Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice.

Articular cartilage (AC) is most susceptible to degeneration in knee osteoarthritis (OA); however, the existing treatments for OA do not target the core link of the pathogenesis-"decreased tissue cell function activity and extracellular matrix (ECM) metabolic disorders" for effective intervention. iMSC hold lower heterogeneity and great promise in biological research and clinical applications. Rps6ka2 may play an important role in the iMSC to treat OA. In this study, the CRISPR/Cas9 gene editing Rps6ka2-/- iMSC were obtained. Effect of Rps6ka2 on iMSC proliferation and chondrogenic differentiation was evaluated in vitro. An OA model was constructed in mice by surgical destabilization of medial meniscus (DMM). The Rps6ka2-/- iMSC and iMSC were injected into the articular cavity twice-weekly for 8 weeks. In vitro experiments showed that Rps6ka2 could promote iMSC proliferation and chondrogenic differentiation. In vivo results further confirmed that Rps6ka2 could improve iMSC viability to promote ECM production to attenuate OA in mice.

Deletion of Runx2 in condylar chondrocytes disrupts TMJ tissue homeostasis.

Runt-related transcription factor-2 (Runx2) is essential for chondrocyte maturation during cartilage development and embryonic mandibular condylar development. The process that chondrocytes, especially a subgroup of hypertrophic chondrocytes (HC), could transform into bone cells in mandibular condyle growth makes chondrocytes crucially important for normal endochondral bone formation. To determine whether Runx2 regulates postnatal condylar cartilage growth and tissue homeostasis, we deleted Runx2 in chondrocytes in postnatal mice and assessed the consequences on temporomandibular joint (TMJ) cartilage growth and remodeling. The cell lineage tracing data provide information demonstrating the role of chondrocytes in subchondral bone remodeling. The histologic and immunohistochemical data showed that Runx2 deficiency caused condylar tissue disorganization, including loss of HC and reduced hypertrophic zone, reduced proliferative chondrocytes, and decreased cartilage matrix production. Expression of Col10a1, Mmp13, Col2a1, Aggrecan, and Ihh was significantly reduced in Runx2 knockout mice. The findings of this study demonstrate that Runx2 is required for chondrocyte proliferation and hypertrophy in TMJ cartilage and postnatal TMJ cartilage growth and homeostasis, and that Runx2 may play an important role in regulation of chondrocyte-derived subchondral bone remodeling.

CHIP regulates bone mass by targeting multiple TRAF family members in bone marrow stromal cells.

Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal (BMS) cells derived from Chip-/- mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip-/- mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.

Specific Deletion of ß-Catenin in Col2-Expressing Cells Leads to Defects in Epiphyseal Bone.

The role of canonical Wnt/ß-catenin signaling in postnatal bone growth has not been fully defined. In the present studies, we generated ß-catenin conditional knockout (KO) mice and deleted ß-catenin in Col2-expressing chondrocytes and mesenchymal progenitor cells. Findings from analyzing the ß-cateninCol2CreER KO mice revealed severe bone destruction and bone loss phenotype in epiphyseal bone, probably due to the increase in osteoclast formation and the accumulation of adipocytes in this area. In addition, we also found bone destruction and bone loss phenotype in vertebral bone in ß-cateninCol2CreER KO mice. These findings indicate that ß-catenin signaling plays a critical role in postnatal bone remodeling. Our study provides new insights into the regulation of epiphyseal bone homeostasis at postnatal stage.

Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

Deregulation of bone forming cells in bone diseases and anabolic effects of strontium-containing agents and biomaterials.

Age-related bone loss and osteoporosis are associated with bone remodeling changes that are featured with decreased trabecular and periosteal bone formation relative to bone resorption. Current anticatabolic therapies focusing on the inhibition of bone resorption may not be sufficient in the prevention or reversal of age-related bone deterioration and there is a big need in promoting osteoblastogenesis and bone formation. Enhanced understanding of the network formed by key signaling pathways and molecules regulating bone forming cells in health and diseases has therefore become highly significant. The successful development of agonist/antagonist of the PTH and Wnt signaling pathways are profits of the understanding of these key pathways. As the core component of an approved antiosteoporosis agent, strontium takes its effect on osteoblasts at multilevel through multiple pathways, representing a good example in revealing and exploring anabolic mechanisms. The recognition of strontium effects on bone has led to its expected application in a variety of biomaterial scaffolds used in tissue engineering strategies aiming at bone repairing and regeneration. While summarizing the recent progress in these respects, this review also proposes the new approaches such as systems biology in order to reveal new insights in the pathology of osteoporosis as well as possible discovery of new therapies.

