In-vivo induction of chondrocytes from articular cartilage stem cells by defined factors Linkai Zhu 4/6/16 Targeting OA

Osteoarthritis Image credit : http://www.med.nyu.edu/medicine/labs/abramsonlab

Osteoarthritis (OA) vs. Rheumatoid arthritis (RA) Pap et al., 2015

Molecular mechanism of osteoarthritis Loss of chondrocyte phenotypic stability is major step in OA progression. OA stress: genetics, injury, mechanical load and inflammation Increased MMPs and ADAMTS (extracellular protease enzymes), ECM degradation and release of mediators Epigenetic changes, loss of CXCR2 and FGF-2 Activation of innate immunity via TLRs Sensitization to inflammation via IL-1 Pap et al., 2015

Molecular mechanism of osteoarthritis TLR4 mediated responses decrease ECM synthesis of chondrocytes. Increased MMPs (matrix metalloproteinases) expression Decreased aggrecan (integral part of the ECM in cartilagenous tissue) and type II collagen synthesis Gómez et al., 2014

Molecular mechanism of osteoarthritis Cytokines production in OA TNF, IL‑1β and IL‑6 produced by chondrocytes, macrophages, T cells and osteophytes Pro-MMPs released by synoviocytes and macrophages Chevalier et al., 2013

Articular cartilage stem cells Under injury, the cartilage would regenerate the lost chondrocytes by articular cartilage stem cells. intact cartilage may be barrier and ECM should be degraded to induce migration of embedded cells. Jiang et al., 2015

Progression of OA An imbalance between anabolism (chondrogenesis and migration) catabolism (ECM breakdown for chondrocytes migration) of the articular cartilage, particularly by an increase in catabolism. The local microenvironment break down (overwhelming immune response).

Hypothesis: The low regenerative ability of articular cartilage stem cells could be because of poor cellular signaling communication for ACSCs (lack of blood vessels). By ectopically generating suitable microenvironment with gradients of defined factors (Needs further screen), we would be able to guide the migration of both chondrocytes and ACSCs and promote their proliferation rate.

In-vitro induction of chondrocytes

Isolation and preparation of cartilage explant specimens Methods Isolation and preparation of cartilage explant specimens femoral trochlea (8 human donors, knee arthroplasty due to primary osteoarthritis; mean age 69.88 years and range 53-80 years) central groove created to expose deeper cartilage layers briefly washed (native cartilage) and thorough washed (cleansed cartilage, minimize cartilage attaching cells)

Superficial zone facing up in 500 µl DMEM, 10% FCS and 1% Pen-Strep Methods Culturing Superficial zone facing up in 500 µl DMEM, 10% FCS and 1% Pen-Strep 5 µl DMEM containing either 200 µg/ml HMGB-1, 10 mg/ml TFF3, 10 µg/ml BMP-2 or 10 µg/ml TGF-β1 applied to the groove of the explants (100X bioactive concentration) Unspecific stimulus: 50 µl/ml cell lysate from primary human articular chondrocytes Co-culture with pieces of the synovial membrane

Results cleansed naive Attaching cells Retained integrity

Cartilage-adjoining cells: fibroblastic morphology Results No cell on the surface Cartilage-adjoining cells: fibroblastic morphology Neither elicited outgrowth of matrix-embedded chondrocytes, nor significantly increased the number of cells adjoining “native cartilage” specimens.

Results Influence of three-dimensional matrices on the number of cartilage-adjoining cells. No change in cellular outgrowth and the number of cartilage-adjoining cells.

Results Influence of three-dimensional matrices on the number of cartilage-adjoining cells. Without an additional matrix, the co-culture with synovial membrane significantly increased the number of cartilage-adjoining cells oriented in a cell layer (b). In co-cultures with application of a fibrin matrix (c) or a collagen matrix (d), the adjoining cells were distributed three-dimensionally within matrices. From this study, the attaching mesenchymal cells may contribute to cartilage regeneration in normal in-vivo environment.

In-vivo induction of chondrocytes Existence of articular cartilage stem cells (ACSCs) in vivo and in situ for the first time; ACSCs were activated and exhibited a transient proliferative response in early OA as an initial attempt for self-repair; ACSCs’ activation were maintained by NF-jB pathway inhibitor, which induced cartilage regeneration, and protected articular cartilage from injury inan OA animal model.

Methods Labeling and detection of ACSCs in-vivo using BrdU (quiescent) and Ki67 (active) OA animal model generated by medial collateral ligament (MCL) transfection and medial meniscal tear of the knee joints NF-ĸB pathway inhibitor (10µM, treatment) and DMSO (control) 1 day after the surgery (10 µl per joint per rat twice a week) The portion of ACSCs characterization was excluded.

ACSCs were existent and quiescent in normal mature mouse cartilage Results ACSCs were existent and quiescent in normal mature mouse cartilage

Results

Results ACSCs were activated in early OA, but the active ACSCs loss gradually as the development of OA progresses. The highest frequencies of Ki671/BrdU1 ACSCs were found in the superficial zone (SZ) of the cartilage Articular cartilage stem cells (ACSCs) were activated in the context of osteoarthritis (OA). (A): BrdU1 ACSCs were detected on the cartilage of the sham group, but no Ki67 expression was observed in ACSCs. (B–D, H): In OA group, Ki67 was gradually detected in BrdU1 ACSCs as OA progressed and maximized at 14 days after the OA-inducing surgical operation. (E–G, H): The number of transient proliferating ACSCs (Ki671/BrdU1 ACSCs) visualized as cartilage degeneration significantly decreased. (A–G, I): The cartilage degeneration and The Osteoarthritis Research Society grade showed the OA development. Cyan nuclear represent BrdU1 ACSCs and white nuclear represent Ki671/BrdU1 ACSCs. *, p<.05; **, p<.01; ***, p<.001 between two groups.

