Induction of iPSC-derived Prg4-positive cells with characteristics of superficial zone chondrocytes and fibroblast-like synovial cells

doi:10.21203/rs.3.rs-1400781/v2

Download PDF

Research Article

Induction of iPSC-derived Prg4-positive cells with characteristics of superficial zone chondrocytes and fibroblast-like synovial cells

https://doi.org/10.21203/rs.3.rs-1400781/v2

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background: Lubricin, a proteoglycan encoded by the PRG4 gene, is synthesised by superficial zone (SFZ) chondrocytes and synovial cells. It reduces friction between joints and allows the smooth sliding of tendons. Although lubricin has been shown to be effective against osteoarthritis and synovitis in animals, its clinical application remains untested. In this study, we aimed to induce lubricin-expressing cells from pluripotent stem cells (iPSCs) and applied them locally via cell transplantation.

Methods: To generate iPSCs, OCT3/4, SOX2, KLF4, and L-MYC were transduced into fibroblasts derived from Prg4-mRFP1 transgenic mice. We established a protocol for the differentiation of iPSC-derived Prg4-mRFP1-positive cells and characterised their mRNA expression profile. Finally, we injected Prg4-mRFP1-positive cells into the paratenon surrounding the Achilles tendons and knee joints of severe combined immunodeficiency mice and assessed lubricin expression.

Results: Wnt3a, activin A, TGF-β1, and bFGF were applied to induce the differentiation of iPSC-derived Prg4-mRFP1-positive cells. Markers related to SFZ chondrocytes and fibroblast-like synovial cells (FLSs) were expressed during differentiation. RNA-sequencing indicated that iPSC-derived Prg4-mRFP1-positive cells manifested expression profiles typical of SFZ chondrocytes and FLSs. Transplanted iPSC-derived Prg4-mRFP1-positive cells survived around the Achilles tendons and in knee joints.

Conclusions: The present study describes a protocol for the differentiation of iPSC-derived Prg4-positive cells with characteristics of SFZ chondrocytes and FLSs. Transplantation of lubricin-expressing cells offers promise as a therapy against arthritis and synovitis.

iPSCs

Prg4

superficial zone chondrocytes

fibroblast-like synovial cells

Lubricin is a high-molecular weight proteoglycan encoded by the PRG4 gene. It is expressed in superficial zone (SFZ) chondrocytes of articular cartilage, synovial cells, tenocytes, and the tendon sheath [1–4]. It diminishes the friction between joints and allows for smooth sliding of tendons [5–9]. Osteoarthritis causes increased chondrocyte destruction, joint friction, and synovial inflammation. When articular cartilage is destroyed in a localised superficial area, chondrocytes are replaced by fibrous cartilage tissue. When the same happens in a large and deep area, chondrocytes do not reproduce and the subchondral bone becomes exposed. Tenosynovitis causes increased synovial inflammation and adhesion of the tendon. Subsequently, prolonged tenosynovitis causes scarring of the tendon, which results in loss of its smooth sliding.

In the early stage of osteoarthritis, with only synovial inflammation, symptoms can be alleviated via intra-articular injection of hyaluronic acid or synovectomy. Similarly, mild tenosynovitis can be alleviated via injection of steroids in the tendon sheath. Instead, in the case of deep cartilage defect in a limited area, autologous cartilage transplantation can be performed [10, 11]. An alternative therapy is cartilage tissue regeneration from mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) [12–18]. Owing to their pluripotency and versatility, iPSCs have been applied in regenerative medicine for several tissue and organs, including cartilage and tendon [19–22].

The therapeutic effect of lubricin administration has also garnered increasing interest. Intra-articular injections of partial recombinant human lubricin (rhPRG4) prevented cartilage degeneration in a rat model of osteoarthritis [6], and full-length rhPRG4 mitigated cartilage damage following destabilisation of the medial meniscus in a minipig model [23]. In addition to lubricating joints and tendons, rhPRG4 exhibited anti-inflammatory effect by inhibiting cytokine (interleukin-1β and tumour necrosis factor-α) secretion from osteoarthritis fibroblasts and cytokine-induced proliferation of synovial cells [24, 25]. However, according to the experimental results, repeated injections of rhPRG4 are likely required to obtain treatment effect. Moreover, rhPRG4-based therapy has not been applied clinically. Here, we hypothesised that a therapeutic strategy based on injecting iPSC-derived lubricin-expressing cells could provide an effective treatment for osteoarthritis and tenosynovitis.

