Generation of Induced Pluripotent Stem Cell-Derived iTenocytes <em>via</em> Combined Scleraxis Overexpression and 2D Uniaxial Tension

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to ...

Cedars-Sinai Medical Center

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.

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

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.

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

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.

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

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.

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

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, iMSC ) 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 iMSC and outcomes were assessed in vitro via gene expression analysis and immunofluorescence. In vivo, rat Achilles tendon defects were repaired with iMSC -seeded microgrooved scaffolds, microgrooved scaffolds only, or suture only and assessed via gait, gene expression, biomechanical testing, histology, and immunofluorescence. iMSC -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 + iMSC -treated group compared to the controls. Tensile testing of the tendons demonstrated improved biomechanical properties of the Scaffold + iMSC group compared with the controls. Histology and immunofluorescence demonstrated more regular tissue formation in the Scaffold + iMSC group. This study demonstrates the potential of 3D-printed scaffolds with cell-instructive surface topography seeded with iMSC 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.

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

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  is essential to address the current challenges in developing a successful cell-based tendon therapy.

Angela Papalamprou

Orthopaedic Stem Cell Research Laboratory,
Board of Governors Regenerative Medicine Institute,
Department of Biomedical Sciences

Cedars-Sinai Medical Center

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

Angela Papalamprou

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Orthopaedic Stem Cell Research Laboratory,Board of Governors Regenerative Medicine Institute,Department of Biomedical Sciences

Cedars-Sinai Medical Center

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

Orthopaedic Stem Cell Research Laboratory,
Board of Governors Regenerative Medicine Institute,
Department of Biomedical Sciences