Chondrogenic differentiation of induced pluripotent stem cells from osteoarthritic chondrocytes in alginate matrix

Abstract

Induced pluripotent stem cells (iPSCs) have the potential to revolutionise cell therapy; however, it remains unclear whether iPSCs can be generated from human osteoarthritic chondrocytes (OCs) and subsequently induced to differentiate into chondrocytes. In the present study, we investigated the differentiation potential of OCs into iPSCs using defined transcription factors and explored the possibility of using these OC-derived iPSCs for chondrogenesis. Our study demonstrates that iPSCs can be generated from OCs and that these iPSCs are indistinguishable from human embryonic stem cells (hESCs). To promote chondrogenic differentiation, we used lentivirus to transduce iPSCs seeded in alginate matrix with transforming growth factor-β1 (TGF-β1) and then in vitro co-cultured these iPSCs with chondrocytes. Gene expression analysis showed that this combinational strategy promotes the differentiation of the established iPSCs into chondrocytes in alginate matrix. Increased expression of cartilage-related genes, including collagen II, aggrecan, and cartilage oligomeric matrix protein (COMP), and decreased gene expression of the degenerative cartilage marker, vascular endothelial growth factor (VEGF), were observed. The histological results revealed a dense sulphated extracellular matrix in the co-culture of TGF-β1-transfected iPSCs with chondrocytes in alginate matrix. Additionally, in vivo chondroinductive activity was also evaluated. Histological examination revealed that more new cartilage was formed in the co-culture of TGF-β1-transfected iPSCs with chondrocytes in alginate matrix. Taken together, our data indicate that iPSCs can be generated from OCs by defined factors and the combinational strategy results in significantly improved chondrogenesis of OC-derived iPSCs. This work adds to our understanding of potential solutions to osteoarthritic cell replacement problem.

Fig. 1.

Normal and osteoarthritic cartilage-derived cells. (A) Representative image of tissue from healthy AC. (B) Representative image of tissue from osteoarthritic cartilage. (C) Representative image of normal chondrocytes. (D) Representative image of OCs. Scale bars: 500 μm (A & B), 100 μm (C & D).

Fig. 2.

Generation of iPSCs from human OCs. (A) RT-PCR revealed that human OC-derived iPSCs (C1 and C2) expressed hESC marker genes, while the other iPSCs (C3) did not. (B) The iPSC colonies were observed 2 weeks after transduction (phase contrast image (a) and fluorescence image (b), and the expression of pluripotent markers of human OC-derived iPSCs was analysed by ALP (c) and immunofluorescence staining against NANOG (d,) SSEA-1 (e), SSEA-4 (f), TAR-60 (g), and TAR-81 (h). Scale bars: 200 μm.

Fig. 3.

Differentiation of iPSCs from human OCs in vitro and in vivo. (A) EBs were fonned from human OC-derived iPSCs at day 2 (a), and differentiation markers were detected by immunofluorescent staining of AFP (b), nestin (c) and desmin (d. (B) Direct differentiation of human OC-derived iPSCs into cell lineages of all three germ layers in vitro. Differentiated cells positive for PDX-1, PAX-6, NKX2.2. and insulin (endodenn); GATA-4, NKX2.5, MLC-2A, and MLC-2V (mesoderm); NSE, NF-M, MBP, GAD, and nestin (ectoderm) as detected by RT-PCR. (C) Teratomas fonned 4–6 weeks after human OC-derived iPSCs (clone 1, C1) were injected subcutaneously into four locations on nonobese diabetes/severe-combined immunodeficient (NOD/SCID) mice. Various tissues of the three germ layers were detected by H&E staining, including gut-like epithelium (a), respiratory-like epithelium (b), bone (c), muscle (d), and neural tissue (e). Scale bars: 200 μm.

Fig. 4.

In vitro chondrogenic differentiation of OC-derived iPSCs. (A) Representative images of OC-derived iPSCs with lentivirus-transduced TGF-β1 at day 5 (a and b) and 14 (c) shown by fluorescence, and a western blot against TGF-β1 in the nontransfected and transfected iPSCs (d). (B) Toluidine blue staining of iPSCs / Alginate (a), iPSCs / Alginate / chondrocytes (b), iPSCs / TGF-β1 / Alginate (c) and iPSCs / TGF-β1 / Alginate / chondrocytes (d). (C) Real-time PCR results showing gene expression for the cartilage markers collagen II, aggrecan and COMP as well as the degenerative cartilage marker VEGF. (^a) indicates that expression levels are statistically significant different from iPSCs/ TGF-β1/chondrocytes in alginate matrix. Scale bars: 200 μm (A), 100 μm (B).

Fig. 5.

Immunohistochemical analysis of ectopic cartilage formation of OC-derived iPSCs. Collagen II staining of iPSCs/Alginate (A), iPSCs/Alginate/chondrocytes (B), iPSCs/TGF-β1/Alginate (C) and iPSCs /TGF-β1/Alginate/chondrocytes (D). Scale bars: 100 μm.