Employing epigenetic memory and native instructive stimuli to stimulate iPS-NLC differentiation

Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and are considered a promising candidate for cell-based therapies. However, NCs are scarcely available as they are lost early in life, and attempts at in vitro expansion have failed because NCs lose their specific phenotype. The production of notochordal-like cells (NLCs) from human induced pluripotent stem cells (hiPSCs) is a viable alternative. Therefore, this study aimed to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD-progenitor cells (TIE2+-cells) and the instructive capacity of decellularized notochordal cell-derived matrix (dNCM)2 to improve hiPSC differentiation towards mature, healthy matrix-producing NLCs. hiPSCs were generated from TIE2+-IVD cells of three adult donors. As a comparison donor-matched minimally invasive peripheral blood mononuclear (PBM)-derived iPSCs were used. Firstly, hiPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)1 for 2 days. Thereafter, the cells were further driven towards the NC-lineage by transfection with synthetic NOTO mRNA1 and matured by switching to a 3D-cell pellet culture in discogenic medium containing 10ng/mL TGF-β1 or 3mg/mL dNCM until day 28. Read-outs included cell morphology, gene and protein expression and matrix deposition. Both TIE2+- and PBM-cell derived hiPSC showed successful differentiation towards mesendodermal progenitors following Wnt-activation on day 2, indicated by the cells moving out of the colonies after CHIR stimulation. Accordingly, a decreased gene expression of pluripotency markers (OCT4, SOX2, NANOG), and upregulation of Wnt and Nodal signaling (LEF1, NODAL) and mesendodermal markers (FOXA2, TBXT) was detected, compared to mTeSR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. On day 3, we detected a significant increase in NOTO mRNA levels in all donor lines after transfection compared to untransfected cell pellets. 3D-pellets of all donor lines showed glycosaminoglycan (GAG)- and collagen type II-rich areas after dNCM- but not TGF-β1-treatment on day 28. This was confirmed with the DMMB-assay, showing a significantly increased GAG content in the 3D-pellets treated with dNCM compared to TGF-β1. Next to that, TIE2+-cell derived iPSC pellets contained a significantly higher GAG content after dNCM-treatment compared to the PBM-cell derived hiPSC pellets. Immunohistochemical evaluation showed a heterogeneous cell population including cells positive for chondrogenic- (ACAN, SOX9), NPC/NC- (panKRT, T), and IVD progenitor- markers (CD24, TIE2). In conclusion, using tissue-specific TIE2+-cell derived hiPSCs combined with dNCM-treatment may allow for an improved differentiation capacity indicated by the increased deposition of GAG and collagen type II-rich matrix. However, the obtained cell population is still very heterogeneous and further transcriptome analysis could unravel whether the 3D-pellets contain cells which were successfully driven towards the notochordal-lineage and how these can be enriched based on unique NC-specific markers. Next to that, delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the observed differences in their differentiation capacity. These insights could be used for further optimization of iPS-NLC differentiation and allow for a more purified population of mature, healthy matrix-producing NLCs. This work was funded by Horizon 2020 (no. 825925) and the Dutch Arthritis Society (LLP22). References 1Colombier, P. et al. (2020). NOTO transcription factor directs human induced pluripotent stem cell-derived mesendoderm progenitors to a notochordal fate. Cells, 9(2), 509. 2Bach, Frances C., et al. "Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside." Oncotarget 9.41 (2018): 26507

Чверть століття на ниві освіти

Наталія Купріянівна Місяць – відомий український вчений-мовознавець, викладач та організатор освіти на Житомирщині. Чверть століття, починаючи вже з далекого 1975 року, життя та діяльність Наталії Куприянівні тісно пов’язані з філологічним факультетом Житомирського едуніверситету імені Івана Франка

A combined western and bead-based multiplex platform to characterize extracellular vesicles