Generation of iPSCs and maintenance

First, transgenic mice expressing monomeric red fluorescent protein (mRFP1) under the control of Prg4 promoter were generated by injection of the transgene into the pronuclei of fertilised eggs from C57BL/6 mice (Charles River). To establish iPSCs, fibroblasts derived from Prg4-mRFP1 transgenic mice were infected with pMX-hOCT3/4, pMX-hSOX2, pMX-hKLF4, and pMX-Hu-L-MYC retroviral vectors (Addgene). The established iPSC clones were maintained in ESC medium consisting of KnockOut DMEM (Gibco), 15% foetal bovine serum (Thermo Scientific), 2 mM L-glutamine (Wako), 1% non-essential amino acids (Wako), and supplemented with 1000 units/mL human recombinant leukaemia inhibitory factor (LIF; Wako), 0.1 mM 2-mercaptoethanol (Gibco), 50 μg/mL L-ascorbic acid (Sigma-Aldrich), 50 units/mL penicillin (Wako), and 50 μg/mL streptomycin (Wako).

Maintenance of ESCs

ESCs (V6.5) were maintained in ESC medium (KnockOut DMEM containing 15% foetal bovine serum, 2 mM L-glutamine, and 1% non-essential amino acids), supplemented with 1000 units/mL LIF, 0.1 mM 2-mercaptoethanol, 50 units/mL penicillin, and 50 μg/mL streptomycin.

Teratoma formation and histological analysis

We injected iPSCs into the subcutaneous tissue of severe combined immunodeficiency (SCID) mice (Charles River). Three weeks after injection, the mice were sacrificed and teratomas were dissected, fixed in 4% paraformaldehyde overnight, and embedded in paraffin. Semi-serial sections were stained with hematoxylin and eosin (H&E).

Induction of iPSC-derived Prg4-positive cells

Before starting the induction of Prg4-positive cells, iPSCs were cultured for 4 days in iPSC maintenance medium in a humidified atmosphere with 5% CO₂ at 37 ℃ until they became subconfluent. Initially, iPSCs were differentiated as embryoid bodies on 96-well plates (Nunclon Sphera; Thermo Scientific) in standard high-glucose DMEM containing 10% foetal bovine serum, penicillin, and streptomycin for 2 days. On day 2, the medium was changed to standard medium containing 9 ng/mL activin A (Peprotech) and 25 ng/mL Wnt3a (R&D) for mesoderm differentiation. On day 3, the medium was changed to standard medium containing 10 ng/mL basic fibroblast growth factor (bFGF; Wako), and the cells were cultured continuously for 2 days. On day 5, embryoid bodies were dissociated and reseeded as a monolayer on collagen-coated 6-well plates containing standard medium supplemented with 1% insulin-transferring-selenium (ITS; Gibco), 10 ng/mL transforming growth factor beta 1 (TGF-β1; Cell Signaling), and 10 ng/mL bFGF. Medium was changed every other day and differentiated cells were cultured until day 21.

Fluorescence-assisted cell sorting analysis

Prg4-mRFP1-positive cells were selected by fluorescence-activated cell sorting (FACS) on a BD FACS Aria flow cytometer (BD Biosciences). We used ESCs-derived differentiated cells as a negative control. Data were analysed with DIVA software (BD Biosciences), which identified iPSC-derived Prg4-mRFP1-positive cells as having a higher PE-Texas Red signal than negative control ESC-derived cells.

Quantitative real-time PCR

Total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen) and reverse-transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using TB Green Premix Ex Taq Ⅱ (TaKaRa). The expression of target genes was normalised to that of reference genes (Actb and Gapdh) and is presented as the mean ± standard deviation based on three independent experiments. Primers used for qRT-PCR are listed in Table 1.

Injection of iPSC-derived Prg4-mRFP1-positive cells in the paratenon surrounding the Achilles tendon and knee joint of SCID mice

We injected iPSC-derived Prg4-mRFP1-positive cells at 1 × 10⁴ cells/10 μL in phosphate-buffered saline in the paratenon surrounding the Achilles tendons and knee joints of SCID mice (Charles River). Three days after injection, the mice were sacrificed, and the Achilles tendons and knee joints were dissected and fixed in 4% paraformaldehyde overnight.

