tPR) (1%, 2.5%, and 5%) or platelet-poor plasma (PPP 5%). Cell proliferation and differentiation was measured on days 0, 3, and 7. Proliferation increased significantly in groups A and B with tPR, and non-significantly in group C. The generation times of MSCs of elderly patients were significantly increased in group C compared to groups A and B. Addition of 1% or 2.5% tPR significantly reduced population doubling times of all age groups. Adding tPR stimulates the proliferation rate of MSCs independent of donor age. For juvenile and middle-aged patients this influence was significant. Cells differentiation into osteoblasts was not influenced by addition of tPR.

RBPjκ-dependent Notch signaling regulates mesenchymal progenitor cell proliferation and differentiation during skeletal development

The Notch pathway has recently been implicated in mesenchymal progenitor cell (MPC) differentiation from bone marrow-derived progenitors. However, whether Notch regulates MPC differentiation in an RBPjkappa-dependent manner, specifies a particular MPC cell fate, regulates MPC proliferation and differentiation during early skeletal development or controls specific Notch target genes to regulate these processes remains unclear. To determine the exact role and mode of action for the Notch pathway in MPCs during skeletal development, we analyzed tissue-specific loss-of-function (Prx1Cre; Rbpjk(f/f)), gain-of-function (Prx1Cre; Rosa-NICD(f/+)) and RBPjkappa-independent Notch gain-of-function (Prx1Cre; Rosa-NICD(f/+); Rbpjk(f/f)) mice for defects in MPC proliferation and differentiation. These data demonstrate for the first time that the RBPjkappa-dependent Notch signaling pathway is a crucial regulator of MPC proliferation and differentiation during skeletal development. Our study also implicates the Notch pathway as a general suppressor of MPC differentiation that does not bias lineage allocation. Finally, Hes1 was identified as an RBPjkappa-dependent Notch target gene important for MPC maintenance and the suppression of in vitro chondrogenesis.

Folinic acid attenuates methotrexate chemotherapy‐induced damages on bone growth mechanisms and pools of bone marrow stromal cells

Chemotherapy often induces bone growth defects in pediatric cancer patients; yet the underlying cellular mechanisms remain unclear and currently no preventative treatments are available. Using an acute chemotherapy model in young rats with the commonly used antimetabolite methotrexate (MTX), this study investigated damaging effects of five once-daily MTX injections and potential protective effects of supplementary treatment with antidote folinic acid (FA) on cellular activities in the tibial growth plate, metaphysis, and bone marrow. MTX suppressed proliferation and induced apoptosis of chondrocytes, and reduced collagen-II expression and growth plate thickness. It reduced production of primary spongiosa bone, volume of secondary spongiosa bone, and proliferation of metaphyseal osteoblasts, preosteoblasts and bone marrow stromal cells, with the cellular activities being most severely damaged on day 9 and returning to or towards near normal levels by day 14. On the other hand, proliferation of marrow pericytes was increased early after MTX treatment and during repair. FA supplementation significantly suppressed chondrocyte apoptosis, preserved chondrocyte proliferation and expression of collagen-II, and attenuated damaging effects on production of calcified cartilage and primary bone. The supplementation also significantly reduced MTX effects on proliferation of metaphyseal osteoblastic cells and of bone marrow stromal cells, and enhanced pericyte proliferation. These observations suggest that FA supplementation effectively attenuates MTX damage on cellular activities in producing calcified cartilage and primary trabecular bone and on pools of osteoblastic cells and marrow stromal cells, and that it enhances proliferation of mesenchymal progenitor cells during bone/bone marrow recovery.

Bortezomib Induces Proliferation of Mesenchymal Progenitor Cells and Promotes Differentiation towards Osteoblastic Lineage.