In regenerative medicine, extracellular vesicles (EVs) are considered as a promising cell-free approach. EVs are lipid bilayer-enclosed vesicles secreted by cells and are key players in intercellular communication. EV-based therapeutic approaches have unique advantages over the use of cell-based therapies, such as a high biological, but low immunogenic and tumorigenic potential. To analyze the purity and biochemical composition of EV preparations, the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins coisolated/recovered with EVs. Traditional methods for EV characterization, such as Western blotting, require a relatively high EV sample/protein input for the analysis of one protein. We here evaluate a combined Western and bead-based multiplex platform, called DigiWest, for its ability to detect simultaneously multiple EV markers in an EV-containing sample with inherent low protein input. DigiWest analysis was performed on EVs from various sources and species, including mesenchymal stromal cells, notochordal cells, and milk, from human, pig, and dog. The study established a panel of nine antibodies that can be used as cross-species for the detection of general EV markers and coisolates in accordance with the ISEV guidelines. This optimized panel facilitates the parallel evaluation of EV-containing samples, allowing for a comprehensive characterization and assessment of their purity. The total protein input for marker analysis with DigiWest was 1 μg for all nine antibodies, compared with ∼10 μg protein input required for traditional Western blotting for one antibody. These findings demonstrate the potential of the DigiWest technique for characterizing various types of EVs in the regenerative medicine field

Mechanical characterization of a novel biomimetic artificial disc for the cervical spine

A novel biomimetic artificial intervertebral disc (bioAID) replacement implant has been developed containing a swelling hydrogel representing the nucleus pulposus, a tensile strong fiber jacket as annulus fibrosus and titanium endplates with pins to primarily secure the device between the vertebral bodies. In this study, the design safety of this novel implant was evaluated based on several biomechanical parameters, namely compressive strength, shear-compressive strength, risk of subsidence and device expulsion as well as identifying the diurnal creep-recovery characteristics of the device. The bioAID remained intact up to 1 kN under static axial compression and only 0.4 mm of translation was observed under a compressive shear load of 20 N. No subsidence was observed after 0.5 million cycles of sinusoidal compressive loading between 50 and 225 N. After applying 400 N in antero-posterior direction under 100 N axial compressive preload, approximately 2 mm displacement was found, being within the range of displacements reported for other commercially available cervical disc replacement devices. The diurnal creep recovery behavior of the bioAID closely resembled what has been reported for natural intervertebral discs in literature. Overall, these results indicate that the current design can withstand (shear-compression loads and is able to remain fixed in a mechanical design resembling the vertebral bodies. Moreover, it is one of the first implants that can closely mimic the poroelastic and viscoelastic behavior of natural disc under a diurnal loading pattern

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

INTRODUCTION: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration. METHODS: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age. RESULTS: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells. CONCLUSIONS: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration

Semi-synthetic degradable notochordal cell-derived matrix hydrogel for use in degenerated intervertebral discs: Initial in vitro characterization

Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs

Delayed endochondral ossification in early medial coronoid disease (MCD): a morphological and immunohistochemical evaluation in growing Labrador retrievers

Medial coronoid disease (MCD) is a common joint disease of dogs. It has a multifactorial aetiology, but the relationship between known causal factors and the disease has yet to be elucidated. As most of the published literature is clinical and it reports changes associated with advanced disease, it is not known whether the changes reflect the cause or consequences of the condition. The aim of this study was to investigate early micromorphological changes occurring in articular cartilage and to describe the postnatal development of the medial coronoid process (MCP) before MCD develops. Three litters of MCD-prone young Labrador retrievers were purpose-bred from a dam and two sires with MCD. Comparisons of the micromorphological appearance of the MCP in MCD-negative and MCD-positive joints demonstrated that MCD was initially associated with a disturbance of endochondral ossification, namely a delay in the calcification of the calcifying zone, without concurrent abnormalities in the superficial layers of the joint cartilage. Cartilage canals containing patent blood vessels were only detected in dogs <12 weeks old, but the role of these channels in impaired ossification requires further investigation. Retained hyaline cartilage might ossify as the disease progresses, but weak areas can develop into cracks between the retained cartilage and the subchondral bone, leading to cleft formation and fragmentation of the MCP