Histological analysis

All tissues were fixed in 4% paraformaldehyde overnight at 4 ℃ and embedded in paraffin. The knee joint tissues of Prg4-mRFP1 transgenic mice and SCID mice (Charles River) which were injected iPSC-derived Prg4-mRFP1-positive cells were fixed in 4% paraformaldehyde overnight at 4 ℃ and then decalcified in EDTA (G-Chelate Mild; NIPPON Genetics) for 2 weeks at 4 ℃. Semi-serial sections were stained with H&E. For immunohistochemistry, sections were stained with a rat monoclonal anti-RFP antibody (1:200, 5f8; Chromotek), anti-rat Histofine simple stain mouse MAX PO (414311; Nichirei Bioscience), and DAB substrate (K3467; DAKO). For immunofluorescence, sections were stained with an Alexa 594-conjugated anti-RFP antibody (1:2000, 150160; Abcam). Cell nuclei were stained with DAPI (1:500; Cell Signaling).

RNA-sequencing analysis

To determine their expression profile, iPSC-derived Prg4-mRFP1-positive cells sorted by FACS (#2-9) were analysed on a next-generation sequencer. A heatmap summarising the genes related to the differentiation process was generated based on markers related to myogenesis, mesoderm, macrophage-like synovial cells (MLSs), pluripotency, chondrogenesis, joint interzone, fibroblast-like synovial cells (FLSs), and SFZ cells. The obtained profiles were compared to ESC (SRS1026767 from the SRA database), mesoderm (ERR2179979 from SRA), chondrocyte (GSE92641 from the GEO database), FLSs (GSE142607 from GEO), MLSs (GSE142607 from GEO), and muscle (GSE152756 from GEO) RNA-sequencing datasets.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5 software. One-way ANOVA with Turkey’s post-test was used for statistical analysis. A P value < 0.05 was considered statistically significant. Data are presented as the mean ± standard deviation based on three independent experiments.

Generation of Prg4-mRFP1 transgenic mice and iPSCs harbouring the Prg4-mRFP1 reporter system

To create the Prg4-mRFP1 transgene, we inserted the mRFP1 expression cassette, which contained a Kozak sequence and poly-adenylation signal, 3 kb downstream of the Prg4 promoter (Fig. 1a). The transgenes were injected in the pronuclei of fertilised eggs from C57BL/6 mice to generate Prg4-mRFP1 transgenic mice. To verify the successful construction of transgenic mice, mRFP1 fluorescence was detected around the Achilles and patellar tendons (Fig. 1b); whereas immunohistochemistry showed mRFP1 expression in tenocytes, tendon synovium, and SFZ chondrocytes of the articular cartilage (Fig. 1c). These results suggested that the Prg4-mRFP1 expression system was active in vivo and could be used to monitor the production of lubricin. Next, we generated iPSCs harbouring the Prg4-mRFP1 reporter system. iPSC-like cells were established from fibroblasts of Prg4-mRFP1 transgenic mice by retroviral transduction with OCT3/4, SOX2, KLF4, and L-MYC. Six iPSC-like clones were selected, and qRT-PCR revealed a comparable expression of pluripotency-related genes between iPSCs and ESCs (Fig. 2a). Clone iPSC (#2–9) was the most similar to ESCs (Fig. 2b) and was chosen for transplantation into subcutaneous tissue of SCID mice. Three weeks after transplantation, teratomas comprising ectodermal, mesodermal, and endodermal tissues could be observed (Fig. 2c), confirming the pluripotency of the established iPSCs.

Differentiation of iPSC-derived Prg4-positive cells

Next, we established a protocol for the differentiation of iPSC-derived Prg4-positive cells (Fig. 3a). First, iPSCs (#2–9) were differentiated as embryoid bodies for 2 days. On day 2, activin A and Wnt3a were added to promote differentiation of the primitive streak [24, 26–28], followed by that of the paraxial mesoderm and somites [29, 30]. Between day 3 and day 5, embryoid bodies were cultured in the presence of bFGF to promote cell proliferation and differentiation of the sclerotome. On day 5, embryoid bodies were dissociated and reseed as a monolayer supplemented with ITS, TGF-β1, and bFGF. TGF-β1 and bFGF have been shown to promote the differentiation of SFZ chondrocytes and synovial cells from the joint interzone [29, 31–36]. On day 21, mRFP1 fluorescence was detected in iPSC-derived differentiated cells, confirming the successful generation of Prg4-positive cells from iPSCs (Fig. 3b).