IL-1β Suppressed the TGF-β1-Induced Chondrogenesis in Rat ACSCs Results IL-1β Suppressed the TGF-β1-Induced Chondrogenesis in Rat ACSCs The proinflammatory cytokines IL-1β inhibited the TGF-b1-induced ACSCs chondrogenesis by suppressing the expression of the transcription factor (SOX9), extracellular matrix (ECM) (COL Ila and aggrecan), and related synthetase (hyaluronansynthetase 2), and these inhibitory effects on ACSCs chondrogenesis were dose-dependent. Figure 4. IL-1β suppressed the TGF-β1 induced chondrogenesis in rat articular cartilage stem cells. (A): Dose dependence of the IL-1β-inhibited effects on aggregates cultured with increasing concentrations of IL-1β in the presence and absence of TGF-β1, which was also demonstrated by (B) pellet diameter. (C): Toluidine blue staining. (D): qPCR analysis of the four cartilage markers, namely, SOX9, COL IIa, aggrecan, and Has 2. The values are the mean and SEM of triplicate experiments. *, p < .05; ***, p < .001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Results NF-ĸB Pathway Plays an Essential Role in IL-1β -Induced Inhibition of ACSCs Chondrogenesis NF-κB pathway has a key role in IL-1β-induced inhibition of articular cartilage stem cells chondrogenesis. (A): PAI-1 was significantly stimulated by TGF-β1 and suppressed by IL-1β in a dose-dependent manner. (B): TGF-β1 downregulated the Smad7 expression, whereas IL-1β significantly reversed the effect of TGF-β1 in a dose-dependent manner. Notably, 10 ng/ml of IL-1β completely offset 10 ng/ml of TGF-β1 suppression on Smad7 and significantly increased the Smad7 expression compared with the control (no TGF-β1) group (approximately 1.4-fold) and the TGF-β1 treatment-only group (approximately 2.6-fold). (C): Conversely, TGF-βRII was significantly induced by TGF-β1 and suppressed by IL-1β in a dose-dependent manner. The results are expressed as the percentage of controls after normalization against GAPDH. (D): Western blot results of Smad7 and TGF-βRII showed the same trend as that of qPCR results. (E): The statistics of the Western blot results. (F, G): The NF-κB pathway was inhibited by BAY11-7082. (F): The suppression of p65 transport into the nucleus induced by IL-1β. (G): MMP13 downregulation. The values are the mean and SEM of triplicate experiments. *, p < .05; **, p < .01; ***, p < .001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Results NF-ĸB Pathway Inhibitor Could Restore TGF-β1-Induced Chondrogenesis Inhibited by IL-1β NF-κB pathway inhibitor restored TGF-β1-induced chondrogenesis inhibited by IL-1β. (A–C): BAY11-7082 distinctly restored TGF-β1-induced chondrogenesis inhibited by IL-1β in a dose-dependent manner. (A): Naked eye pellet size, (B) pellet diameter, and (C) qPCR analysis of SOX9, COL IIa, aggrecan, and Has 2. (D): The level of PAI-1 suppressed by IL-1β, and restored by BAY11-7082 in a dose-dependent manner. (E): TGFβ-1 downregulated Smad7 expression and IL-1β markedly reversed the effect of TGF-β1. However, the IL-1β effect could be reversed by BAY11-7082. (F): Conversely, TGF-βRII was significantly induced by TGF-β1, suppressed by IL-1β, and restored by BAY11-7082 in a dose-dependent manner. (G, H): Western blot results of Smad7 and TGF-βRII, which showed the same trend as that of the qPCR results. (H): The statistics of the Western blot results. The values are the mean and SEM of triplicate experiments. *, p < .05; **, p < .01; ***, p < .001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Results Inhibition of NF-ĸB Signaling Can Induce ACSCs Activation and Retard the Progression of Experimental OA The NF-ĸB pathway is crucial for regulating ACSCs activity and blocking the NF-ĸB pathway to induce ACSCs activation may be a possible method for OA therapy. Inhibition of NF-κB signaling induced articular cartilage stem cells (ACSCs) activation and retarded the progression of experimental osteoarthritis (OA). (A–I): The BrdU+ ACSCs were gradually activated (Ki67+/BrdU+ ACSCs) from the resting state in early OA, maximized in about the 14th day (A, B) in the DMSO group and the 30th day (E–G) in the BAY11-7082 treatment group after the OA-inducing surgical operation. (B–D, G–J): The Ki67+/BrdU+ ACSCs in the DMSO group (after 14 days postoperation) and in the BAY11-7082 treatment group (after 30 days postoperation) gradually reduced with the progression of OA. The Ki67+/BrdU+ ACSCs maintained approximately 25% on the 90th day in the BAY11-7082 group (H, I) in contrast to 7% in the DMSO group (D, I). (A–H, J): The cartilage degeneration and The Osteoarthritis Research Society grade were more improved in the BAY11-7082 treatment group compared with the DMSO group. Cyan nuclear represent BrdU+ ACSCs and white nuclear represent Ki67+/BrdU+ ACSCs. ***, p < .001 between two groups.

Specific aims Down regulate IL-1β, IL-6 and TNF to slow the process of ECM degradation in OA. Induce the migration of chondrocytes and ACSCs to the superficial zone.

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