Patient‐specific 3D‐printed shelf implant for the treatment of hip dysplasia: anatomical and biomechanical outcomes in a canine model

A solution for challenging hip dysplasia surgery could be a patient-specific 3D-printed shelf implant that is positioned extra-articular and restores the dysplastic acetabular rim to normal anatomical dimensions. The anatomical correction and biomechanical stability of this concept were tested in a canine model that, like humans, also suffers from hip dysplasia. Using 3D reconstructed computed tomography images the 3D shelf implant was designed to restore the radiological dysplastic hip parameters to healthy parameters. It was tested ex vivo on three dog cadavers (six hips) with hip dysplasia. Each hip was subjected to a biomechanical subluxation test, first without and then with the 3D shelf implant in place. Subsequently, an implant failure test was performed to test the primary implant fixation. At baseline, the dysplastic hips had an average Norberg angle of 88 ± 3° and acetabular coverage of 47 ± 2% and subluxated at an average of 83 ± 2° of femoral adduction. After adding the patient-specific shelf implants the dysplastic hips had an average Norberg angle of 122 ± 2° and acetabular coverage of 67 ± 3% and subluxated at an average of 117 ± 2° of femoral adduction. Implant failure after primary implant fixation occurred at an average of 1330 ± 320 Newton. This showed that the patient-specific shelf implants significantly improved the coverage and stability of dysplastic hips in a canine model with naturally occurring hip dysplasia. The 3D shelf is a promising concept for treating residual hip dysplasia with a straightforward technology-driven approach; however, the clinical safety needs to be further investigated in an experimental proof-of-concept animal study

Biomechanical evaluation of a novel biomimetic artificial intervertebral disc in canine cervical cadaveric spines

Background Context Cervical disc replacement (CDR) aims to restore motion of the treated level to reduce the risk of adjacent segment disease (ASD) compared with spinal fusion. However, first-generation articulating devices are unable to mimic the complex deformation kinematics of a natural disc. Thus, a biomimetic artificial intervertebral CDR (bioAID), containing a hydroxyethylmethacrylate (HEMA)—sodium methacrylate (NaMA) hydrogel core representing the nucleus pulposus, an ultra-high-molecular-weight-polyethylene fiber jacket as annulus fibrosus, and titanium endplates with pins for primary mechanical fixation, was developed. Purpose To assess the initial biomechanical effect of the bioAID on the kinematic behavior of the canine spine, an ex vivo biomechanical study in 6-degrees-of-freedom was performed. Study Design A canine cadaveric biomechanical study. Methods Six cadaveric canine specimens (C3-C6) were tested in flexion-extension (FE), lateral bending (LB) axial rotation (AR) using a spine tester in three conditions: intact, after C4-C5 disc replacement with bioAID, and after C4-C5 interbody fusion. A hybrid protocol was used where first the intact spines were subjected to a pure moment of ±1 Nm, whereafter the treated spines were subjected to the full range of motion (ROM) of the intact condition. 3D segmental motions at all levels were measured while recording the reaction torsion. Biomechanical parameters studied included ROM, neutral zone (NZ), and intradiscal pressure (IDP) at the adjacent cranial level (C3-C4). Results The bioAID retained the sigmoid shape of the moment-rotation curves with a NZ similar to the intact condition in LB and FE. Additionally, the normalized ROMs at the bioAID-treated level were statistically equivalent to intact during FE and AR while slightly decreased in LB. At the two adjacent levels, ROMs showed similar values for the intact compared to the bioAID for FE and AR and an increase in LB. In contrast, levels adjacent to the fused segment showed an increased motion in FE and LB as compensation for the loss of motion at the treated level. The IDP at the adjacent C3-C4 level after implantation of bioAID was close to intact values. After fusion, increased IDP was found compared with intact but did not reach statistical significance. Conclusion This study indicates that the bioAID can mimic the kinematic behavior of the replaced intervertebral disc and preserves that for the adjacent levels better than fusion. As a result, CDR using the novel bioAID is a promising alternative treatment for replacing severely degenerated intervertebral discs