Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs

Abstract

Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor β1 (TGF-β1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.

Graphical abstract

Figure 1

Optimized Protocol for Differentiating hiPSCs toward Chondrocytes (A) Images of hiPSC-derived cells induced in the presence of the indicated supplement on day 14. Top panels: phase view. Bottom panels: GFP fluorescence view. The right panels show images of cells derived from hiPSCs that did not bear the COL11A2-EGFP transgene cultured in the presence of ABTG supplementation. Scale bars, 50 μm. (B) FACS analysis of COL11A2-EGFP-positive cells in the iPSC-derived cell culture in the presence of the indicated supplements on day 14. The error bars denote the means ± SD of three individual experiments. ^∗∗p < 0.01. (C) Phase and GFP fluorescence images of the COL11A2-EGFP hiPSC-derived cell culture under ABTG supplementation. Scale bars, 50 μm. (D) Images of the hiPSC-derived particles on day 56 in 3.5-cm dishes. Scale bar, 5 mm. (E) Schematic representation of the protocol for differentiating hiPSCs toward chondrocytes. See also Figure S1.

Figure 2

Histological Analysis of hiPSC-Derived Particles in Suspension Culture Semiserial sections were stained with H&E and safranin O-fast green-iron hematoxylin and immunostained with anti-type II collagen antibodies, anti-type I collagen antibodies, anti-SOX9 antibodies, and anti-GFP antibodies, as indicated. Scale bars, 50 μm. (A) A particle 28 days after the start of differentiation of hiPSCs (day 28). The second and fourth panels are magnifications of the boxed region. (B) A particle on day 42. Bottom panels are magnifications of the boxed region. (C) A particle on day 70. The medium was switched from chondrogenic medium to conventional medium on day 42. Bottom panels are magnifications of the boxed region. (D) Histological sections of particles obtained on days 56 and 70 were immunostained with anti-SOX9 antibodies. (E) Histological sections of particles obtained on day 56 were immunostained with anti-GFP antibodies. (F) The ratio of the number of SOX9-positive cells per total cells during the maturation of particles. Cells were counterstained with hematoxylin. The numbers of cells in the particles, except for the surface layers, were counted. n = 3 particles. The error bars denote the means ± SD. See also Figures S2 and S3 and Table S1.

Figure 3

Analysis of Marker Gene Expression and Growth and Death of hiPSC-Derived Cells during Differentiation We collected whole cells during the adhesion culture until day 14. From day 15, we collected only particles, not cells attached to the bottom of the dishes. (A) Real-time RT-PCR expression analysis of marker genes for pluripotency and the development of the mesoderm, chondrocytes, fibroblasts, and osteoblasts. RNA expression levels were normalized to the level of β-ACTIN (ACTB) expression. n = 3 technical replicates. The data are representative of two independent experiments. (B) Growth and death of hiPSC-derived cells. The collected cells were subjected to collagenase digestion to obtain a single-cell suspension. Cell numbers were counted after the addition of trypan blue. Cells that did not incorporate trypan blue were considered alive. Cell survival rates indicate the number of live cells divided by the total number of cells. n = 3 dishes. The error bars denote the means ± SD. See also Figures S4 and S5 and Tables S2 and S3.

Figure 4

Transplantation of hiPSC-Derived Particles on Day 28, 42, or 70 into the Subcutaneous Spaces of SCID Mice Mice were sacrificed 12 weeks after transplantation. Histological analysis of the transplanted sites was performed. (A) Semiserial sections were stained with H&E and safranin O-fast green-iron hematoxylin and immunostained with anti-type II and anti-type I collagen antibodies. Scale bars, 500 μm. (B) Magnified images of day-42 particle progeny in the boxed regions of (A). Semiserial sections were immunostained with anti-vimentin antibodies that recognize only human vimentin. The blue color reflects DAPI. Scale bars, 50 μm. (C) Magnified images of day-28, 42, and 70 progenies stained with safranin O in the boxed regions of (A). Semiserial sections were immunostained with anti-type X collagen antibody. The blue color reflects DAPI. Scale bars, 50 μm. (D) RNAs were extracted from various organs of SCID mice that received hiPSC-derived particles on day 42 and subjected to real-time RT-PCR to amplify human and murine β-actin mRNAs. n = 3 mice. The error bars denote the means ± SD. Transplant, transplanted site; Surrounding fat, fat tissue surrounding the transplanted site; Intraperitoneal, intraperitoneal tissue; Groin, groin lymph nodes; Axillary, axillary lymph nodes; Cervical, cervical lymph nodes; MEF, murine embryonic fibroblasts; HDF, human dermal fibroblasts. See also Figure S6 and Tables S1–S3.

Figure 5

Long-Term Observations of hiPSC-Derived Particles on Day 42 Transplanted into the Subcutaneous Space of SCID Mice Mice were sacrificed 12 months after transplantation. Semiserial sections were stained with H&E and safranin O-fast green-iron hematoxylin and immunostained with anti-type X collagen antibodies. Magnified images of the boxed regions are shown in panels to the immediate right. Scale bar, 500 μm (left) and 50 μm (middle and right). See also Table S1.

Figure 6

Orthotopic Transplantation of hiPSC-Derived Cells into SCID Rats hiPSC-derived cartilaginous particles obtained on day 28 were transplanted into defects created in the articular cartilage of the distal femurs of SCID rats. The transplanted sites (A and B) and various organs (C) were collected. (A and B) Histological analysis of the transplanted sites at 1 and 4 weeks after transplantation. Semiserial sections were stained with H&E and toluidine blue and immunostained with anti-vimentin antibodies that recognize only human vimentin and anti-type II collagen antibodies. The blue color reflects DAPI. Magnified images of the boxed regions in (A) are shown in (B). Scale bars, 50 μm. (C) RNAs were extracted from various organs at 4 and 12 weeks after transplantation and subjected to real-time RT-PCR to amplify human and rat β-actin mRNAs. n = 3 rats. The error bars denote the means ± SD. Bone, bone of the femoral diaphysis; Surrounding fat, fat tissue surrounding the transplanted sites; Intraperitoneal, intraperitoneal tissue; Groin, groin lymph nodes; Axillary, axillary lymph nodes; Cervical, cervical lymph nodes; MEF, murine embryonic fibroblasts; HDF, human dermal fibroblasts. See also Figure S6 and Tables S1–S3.

Figure 7

hiPSC-Derived Cartilaginous Particles Fixed Articular Defects in Mini-pigs (A) hiPSC-derived cartilaginous particles (approximately ten) obtained on day 56 were transplanted into defects created in the articular cartilage of the distal femurs of mini-pigs and fixed with fibrin glue. (B) Histological analysis of the transplanted sites at four weeks after transplantation. Semiserial sections were stained with H&E and safranin O-fast green-iron hematoxylin and immunostained with anti-vimentin antibodies that recognize only human vimentin. The blue color reflects DAPI. Magnified images of the solid boxed regions are shown in central panels. Magnified images of the dotted boxed regions are shown in right panels. Arrows indicate the boundary between native cartilage and transplanted cartilage. Scale bars, 500 μm (left) and 50 μm (right).