Analysis of extracellular vesicles as a potential index for monitoring differentiation of neural lineage cells from induced pluripotent stem cells

https://doi.org/10.1016/j.jbiosc.2021.06.004Get rights and content

To improve cell production efficacy, it is important to evaluate cell conditions during culture. Extracellular vesicles (EVs) secreted from various cells are involved in stem cell differentiation. As EVs carry information about their source cells, we hypothesized that they may serve as a noninvasive index of cell conditions. We evaluated changes in EV morphology, concentration, and microRNA (miRNA) and protein expression in culture supernatants during the differentiation of induced pluripotent stem cells (iPSCs) into neural lineage cells, for application in regenerative medicine for Parkinson's disease. We observed EVs (50–150 nm) in culture supernatants of iPSCs and differentiated cells. The EVs expressed the exosome markers CD63, CD81, and CD9. Throughout differentiation, the EV concentration in the supernatants decreased, and EV miRNA and protein expression changed substantially. Especially, miR-106b, involved in neural stem cell differentiation and normal brain development, was considerably downregulated. CD63 expression correlated with the CORIN-positive cell rate, which is an index of differentiation. Thus, EV concentration and miRNA and protein expression may reflect the differentiation status of iPSCs. These findings pave the way for the development of novel and sensitive cell culture monitoring methods.

Section snippets

Extracellular matrix coating of culture plates

Six-well cell culture plates (AGC, Tokyo, Japan) were precoated with LM511-E8 (Nippi, Tokyo, Japan), an extracellular matrix (ECM) fragment, at 0.5 mg/cm2 and incubated at 37 °C for 1 h just before cell seeding. LM511-E8 was diluted in Dulbecco's phosphate-buffered saline without magnesium and calcium (PBS(−)).

Human iPSC culture

Human iPSCs (201B7-Ff; Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan) were maintained on an LM511-E8 ECM layer in StemFit AK-03N medium (Ajinomoto, Tokyo,

Morphological analysis of EVs in culture supernatant during the differentiation of iPSCs into neural lineage cells

EVs have been reported in culture supernatants of mesenchymal SCs and other cell types (23). We concentrated EVs from iPSC culture supernatants using several methods, including ultracentrifugation, polymer precipitation, filtration, and phosphatidylserine affinity-based concentration (27, 28, 29, 30). Concentrated (100-fold) pellet fractions obtained from cell culture supernatants (hereinafter called EV fractions) were negatively stained and observed using TEM. Numerous particles with

Discussion

We demonstrated that neural marker gene CORIN, LMX1A, and FOXA2 expression was induced, whereas the expression of the pluripotent marker gene NANOG was repressed, during the differentiation of iPSCs into neural lineage cells (Fig. 1F). These results suggested that cell conditions change drastically during neural induction for 12 days. However, cell morphology did not obviously change during differentiation (Fig. S1), indicating that optical microscopy is not suitable for monitoring cell

Acknowledgments

This work was supported by Japan Agency for Medical Research and Development (grant number: JP20be0404010) and the KBIC Research Grants-in-Aid for Young Researchers. We would like to thank Satoshi Mimura, Ph.D. and Masayo Hanaoka for technical assistance. We wish to thank Manabu Yoshikawa, Ph.D. and Ayaka Fujiki at Sumitomo Dainippon Pharma Co., Ltd. for technical guidance. We thank The Center for iPS Cell Research and Application, Kyoto University for providing human iPSCs.

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