Gene expression analysis of iPSC-derived Prg4-mRFP1-positive cells by qRT-PCR

To investigate the differentiation state of iPSC-derived cells generated with our protocol, we analysed the expression of target mRNAs by qRT-PCR over time. Prg4 is expressed in SFZ chondrocytes and synovial cells [1–4]. The latter are classified mainly into FLSs and MLSs [37, 38]. FLSs, which are of mesenchymal origin, secrete lubricin and play a critical role in normal joint homeostasis [38, 39]. Both SFZ chondrocytes and FLSs differentiate from the joint interzone during the development of synovial joints [40–42]. Therefore, we confirmed the expression of genes representative of the joint interzone (Wnt9a and Wnt16) [40–42], SFZ (Prg4, Erg, Tnc, and Runx1) [43–47], and FLSs (Cdh11, Col4a, Vcam1, Icam1, and Cd248) [48–54]. Expression of Wnt9a and Wnt16 was significantly higher on day 12, but dropped on day 21 (Fig. 4a). Consistent with mRFP1 fluorescence on day 21, Prg4 expression was significantly increased on day 21, together with that of SFZ-related genes Erg and Tnc (Fig. 4b) and FLS-related genes Cdh11, Col4a, and Vcam1 (Fig. 4c). Our protocol also caused the differentiation of ESCs up until day 21, which we verified by qRT-PCR. ESC-derived differentiated cells displayed a similar gene expression profile as iPSC-derived Prg4-positive cells (Fig. 5). These results suggested that our differentiation protocol successfully induced Prg4-positive cells from pluripotent stem cells and equipped them with SFZ chondrocyte and FLS characteristics.

RNA-sequencing analysis of FACS-sorted iPSC-derived Prg4-mRFP1-positive cells

Having demonstrated the successful induction of Prg4-mRFP1-positive cells by our differentiation protocol, we proceeded with their further characterisation. Based on FACS, mRFP1-positive cells constituted 10% of the cells differentiated from the iPSC (#2–9) clone (Fig. 6a). RNA-sequencing of mRFP1-positive cells obtained by FACS revealed that iPSC-derived Prg4-mRFP1-positive cells lost the properties of pluripotent stem cells, mesoderm, and joint interzone; however, they showed the properties of SFZ chondrocytes and FLSs but not those of deep zone chondrocytes (Fig. 6b).

Injection of iPSC-derived Prg4-mRFP1-positive cells in the paratenon around the Achilles tendons and knee joints of SCID mice

Finally, we injected iPSC-derived Prg4-mRFP1-positive cells obtained by FACS in the paratenon surrounding the Achilles tendons and knee joints of SCID mice. Immunohistochemical analysis showed that, 3 days after injection, some iPSC-derived Prg4-mRFP1-positive cells were still present around the Achilles tendons and in the knee joint (Fig. 7). Notably, no teratomas were observed in SCID mice at 3 weeks after cell transplantation (data not shown). These results indicated that transplanted iPSC-derived Prg4-mRFP1-positive cells could produce lubricin in vivo for at least 3 days without risk of tumour formation.

Lubricin is believed to be an effective therapeutic agent for osteoarthritis and synovitis. However, clinical application of recombinant or synthesised lubricin has not been attempted yet. In this study, we successfully developed a protocol to induce lubricin-expressing cells from pluripotent stem cells and confirmed their differentiation into Prg4-expressing cells that shared the characteristics of SFZ chondrocytes and FLSs from the joint interzone.

Prg4 is expressed in SFZ chondrocytes, synovial cells, synovial fluid, tendons, and tendon sheath [3, 4]. Such localisation was confirmed in our Prg4-mRFP1 transgenic mice. First, iPSCs were established by transducing ear tip mouse fibroblasts with all four Yamanaka factors [55]. Second, we devised a protocol for the differentiation of iPSC-derived Prg4-positive cells. We focused on differentiation into SFZ cells and FLSs, which formed the synovial joint rather than cartilage. To induce the primitive streak, and subsequent differentiation into paraxial mesoderm and somites, we followed previous reports on embryoid body formation in suspension or adhesion for 4–6 days [11, 12, 15]. To maximise differentiation, we chose embryoid body formation in suspension for 5 days. Based on known cytokines involved in this process [12, 14, 15], we selected Wnt3a, activin A, and bFGF to promote differentiation of the sclerotome as a precursor to the synovial joint. Although useful for induction of the primitive streak, bone morphogenetic protein (BMP) 4 was not applied in this case as it could not lead to differentiation of the paraxial mesoderm.

