Cartilage-derived Stem Cells Research Articles

From regeneration to osteoarthritis in the knee joint: The role shift of cartilage-derived progenitor cells.

A mount of growing evidence has proven that cartilage-derived progenitor cells (CPCs) harbor strong proliferation, migration, andmultiple differentiation potentials over the past 2 decades. CPCs in the stage of immature tissue play an important role in cartilage development process and injured cartilage repair in the young and active people. However, during maturation and aging, cartilage defects cannot be completely repaired by CPCs in vivo. Recently, tissue engineering has revealed that repaired cartilage defects with sufficient stem cell resources under good condition and bioactive scaffolds in vitro and in vivo. Chronic inflammation in the knee joint limit the proliferation and chondrogenesis abilities of CPCs, which further hampered cartilage healing and regeneration. Neocartilage formation was observed in the varus deformity of osteoarthritis (OA) patients treated with offloading technologies, which raises the possibility that organisms could rebuild cartilage structures spontaneously. In addition, nutritionmetabolismdysregulation, including glucose and free fatty acid dysregulation, could influence both chondrogenesis and cartilage formation. There are a few reviews about the advantages of CPCs for cartilage repair, but few focused on the reasons why CPCs could not repair the cartilage as they do in immature status. A wide spectrum of CPCs was generated by different techniques and exhibited substantial differences. We recently reported that CPCs maybe are as internal inflammation sources during cartilage inflammaging. In this review, we further streamlined the changes of CPCs from immature development to maturation and from healthy status to OA advancement. The key words including "cartilage derived stem cells", "cartilage progenitor cells", "chondroprogenitor cells", "chondroprogenitors" were set for latest literature searching in PubMed and Web of Science. The articles were then screened through titles, abstracts, and the full texts in sequence. The internal environment including long-term inflammation, extendedmechanical loading, and nutritional elements intake and external deleterious factors were summarized. Taken together, these results provide a comprehensive understanding of the underlying mechanism of CPC proliferation and differentiation during development, maturation, aging, injury, and cartilage regeneration in vivo.

Single Cell Transcriptomic of Cartilage Reveals Variable Trajectory of CSPCs in the Progression of Osteoarthritis

Background: Osteoarthritis (OA) is characterized as cartilage destruction and lack of specific drugs. Cartilage-derived stem cells or progenitor cells (CSPCs) as multipotent cells play an essential role in cartilage renewal. However, alteration of CSPCs in the process of osteoarthritis remains elusive. Thus, understanding the changing patterns of CSPCs in the pathologic process of OA is greatly highlighted to provide a new insight on stem cell therapy in osteoarthritis (OA). Methods: We downloaded single cell transcriptomic data of patients with OA from GEO database and performed them on Unbiased clustering of the cells based on gene expression pattern by using Seurat package. By using specific cell markers, we identified CSPCs from all chondrocyte. We further explored the dynamics of CSPCs in the progression of OA and validated the scRNA-seq data in vitro with qPCR, immunofluorescence and western blotting. Findings: By using pan stem-cell markers, we traced CSPCs through whole stages. Specifically, we found that the heterogeneity of CSPCs was tightly associated with the progressing of OA. Our results indicates that CSPCs displayed significantly reduced differentiation capacity in the late stage of OA, while the proliferation capacity tends to increase. We also found that genes implicating in fibrosis, cell motility and extracellular matrix (ECM) remodeling were upregulated in CSPCs upon the progression of OA. Interpretation: Our study uncovers a dynamic of stem cell in progression of OA and may inform the development of stem cell therapy for OA. Funding Information: This work was supported by grants from the National Natural Science Foundation of China (81771651) and Xu Jun′s expert work station (202005AF150050). Declaration of Interests: The authors confirm that there are no conflicts of interest. Ethics Approval Statement: All of the animal experiments in this study were approved by the Animal Care and Use Committee of Tongji University School of Medicine.

