Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension.

Today's challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineage.

During vertebrate embryogenesis, axial tendons develop from the paraxial mesoderm and differentiate through specific developmental stages to reach the syndetome stage. While the main roles of signaling pathways in the earlier stages of the differentiation have been well established, pathway nuances in syndetome specification from the sclerotome stage have yet to be explored. Here, we show stepwise differentiation of human iPSCs to the syndetome stage using chemically defined media and small molecules that were modified based on single cell RNA-sequencing and pathway analysis. We identified a significant population of branching off-target cells differentiating towards a neural phenotype overexpressing Wnt. Further transcriptomics post-addition of a WNT inhibitor at the somite stage and onwards revealed not only total removal of the neural off-target cells, but also increased syndetome induction efficiency. Fine-tuning tendon differentiation in vitro is essential to address the current challenges in developing a successful cell-based tendon therapy.

iPSC-derived tenocytes seeded on microgrooved 3D printed scaffolds for Achilles tendon regeneration.

Tendons and ligaments have a poor innate healing capacity, yet account for 50% of musculoskeletal injuries in the United States. Full structure and function restoration postinjury remains an unmet clinical need. This study aimed to assess the application of novel three dimensional (3D) printed scaffolds and induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) overexpressing the transcription factor Scleraxis (SCX, iMSCSCX+ ) as a new strategy for tendon defect repair. The polycaprolactone (PCL) scaffolds were fabricated by extrusion through a patterned nozzle or conventional round nozzle. Scaffolds were seeded with iMSCSCX+ and outcomes were assessed in vitro via gene expression analysis and immunofluorescence. In vivo, rat Achilles tendon defects were repaired with iMSCSCX+ -seeded microgrooved scaffolds, microgrooved scaffolds only, or suture only and assessed via gait, gene expression, biomechanical testing, histology, and immunofluorescence. iMSCSCX+ -seeded on microgrooved scaffolds showed upregulation of tendon markers and increased organization and linearity of cells compared to non-patterned scaffolds in vitro. In vivo gait analysis showed improvement in the Scaffold + iMSCSCX+ -treated group compared to the controls. Tensile testing of the tendons demonstrated improved biomechanical properties of the Scaffold + iMSCSCX+ group compared with the controls. Histology and immunofluorescence demonstrated more regular tissue formation in the Scaffold + iMSCSCX+ group. This study demonstrates the potential of 3D-printed scaffolds with cell-instructive surface topography seeded with iMSCSCX+ as an approach to tendon defect repair. Further studies of cell-scaffold constructs can potentially revolutionize tendon reconstruction by advancing the application of 3D printing-based technologies toward patient-specific therapies that improve healing and functional outcomes at both the cellular and tissue level.

Directing iPSC differentiation into iTenocytes using combined scleraxis overexpression and cyclic loading.

Regenerative therapies for tendon are falling behind other tissues due to the lack of an appropriate and potent cell therapeutic candidate. This study aimed to induce tenogenesis using stable Scleraxis (Scx) overexpression in combination with uniaxial mechanical stretch of iPSC-derived mesenchymal stromal-like cells (iMSCs). Scx is the single direct molecular regulator of tendon differentiation known to date. Bone marrow-derived (BM-)MSCs were used as reference. Scx overexpression alone resulted in significantly higher upregulation of tenogenic markers in iMSCs compared to BM-MSCs. Mechanoregulation is known to be a central element guiding tendon development and healing. Mechanical stimulation combined with Scx overexpression resulted in morphometric and cytoskeleton-related changes, upregulation of early and late tendon markers, and increased extracellular matrix deposition and alignment, and tenomodulin perinuclear localization in iMSCs. Our findings suggest that these cells can be differentiated into tenocytes and might be a better candidate for tendon cell therapy applications than BM-MSCs.

Neural crest-derived mesenchymal progenitor cells enhance cranial allograft integration.

Replacement of lost cranial bone (partly mesodermal and partly neural crest-derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow-derived mesenchymal stromal cells (mesoderm-derived BM-MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell-mesenchymal progenitor cells (iNCC-MPCs) improves implant-to-bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC-MPCs. BM-MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (µCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC-MPC-Luc2 vs BM-MSC-Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, µCT analysis showed enhanced structural parameters in the iNCC-MPC-Luc2 group and increased bone volume in the BM-MSC-Luc2 group compared to controls. Histology demonstrated improved integration of iNCC-MPC-Luc2 allografts compared to BM-MSC-Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft-host interphase in cell-seeded groups. The iNCC-MPC-Luc2 group also demonstrated improved biomechanical properties compared to BM-MSC-Luc2 implants and cell-free controls. Our results show an improved integration of iNCC-MPC-Luc2-coated allografts compared to BM-MSC-Luc2 and controls, suggesting the use of iNCC-MPCs as potential cell source for cranial bone repair.

