Alterations of Glycosphingolipid Glycans and Chondrogenic Markers during Differentiation of Human Induced Pluripotent Stem Cells into Chondrocytes

Abstract

Due to the limited intrinsic healing potential of cartilage, injury to this tissue may lead to osteoarthritis. Human induced pluripotent stem cells (iPSCs), which can be differentiated into chondrocytes, are a promising source of cells for cartilage regenerative therapy. Currently, however, the methods for evaluating chondrogenic differentiation of iPSCs are very limited; the main techniques are based on the detection of chondrogenic genes and histological analysis of the extracellular matrix. The cell surface is coated with glycocalyx, a layer of glycoconjugates including glycosphingolipids (GSLs) and glycoproteins. The glycans in glycoconjugates play important roles in biological events, and their expression and structure vary widely depending on cell types and conditions. In this study, we performed a quantitative GSL-glycan analysis of human iPSCs, iPSC-derived mesenchymal stem cell like cells (iPS-MSC like cells), iPS-MSC-derived chondrocytes (iPS-MSC-CDs), bone marrow-derived mesenchymal stem cells (BMSCs), and BMSC-derived chondrocytes (BMSC-CDs) using glycoblotting technology. We found that GSL-glycan profiles differed among cell types, and that the GSL-glycome underwent a characteristic alteration during the process of chondrogenic differentiation. Furthermore, we analyzed the GSL-glycome of normal human cartilage and found that it was quite similar to that of iPS-MSC-CDs. This is the first study to evaluate GSL-glycan structures on human iPS-derived cartilaginous particles under micromass culture conditions and those of normal human cartilage. Our results indicate that GSL-glycome analysis is useful for evaluating target cell differentiation and can thus support safe regenerative medicine.

Keywords: aminolysis-SALSA; cartilage injury; chondrocytes; glycomics; glycosphingolipid; human induced pluripotent stem cells.

Figure 1

Evaluation of the pluripotent stem cell markers and GSL-glycan profiles of on feeder and feeder-free human iPSCs. (A) Representative microscopic findings of undifferentiated iPSCs 201B7 and 606A1. Both iPS cell lines formed typical colonies on feeder cells (a,e) and were stained with blue alkaline phosphatase (blue AP) (b,f). Under feeder-free conditions, typical iPSC colonies were observed (c,g) and confirmed by blue AP staining (d,h). Scale bars = 500 μm. (B) Gene expression analyses of pluripotent stem cell markers such as NANOG, OCT3/4, and SOX2 in iPSCs on feeder at passage 2 (iPS P2), and feeder-free iPSCs at passage 3 (iPS FFP3). Values are presented as means ± SD. (C) MALDI-TOF MS spectra showing GSL-glycans on feeder-free human iPSCs 201B7 (a) and 606A1 (b). Red asterisks (*) and dashed red boxes indicate free oligosaccharides and undifferentiated iPSC-specific GSL-glycans, respectively.

Figure 2

Disappearance of pluripotent stem cell markers and appearance of mesodermal markers during differentiation of iPSCs into iPSC-derived mesenchymal stem cell like cells (iPS-MSC). (A) Morphology of the iPS-MSC like cells differentiated from iPS cell lines (201B7 and 606A1) at passage 3 and 6, and human BMSCs at passage 3. Scale bars = 500 μm. (B) Gene expression analyses of pluripotent stem cell markers such as NANOG, OCT3/4, and SOX2 in feeder-free iPSCs at passage 3 (iPS FFP3), iPS-MSCs at passages 3 and 6 (iPS-MSC P3 and P6), and BMSCs (passage 3). Values are presented as means ± SD. (C) Expression of surface antigens in iPS-MSC like cells derived from both iPSC lines, and BMSCs as determined by fluorescence activated cell sorter (FACS) analysis. Representative FACS profiles of iPS-MSC like cells (P6) and BMSCs (P3) associated with the mesenchymal phenotype (red font: positive antibodies against CD44, CD73, CD90, and CD105; blue font: cocktail of negative antibodies against CD34, CD11b, CD19, CD45, and HLA-DR). Red profiles: population stained with positive antibodies; blue profile: population stained with negative antibodies; black profile: population stained with isotype control.

Figure 3

MALDI-TOF MS spectra of GSL-glycans. 201B7 iPS-MSC like cells (a), 606A1 iPS-MSC like cells (b), and human BMSCs (c).

Figure 4

Evaluation of GSL-glycan profiles on iPS-MSC-CDs, BMSC-CDs, and human cartilage. (A) Alcian blue staining revealed gradual accumulation of proteoglycan-rich matrix during micromass cultures of iPS-MSCs and BMSCs. Scale bars = 1 mm. (B) Representative macroscopic findings of cartilage particles on day 21 of micromass cultures in a 24-well plate (a,b,c). a: 201B7, b: 606A1, c: collection of cartilage particles (606A1) in a 6 cm dish, d: hemispherical transparent cartilage particles. (C) qRT-PCR gene expression analyses of iPS-MSC-CDs (201B7 and 606A1) and BMSC-CDs on days 1, 7, 14, and 21 of chondrogenic differentiation. Expressions of chondrogenic markers (SOX9, COL2A1, ACAN, and COL1A1), adipogenic markers (PPARγ), and osteogenic markers (RUNX2) were evaluated. Values are presented as means ± SD. (D) MALDI-TOF MS spectra of GSL-glycans of 201B7 iPS-MSC-CDs at day 21 (a), 606A1 iPS-MSC-CDs at day 21 (b), BMSC-CDs at day 21 (c) and human cartilage (d). (E). Relative levels of GSL-glycans in 201B7 iPS-MSC-CDs at day 21, 606A1 iPS-MSC-CDs at day 21, BMSC-CDs at day 21 and human cartilage.

Figure 5

Correlation analysis between GSL-glycans of human cartilage and chondrogenic differentiated cells. (A) 201B7 iPS-MSC-CDs at day 21. (B) 606A1 iPS-MSC-CDs at day 21. (C) BMSC-CDs at day 21.