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.

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.

Advanced Glycation End Product Inhibitor Pyridoxamine Attenuates IVD Degeneration in Type 2 Diabetic Rats.

Type 2 diabetes mellitus (T2DM) is associated with advanced glycation end product (AGE) enrichment and considered a risk factor for intervertebral disc (IVD) degeneration. We hypothesized that systemic AGE inhibition, achieved using pyridoxamine (PM), attenuates IVD degeneration in T2DM rats. To induce IVD degeneration, lumbar disc injury or sham surgery was performed on Zucker Diabetic Sprague Dawley (ZDSD) or control Sprague Dawley (SD) rats. Post-surgery, IVD-injured ZDSD rats received daily PM dissolved in drinking water or water only. The resulting groups were SD uninjured, SD injured, ZDSD uninjured, ZDSD injured, and ZDSD injured + PM. Levels of blood glycation and disc degeneration were investigated. At week 8 post-surgery, glycated serum protein (GSP) levels were increased in ZDSDs compared to SDs. PM treatment attenuated this increase. Micro-MRI analysis demonstrated IVD dehydration in injured versus uninjured SDs and ZDSDs. In the ZDSD injured + PM group, IVD dehydration was diminished compared to ZDSD injured. AGE levels were decreased and aggrecan levels increased in ZDSD injured + PM versus ZDSD injured rats. Histological and immunohistochemical analyses further supported the beneficial effect of PM. In summary, PM attenuated GSP levels and IVD degeneration processes in ZDSD rats, demonstrating its potential to attenuate IVD degeneration in addition to managing glycemia in T2DM.

A Translational Porcine Model for Human Cell-Based Therapies in the Treatment of Posttraumatic Osteoarthritis After Anterior Cruciate Ligament Injury.

BACKGROUND: There is a high incidence of posttraumatic osteoarthritis (PTOA) after anterior cruciate ligament (ACL) injury, and these injuries represent an enormous health care economic burden. In an effort to address this unmet clinical need, there has been increasing interest in cell-based therapies. PURPOSE: To establish a translational large animal model of PTOA and demonstrate the feasibility of intra-articular human cell-based interventions. STUDY DESIGN: Descriptive laboratory study. METHODS: Nine Yucatan mini-pigs underwent unilateral ACL transection and were monitored for up to 12 weeks after injury. Interleukin 1 beta (IL-1ß) levels and collagen breakdown were evaluated longitudinally using enzyme-linked immunosorbent assays of synovial fluid, serum, and urine. Animals were euthanized at 4 weeks (n = 3) or 12 weeks (n = 3) after injury, and injured and uninjured limbs underwent magnetic resonance imaging (MRI) and histologic analysis. At 2 days after ACL injury, an additional 3 animals received an intra-articular injection of 107 human bone marrow-derived mesenchymal stem cells (hBM-MSCs) combined with a fibrin carrier. These cells were labeled with the luciferase reporter gene (hBM-MSCs-Luc) as well as fluorescent markers and intracellular iron nanoparticles. These animals were euthanized on day 0 (n = 1) or day 14 (n = 2) after injection. hBM-MSC-Luc viability and localization were assessed using ex vivo bioluminescence imaging, fluorescence imaging, and MRI. RESULTS: PTOA was detected as early as 4 weeks after injury. At 12 weeks after injury, osteoarthritis could be detected grossly as well as on histologic analysis. Synovial fluid analysis showed elevation of IL-1ß shortly after ACL injury, with subsequent resolution by 2 weeks after injury. Collagen type II protein fragments were elevated in the synovial fluid and serum after injury. hBM-MSCs-Luc were detected immediately after injection and at 2 weeks after injection using fluorescence imaging, MRI, and bioluminescence imaging. CONCLUSION: This study demonstrates the feasibility of reproducing the chondral changes, intra-articular cytokine alterations, and body fluid biomarker findings consistent with PTOA after ACL injury in a large animal model. Furthermore, we have demonstrated the ability of hBM-MSCs to survive and express transgene within the knee joint of porcine hosts without immunosuppression for at least 2 weeks. CLINICAL RELEVANCE: This model holds great potential to significantly contribute to investigations focused on the development of cell-based therapies for human ACL injury-associated PTOA in the future (see Appendix Figure A1, available online).

Electrospun, synthetic bone void filler promotes human MSC function and BMP-2 mediated spinal fusion.

INTRODUCTION: Synthetic bone grafts are often used to achieve a well-consolidated fusion mass in spinal fusion procedures. These bone grafts function as scaffolds, and ideally support cell function and facilitate protein binding. OBJECTIVE: The aim was to characterize an electrospun, synthetic bone void filler (Reb) for its bone morphogenetic protein (BMP)-2 release properties and support of human mesenchymal stem cell (hMSC) function in vitro, and its efficacy in promoting BMP-2-/bone marrow aspirate-(BMA)-mediated posterolateral spinal fusion (PLF) in vivo. METHODS: BMP-2 release kinetics from Reb versus standard absorbable collagen sponge (ACS) was determined. hMSC adhesion and proliferation on Reb was tested using cell counting, fluorescence microscopy and MTS. Cell osteogenic differentiation was quantified via cellular alkaline phosphatase (ALP) activity. For in vivo analysis, 18 Lewis rats were treated during PLF surgery with the following groups: (I) Reb + BMA, (II) Reb + BMA + BMP-2 and (III) BMA. A safe, minimally effective dose of BMP-2 was used. Fusion consolidation was followed for 3 months using radiography and micro-CT. After sacrifice, fusion rate and biomechanical stiffness was determined using manual palpation, biomechanical tests and histology. RESULTS: In vitro, BMP-2 release kinetics were similar between Reb versus ACS. MSC proliferation and differentiation were increased in the presence of Reb. At 3 months post-surgery, fusion rates were 29% (group I), 100% (group II), and 0% (group III). Biomechanical stiffness was higher in group II versus I. Micro-CT showed an increased bone volume and connectivity density in group II. Trabecular thickness was increased in group I versus II. H&E staining showed newly formed bone in group II only. CONCLUSIONS: Reb possesses a high protein binding affinity and promotes hMSC function. Combination with BMA and minimal dose BMP-2 allowed for 100% bone fusion in vivo. This data suggests that a minimally effective dose of BMP-2 can be used when combined with Reb.