Transplantation of tendon-derived stem cells pre-treated with connective tissue growth factor and ascorbic acid in vitro promoted better tendon repair in a patellar tendon window injury rat model.

BACKGROUND AIMS: Treatment of tendon-derived stem cells (TDSCs) with connective tissue growth factor (CTGF) and ascorbic acid promoted their tenogenic differentiation. We investigated the effects of TDSCs pre-treated with CTGF and ascorbic acid on tendon repair in a patellar tendon window injury rat model. METHODS: Green fluorescent protein-TDSCs (GFP-TDSCs) were pre-treated with or without CTGF and ascorbic acid for 2 weeks before transplantation. The patellar tendons of rats were injured and divided into three groups: fibrin glue-only group (control group), untreated and treated TDSC group. The rats were followed up until week 16. RESULTS: The treated TDSCs accelerated and enhanced the quality of tendon repair compared with untreated TDSCs up to week 8, which was better than that in the controls up to week 16 as shown by histology, ultrasound imaging and biomechanical test. The fibrils in the treated TDSC group showed better alignment and larger size compared with those in the control group at week 8 (P = 0.004). There was lower risk of ectopic mineralization after transplantation of treated or untreated TDSCs (all P ≤ 0.050). The transplanted cells proliferated and could be detected in the window wound up to weeks 2 to 4 and week 8 for the untreated and treated TDSC groups, respectively. CONCLUSIONS: The transplantation of TDSCs promoted tendon repair up to week 16, with CTGF and ascorbic acid pre-treatment showing the best results up to week 8. Pre-treatment of TDSCs with CTGF and ascorbic acid may be used to further enhance the rate and quality of tendon repair after injury.

Proteomic Analysis of Human Pluripotent Stem Cell-Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation.

BACKGROUND: Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs), but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived VCMs, human fetal and human adult ventricular and atrial CMs. METHODS AND RESULTS: Using two-dimensional-differential-in-gel electrophoresis, 121 differentially expressed (>1.5-fold; P<0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator-activated receptor α signaling in cardiac maturation. Consistently, WY-14643, a peroxisome proliferator-activated receptor α agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold, signifying their maturation. CONCLUSIONS: We conclude that the peroxisome proliferator-activated receptor α and thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.

Immunogenicity and escape mechanisms of allogeneic tendon-derived stem cells.

The immunogenicity of tendon-derived stem cells (TDSCs) has implications for their clinical use for the promotion of tendon repair. The immunogenicity and escape mechanisms of rat patellar TDSCs were examined after allogeneic transplantation. Our results showed that TDSCs exhibited low immunogenicity as evidenced by the following: (i) the incubation of target TDSCs with immunized serum did not show antibody recognition and did not induce the complement-dependent cytotoxicity; (ii) target TDSCs elicited a very low level of lymphocyte proliferation and did not exhibit host lymphocyte-mediated cytotoxicity; and (iii) target TDSCs dose dependently suppressed the phorbol 12-myristate 13-acetate (PMA)- and ionomycin-induced host lymphocyte proliferation. For the mechanistic studies, TDSCs expressed major histocompatibility complex (MHC)-I but a very low level of MHC-II, CD86 and CD80 for the induction of T-cell response. Also, TDSCs were found to express intracellular Fas and FasL. γ-IFN pretreatment did not increase the level of MHC-II and CD86 for the upregulation of immune response. Moreover, the immunosuppressive mediators indoleamine 2,3-dioxygenase (IDO) and transforming growth factor-beta 1 (TGF-ß1) were found not to be involved in the escape mechanism of target TDSCs from host lymphocyte attack. In conclusion, allogeneic TDSCs exhibited low immunogenicity. Allogeneic TDSCs might be used for transplantation.

Allogeneic tendon-derived stem cells promote tendon healing and suppress immunoreactions in hosts: in vivo model.