For chondrogenic differentiation, most studies have employed serum-free medium [13–15] or 1% foetal bovine serum [12], as well as the cytokines ITS, bFGF, BMP2, TGF-β1, TGF-β3, growth differentiation factor 5, TD-198946, and ascorbic acid. Here, we cultured iPSCs in medium containing 10% foetal bovine serum, ITS, bFGF, and TGF-β1 to differentiate Prg4-positive SFZ cells and FLSs of the synovial joint.

To confirm successful differentiation, we analysed mRNA expression of iPSC-derived differentiated cells over time using qRT-PCR. Wnt9a and Wnt16 are important in the joint interzone for the development of synovial joints [40–42]. Wnt16 also supports the phenotype of SFZ progenitor cells and lubricin expression [56]. During differentiation, Wnt9a and Wnt16 showed significant upregulation on day 12, followed by a drop on day 21. This expression dynamics suggested that iPSCs differentiated in the joint interzone on day 12 and advanced to the next differentiation stage thereafter.

Besides Prg4, other genes expressed in SFZ chondrocytes include Erg, Tnc, and Runx1 [43–47]. We confirmed increased expression of Prg4, Erg, and Tnc on day 21, but could hardly detect them on day 12. Cdh11, Col4a, Cd55, Vcam1, Icam1, and Cd248 are expressed in FLSs of synovial joints [48–54]. Here, SFZ chondrocyte markers Prg4, Erg, and Tnc exhibited a similar expression pattern as FLS markers Cdh11, Col4a, and Vcam1. These results suggest that iPSCs differentiated into Prg4-expressing cells via the joint interzone. Analysis of mRNA expression in ESC-derived differentiated cells produced a similar temporal pattern as in iPSCs, suggesting that our protocol was applicable to all pluripotent stem cells. RNA-sequencing confirmed that iPSC-derived Prg4-positive cells (clone #2–9) were more similar to SFZ chondrocytes and FLSs than to deep zone chondrocytes, mesodermal cells, myogenic cells, and MLSs. Therefore, iPSC-derived Prg4-positive cells shared the characteristics of both SFZ chondrocytes and FLSs in synovial joints.

Recent strategies in regenerative therapy favour the use of stem cells rather than autologous cartilage transplantation to replenish missing or damaged cartilage [10, 11]. Examples of such an approach include the generation of articular cartilage using human ESCs [15], the generation of scaffold-free hyaline cartilage from human iPSCs [12], the generation of Col2a1-EGFP iPSCs for monitoring chondrogenic differentiation [13], the formation of stable human articular cartilage using human MSCs [16, 17], cartilage repair using a scaffold-free construct derived from porcine synovial MSCs [18], and specification of chondrocytes and cartilage tissue from murine ESCs [14]. The present study aimed to provide lubrication for arthritic joints and tenosynovitis by transplanting Prg4-expressing cells rather than regenerating damaged cartilage tissue. Hence, we injected iPSC-derived Prg4-mRFP1-positive cells into the paratenon surrounding the Achilles tendon and knee joints of SCID mice and confirmed the cells’ survival and lubricin expression in vivo. A recent study revealed that Prg4-lineage cells in the joint SFZ differentiated into articular chondrocytes [57], suggesting that injection of iPSC-derived Prg4-expressing cells might also have a potential to regenerate articular cartilage.

Several hurdles need to be overcome prior to the clinical application of Prg4-expressing cells. First, using the same protocol, we previously induced tenocyte-like cells from Scx-EGFP iPSCs [58]. Tenocytes share the same developmental process through somite to sclerotome with chondrocytes and synovial cells. Given that Prg4 expression has been confirmed in tendons, iPSC-derived Prg4-positive cells in this study might contain tenocytes. Therefore, the differentiation protocol should be optimised to improve the induction efficiency and purity of Prg4-epressing cells presenting SFZ and FLS characteristics. Second, we need to prolong the survival of these cells after injection. Transplanted Prg4-mRFP1-positive cells were detected in the paratenon surrounding the Achilles tendons and knee joints, but only for up to 3 days. Use of scaffold materials or platelet-rich plasma might offer a physical means for improved temporal survival of iPSC-derived Prg4-expressing cells [59, 60].