Assessing the Resident Progenitor Cell Population and the Vascularity of the Adult Human Meniscus.

PURPOSE: To identify, characterize, and compare the resident progenitor cell populations within the red-red, red-white, and white-white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging. METHODS: Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow-derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin. RESULTS: There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44+CD105+CD29+CD90+ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red-red zone of meniscus were observed spanning toward red-white, sprouting to smaller arterioles and venules. CD31+ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed. CONCLUSIONS: The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone. CLINICAL RELEVANCE: The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.

Xenogeneic cardiac extracellular matrix scaffolds with or without seeded mesenchymal stem cells exhibit distinct in vivo immunosuppressive and regenerative properties.

Cardiac extracellular matrix (cECM) scaffolds are promising biomaterials for reconstructive surgery applications since they possess the structure/function properties of native tissue. Production of cECM scaffolds has been achieved using decellularization approaches, which commonly employ denaturing detergents, such as sodium dodecyl sulfate (SDS). Our antigen removal (AR) method has been shown to remove cellular and nonmyocyte components, while preserving cECM scaffold structure/function relationships. Here, we demonstrate that more human mesenchymal stem cells (MSCs) invaded AR scaffolds compared to SDS controls. Additionally, AR scaffolds stimulated a constructive remodeling response similar to allograft controls, and were transformed to adipose tissue in a xenogeneic rat to mouse subpannicular in vivo model. Conversely, SDS scaffolds showed a chronic inflammatory response that worsened throughout the 12-wk time course preventing constructive remodeling and mirroring the response seen towards xenogeneic tissue. AR scaffolds and xenogeneic controls recellularized with murine MSCs (mMSCs) were also implanted to assess whether mMSCs would offer any additive benefit in overcoming residual scaffold-specific immune responses. Paradoxically, recellularization resulted in chronic inflammatory response in AR-recellularized scaffolds. We conclude that AR cECM scaffolds represent a promising biomaterial, which is accepted by the recipient as self in origin and fosters implantation site appropriate regenerative responses. STATEMENT OF SIGNIFICANCE: We demonstrated that an antigen-removal (AR) approach utilizing principles of differential solubility for production of a xenogeneic rat cardiac extracellular matrix scaffold results in improved recellularization efficiency with human and mouse mesenchymal stem cells (MSCs) in vitro. Furthermore, we tested the immune response to AR scaffolds versus allograft and xenograft controls with or without MSC recellularization using a rat to mouse subcutaneous model. We showed that AR scaffolds and allograft controls resulted in significant adipose tissue transformation after 12weeks. Paradoxically, MSCs had a positive impact in the immune response to xenografts, but had the opposite effect in AR scaffolds, resulting in chronic inflammatory response, which might be attributed to a change of their phenotype following recellularization into scaffolds.

Cardiac Extracellular Matrix Scaffold Generated Using Sarcomeric Disassembly and Antigen Removal.

Xenogeneic cardiac extracellular matrix (cECM) scaffolds for reconstructive cardiac surgery applications have potential to overcome the limitations of current clinically utilized patch materials. A potentially ideal cECM scaffold would be immunologically acceptable while preserving the native cECM niche. Production of such a scaffold necessitates removal of cellular and antigenic components from cardiac tissue while preserving cECM structure/function properties. Existing decellularization methodologies predominantly utilize denaturing detergents which might irreversibly alter cECM material properties. To overcome potential deficiencies of current approaches, the effect of sarcomere relaxation and disassembly on resultant cECM scaffold cellularity was investigated. Additionally, the ability of sequential differential protein solubilization (antigen removal-AR) to reduce cECM scaffold antigenicity was examined. Sarcomeric relaxation and disassembly were necessary to achieve scaffold acellularity. All groups in which AR was employed displayed statistically significant decreases in residual antigenicity regardless of their degree of acellularity. AR combined with sarcomeric disassembly preserved structural, biochemical, mechanical and recellularization properties of the cECM scaffold. However, sodium dodecyl sulfate significantly altered cECM properties. This study demonstrates the importance of solubilizing cellular elements and antigenic components in a stepwise manner for production of a potentially ideal cECM scaffold and may have implications for future tissue engineering and regenerative medicine applications.