A comparative study of mesenchymal stem cell transplantation and NTG-101 molecular therapy to treat degenerative disc disease

Cellular replacement therapy using mesenchymal stem cells (MSCs) and/or the delivery of growth factors are at the forefront of minimally invasive biological treatment options for Degenerative Disc Disease (DDD). In this study, we compared the therapeutic potential of a novel drug candidate, NTG-101 to MSCs, including rat cartilage derived stem cells (rCDSCs), bone marrow stem cells (rBMSCs) and human Umbilical Cord Derived Mesenchymal Stem Cells (hUCMSCs) for the treatment of DDD. We induced DDD using a validated image-guided needle puncture injury in rat-tail IVDs. Ten weeks post-injury, animals were randomized and injected with MSCs, NTG-101 or vehicle. At the end of the study, histological analysis of the IVD-Nucleus Pulposus (NPs) injected with NTG-101 or rCDSCs showed a healthy or mild degenerative phenotype in comparison to vehicle controls. Immunohistochemical analysis revealed strong expression of aggrecan, collagen 2, brachyury and Oct4 in IVD-NPs injected with NTG-101. Our results also demonstrated suppression of inflammation induced p38 and NFκB resulting in inhibition of catabolic genes, but activation of Smad-2/3, Erk-1/2 and Akt-dependent signaling inducing anabolic genes in IVD-NP on treatment with NTG-101. In conclusion, a single injection of NTG-101 into the degenerative disc demonstrated superior benefits compared to stem cell transplantation.

Corneal Repair with Adhesive Cell Sheets of Fetal Cartilage-Derived Stem Cells.

Corneal scarring or disease may lead to severe corneal opacification and consequently, severe loss of vision due to the complete loss of corneal epithelial cells. We studied the use of epithelial cell sheets differentiated from fetal cartilage-derived stem cells (FCSC) to resurface damaged cornea. The FCSC were isolated from the femoral head of immature cartilage tissue. The ability of the FCSCs to differentiate into corneal epithelial cells was evaluated using differentiation media at 2days and 7days post-seeding. A sheet fabricated of FCSCs was also used for the differentiation assay. The results of the in vitro studies were evaluated by immunocytochemistry and Western blots for corneal epithelial cell markers (CK3/12 and Pax6) and limbal epithelial stem cell markers (ABCG2 and p63). To test the material in vivo, an FCSC-sheet was applied as a treatment in a chemically burned rabbit model. The healing ability was observed histologically one week after treatment. The in vitro experiments showed morphological changes in the FCSCs at two and seven days of culture. The differentiated cells from the FCSCs or the FCSC-sheet expressed corneal epithelial cells markers. FCSC were create cell sheet that successfully differentiated into corneal epithelial cells and had sufficient adhesion so that it could be fused to host tissue after suture to the ocular surface with silk suture. The implanted cell sheet maintained its transparency and the cells were alive a week after implantation. These results suggest that carrier-free sheets fabricated of FCSCs have the potential to repair damaged corneal surfaces.

213. Study comparing NTG-101, a growth factor-based therapy with mesenchymal stem cell (MSC) based treatment of degenerative disc disease (DDD) in pre-clinical rodent model

