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Human adipose-derived stromal/stem cells expressing doublecortin improve cartilage repair in rabbits and monkeys.

ABSTRACT: Localized cartilage lesions in early osteoarthritis and acute joint injuries are usually treated surgically to restore function and relieve pain. However, a persistent clinical challenge remains in how to repair the cartilage lesions. We expressed doublecortin (DCX) in human adipose-derived stromal/stem cells (hASCs) and engineered hASCs into cartilage tissues using an in vitro 96-well pellet culture system. The cartilage tissue constructs with and without DCX expression were implanted in the knee cartilage defects of rabbits (n = 42) and monkeys (n = 12). Cohorts of animals were euthanized at 6, 12, and 24 months after surgery to evaluate the cartilage repair outcomes. We found that DCX expression in hASCs increased expression of growth differentiation factor 5 (GDF5) and matrilin 2 in the engineered cartilage tissues. The cartilage tissues with DCX expression significantly enhanced cartilage repair as assessed macroscopically and histologically at 6, 12, and 24 months after implantation in the rabbits and 24 months after implantation in the monkeys, compared to the cartilage tissues without DCX expression. These findings suggest that hASCs expressing DCX may be engineered into cartilage tissues that can be used to treat localized cartilage lesions.

SUBMITTER: Ge D

PROVIDER: S-EPMC8633050 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Human adipose-derived stromal/stem cells expressing doublecortin improve cartilage repair in rabbits and monkeys.

Ge Dongxia D O'Brien Michael J MJ Savoie Felix H FH Gimble Jeffrey M JM Wu Xiying X Gilbert Margaret H MH Clark-Patterson Gabrielle L GL Schuster Jason D JD Miller Kristin S KS Wang Alun A Myers Leann L You Zongbing Z

NPJ Regenerative medicine 20211130 1

Localized cartilage lesions in early osteoarthritis and acute joint injuries are usually treated surgically to restore function and relieve pain. However, a persistent clinical challenge remains in how to repair the cartilage lesions. We expressed doublecortin (DCX) in human adipose-derived stromal/stem cells (hASCs) and engineered hASCs into cartilage tissues using an in vitro 96-well pellet culture system. The cartilage tissue constructs with and without DCX expression were implanted in the kn ...[more]

PMID: 34848747

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Project description:BackgroundTumour homing capacity of engineered human adipose-derived mesenchymal stromal cells (ADMSCs) expressing anti-tumour agents might be the key for a much safer and yet efficient targeted tumour therapy. However, ADMSCs exhibit resistant to most gene transfection techniques and the use of highly efficient viral vectors has several disadvantages primarily concerning safety risk. Here, we optimized the use of highly efficient and safe nucleofection-based transfection using plasmid encoded for TNF-Related Apoptosis Inducing Ligand (TRAIL) into ADMSCs and investigated the potential anti-tumourigenic of TRAIL-expressing ADMSCs (ADMSCs-TRAIL) on selected cancer models in vitro.MethodsDifferent concentration of TRAIL-encoded plasmid and ADMSCs were nucleofected and the percentage of fluorescence cells were analyzed to determine the optimal condition. TRAIL protein and mRNA were validated in nucloeofected ADMSCs using ELISA and RT-PCR respectively. Evaluation of TRAIL specific death receptors were performed on both tumours (A549/lung tumour, LN18/glioblastoma and HepG2/hepatocellular carcinoma) and haematological malignant lines (REH/acute lymphocytic leukaemia, K562/chronic myelogenous leukaemia and KMS-28BM/multiple myeloma) using flow cytometry. ADMSCs-TRAIL was subsequently assessed for anti-tumourigenic properties using both proliferation assay (MTS assay) and apoptosis assay (Annexin-V / Propidium Iodide staining).ResultsNucleofection showed increased total plasmid concentration (2 μg to 8 μg) resulted in significantly higher reporter expression (11.33% to 39.7%) with slight reduction on cells viability (~10%). ADMSCs-TRAIL significantly inhibited ~50% of cell proliferation in LN18, signifying sensitivity of the cell to ADMSCs-TRAIL mediated inhibition. Inhibition of both tumour and malignant lines proliferation by ADMSCs-TRAIL conditioned medium noticed in HepG2, A549 and REH respectively, whereas K562 and KMS-28BM malignant lines exhibit resistant to ADMSCs-TRAIL mediated inhibition. Moreover, we found that native ADMSCs alone were capable of inducing apoptosis in both LN18 and HepG2 tumour lines, despite substantial increased on the percentage of apoptosis by ADMSCs-TRAIL.ConclusionADMSCs-TRAIL selectively inhibit cancer model and markedly induces apoptosis. Through investigation of the specific TRAIL death receptors expression, we saw that the receptors expression did influence the sensitivity of some but not all cancer lines to TRAIL-mediated inhibition. This study provides further insight into the anti-tumourigenic potential of ADMSCs-TRAIL on different cancer models.

