Self‐Assembling Proteins as High‐Performance Substrates for Embryonic Stem Cell Self‐Renewal

Abstract

modeling of disease, drug discovery, the screening of toxicants, and personalized therapies in regenerative medicine for conditions like age-related macular degeneration and Parkinson's disease. [3,4] Human pluripotent stem cells (hPSCs), specifically human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are of critical significance in these pursuits. [5] Biomedical applications of hPSCs require large-scale ex vivo culture. Therapeutic uses require, in addition, xeno-free conditions to avoid risk of zoonotic transmission. Meeting these combined requirements is challenging as hPSCs are typically cultured on costly animal-derived substrates of undefined and variable batch-to-batch composition. In particular, hPSCs are commonly cocultured with mouse fibroblast feeder cells or grown on Matrigel, a complex mixture of extracellular matrix (ECM) proteins secreted by mouse sarcoma cells that consists primarily of laminin, collagen IV, and entactin. [6] Recently, substrates based on the recombinant eukaryotic expression of ECM proteins such as laminin or vitronectin have been introduced. [7,8] These successfully support hPSC self-renewal, [9,10] The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are sought that can enact control over the self-renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large-scale cell production under xeno-free culture conditions using current matrices. Here, a bioactive, recombinant, protein-based polymer, termed ZT Fn , is presented that closely mimics human plasma fibronectin and serves as an economical, xeno-free, biodegradable, and functionally adaptable cell substrate. The ZT Fn substrate supports with high performance the propagation and long-term self-renewal of human embryonic stem cells while preserving their pluripotency. The ZT Fn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications. The generation of substrates that preserve pluripotency in human stem cells and direct their controlled differentiation is intensely pursued. [1,2] Such substrates are of critical value to stem-cell-based applications, such as those concerning the

The ZT Biopolymer: A Self-Assembling Protein Scaffold for Stem Cell Applications

“…The crystal structures of RGD (Z 1212 RGD ; PDB: 6FWX) and KLER (Z 12 KLER ; PDB: 6SDB) modified variants as well as the NMR analysis of the FLAG single-domain variant (Z1 FLAG ) [39] confirmed that Z1 retained its native fold and structural integrity after the modifications and that the modified loop did not affect the long-range conformation of the Z 1212 tandem or its capability to polymerize through interaction with Tel. Furthermore, functional studies demonstrated that FLAG and SSGRGDSS sequences remained accessible and functional upon grafting, respectively supporting molecular and cell-based interactions with good efficiency [39,40]. These results established these Ig domains as successful scaffolds amenable to extensive protein engineering without detriment to their structural integrity, stability or assembly.…”

“…The display of peptidic sequences on the ZT polymer is established, with sequences having been introduced both in Tel and Z 1212 components without preventing polymerization [30,39,40]. Both components permit fusing peptidic sequences to their N- and C-termini and, in addition, Z 1212 permits the introduction of motifs in four internal positions, namely the CD loop of each of its four Ig domains (Figure 1b).…”

“…We have demonstrated that the CD loop can tolerate drastic diversification in length and composition and have established its suitability as a locus for peptide grafting. Specifically, we replaced the native loop in Z1 for a highly charged FLAG affinity tag (DYKDDDDK) [39], a native PxxP SH3-interaction motif (EAMPPTLPHRDWKD) [39], a sequence carrying the integrin-attachment motif GRGDS from fibronectin (SSGRGDSS) [40], and a decorin-mimetic KLER motif (PDB ID: 6SDB; Figure 3a). All variants could be produced recombinantly in bacteria in yields equivalent to those of the wild-type.…”

“…The crystal structure of Z 1212 RGD proved that the RGD motif incorporated in the CD loop of the third tandem Ig mimicked well the flexibility and the local conformation of the RGD motif within the 10 th cell binding domain of native fibronectin, Fn10 (Figure 3b). Size-exclusion chromatography (SEC), SEC-multi angle light scattering, native-polyacrylamide gel electrophoresis (PAGE), and TEM micrographs showed that the introduction of the RGD carrying-sequence at the CD locus did not alter the interaction with Tel or the ensuing polymerization [40]. Subsequent cell assays (below) showed the Z 1212 RGD variant to be functional.…”

“…Such incorporations can comprise natural integrin attachment domains, fluorescent proteins, or even processive enzymes. Examples of such functional chimeras are the fusion of the Fn10 domain from fibronectin (Fn10; 10.5 kDa) [40] and the fusion of yellow fluorescent protein (mCitrineFP; 26.9 kDa) to the C-terminus of Z 1212 (Z 1212 Fn and Z 1212 mYFP , respectively; Figure 4a). SEC showed that the chimeric Z 1212 tandems remained monomeric and highly monodisperse (Figure 4b) [40].…”