The adult heart responds to increased workload with physiologic hypertrophy, cardiac stem cell activation, and new myocyte formation.

AIMS: It is a dogma of cardiovascular pathophysiology that the increased cardiac mass in response to increased workload is produced by the hypertrophy of the pre-existing myocytes. The role, if any, of adult-resident endogenous cardiac stem/progenitor cells (eCSCs) and new cardiomyocyte formation in physiological cardiac remodelling remains unexplored. METHODS AND RESULTS: In response to regular, intensity-controlled exercise training, adult rats respond with hypertrophy of the pre-existing myocytes. In addition, a significant number (∼7%) of smaller newly formed BrdU-positive cardiomyocytes are produced by the exercised animals. Capillary density significantly increased in exercised animals, balancing cardiomyogenesis with neo-angiogenesis. c-kit(pos) eCSCs increased their number and activated state in exercising vs. sedentary animals. c-kit(pos) eCSCs in exercised hearts showed an increased expression of transcription factors, indicative of their commitment to either the cardiomyocyte (Nkx2.5(pos)) or capillary (Ets-1(pos)) lineages. These adaptations were dependent on exercise duration and intensity. Insulin-like growth factor-1, transforming growth factor-ß1, neuregulin-1, bone morphogenetic protein-10, and periostin were significantly up-regulated in cardiomyocytes of exercised vs. sedentary animals. These factors differentially stimulated c-kit(pos) eCSC proliferation and commitment in vitro, pointing to a similar role in vivo. CONCLUSION: Intensity-controlled exercise training initiates myocardial remodelling through increased cardiomyocyte growth factor expression leading to cardiomyocyte hypertrophy and to activation and ensuing differentiation of c-kit(pos) eCSCs. This leads to the generation of new myocardial cells. These findings highlight the endogenous regenerative capacity of the adult heart, represented by the eCSCs, and the fact that the physiological cardiac adaptation to exercise stress is a combination of cardiomyocyte hypertrophy and hyperplasia (cardiomyocytes and capillaries).

Glycogen content regulates peroxisome proliferator activated receptor-∂ (PPAR-∂) activity in rat skeletal muscle.

Performing exercise in a glycogen depleted state increases skeletal muscle lipid utilization and the transcription of genes regulating mitochondrial ß-oxidation. Potential candidates for glycogen-mediated metabolic adaptation are the peroxisome proliferator activated receptor (PPAR) coactivator-1α (PGC-1α) and the transcription factor/nuclear receptor PPAR-∂. It was therefore the aim of the present study to examine whether acute exercise with or without glycogen manipulation affects PGC-1α and PPAR-∂ function in rodent skeletal muscle. Twenty female Wistar rats were randomly assigned to 5 experimental groups (n = 4): control [CON]; normal glycogen control [NG-C]; normal glycogen exercise [NG-E]; low glycogen control [LG-C]; and low glycogen exercise [LG-E]). Gastrocnemius (GTN) muscles were collected immediately following exercise and analyzed for glycogen content, PPAR-∂ activity via chromatin immunoprecipitation (ChIP) assays, AMPK α1/α2 kinase activity, and the localization of AMPK and PGC-1α. Exercise reduced muscle glycogen by 47 and 75% relative to CON in the NG-E and LG-E groups, respectively. Exercise that started with low glycogen (LG-E) finished with higher AMPK-α2 activity (147%, p<0.05), nuclear AMPK-α2 and PGC-1α, but no difference in AMPK-α1 activity compared to CON. In addition, PPAR-∂ binding to the CPT1 promoter was significantly increased only in the LG-E group. Finally, cell reporter studies in contracting C2C12 myotubes indicated that PPAR-∂ activity following contraction is sensitive to glucose availability, providing mechanistic insight into the association between PPAR-∂ and glycogen content/substrate availability. The present study is the first to examine PPAR-∂ activity in skeletal muscle in response to an acute bout of endurance exercise. Our data would suggest that a factor associated with muscle contraction and/or glycogen depletion activates PPAR-∂ and initiates AMPK translocation in skeletal muscle in response to exercise.