Intravenously administered BMSCs reduce neuronal apoptosis and promote neuronal proliferation through the release of VEGF after stroke in rats.

BACKGROUND: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) could ameliorate neurological deficits after stroke in the rodent. OBJECTIVE: The purpose of this study was to investigate the potential mechanisms underlying the neuroprotective effects of implanted BMSCs. METHODS: Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAo) in Sprague-Dawley rats. BMSCs were intravenously transplanted at 24 hours after MCAo. Neurological function was evaluated using modified neurological severity score and Morris water maze test. Immunohistochemistry and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining were performed to detect neuronal apoptosis and proliferation. The protein and mRNA levels of vascular endothelial growth factor (VEGF) were determined by ELISA and reverse transcriptase polymerase chain reaction, respectively. RESULTS: Significant improvement of neurological deficits was found in BMSC-treated rats compared with control animals at 14 and 28 days after MCAo (p<0.05). Histological evaluation showed that BMSCs treatment significantly promoted neuronal survival and proliferation in the ischemic boundary area. The expression of VEGF was predominantly increased in the ischemic hemisphere of BMSC-treated rats compared with the other groups. On the other hand, transduction of VEGF RNAi lentivirus partially attenuated the above described beneficial effects of systemically administered BMSCs. CONCLUSION: Our data suggest that intravenously administrated BMSCs facilitate neurological function, reduce neuronal apoptosis and promote neuronal proliferation through the release of VEGF.

Development and progress of engineering of skeletal muscle tissue.

Engineering skeletal muscle tissue remains still a challenge, and numerous studies have indicated that this technique may be of great importance in medicine in the near future. This article reviews some of the recent findings resulting from tissue engineering science related to the contractile behavior and the phenotypes of muscle tissue cells in different three-dimensional environment, and discusses how tissue engineering could be used to create and regenerate skeletal muscle, as well as the extended applications and the related patents concerned with engineered skeletal muscle.

[Clone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skin].

OBJECTIVE: To screen stable cell clones of CCL20 gene knockdown and assess their interference effects, recombinant lentivirus vectors with CCL20 gene specific shRNA were applied to infect human immortal keratinocyte line (HaCaT). METHODS: The three pHSER-CCL20-shRNA-GFP vectors (pHCG-1 and pHCG-2 were CCL20 gene specific, and pHCG-3 was used as mismatch control) have been previously constructed. The virus packaging cell line 293FT was transfected with these vectors by using CaCl2 methods to produce lentiviral particles. After the viral titers of these three harvested cell supernatants were determined by flow cytometry, HaCaT cells were transfected by these viruses and screened under the pressure of G418. The CCL20 mRNA from HaCaT cell clones and the CCL20 protein levels in the supernatants of HaCaT cell clones were detected by Real-time RT-PCR and ELISA, respectively. RESULTS: The titers of three lentiviruses were 7.08 x 10(5) transduced units (TU)/ml, 1.88 x 10(5) TU /ml and 2.08 x 10(5) TU/ml, respectively. Two HaCaT cell clones from each lentiviral vectors were obtained after G418 screening for 5 - 8 weeks. Four CCL20 gene specific clones showed stable interference effect in both Real-time RT-PCR and ELISA. The mRNA expression and protein level of CCL20 gene specific clones were down regulated significantly. CONCLUSIONS: The four human immortal keratinocyte clones with long term CCL20 gene knockdown have been screened by recombinant lentivirus vectors with CCL20 gene specific shRNA. These clones might be served as seed cells for novel tissue-engineered skin with lower rejection.

SOX9 directly binds CREB as a novel synergism with the PKA pathway in BMP-2-induced osteochondrogenic differentiation.