The present study describes the generation of murine iPSCs harbouring a Prg4-mRFP1 reporter system and the establishment of a protocol for the differentiation of pluripotent stem cells into Prg4-expressing cells exhibiting the characteristics of SFZ chondrocytes and FLSs. Injection of lubricin-producing cells in the joints and paratenon around the tendons could become a viable therapeutic approach for osteoarthritis and tenosynovitis.

bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; ESC, embryonic stem cell; FLS, fibroblast-like synovial cell; iPSC, induced pluripotent stem cell; ITS, insulin-transferring-selenium; LIF, leukaemia inhibitory factor; MLS, macrophage-like synovial cell; MSC, mesenchymal stem cell; qRT-PCR, quantitative real-time PCR; rhPRG4, recombinant human lubricin; RFP, red fluorescent protein; SFZ, superficial zone; TGF-β, transforming growth factor beta.

Ethics approval and consent to participate

All animal experiments were approved by the Ethics Committee of Animal Experiment in Gifu University (Approval number 27-38), and were in compliance with the ARRIVE guidelines. All procedures for experimental animals were conducted in accordance with the guideline for regulations of Gifu University. No human subjects were used in this study.

Consent for publication

Not applicable.

Funding

The authors declare that they have no financial interests and have not received funding associated with this study.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

RNA-seq data of iPSC-derived Prg4-mRFP1-positive cells is available in the SRA database (PRJNA839189).

Authors’ contribution

T.S., S.K., and A.H. performed the experiments. S.K. and H.A. proposed the research project and designed the experiments. T.S., S.K., and H.A. wrote the manuscript. H.A. and Y.I. performed the RNA-sequencing experiments and provided technical advice.

Acknowledgments

We are grateful to Professor Roger Y. Tsien for kindly providing the mRFP1 construct [61].