The medium- to long-term healing effect and infiltration of inflammatory cells, after transplantation of allogeneic tendon-derived stem cell (TDSC) to the rat patellar tendon window wound, were examined. Allogeneic patellar TDSCs derived from a green fluorescent protein rat were used. The outcome of tendon healing and the infiltration of inflammatory cells were examined by histology and immunohistochemistry up to week 16 postinjury. The fate of the transplanted cells was examined by ex vivo fluorescent imaging and immunohistochemistry. Our results showed that the transplantation of allogeneic TDSCs promoted tendon healing with no increased risk of ectopic chondro-ossification up to week 16. A low infiltration of T cells, ED1 macrophages, ED2 macrophages, and mast cells in the window wound was obtained. The transplanted TDSCs were found in the window wound at week 1 and 2, but were absent after week 4 postinjury. In conclusion, allogeneic TDSCs promoted tendon repair in the medium to long term and exhibited weak immunoreactions and anti-inflammatory effects in the hosts after transplantation in a rat model. There was no increased risk of ectopic chondro-ossification after TDSC transplantation. The decrease in the number of transplanted cells with time suggested that allogeneic TDSCs did not promote tendon repair through direct differentiation.

Application of tendon-derived stem cell sheet for the promotion of graft healing in anterior cruciate ligament reconstruction.

BACKGROUND: Both osteointegration and remodeling of graft midsubstance (collectively called graft healing) are slow processes after anterior cruciate ligament (ACL) reconstruction. Tendon-derived stem cells (TDSCs) form a cell sheet after treatment with connective tissue growth factor (CTGF) and ascorbic acid, which exhibits higher tenogenic and maintains high chondro-osteogenic gene expression of TDSCs. No external scaffold is required for cell delivery. HYPOTHESIS: Wrapping the TDSC sheet around the ACL graft would promote early graft healing in a rat model. STUDY DESIGN: Controlled laboratory study. METHODS: Green fluorescent protein (GFP) rat TDSCs were treated with connective tissue growth factor and ascorbic acid to promote cell sheet formation. Rats undergoing unilateral ACL reconstruction were divided into a control group and a TDSC group. The tendon graft was wrapped with the GFP-TDSC sheet before graft insertion in the TDSC group. At weeks 2, 6, and 12 after reconstruction, the samples were harvested for computed tomography imaging and histologic or biomechanical testing. The fate of the transplanted cell sheet was examined by immunohistochemical staining of GFP. RESULTS: There were significantly higher tunnel bone mineral density (BMD) (42.3% increase, P = .047) and bone volume/total volume (BV/TV) (625% increase, P = .009) at the metaphyseal region of the tibial tunnel at week 2 and at the femoral tunnel at week 6 (BMD: 30.8% increase, P = .014; BV/TV: 100% increase, P = .014) in the TDSC group compared with the control group. Only the TDSC group showed a time-dependent increase in tunnel BMD (overall P = .038) and BV/TV (overall P = .015) at the epiphyseal region of the tibial tunnel. Semiquantitative image analysis showed better graft osteointegration and higher intra-articular graft integrity with lower cellularity and vascularity, better cell alignment, and higher collagen birefringence in the TDSC group. The ultimate load at week 2 (52.5% increase, P = .027) and stiffness at week 6 (62% increase, P = .008) were significantly higher in the TDSC group. Cells positive for GFP were observed in all samples in the TDSC group at week 2 but became reduced with time after reconstruction. CONCLUSION: The TDSC sheet improved early graft healing after ACL reconstruction in the rat model. CLINICAL RELEVANCE: The TDSC sheet could potentially be used for the promotion of graft healing in ACL reconstruction.

Tendon stem cells: experimental and clinical perspectives in tendon and tendon-bone junction repair.

Tendon and tendon-bone junction injuries, while heal, have high re-tear rates. Mesenchymal stem cells (MSCs) have great appeal for the promotion of tendon and tendon-bone junction healing because of their high proliferation rate, multi-potency and relative ease of isolation from various tissues. Tendon stem cells have been identified recently and could be an alternative new cell source for tendon and tendon-bone junction repair. In this review, we summarized the in vitro characteristics of tendon stem cells. The evidence supporting the potential use of these cells for tendon and tendon-bone junction repair was presented. In order to therapeutically apply tendon stem cells in the clinical settings, standardization of tendon stem cell culture is essential. Issues relating to the sources, purity, efficacy, safety and delivery of tendon stem cells for tendon and tendon-bone junction repair were summarized and discussed. The direction for future research was suggested.