SOX9 acts as a master transcription factor in osteochondrogenesis, and the phosphorylation by protein kinase A (PKA) has been shown to increase its DNA binding and transactivation activity. The PKA pathway is involved in the complex downstream signaling underlying the BMP-2-mediated osteochondrogenesis. This study therefore aimed at further analyzing the possible cross-talk between the SOX9 and the PKA regulation on the background of BMP-2 stimulation. It was first shown that the removal of the residues serine 64 and 211 of SOX9 diminished, but did not completely deplete, its stimulatory effect on the expression of both osteo- and chondrogenic markers. PKA activators and inhibitors increased and decreased the action of wildtype and mutated SOX9, respectively. Interestingly, the interplay of the SOX9 action with the PKA pathway was further shown to occur through direct physical association between SOX9 and CREB, a prototypical PKA downstream transcription factor. Moreover, the binding was shown to be an active biological event happening on BMP-2 stimulation. The C-terminal domain of SOX9 and amino acid residue serine at position 133 of CREB were identified to be involved in the interaction. The action of SOX9 was enhanced by overexpressing CREB. These results suggest that PKA signaling synergizes with SOX9 at the nuclear and cytoplasmic levels to promote BMP-2-induced osteochondrogenic differentiation.

SDF-1alpha/CXCR4-mediated migration of systemically transplanted bone marrow stromal cells towards ischemic brain lesion in a rat model.

Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) can promote functional recovery of brain after stroke with the mechanism regulating the BMSCs migration to ischemic penumbra poorly understood. Interaction between stromal cell-derived factor-1alpha (SDF-1alpha) and its cognate receptor CXCR4 is crucial for homing and migration of multiple stem cell types. Their potential role in mediating BMSC migration in ischemic brain has not been demonstrated. In this study, ischemic brain lesion model was created in rats by permanent middle cerebral artery occlusion and green fluorescent protein (GFP)-labeled BMSCs were intravenously injected. Immunohistochemical staining showed that BMSCs were able to enter the route from olfactory areas to cortex of the rat brain. Significant recovery of modified Neurological Severity Score was observed at days 14 and 28. Interestingly, the SDF-1alpha mRNA and protein were predominantly localized in the ischemic penumbral, peaked by 3-7 days and retained at least 14 days post-transplantation. On the other hand, the CXCR4 expression by BMSCs was elevated under hypoxia. The pre-treatment with the CXCR4-specific antagonist AMD3100 significantly prevented the migration of BMSCs to the injured brain. Taken together, these observations indicate that systemically administered BMSCs can migrate to the ischemic lesion of brain along with the olfactory-thalamus and hippocampus-cortex route. The interaction of locally produced SDF-1alpha and CXCR4 expressed on the BMSC surface plays an important role in the migration of transplanted cells, suggesting that it might be a potential approach to modulate the expression of the two molecules in order to further facilitate the therapeutic effects using BMSCs.

The performance of a bone-derived scaffold material in the repair of critical bone defects in a rhesus monkey model.

The efficacy and safety of a material derived from human bones in the repair of critical segmental bone defects are evaluated in a rhesus monkey model. Frozen human bones were chemically and physically processed into a partially demineralized and deproteinized material in blocks. The complete tissue-engineered (TE) bone was constructed of the material preseeded with allogeneic bone marrow mesenchymal stem cells (MSCs). The material alone and the TE bone were, respectively, implanted to bridge 2.5cm-long critical defects in right and left radii of 15 monkeys. At weeks 1, 2, 3, 6 and 12 post-implantation, the grafts were collected from three animals and assessed for the local expression of osteogenic markers, histological and roentgenographic features, and immune reactions. It was shown that defects were well repaired with both treatments whereas the bone defects in 2 additional untreated animals remained the same size after 12 weeks. In radii implanted with the TE bones, the repair processes were approximately 3 weeks faster and new bones were formed in a multipoint way. There was neither observable toxic effect nor overt immune rejection in any animals. Taken together, these observations suggest that the TE bone blocks composited of the allogeneic or xenogeneic bone-derived scaffold and allogeneic MSCs may provide an ideal method for repairing large segmental bone defects.

Chondrogenic differentiation alters the immunosuppressive property of bone marrow-derived mesenchymal stem cells, and the effect is partially due to the upregulated expression of B7 molecules.

