Cord blood cell-derived iPSCs as a new candidate for chondrogenic differentiation and cartilage regeneration

Abstract

Background: The native articular cartilage lacks the ability to heal. Currently, ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells are used to regenerate the damaged cartilage. With unlimited self-renewal ability and multipotency, human induced pluripotent stem cells (hiPSCs) have been highlighted as a new replacement cell source for cartilage repair. Still, further research is needed on cartilage regeneration using cord blood mononuclear cell-derived hiPSCs (CBMC-hiPSCs).

Methods: Human iPSCs were generated from CBMCs using the Sendai virus. The characterization of CBMC-hiPSCs was performed by various assays. Embryonic bodies (EBs) were obtained using CBMC-hiPSCs, and outgrowth cells were induced by plating the EBs onto a gelatin-coated plate. Expanded outgrowth cells were detached and dissociated for chondrogenic differentiation. Outgrowth cells were differentiated into chondrogenic lineage with pellet culture. Chondrogenic pellets were maintained for 30 days. The quality of chondrogenic pellets was evaluated using various staining and genetic analysis of cartilage-specific markers.

Results: Reprogramming was successfully done using CBMCs. CBMC-hiPSCs (n = 3) showed high pluripotency and normal karyotype. Chondrogenic pellets were generated from the outgrowth cells derived from CBMC-hiPSC EBs. The generated chondrogenic pellets showed high expression of chondrogenic genetic markers such as ACAN, COMP, COL2A1, and SOX9. The production of extracellular matrix (ECM) proteins was confirmed by safranin O, alcian blue and toluidine blue staining. Expression of collagen type II and aggrecan was detected in the accumulated ECM by immunohistological staining. Chondrogenic pellets showed low expression of fibrotic and hypertrophic cartilage marker, collagen type I and X.

Conclusions: This study reveals the potential of CBMC-hiPSCs as a promising candidate for cartilage regeneration.

Keywords: Cartilage regeneration; Chondrocytes; Cord blood mononuclear cells; Induced pluripotent stem cells; Regenerative medicine.

Fig. 1

Characterization of three CBMC-hiPSC lines. a Morphology of the three generated CBMC-hiPSC lines. b CBMC-hiPSC colonies positively stained with alkaline phosphatase. c Relative expression of pluripotent markers in each CBMC-hiPSC line. Primary CBMC was used as a control. d Immunofluorescence staining image of the generated CBMC-hiPSC lines. All scale bars represent 200 μm. CBMC-hiPSC cord blood mononuclear cell-derived human induced pluripotent stem cell

Fig. 2

Chondrogenic pellet generation using CBMC-hiPSCs. a Scheme of chondrogenic pellet generation. b Morphology of CBMC-hiPSC. c Morphology of generated EBs. d Image of outgrowth cells derived from EBs attached to a gelatin-coated culture dish. e Image of chondrogenic pellets. All scale bars represent 200 μm. EB embryoid body, hiPSC human induced pluripotent stem cell

Fig. 3

Genetic characterization of chondrogenic pellet generated from CBMC-hiPSCs. The expression of COL2A1, ACAN, COMP, and SOX9 in day 10, 20, and 30 chondrogenic pellets. Data was obtained using RT-PCR and band intensity was evaluated. (^{*, +} p < 0.05, ^**, ⁺⁺ p < 0.01, ^{***, +++} p < 0.001). ACAN aggrecan gene, COL2A1 collagen type II gene, COMP cartilage oligomeric matrix protein gene, hiPSC human induced pluripotent stem cell, SOX9 sex-determining region Y-box 9 gene

Fig. 4

Histological analysis of CBMC-hiPSC-derived chondrogenic pellet. Image of pellets stained by safranin O, alcian blue, and toluidine blue on day 10, 20, and 30. All scale bars represent 100 μm. BMSC bone marrow-derived mesenchymal stem cell

Fig. 5

Immunohistological analysis of CBMC-hiPSC-derived chondrogenic pellets. a Image of pellet harvested at various time points stained with antibody against collagen type II and aggrecan. b Image of pellet stained with antibody against collagen type I. All scale bars represent 100 μm. BMSC bone marrow-derived mesenchymal stem cell

Fig. 6

Further analysis of genetic markers in chondrogenic pellets derived from CBMC-hiPSCs and MSCs. a The expression of fibrotic cartilage representative gene, COL1A1 and hypertrophic marker, COL10 at various time points. b The ratio of COL2A1 and COL1A1 at day 10, 20, and 30. c The relative expression of ACAN, COMP, COL2A1, SOX9, COL1A1, and COL10 in chondrogenic pellets derived from BMSC and CBMC-hiPSC on day 30. (^*,+ p < 0.05, ^**,++ p < 0.01^{, ***,+++} p < 0.001). ACAN aggrecan gene, COL10 collagen type 10 gene, COL1A1 collagen type I gene, COL2A1 collagen type II gene, COMP cartilage oligomeric matrix protein gene, iPSC induced pluripotent stem cell, MSC mesenchymal stem cell, SOX9 sex-determining region Y-box 9 gene