Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

Abstract

The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP- cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.

Fig. 1.

Characterization of iPSCs. (A) Pluripotency markers (top left to right, bottom left to right; scale bars: 20 µm): bright field, DAPI, alkaline phosphatase, nanog, Oct4/Pou5f1, SSEA1. (B) Undifferentiated iPSCs formed teratomas (Upper Left) with endoderm (gland, Upper Right), mesoderm (muscle, Lower Left), and ectoderm (neural rosette, Lower Right). (C) Karyogram from G banding, arrow indicates deletion.

Fig. 2.

Monolayer expansion of sorted cells. (A) Passage 2 cells after sorting for GFP+ (left) or GFP− (right). (Scale bar: 100 µm.) (B) Cumulative fold increase in cell number (log scale). Asterisk indicates P < 0.05 to GFP− of same passage, n ≥ 3 per group, mean ± SEM. (C) RT-PCR. Fold increase normalized to passage 1 GFP+ cells, asterisk indicates P < 0.05 to GFP− of same passage and pound indicates P < 0.05 to passage 1 of own cell type, n ≥ 3 per group, mean ± SEM.

Fig. 3.

Glycosaminoglycans (GAGs) production. (A) Safranin-O/Fast-Green/Hematoxylin stained section from pellets of GFP+ or GFP− cells passaged once (Left), twice (Center), or three times (Right) after sorting. (Scale bar: 100 µm.) (B and C) Total GAGs per pellet (B) andGAGs per DNA by DMB assay (C), asterisk indicates P < 0.05 to GFP− of same passage and pound indicates P < 0.05 to passage 1 of own cell type, n ≥ 4 per group, mean ± SEM.

Fig. 4.

Collagen production. (A) Immunohistochemistry for type II, type VI, type X, and type I collagen (as labeled). (Scale bar: 100 µm.) (B) Quantification of type II (Left) or type I collagen (Right) by ELISA, presented as total content or normalized to DNA content. Asterisk indicates P < 0.05 to GFP− of same passage and pound indicates P < 0.05 to passage 1 of own cell type, n ≥ 4 per group, mean ± SEM.

Fig. 5.

Mechanical characterization of pellets via AFM. (A) Bulk elastic modulus measurements of GFP+ and GFP− pellets, n ≥ 5, mean ± SEM. (B) Regional elastic modulus values from cryosections, n ≥ 4, mean ± SEM, pound symbol indicates P < 0.05 by region and asterisk indicates P < 0.05 by cell type. (C) Picrosirius red stained section of GFP+ pellet depicting regions tested: 0–40 μm (between solid lines) and 100–300 μm (between dashed lines). (Scale bars: 100 µm.) (D) Polarized light micrograph of section in C. (E) Correlation between regional elastic modulus values and percent collagen alignment within those regions for pellets from GFP+ (Left) and GFP− (Right) cells.

Fig. 6.

In vitro cartilage defect repair. (A) Cored cartilage explants filled with agarose only, GFP+ cells in agarose, GFP− cells in agarose, or replaced cartilage (as labeled). (Scale bars: 100 μm.) (B) Shear strength of repair by the maximum stress as the inner core is pushed out of the explant, n ≥ 5 per group, mean ± SEM, asterisk indicates P < 0.05 to cartilage, pound indicates P < 0.05 to agarose, and ampersand indicates P < 0.05.