BACKGROUND CONTEXT Currently there is significant interest in the use of cellular therapies (such as stem cells) to treat degenerative disc disease (DDD). We recently explored the potential use of bone marrow derived stem cells (BMSCs) and cartilage-derived stem cells (CDSCs) in a small animal model of DDD as compared to our recently developed molecular therapy ‘NTG-101’. PURPOSE To compare the therapeutic efficacy of NTG-101 with BMSCs or CDSCs to treat DDD in rodent model. STUDY DESIGN/SETTING In vivo laboratory experimentation involving female Wistar rats aged 12-weeks. PATIENT SAMPLE No human subjects. Animal Care approved study involving female Wistar rats using validated rat-tail injury and injection model. OUTCOME MEASURES Histology, immunohistochemistry, Western blotting, gene expression. METHODS CDSCs were derived from the tibial and femoral plateaus of Wistar rats and sphere forming cells were passaged further in serum free, stem cell specific media. BMSCs were obtained from the femurs of these animals and cultured as adherent cells in media containing 8% fetal bovine serum (FBS) following standard protocols. Stemness was confirmed by differentiation into multiple lineages. In parallel, we induced DDD in caudal intervertebral discs (IVDs) of 12 weeks old Wistar rats (N=30) using image-guided needle puncture injury. Ten weeks later, animals were randomized in six treatment groups (n=5 animals/group) and received an intradiscal injection (8µl) containing:G1-phosphate buffered saline (PBS, 1X, pH=7.2); G2-NTG-101(1X);G3-CDSCs (150,000 cells/disc) suspended in PBS; G4-BMSCs (150,000 cells/disc) suspended in PBS;G5-CDSCs (150,000 cells/disc) suspended in NTG-101(1X); G6-BMSCs (150,000 cells/disc) suspended in NTG-101(1X). At the end of experiment (ie, 10 weeks post-injection, 20 weeks post-injury), animals were humanely euthanized and IVDs were harvested for histology, immunohistochemistry and Western blotting. RESULTS Gene expression analysis using quantitative real time PCR confirmed expression of pluripotency (Oct4, Nanog, Sox2) and mesenchymal stem cell surface markers (CD29, CD44, CD90, CD105, CD133, CD166) in all stem cells. CDSC and BMSCs differentiation into chondrogenic, osteogenic and adipogenic lineages was confirmed, thereby validating their stemness potential. Discs injected with PBS developed a degenerative phenotype with the loss of NP morphology, cellularity and the development of a degenerative fibrocartilaginous phenotype. On the other hand, IVDs injected with NTG-101 or CDSCs alone showed a significant recovery. The morphology and cellularity of discs injected with BMSCs alone or in combination with NTG-101 was not significantly different from those discs injected with PBS. NTG-101 injected discs showed a healthy NP rich in notochordal and resident stem cells as revealed by nuclear Brachyury and Oct4 respectively, whereas discs injected with stem cells alone or stem cells plus NTG-101 did not. CONCLUSIONS Our preclinical data demonstrated that single intradiscal injection of NTG-101 is a superior and effective therapy for regeneration of IVD-NP in comparison to mesenchymal stem cell-based therapies for treatment of DDD. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

122. Cartilage derived stem cells: a novel cellular replacement therapy to treat degenerative disc disease

BACKGROUND CONTEXT Degenerative disc disease (DDD) involves the loss of viable cells and degradation of the extracellular matrix (ECM) within the avascular, ischemic and hypoxic intervertebral disc (IVD) nucleus pulposus (NP). We have previously demonstrated that injection of a molecular therapeutic (NTG-101) containing TGF-β1 and CTGF can mediate the progression of DDD in a rat and canine model. However, patients suffering from more advanced degenerative disc may require cellular replacement. Bone marrow derived mesenchymal stem cells (MSCs) originate from a highly vascularized and immune competent niche but are incompatible with the harsh IVD NP environment. Therefore, we harvested a novel source of articular cartilage derived stem cells (CDSCs) from the hypoxic, ischemic and avascular cartilage niche that may be compatible with the severe NP tissue compartment and transplanted them into the NP alone and in combination with NTG-101. PURPOSE To identify and characterize a novel source of stem cells (CDSCs) and determine the efficacy of CDSC intradiscal transplantation alone or in combination with a molecular therapeutic 'NTG-101′ in our validated rat-tail model of DDD. STUDY DESIGN/SETTING In vivo laboratory experimentation involving female Wistar rats aged 12 weeks. PATIENT SAMPLE No human subjects. Animal Care approved study involving female Wistar rats using validated rat-tail injury and injection model. OUTCOME MEASURES Histology, immunohistochemistry, Western blotting. METHODS Cells sourced from the tibial and femoral plateaus of Wistar rats, were expanded in free-floating culture and sphere forming cells collected and passaged under clonal conditions. Stemness was confirmed by differentiation into chondrogenic, osteogenic, adipogenic and neurogenic lineages. DDD was induced within five rat-tail IVDs/animal in three groups of seven animals using image-guided needle puncture injury. Ten weeks later, 8ul/IVD phosphate buffered saline (PBS) was injected into the IVD NPs of five discs/animal (7 animals/group). Other groups of animals with identical disc injuries received injections of 150,000 CDSCs suspended in 8ul PBS, or 150,000 CDSCs suspended within 8ul NTG-101. The animals were humanely sacrificed 10 weeks later, and normal and treated IVDs were assayed by histological, immunohistochemical and Western blotting to determine levels of aggrecan, collagen type 2, Oct4, MMP-3, MMP-13 and COX-2. RESULTS CDSCs have stemness characteristics as defined by their differentiation into multiple cellular lineages. PBS-injected IVDs develop a degenerative phenotype whereas CDSC and CDSC+NTG-101 injected discs retain robust cellularity, maintenance of disc height and size of the NP. Immunohistochemical analyses demonstrated that PBS-injected IVDs become deficient in collagen type 2, aggrecan and Oct4 but show increased levels of matrix degrading (MMP-3 and -13) and pro-inflammatory (COX2) markers. IVD's that received CDSCs or CDSCs+NTG-101 showed undetectable levels of COX-2, MMP-3 and MMP-13 however Oct4 was expressed at levels like healthy controls. Western blotting supported the immunohistochemical results with PBS-injected discs showing low levels of Oct4, but markedly increased levels of COX-2, MMP-3 and MMP-13. CONCLUSIONS These results clearly support the use of CDSCs as a source of cellular therapy that is enhanced by suspension within a molecular therapeutic (NTG-101) for the treatment of advanced DDD. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Chondrogenic induction of human osteoarthritic cartilage-derived mesenchymal stem cells activates mineralization and hypertrophic and osteogenic gene expression through a mechanomiR

