Impact of Fibronectin Knockout on Proliferation and Differentiation of Human Infrapatellar Fat Pad-Derived Stem Cells.

Yiming Wang,Zuoqin Yan,Yawen Fu,Ming Pei,Xiao-Bing Zhang

doi:10.3389/fbioe.2019.00321

Abstract

Fibronectin plays an essential role in tissue development and regeneration. However, the effects of fibronectin knockout (FN1-KO) on stem cells' proliferation and differentiation remain unknown. In this study, CRISPR/Cas9 generated FN1-KO in human infrapatellar fat pad-derived stem cells (IPFSCs) was evaluated for proliferation ability including cell cycle and surface markers as well as stemness gene expression and for differentiation capacity including chondrogenic and adipogenic differentiation. High passage IPFSCs were also evaluated for proliferation and differentiation capacity after expansion on decellularized ECM (dECM) deposited by FN1-KO cells. Successful FN1-KO in IPFSCs was confirmed by Sanger sequencing and Inference of CRISPR Edits analysis (ICE) as well as immunostaining for fibronectin expression. Compared to the GFP control, FN1-KO cells showed an increase in cell growth, percentage of cells in the S and G2 phases, and CD105 and CD146 expression but a decrease in expression of stemness markers CD73, CD90, SSEA4, and mesenchymal condensation marker CDH2 gene. FN1-KO decreased both chondrogenic and adipogenic differentiation capacity. Interestingly, IPFSCs grown on dECMs deposited by FN1-KO cells exhibited a decrease in cell proliferation along with a decline in CDH2 expression. After induction, IPFSCs plated on dECMs deposited by FN1-KO cells also displayed decreased expression of both chondrogenic and adipogenic capacity. We concluded that FN1-KO increased human IPFSCs' proliferation capacity; however, this capacity was reversed after expansion on dECM deposited by FN1-KO cells. Significance of fibronectin in chondrogenic and adipogenic differentiation was demonstrated in both FN1-KO IPFSCs and FN(–) matrix microenvironment.

Highlights

As a connective tissue, articular cartilage is susceptible to damage caused by trauma or osteoarthritis (OA)
We found that FN1-KO increased infrapatellar fat pad-derived stem cells (IPFSCs)’ proliferation but decreased the proliferation of high passage IPFSCs grown on FN(–) decellularized extracellular matrix (ECM) (dECM)
FN1-KO had a negative effect on chondrogenic and adipogenic differentiation of IPFSCs, which was reflected in high passage IPFSCs grown on FN(–) dECM

Summary

Introduction

Articular cartilage is susceptible to damage caused by trauma or osteoarthritis (OA). An increasing body of evidence indicates that mesenchymal stem cells (MSCs) have great potential for cartilage engineering and regeneration (Jones and Pei, 2012; Pizzute et al, 2015). After first being isolated from bone marrow (Friedenstein et al, 1976), MSCs have been found in a variety of tissues including infrapatellar fat pad (IPFP) (Sun et al, 2018a). IPFP-derived MSCs (IPFSCs) are accessible and have better chondrogenic potential than bone marrow-derived MSCs (BMSCs) (Hindle et al, 2017). IPFSCs from OA patients were shown to possess comparable chondrogenic potential as those from non-OA donors (Liu et al, 2014), supporting the feasibility of using patients’ autologous cells for regeneration. MSCs including IPFSCs were reported to inevitably suffer from cell senescence due to in vitro expansion or donor age (Li and Pei, 2012; Lynch and Pei, 2014)

Methods

Results

Conclusion