Stem cells play an important role in tissue engineering and clinical regeneration therapy due to their ability to self-replicate and potential to differentiate into multiple cell lines, called stemness (Keating 2012). The clinical demand for stem cells from various sources far exceeds their supply (Van Zant and Liang 2012). In this regard, an effective solution may be to obtain stem cells through in vitro culture. However, multiple passages of in vitro cultivation gradually decrease the therapeutic properties of stem cells by weakening their proliferation and multipotent differentiation potential, eventually leading to treatment failure (Truong et al. 2019; Zaim et al. 2012). Therefore, developing more substances that can maintain or even enhance the stemness of stem cells during large-scale in vitro culture is important for their clinical applications.
Extracellular matrix (ECM), a noncellular three-dimensional macromolecular network composed of collagens, fibronectin, elastin, and several other glycoproteins (Ishihara et al. 2014; Isomursu et al. 2019), exerts essential functions on the proliferation and differentiation of stem cells (Du et al. 2011). Extensive research and testing have shown that collagen promotes stem cell migration and adhesion (Sorushanova et al. 2019). Fibronectin promotes cell adhesion similar to collagen, and it also fixes collagen to maintain the ECM stability. In addition, the ECM communicates with cells through integrins located on the surface of the cell membrane, such as αvβ3, αvβ1, and αvβ6, via Arg-Gly-Asp (RGD) sequences (Pierschbacher and Ruoslahti 1984). FN10, a part of the fibronectin type III module, contains RGD sequences that specifically bind αvβ1 to guide the fate of the cells (Bharadwaj et al. 2017; Hocking et al. 1996). Moreover, collagen and fibronectin support the growth of human embryonic stem cells (hESC) without the use of a feeder layer under certain conditions (Lu et al. 2006) and play a role in maintaining the stemness of stem cells (Akhir and Teoh 2020; Thaweekitphathanaphakdee et al. 2019). Notably, as the main strain matrix element, collagen relies on fibronectin, fibronectin-bound, and collagen-bound integrins to complete its corresponding functions (Kadler et al. 2008; Kubow et al. 2015). Meanwhile, natural collagen and fibronectin from animal sources have some problems such as unstable quality and difficult dissolution(Davison-Kotler et al. 2019).In contrast, the recombinant proteins based on collagen and fibronectin and prepared by synthetic biology possess the excellent characteristics of high yield, easy amplification, low cost, and not easily contaminated by mammalian pathogens. These characteristics are slowly being recognized as contributors leading to the future application of recombinant proteins based on collagen and fibronectin in regenerative medicine (Ferrer-Miralles and Villaverde 2013). Therefore, it is necessary to use genetic engineering technology to synthesize a novel functional protein that acts as a biomimetic ECM for stem-cell therapy.
Integrin plays an important role in the long-term ex vivo culture of stem cells. As a member of the integrin family, integrin β3 is a transmembrane receptor that mediates interactions between cells and the ECM and which widely exists on the surface of hematopoietic stem cells and bone marrow mesenchymal stem cells (Pei et al. 2011; Umemoto et al. 2012). The integrin-αvβ3 found on hematopoietic stem cells (HSCs) plays an important role in maintaining stem cell activity by regulating stem cell function and affecting stemness through signal transduction (Umemoto et al. 2012; de Graaf and Metcalf 2011; Ishihara et al. 2014). These integrin functions are dependent on specific ECM ligand–receptor interactions and the specific molecular interactions that lead to cytoskeletal changes that result in different migratory behavior or changes in growth and differentiation (Hynes 2002; Isomursu et al. 2019). It is interesting to design innovative proteins based on the functional characteristics of the principal ECM structural proteins that contain specific ECM ligands, such as RGD, and which can bind with integrin β3 to de-anchor and adsorb cells.
Here, we reported a recombinant protein FCP improved according to the early exploration of our laboratory. FCP structure and functions, including binding to integrin β3, were predicted and recombinant fusion expression assays were conducted. Experiments verified that FCP promoted cell migration and adhesion and maintained cell stemness by binding integrin β3. Its effects could be blocked both by integrin β3 silencing and employing the integrin β3 inhibitor Cilengitide. Therefore, FCP may be used in the in vitro culture of stem cells, such as hematopoietic stem cells and other stem cells with high expression of integrin β3, or even in the field of tissue regeneration engineering.
Vectors pPICZαA (Invitrogen, Guangzhou, China) and P. pastoris strain X33 (Invitrogen, Guangzhou, China, Invitrogen™ C18000, ATCC® 28,485™) were used for cloning and heterologous expression. PCR purification kits, gel extraction kits, and micro preparation kits were purchased from Tiangen (Beijing, China). CC (a collagen-like protein, designed from COLA1, ID: BC036531.2, 2120-2164nt) was artificially synthesized by GL Biochem (Shanghai, China). FN10(ID: U42594.1, 633-770nt) was recombinantly expressed in our Lab (Fig. S2).
ECV304 (Human umbilical vein endothelial cells, ATCC® CRL-1998) and ECV304-eGFP (Human umbilical vein endothelial cells-enhanced green fluorescent protein, ATCC® PCS-100–010) cells were purchased from the Chinese Academy of Sciences (Shanghai, China). They were cultured in Roswell Park Memorial Institute 1640 medium (RPM