To investigate the immunosuppressive properties of MSCs, in the present study we examined the immunogenicity of undifferentiated and trilineage-differentiated (chondrocytes, osteoblasts, and adipocytes) rat bone marrow-derived MSCs under xenogeneic conditions. After chondrogenic differentiation, rat bone marrow-derived MSCs stimulated human dendritic cells (hDCs) derived from peripheral blood monocytes, leading to eight- and fourfold higher lymphocyte proliferation and cytotoxicity than that of undifferentiated MSCs. The chondrogenic-differentiated MSCs were chemotactic to hDCs in Dunn chamber chemotaxis system and were rosetted by hDCs in rosette assays. Flow cytometry analysis revealed that chondrogenic-differentiated MSCs had promoted hDC maturation, causing higher CD83 expression in hDCs, whereas undifferentiated MSCs and osteogenic- and adipogenic-differentiated MSCs showed an inhibitory effect on hDC maturation. The costimulatory B7 molecules were upregulated only in the chondrogenic-differentiated MSCs. After blocking B7 molecules with specific monoclonal antibodies in the chondrogenic-differentiated MSCs, CD83 expression of cocultured hDCs was greatly reduced. In conclusion, chondrogenic differentiation may increase the immunogenicity of MSCs, leading to stimulation of dendritic cells. The upregulated expression of B7 molecules on the chondrogenic-differentiated MSCs may be partially responsible for this event.

Current understanding of dystrophin-related muscular dystrophy and therapeutic challenges ahead.

OBJECTIVE: To review the recent research progress in dystrophin-related muscular dystrophy includes X-linked hereditary Duchenne and Becker muscular dystrophies (DMD and BMD). DATA SOURCES: Information included in this article was identified by searches of PUBMED and other online resources using the key terms DMD, dystrophin, mutations, animal models, pathophysiology, gene expression, stem cells, gene therapy, cell therapy, and pharmacological. Study selection Mainly original milestone articles and timely reviews written by major pioneer investigators of the field were selected. RESULTS: The key issues related to the genetic basis and pathophysiological factors of the diseases were critically addressed. The availabilities and advantages of various animal models for the diseases were described. Major molecular and cellular therapeutic approaches were also discussed, many of which have indeed exhibited some success in pre-clinical studies but at the same time encountered a number of technical hurdles, including the efficient and systemic delivery of a functional gene and myogenic precursor/stem cells to repair genetic defects. CONCLUSIONS: Further understanding of pathophysiological mechanisms at molecular levels and regenerative properties of myogenic precursor/stem cells will promote the development of multiple therapeutic strategies. The combined use of multiple strategies may represent the major challenge as well as the greatest hope for the therapy of these diseases in coming years.

The Drosophila ortholog of the endolysosomal membrane protein, endolyn, regulates cell proliferation.

Endolyn (CD164) is a sialomucin that regulates the proliferation, adhesion, and migration of human haematopoietic stem and progenitor cells. This molecule is predominately localized in endocytotic compartments, where it may contribute to endolysosomal biogenesis and trafficking. In order to more closely define the function of endolyn from an evolutionary view-point, we first analyzed endolyn orthologs in species ranging from insects, fish, and birds to mammals. The predicted molecular structures of the endolyn orthologs from these species are well conserved, particularly with respect to significant O-linked glycosylation of the extracellular domain, and the high degree of amino acid similarities within their transmembrane and cytoplasmic domains, with the latter possessing the lysosomal target signal, YXXphi. Focusing on Drosophila, our studies showed that the subcellular distribution of endolyn in non-polarized Drosophila S2 cells resembles that of its human counterpart in hematopoietic cells, with its predominant localization being within intracellular vesicles, while a small fraction occurs on the cell surface. Both Y --> A and L --> A mutations in the YHTL motif perturbed the normal subcellular distribution of Drosophila endolyn. Interestingly, embryonic and early larval development was often arrested in endolyn-deficient Drosophila mutants. This may partly be due to the role of endolyn in regulating cell proliferation, since knock-down of endolyn expression in S2 cells resulted in up to 50% inhibition of cell growth, with a proportion of cells undergoing apoptosis. Taken together, these results demonstrate that endolyn is an evolutionarily conserved sialomucin fundamentally involved in cell proliferation in both the human and Drosophila melanogaster.