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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

Pancreatic neuroendocrine tumors (PNETs), a group of endocrine tumors arising in the pancreas, are among the most common neuroendocrine tumors. The genetic causes of familial and sporadic PNETs are somewhat understood, but their molecular pathogenesis remains unknown. Most PNETs are indolent but have malignant potential. The biological behavior of an individual PNET is unpredictable; higher tumor grade, lymph node and liver metastasis, and larger tumor size generally indicate a less favorable prognosis. Endocrine testing, imaging, and histological evidence are necessary to accurately diagnose PNETs. A 4-pronged aggressive treatment approach consisting of surgery, locoregional therapy, systemic therapy, and complication control has become popular in academic centers around the world. The optimal application of the multiple systemic therapeutic modalities is under development; efficacy, safety, availability, and cost should be considered when treating a specific patient. The clinical presentation, diagnosis, and treatment of specific types of PNETs and familial PNET syndromes, including the novel Mahvash disease, are summarized.

Pancreatic neuroendocrine tumors: biology, diagnosis,and treatment.

Visual vignette.

Intervertebral disc (IVD) degeneration is often associated with low back pain and radiating leg pain. The purpose of this study is to develop a reproducible and standardized preclinical model of painful lumbar IVD degeneration by evaluation of structural and behavioral changes in response to IVD injury with increasing needle sizes. This model can be used to develop new therapies for IVD degeneration.

Optimization of a rat lumbar IVD degeneration model for low back pain.

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.

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

In regenerative medicine, experimental animal models are commonly used to study potential effects of human cells as therapeutic candidates. Although some studies describe certain cells, such as mesenchymal stromal cells (MSC) or human primary cells, as hypoimmunogenic and therefore unable to trigger strong inflammatory host responses, other studies report antibody formation and immune rejection following xenotransplantation. Accordingly, the goal of our study was to test the cellular retention and survival of human-induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) and primary nucleus pulposus cells (NPCs) following their xenotransplantation into immune-privileged knee joints (14 days) and intervertebral discs (IVD; 7 days) of immunocompromised Nude and immunocompetent Sprague Dawley (SD) rats. At the end of both experiments, we could demonstrate that both rat types revealed comparably low levels of systemic IL-6 and IgM inflammation markers, as assessed via ELISA. Furthermore, the number of recovered cells was with no significant difference between both rat types. Conclusively, our results show that xenogeneic injection of human iMSC and NPC into immunoprivileged knee and IVD sites did not lead to an elevated inflammatory response in immunocompetent rats when compared to immunocompromised rats. Hence, immunocompetent rats represent suitable animals for xenotransplantation studies targeting immunoprivileged sites.

Article	Year
Pancreatic neuroendocrine tumors: biology, diagnosis,and treatment. Chinese journal of cancer\| PubMed ID: 23237225	2013
Visual vignette. Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists\| PubMed ID: 24126233	2014
Optimization of a rat lumbar IVD degeneration model for low back pain. JOR spine\| PubMed ID: 32613167	2020
Directing iPSC differentiation into iTenocytes using combined scleraxis overexpression and cyclic loading. Journal of orthopaedic research : official publication of the Orthopaedic Research Society\| PubMed ID: 36203346	2023
iPSC-derived tenocytes seeded on microgrooved 3D printed scaffolds for Achilles tendon regeneration. Journal of orthopaedic research : official publication of the Orthopaedic Research Society\| PubMed ID: 36961351	2023
Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineage. bioRxiv : the preprint server for biology\| PubMed ID: 37090543	2023
Retention of Human iPSC-Derived or Primary Cells Following Xenotransplantation into Rat Immune-Privileged Sites. Bioengineering (Basel, Switzerland)\| PubMed ID: 37760151	2023

Victoria Yu

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