Jay, G.D. and K.A. Waller, The biology of lubricin: near frictionless joint motion. Matrix Biology, 2014. 39: p. 17-24.
Marcelino, J., et al., CACP, encoding a secreted proteoglycan, is mutated in camptodactyly-arthropathy-coxa vara-pericarditis syndrome. Nature genetics, 1999. 23(3): p. 319.
Kozhemyakina, E., et al., Identification of a Prg4-expressing articular cartilage progenitor cell population in mice. Arthritis Rheumatol, 2015. 67(5): p. 1261-73.
Rhee, D.K., et al., The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth. Journal of Clinical Investigation, 2005. 115(3): p. 622-631.
Waller, K.A., et al., Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis. Proc Natl Acad Sci U S A, 2013. 110(15): p. 5852-7.
Flannery, C.R., et al., Prevention of cartilage degeneration in a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin. Arthritis Rheum, 2009. 60(3): p. 840-7.
Lawrence, A., et al., Synthesis and characterization of a lubricin mimic (mLub) to reduce friction and adhesion on the articular cartilage surface. Biomaterials, 2015. 73: p. 42-50.
Hayashi, M., et al., The effect of lubricin on the gliding resistance of mouse intrasynovial tendon. PLoS One, 2013. 8(12): p. e83836.
Taguchi, M., et al., Lubricin surface modification improves extrasynovial tendon gliding in a canine model in vitro. J Bone Joint Surg Am, 2008. 90(1): p. 129-35.
Brittberg, M., et al., Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New england journal of medicine, 1994. 331(14): p. 889-895.
Roberts, S., et al., Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation. Knee, 2009. 16(5): p. 398-404.
Yamashita, A., et al., Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs. Stem Cell Reports, 2015. 4(3): p. 404-18.
Saito, T., et al., Generation of Col2a1-EGFP iPS cells for monitoring chondrogenic differentiation. PLoS One, 2013. 8(9): p. e74137.
Craft, A.M., et al., Specification of chondrocytes and cartilage tissues from embryonic stem cells. Development, 2013. 140(12): p. 2597-2610.
Craft, A.M., et al., Generation of articular chondrocytes from human pluripotent stem cells. Nature biotechnology, 2015. 33(6): p. 638-645.
Nakagawa, Y., et al., Cartilage derived from bone marrow mesenchymal stem cells expresses lubricin in vitro and in vivo. PLoS One, 2016. 11(2): p. e0148777.
Ng, J.J., et al., Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage. Proc Natl Acad Sci U S A, 2017. 114(10): p. 2556-2561.
Ando, W., et al., Cartilage repair using an in vitro generated scaffold-free tissue-engineered construct derived from porcine synovial mesenchymal stem cells. Biomaterials, 2007. 28(36): p. 5462-5470.
Sugita, S., et al., HLA-Matched Allogeneic iPS Cells-Derived RPE Transplantation for Macular Degeneration. J Clin Med, 2020. 9(7).
Suzuki, K., et al., Therapeutic efficacy of large aligned cardiac tissue derived from induced pluripotent stem cell in a porcine ischemic cardiomyopathy model. J Heart Lung Transplant, 2021. 40(8): p. 767-777.
Chen, X., et al., Integration Capacity of Human Induced Pluripotent Stem Cell-Derived Cartilage. Tissue Eng Part A, 2019. 25(5-6): p. 437-445.
Nakajima, T., et al., Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture. Nat Commun, 2021. 12(1): p. 5012.
Waller, K.A., et al., Intra-articular recombinant human proteoglycan 4 mitigates cartilage damage after destabilization of the medial meniscus in the Yucatan minipig. The American journal of sports medicine, 2017. 45(7): p. 1512-1521.
Al‐Sharif, A., et al., Lubricin/proteoglycan 4 binding to CD44 receptor: a mechanism of the suppression of proinflammatory cytokine–induced synoviocyte proliferation by lubricin. Arthritis & rheumatology, 2015. 67(6): p. 1503-1513.
Iqbal, S.M., et al., Lubricin/Proteoglycan 4 binds to and regulates the activity of Toll-Like Receptors In Vitro. Sci Rep, 2016. 6: p. 18910.
Umeda, K., et al., Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells. Sci Rep, 2012. 2: p. 455.
Gadue, P., et al., Wnt and TGF-β signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proceedings of the National Academy of Sciences, 2006. 103(45): p. 16806-16811.
Oldershaw, R.A., et al., Directed differentiation of human embryonic stem cells toward chondrocytes. Nat Biotechnol, 2010. 28(11): p. 1187-94.
Nowotschin, S., et al., Interaction of Wnt3a, Msgn1 and Tbx6 in neural versus paraxial mesoderm lineage commitment and paraxial mesoderm differentiation in the mouse embryo. Dev Biol, 2012. 367(1): p. 1-14.
Tanaka, M., et al., BMP inhibition stimulates WNT-dependent generation of chondrogenic mesoderm from embryonic stem cells. Stem cell research, 2009. 3(2-3): p. 126-141.
Goldring, S.R. and M.B. Goldring, Biology of the normal joint, in Kelley's textbook of rheumatology. 2013, Elsevier. p. 1-19. e6.
Kimelman, D. and M. Kirschner, Synergistic induction of mesoderm by FGF and TGF-β and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell, 1987. 51(5): p. 869-877.
Spagnoli, A., et al., TGF-beta signaling is essential for joint morphogenesis. J Cell Biol, 2007. 177(6): p. 1105-17.
Niikura, T. and A.H. Reddi, Differential regulation of lubricin/superficial zone protein by transforming growth factor beta/bone morphogenetic protein superfamily members in articular chondrocytes and synoviocytes. Arthritis Rheum, 2007. 56(7): p. 2312-21.
