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

Liang Xu,Yuan Tian,Tomohiro Onodera,Norimasa Iwasaki,Kazutoshi Hontani,Takuji Miyazaki,Jun-Ichi Furukawa,Kentaro Homan,Hisatoshi Hanamatsu,Rikiya Baba

doi:10.3390/biom10121622

Liang Xu, Yuan Tian + Show 8 more

Open Access

https://doi.org/10.3390/biom10121622

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Journal: Biomolecules	Publication Date: Dec 1, 2020
Citations: 10	License type: CC BY 4.0

Affiliation: Hokkaido University

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.

Highlights

The regeneration of articular cartilage remains a major challenge in the field of orthopedics
The expression level of globo- and (n) lacto-series GSLs diminishes rapidly upon differentiation of induced pluripotent stem cells (iPSCs), whereas expression of ganglioside GSLs such as GM3, GM2, and GD3 is elevated [14]. These results suggest that GSL-glycan profiles are highly cell type-specific, and that cellular GSL-glycomic analysis could be useful for evaluating the chondrogenic differentiation of iPSCs
Maintenance of human iPSCs typically requires the absence of a murine embryonic fibroblast (MEF) feeder layer to further enhance human iPSC adhesion and growth, iPSCs cultured on a feeder layer might increase the risk of MEF contamination

Summary

Introduction

The regeneration of articular cartilage remains a major challenge in the field of orthopedics. This problem is important because articular cartilage has very limited intrinsic healing potential; injuries to this tissue can eventually lead to osteoarthritis [1]. The predominant technologies for evaluation of chondrogenic differentiation of iPSCs are based on chondrogenic markers such as Sox, type II collagen, and aggrecan, along with the histological analysis of extracellular matrix (ECM) components [5,6,7]. Despite the fact that histological analysis of extracellular matrix (ECM) components is usually performed for evaluation of cartilage formation [7], it remains unclear what extent of matching is required for acceptable allogeneic transplantation of chondrocytes or cartilage. To achieve a more comprehensive and diverse evaluation of chondrogenic differentiation, new methods must be developed

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