Recombinant Extracellular Matrix Protein Fragments Support Human Embryonic Stem Cell Chondrogenesis.

Aixin Cheng,Andrew K Baldwin,Stuart A Cain,Susan J Kimber,Paweena Uppanan,Cay M Kielty,Pinyuan Tian

doi:10.1089/ten.tea.2017.0285

Abstract

We previously developed a 14-day culture protocol under potentially GMP, chemically defined conditions, to generate chondroprogenitors from human embryonic stem cells (hESCs). In vivo work has confirmed the cartilage repair capacity of these cells in a nude rat osteochondral defect model. Aiming to enhance hESC-chondrogenesis, we screened a range of extracellular matrix (ECM) molecules for their ability to support differentiation of hESCs toward chondrocytes. We identified two novel ECM protein fragments that supported hESC-chondrogenesis: Fibronectin III (fibronectin 7-14 protein fragments, including the RGD domain, syndecan-binding domain, and heparin-binding domain) and fibrillin-1 (FBN1) fragment PF8 (encoded by exons 30–38, residues 1238–1605, which contains the RGD motif but not heparin-binding site). These two protein fragments support hESC-chondrogenesis compared with the substrates routinely used previously (a mixture of fibronectin and gelatin) in our directed chondrogenic protocol. We have identified recombinant fibronectin fragment (FN III) and FBNI fragment (PF8) as alternative coating substrates to promote expression of genes known to regulate chondrocytes and code for chondrocyte ECM components. These recombinant protein fragments are likely to have better batch to batch stability than full-length molecules, especially where extracted from tissue/serum.

Highlights

The articular cartilage covering of synovial joints disperses loading forces to the underlying subchondral bone and provides a low-friction interface to facilitate joint movement
To screen extracellular matrix (ECM) components that support hESCchondrogenesis, a range of ECM proteins and protein fragments was coated onto 96-well tissue culture plate (TCP) (Fig. 2, Supplementary Table S1)
We produced an array of recombinant extracellular proteins and protein fragments, including FN and fibrillin-1 fragments, which were screened for human embryonic stem cells (hESCs) culture expansion and for generation of Safranin O positive cells in the directed differentiation protocol (DDP) to generate chondroprogenitors

Summary

Introduction

The articular cartilage covering of synovial joints disperses loading forces to the underlying subchondral bone and provides a low-friction interface to facilitate joint movement. After the closure of the growth plate, the articular cartilage loses regenerative capacity due to the lack of blood supply.[1] Chondrocytes, the only cell type in cartilage, have a low proliferation rate and produce all the extracellular matrix (ECM) in the cartilage, mainly proteoglycans and other noncollagenous molecules in adult cartilage, as turnover of the type II collagen network is very slow.[2] These characteristics contribute to a lack of self-repair capacity in the event of disease or trauma, when the integrity of cartilage is disrupted.[3]. Additional components include a collection of other molecules, such as link protein, COMP, matrilins, decorin, and hyaluronic acid, which act as bridging molecules and by binding to collagens regulate the fibrillogenesis and assembly of the cartilage network.[9,10,11,12] Aggrecan is the major cartilage-specific proteoglycan core protein to which glycosaminoglycan (GAG)

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