BackgroundWhile bone marrow-derived mesenchymal stem cells (BMSC) are established sources for stem cell-based cartilage repair therapy, articular cartilage-derived mesenchymal stem cells from osteoarthritis patients (OA-MSC) are new and potentially attractive candidates. We compared OA-MSC and BMSC in chondrogenic potentials, gene expression, and regulation by miR-365, a mechanical-responsive microRNA in cartilage (Guan et al., FASEB J 25: 4457–4466, 2011).MethodsTo overcome the limited number of OA-MSC, a newly established human OA-MSC cell line (Jayasuriya et al., Sci Rep 8: 7044, 2018) was utilized for analysis and comparison to BMSC. Chondrogenesis was induced by the chondrogenic medium in monolayer cell culture. After chondrogenic induction, chondrogenesis and mineralization were assessed by Alcian blue and Alizarin red staining respectively. MiRNA and mRNA levels were quantified by real-time PCR while protein levels were determined by western blot analysis at different time points. Immunohistochemistry was performed with cartilage-specific miR-365 over-expression transgenic mice.ResultsUpon chondrogenic induction, OA-MSC underwent rapid chondrogenesis in comparison to BMSC as shown by Alcian blue staining and activation of ACAN and COL2A1 gene expression. Chondrogenic induction also activated mineralization and the expression of hypertrophic and osteogenic genes in OA-MSC while only hypertrophic genes were activated in BMSC. MiR-365 expression was activated by chondrogenic induction in both OA-MSC and BMSC. Transfection of miR-365 in OA-MSC induced chondrogenic, hypertrophic, and osteogenic genes expression while miR-365 inhibition suppressed the expression of these genes. Over-expression of miR-365 upregulated markers of OA-MSC and hypertrophy and increased OA scores in adult mouse articular cartilage.ConclusionsInduction of chondrogenesis can activate mineralization, hypertrophic, and osteogenic genes in OA-MSC. MiR-365 appears to be a master regulator of these differentiation processes in OA-MSC during OA pathogenesis. These findings have important implications for cartilage repair therapy using cartilage derived stem cells from OA patients.