Project description:Human adipose-derived stromal cells (hASCs) have the proven capacity to ossify skeletal defects. The mechanisms whereby hASCs stimulate bone repair are not fully understood. In this study, we examined the potential for hASCs to stimulate autogenous repair of a mouse calvarial defect. Immunofluoresence, osteogenic stains, and surface electron microscopy were used to demonstrate osteogenic differentiation of hASCs. hASCs were engrafted into 4 mm calvarial defects in athymic mice using an osteoconductive scaffold. Analysis included microcomputed tomography, histology, in situ hybridization, and quantitative real-time-polymerase chain reaction. Next, the in vitro interaction between hASCs and mouse calvarial osteoblasts (mOBs) was assessed by the conditioned medium and coculture assays. The medium was supplemented with Hedgehog signaling modifiers, including recombinant N-terminal Sonic hedgehog, smoothened agonist, and cyclopamine. Finally, cyclopamine was delivered in vivo to hASC-engrafted defects. Significant calvarial healing was observed among hASC-engrafted defects compared with control groups (no treatment or scaffold alone) (*P<0.05). hASCs showed evidence of stimulation of host mouse osteogenesis, including (1) increased expression of bone markers at the defect edge by in situ hybridization, and (2) increased host osteogenic gene expression by species-specific quantitative real-time polymerase chain reaction. Using the conditioned medium or coculture assays, hASCs stimulated mOB osteogenic differentiation, accompanied by Hedgehog signaling activation. N-terminal Sonic hedgehog or smoothened agonist replicated, while cyclopamine reversed, the pro-osteogenic effect of the conditioned medium on mOBs. Finally, cyclopamine injection arrested bone formation in vivo. hASCs heal critical-sized mouse calvarial defects, this is, at least in part, via stimulation of autogenous healing of the host defect. Our studies suggest that hASC-derived Hedgehog signaling may play a paracrine role in skeletal repair.

Project description:PurposeTo compare the pain relief and cartilage repair status of patients with knee osteoarthritis who received arthroscopic treatment with or without stromal vascular fraction (SVF) implantation.MethodsWe retrospectively evaluated the patients who were examined with 12-month follow-up magnetic resonance imaging (MRI) after arthroscopic treatment for knee osteoarthritis from September 2019 to April 2021. Patients were included in this study if they had grade 3 or 4 knee osteoarthritis according to the Outerbridge classification in MRI. The visual analog scale (VAS) was used for pain assessment over the follow-up period (baseline and at 1-, 3-, 6-, and 12-month follow-ups). Cartilage repair was evaluated using follow-up MRIs based on Outerbridge grades and the Magnetic Resonance Observation of Cartilage Repair Tissue scoring system.ResultsAmong 97 patients who received arthroscopic treatment, 54 patients received arthroscopic treatment alone (conventional group) and 43 received arthroscopic treatment along with SVF implantation (SVF group). In the conventional group, the mean VAS score decreased significantly at 1-month post-treatment compared with baseline (P < .05), and gradually increased from 3 to 12 months' post-treatment (all P < .05). In the SVF group, the mean VAS score decreased until 12 months post-treatment compared with baseline (all P < .05 except P = .780 in 1-month vs 3-month follow-ups). Significantly greater pain relief was reported in the SVF group than in the conventional group at 6 and 12 months' post-treatment (all P < .05). Overall, Outerbridge grades were significantly greater in the SVF group than in the conventional group (P < .001). Similarly, mean Magnetic Resonance Observation of Cartilage Repair Tissue scores were significantly greater (P < .001) in the SVF group (70.5 ± 11.1) than in the conventional group (39.7 ± 8.2).ConclusionsThe results regarding pain improvement and cartilage regeneration and the significant correlation between pain and MRI outcomes at 12-months follow-up indicate that the arthroscopic SVF implantation technique may be useful for repairing cartilage lesions in knee osteoarthritis.Level of evidenceLevel III, retrospective comparative study.

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Human adipose-derived stromal/stem cells expressing doublecortin improve cartilage repair in rabbits and monkeys.

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Human adipose-derived stromal/stem cells expressing doublecortin improve cartilage repair in rabbits and monkeys.

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