Proteomic Analysis of Exosomes during Cardiogenic Differentiation of Human Pluripotent Stem Cells.

Preeti Ashok,Emmanuel S Tzanakakis

doi:10.3390/cells10102622

Abstract

Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases.

Highlights

IntroductionExosomes mediate the intercellular transfer of information in the form of RNA and proteins in response to physiological processes under normal and diseased states
Cardiac mesoderm cells emerging after 72 h of differentiation (24 h of Wnt suppression) expressing PDGFRA and NKX2.5; Stage 3—CM-like cells expressing cardiac Troponin T
The rise and fall in expression of the above markers at various stages were monitored by qPCR (Figure 1B)

Summary

Introduction

Exosomes mediate the intercellular transfer of information in the form of RNA and proteins in response to physiological processes under normal and diseased states. The protein content of cell- and body fluid-derived exosomes and its potential effects on cell physiology have remained surprisingly unexplored [4,5,6,7], especially when considering that microRNAs may be present within exosomes in suboptimal quantities to induce changes [8]. Human-induced pluripotent stem cells (hiPSCs) overexpressing ALIX secrete exosomes with higher protein content and decelerate aging-related damage upon addition to skin fibroblasts [9]. Mesenchymal stem cells (MSCs) release exosomes containing proteins, which influence NF-κB signaling that promotes angiogenesis in HUVEC cells [10]

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Conclusion