Protein Expression of AEBP1, MCM4, and FABP4 Differentiate Osteogenic, Adipogenic, and Mesenchymal Stromal Stem Cells.

Thorben Sauer,Julia Horn,Moustapha Kassem,Arndt-Peter Schulz,Ivo Kwee,Justyna M Kowal,Michael Kohl,Sonja Hartwig,Timo Gemoll,Giulia Facchinetti,Svitlana Rozanova,Jens K Habermann,Hagen Schmal

doi:10.3390/ijms23052568

Abstract

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future.

Highlights

Bone marrow stromal cells (MSCs) were first detected by Friedenstein in murine bone marrow cultures [1] and subsequently gained enormous attention regarding their medical utility including cellular therapy applications
The individual Mesenchymal stem cells (MSCs) isolates (n = 5), each derived from one single individual, were characterized in vitro (Supplementary Table S1), and have been published elsewhere [32,38]: using a colony-forming unit fibroblast (CFU-F)
The cells were further characterized by their MSC surface marker expression recommended by the International Society for Cellular Therapy (ISCT [39])

Summary

Introduction

Bone marrow stromal cells (MSCs) were first detected by Friedenstein in murine bone marrow cultures [1] and subsequently gained enormous attention regarding their medical utility including cellular therapy applications. MSCs have been proven to be effective treatments in many diseases, e.g., cardiovascular diseases [6,7], musculoskeletal diseases [8], neurological diseases [9], immune system defects [10,11], cancer [12] and tissue regeneration in large bone defects [13–18]. Their clinical efficacy was recently tested as a therapeutic approach for patients with a COVID-19 infection [19]. MSCs show cellular heterogeneity in the context of in vitro osteoblast differentiation, resulting in heterogenous bone formation capacity in vivo [24,28–34] and certain bone diseases such as osteoporosis [35–37]

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Conclusion