Cuellar, A. and A.H. Reddi, Stimulation of Superficial Zone Protein/Lubricin/PRG4 by Transforming Growth Factor-beta in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans. Tissue Eng Part A, 2015. 21(13-14): p. 1973-81.
Miyatake, K., et al., Modulation of superficial zone protein/lubricin/PRG4 by kartogenin and transforming growth factor-β1 in surface zone chondrocytes in bovine articular cartilage. Cartilage, 2016. 7(4): p. 388-397.
IWANAGA, T., et al., Morphology and functional roles of synoviocytes in the joint. Archives of histology and cytology, 2000. 63(1): p. 17-31.
Mor, A., S.B. Abramson, and M.H. Pillinger, The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction. Clin Immunol, 2005. 115(2): p. 118-28.
Kiener, H.P., et al., Synovial fibroblasts self‐direct multicellular lining architecture and synthetic function in three‐dimensional organ culture. Arthritis & Rheumatism, 2010. 62(3): p. 742-752.
Guo, X., et al., Wnt/beta-catenin signaling is sufficient and necessary for synovial joint formation. Genes Dev, 2004. 18(19): p. 2404-17.
Hartmann, C. and C.J. Tabin, Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing appendicular skeleton. Cell, 2001. 104(3): p. 341-351.
Brunt, L., E. Kague, and C. Hammond, Developmental Insights into Osteoarthritis Increase the Applicability of New Animal Models. J Musculoskelet Disord Treat, 2016. 2: p. 017.
Ohta, Y., et al., Articular cartilage endurance and resistance to osteoarthritic changes require transcription factor Erg. Arthritis Rheumatol, 2015. 67(10): p. 2679-90.
Iwamoto, M., et al., Transcription factor ERG and joint and articular cartilage formation during mouse limb and spine skeletogenesis. Dev Biol, 2007. 305(1): p. 40-51.
Koyama, E., et al., A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis. Dev Biol, 2008. 316(1): p. 62-73.
Yasuhara, R., et al., Roles of beta-catenin signaling in phenotypic expression and proliferation of articular cartilage superficial zone cells. Lab Invest, 2011. 91(12): p. 1739-52.
LeBlanc, K.T., et al., Runx1 activities in superficial zone chondrocytes, osteoarthritic chondrocyte clones and response to mechanical loading. Journal of cellular physiology, 2015. 230(2): p. 440-448.
Smith, M.D., Suppl 1: the normal synovium. The open rheumatology journal, 2011. 5: p. 100.
Bartok, B. and G.S. Firestein, Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev, 2010. 233(1): p. 233-55.
Valencia, X., et al., Cadherin-11 provides specific cellular adhesion between fibroblast-like synoviocytes. The Journal of experimental medicine, 2004. 200(12): p. 1673-1679.
Mizuno, M., et al., Specific markers and properties of synovial mesenchymal stem cells in the surface, stromal, and perivascular regions. Stem cell research & therapy, 2018. 9(1): p. 1-11.
Edwards, J.C., Fibroblast biology Development and differentiation of synovial fibroblasts in arthritis. Arthritis Research & Therapy, 2000. 2(5): p. 344.
Hardy, R.S., et al., Characterisation of fibroblast-like synoviocytes from a murine model of joint inflammation. Arthritis research & therapy, 2013. 15(1): p. 1-15.
Pollock, L., P. Lalor, and P. Revell, Type IV collagen and laminin in the synovial intimal layer: an immunohistochemical study. Rheumatology international, 1990. 9(6): p. 277-280.
Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. cell, 2006. 126(4): p. 663-676.
Nalesso, G., et al., WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis. Ann Rheum Dis, 2017. 76(1): p. 218-226.
Kawashima, K., et al., Heparan sulfate deficiency leads to hypertrophic chondrocytes by increasing bone morphogenetic protein signaling. Osteoarthritis Cartilage, 2020. 28(11): p. 1459-1470.
Komura, S., et al., Induced pluripotent stem cell-derived tenocyte-like cells promote the regeneration of injured tendons in mice. Sci Rep, 2020. 10(1): p. 3992.
Choi, S., et al., Intra-Articular Injection of Alginate-Microencapsulated Adipose Tissue-Derived Mesenchymal Stem Cells for the Treatment of Osteoarthritis in Rabbits. Stem Cells Int, 2018. 2018: p. 2791632.
Bastos, R., et al., Intra-articular injections of expanded mesenchymal stem cells with and without addition of platelet-rich plasma are safe and effective for knee osteoarthritis. Knee Surgery, Sports Traumatology, Arthroscopy, 2018. 26(11): p. 3342-3350.
Campbell, R.E., et al., A monomeric red fluorescent protein. Proceedings of the National Academy of Sciences, 2002. 99(12): p. 7877-7882.

Table 1 is available in the Supplementary Files section

No competing interests reported.

Table20220513.pdf

Download PDF

Editorial decision: Major revision
20 Jun, 2022
Reviews received at journal
07 Jun, 2022
Reviewers agreed at journal
31 May, 2022
Reviewers invited by journal
28 May, 2022
Editor assigned by journal
28 May, 2022
Editor invited by journal
25 May, 2022
Submission checks completed at journal
25 May, 2022
First submitted to journal
12 May, 2022

You are reading this latest preprint version

Induction of iPSC-derived Prg4-positive cells with characteristics of superficial zone chondrocytes and fibroblast-like synovial cells

Status:

Version 2

Abstract

Figures

Background

Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Table

Additional Declarations

Supplementary Files

Status:

Version 2