Study on the effect of adiponectin on the differentiation of cartilage derived stem cells into chondrocytes

Objective To observe the effect of adiponectin on the differentiation of cartilage derived stem cells into chondrocytes from the articular cartilage of patients with advanced osteoarthritis. Methods The remaining cartilage tissue was collected from 5 patients with advanced osteoarthritis underwent total knee arthroplasty, then cultured and selected chondroprogenitors, to observe and compare the expression of type Ⅰ, type Ⅱ collagen, Aggrecan and sex determining region Y box-9 (SOX-9) by immunohistochemistry and the related genes expression between simple cartilage induced group and the group with adiponectin. Results The chondroprogenitors selected from articular cartilage of patients with advanced osteoarthritis can differentiate into chondrocytes under appropriate conditions; when in chondrogenic medium added adiponectin, compared with the group without adiponectin, the gene and protein expression of type Ⅱ collagen and Aggrecan decreased (type Ⅱ collagen: 1.004±0.125 vs. 0.588±0.101, P=0.001; Aggrecan: 1.002±0.050 vs. 0.584±0.138, P=0.000), the SOX-9 downregulation was not obvious (0.998±0.080 vs. 0.946±0.149, P=0.513), but the expression of type I collagen increased significantly (2.290±0.349 vs. 1.010±0.059, P=0.000). Conclusion Adiponectin can inhibit chondrogenic potential of chondroprogenitors. Key words: Adiponectin; Chondrogprogenitors; Chondrogenic differentiation; Sex determining region Y box-9

Isolation and identification of stem cells in different subtype of cartilage tissue

Background: Cartilage tissue engineering provided a promising therapy for the repair of cartilage defects, and seeding cells play a vital role in cartilage regeneration. Chondrocytes and bone marrow-derived mesenchymal stem cells (BMSCs) were reported to be the ideal seeding cells, but ‘dedifferentiation’ and ‘unstable phenotype’ of tissue-engineered cartilage constructed by the two cell type hamper their clinical application. Recently, cartilage tissue was reported to possess a stem cell population, which may be a more superior cell source in cartilage tissue engineering.Methods: In current study, we isolated a cell population from different subtype of cartilage tissue via a differential adhesion assay to fibronectin.Results: Flow cytometry analysis demonstrates the cell lines expressed mesenchyme stem cell positive surface marker such as CD29 and CD90. Meanwhile, the cells are highly proliferative and multipotent. Reverse transcription–PCR detection showed the cell population expressed osteogenic and adipogenic differentiation under different induction conditions. More interesting, monolayer cells underwent chondrogenic differentiation in the presence of dexamethasone and insulin-like growth factor 1. In addition, the expression of chondrogenic genes in cartilage-derived stem cells (CSCs) was higher than those in BMSCs.Conclusion: CSC may become an ideal seeding cell in cartilage tissue engineering, owing to its stemness and chondrogenic characteristics.

Articular Cartilage-Derived Stem Cells: Identification, Characterisation and their Role in Spontaneous Repair

The poor reparative potential of articular cartilage is largely attributed to its avascular and aneural status combined with low cellularity; chondrocytes only occupy 10% of the tissue volume. Consequently, there have been a number of strategies developed to augment repair most notably through microfracture and Autologous Chondrocyte Implanation (ACI). However, both of these techniques have limitations. In the case of microfracture, the repair tissue is often fibrocartilaginous and in ACI, the number of cells that can be generated that maintain chondrogenicity is limited thus restricting the size of defect that can be treated. Consequently, there has been increasing interest in the use of Mesenchymal Stem Cells (MSCs) for the treatment of cartilage defects. Here, we discuss the isolation and characterization of a tissue-specific stem cell population from articular cartilage and the potential for its use in intrinsic and extrinsic repair of articular cartilage lesions. Significantly, unlike MSCs isolated from bone marrow, upon differentiation into the chondrogenic lineage, these cells fail to terminally differentiate i.e. are not endochondral, failing to synthesize both collagen type X and alkaline phosphatase. Senescence of chondrocytes following injury or as part of aging is hypothesized to be integral to degenerative disease and correlates with significant telomere erosion within chondrocytes. Articular cartilage-derived stem cells exhibit detectable telomerase activity and exhibit reduced erosion of telomeres; therefore, we hypothesize maintenance of this population is critical to tissue homeostasis.