Bortezomib is a first-in-class proteasome inhibitor approved for the therapy of relapsed, refractory multiple myeloma (MM). Two large registration trials (SUMMIT and APEX) of bortezomib in MM revealed an increase in the serum levels of bone-specific alkaline phosphatase (b-ALP) and osteocalcin (ocn) in bortezomib-responsive patients, raising the prospect that bortezomib may influence the bone marrow (BM) microenvironment in association with its anti-myeloma effect,. However, the precise cellular target of bortezomib within the BM milieu remains unknown. We hypothesized that the observed rise in b-ALP and ocn was the result of direct effects of bortezomib on osteoblast-lineage committed cells. The effect of bortezomib on osteoblastic cells was first evaluated in in vitro. When bortezomib was added to freshly isolated primary BM mononuclear cells, the CFU-Ob (osteoblastic colony-forming units) was unchanged, but the colony size was increased, with a maximum effect observed at 1 nM. In particular, bortezomib increased the number of CD45−/CD51+ cells 1.8 fold (P<0.05), while no change was noted in the CD45+ (hematopoietic) cells. These CD45−/CD51+ cells expressed Collagen-1 and b-ALP, rapidly formed VanKossa+ bone nodules in vitro and lacked the expression of Sca1 and Collagen-II, suggesting that they were of osteoblastic lineage. When mice were treated with bortezomib (0.1mg/kg biw for 3 weeks), a significant increase in CD45−/CD51+ cells was observed in the bone marrow suggesting bortezomib increases the number of osteoblast-lineage cells in vivo. Next, we sought to identify the target cell for bortezomib activity. We treated CD45− BMSCs with bortezomib in vitro for 7 days and then exposed these cells to either adipogenic or osteogenic media (with bortezomib now removed). Interestingly, both differentiated osteoblasts (i.e Van-Kossa+ cells) and adipocytes (Oil-Red staining cells) were increased, suggesting that bortezomib increases the proliferation of a bipotent MPC. We then studied the impact of bortezomib on the commitment of MPCs towards osteoblastic or adipocytic lineages by culturing CD45− cells in osteogenic and adipogenic media with concurrent treatment with bortezomib. In these conditions, we found an increased number of Alk-Phos + osteoblasts in bortezomib-treated cells and a decreased number of adipocytes in adipogenic media. These data suggest that bortezomib (a) increases the proliferation of uncommitted MPCs and (b) promotes the differentiation towards osteoblastic lineage. To investigate the mechanism of bortezomib action, we studied the effect of forskolin, a cyclic AMP analogue known to accelerate the ubiquitin-mediated degradation of RUNX-2, the master-regulator of osteogenic differentiation. Forskolin treatment abrogated the effect of bortezomib on CD45− BMSCs, suggesting that bortezomib activity is influenced by the adenylate cyclase pathway, possibly through bortezomib-mediated inhibition of RUNX-2 degradation. Bortezomib alters primitive mesenchymal cell function causing increased osteoblast lineage cells.

Targeted disruption of Mig-6 in the mouse genome leads to early onset degenerative joint disease.

Degenerative joint disease, also known as osteoarthritis, is the most common joint disorder in human beings. The molecular mechanism underlying this disease is not fully understood. Here, we report that disruption of mitogen-inducible gene 6 (Mig-6) in mice by homologous recombination leads to early onset degenerative joint disease, which is revealed by simultaneous enlargement and deformity of multiple joints, degradation of articular cartilage, and the development of bony outgrowths or osteophyte formation within joint space. The osteophyte formation appears to be derived from proliferation of mesenchymal progenitor cells followed by differentiation into chondrocytes. Absence of the Rag2 gene does not rescue the joint phenotype, excluding a role for the acquired immune system in the development of this disease. Our results provide insight into the mechanism of osteoarthritis by showing that loss of Mig-6 leads to early onset of this disease, implying that this gene or its pathway is important in normal joint maintenance. Because of the striking similarity of osteoarthritis in humans and mice, the Mig-6 mutant mouse should provide a useful animal model for studying the mechanism of this disease and for testing drugs or therapies for treating osteoarthritis.

Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells.

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.