Developing cell culture materials is a challenging task for the chemistry community, but its successful realization would allow cellular behavior to be directly influenced in order to optimize their applications in regenerative medicine. By tuning the properties of such materials, i.e., stiffness or the presence of chemical inducers like recognition motifs, it is possible to tailor the stem cell's microenvironment to give information regarding proliferation and differentiation. In nature, stem cells are arranged in a defined microenvironment, referred to as stem cell niche, which regulates the stem cell's behavior based on input from all of the components that comprise it. The stem cell niche theory was developed by Schofield (1978) and describes how stem cells are supported or influenced by the defined microenvironment, by means of physical interactions between the stem cells and other cells, the secretion of signal molecules, or the presence of molecules on the surface of other cells, like integrins.
The extracellular matrix (ECM) is the major and most important part of the stem cell niche. Numerous studies have shown that ECMs influence stem cell's fate. The composition of natural ECM can differ and depends on its tissue of origin. It consists of different adhesion proteins like collagen, fibronectin, and laminin, to which the stem cells are exposed. These adhesion proteins bind to integrins on the cell surface via cell-binding epitopes. Cell-binding epitopes are small peptide sequences derived from adhesion proteins, namely, RGD from collagen, RGDS from fibronectin and IKVAV and YIGSR from laminin. The stem cell senses the environment in which it is embedded and is able to modify its form and function accordingly; this is its main mechanism of proliferation and differentiation. Understanding this mechanism in detail is of paramount importance to the development of new biomaterials for stem cell biology.
Stem cells have specific characteristics that differ from those of other cells in the body. In particular, they are able to differentiate into individual cell-types and contribute to the regeneration of tissues. Additionally, the unlimited self-regeneration by asymmetric cell division into a specialized and an unspecialized daughter cell constitutes one of the main characteristics of stem cells. Two different types are known: embryonic stem cells (ESCs) and adult stem cells (ASCs). ESCs are derived from the inner cell mass of the blastocyst and have a pluripotent differentiation potential. Induced pluripotent stem cells (iPSCs) are a nearly inexhaustible source of pluripotent stem cells. They are directly reprogrammed from fibroblasts by introduction of four growth factors Oct3/4, Sox2, c-Myc, and Klf4. iPSCs possess the properties similar to ESCs and are therefore an auspicious cell species to study the differentiation potential of artificial cell culture substrates for differentiation of pluripotent stem cell lines. ASCs are unspecialized, multipotent cells derived from adult tissues and can differentiate into restricted types of specialized cells. The most common type of ASCs are mesenchymal stem cells (MSCs) ASCs require a defined microenvironment in an adult organism to maintain their stem cell properties.
Over the last decade, the importance of identifying and exploring ECMs and their niches has been increasingly recognized. Peptides and peptide derivatives are excellent candidates for the development of 2D and 3D cell-culture materials, thanks to their biodegradability and biocompatibility, and make it possible to tailor biomaterials for different applications in tissue engineering and regenerative medicine. Specific types of peptide-based materials possess specific advantages: whereas 2D scaffolds present highly sensitive ligands bound to an inert substrate, elasticity and stiffness are key in obtaining differentiation signals for stem cells embedded in 3D scaffolds. The properties of each peptide-based material can be adjusted by the primary structure, and they are generally easy to procure by means of standard peptide synthesis protocols. Peptide structures, folding processes and stability are also for the most part well-established in the literature.
Stem cells are sensitive toward the physical parameters of the surrounding microenvironment and are able to adapt their differentiation lineage according to the topology, polarity or the elasticity of the surface.
Non-peptidic topological cues influencing stem cells include surfaces presenting different chemical functionalities; for example, glass silane modified substrates with diverse functional groups (methyl-, amino, silane-, hydroxy-, and carboxy-groups) on their surfaces were established to culture hMSCs. Methyl-substituted surfaces lead to maintenance of the hMSC phenotype, amino-surfaces promote and maintain osteogenesis, and hydroxyl- and carboxyl-groups promote and maintain chondrogenesis. Stiffness is one of the main parameters for directing the fate of a stem cell, and it is expressed by an elastic modulus. Stem cells are mechanically sensitive, thus their fate is directed by the stiffness of the environment into which it is embedded (see Figure 1). Examples of stiffness cues include the development of biomaterials for directed stem cell differentiation in 2D and 3D cell cultures. For example, polyethylene glycol silica nanocomposite gels containing RGD-peptides and exhibiting different degrees of stiffness were investigated regarding hMSC differentiation. Stiffer gels showed higher expression levels of Runx2, an early bone differentiation transcription factor, compared to gels having low or intermediate stiffness.
Figure 1. Schematic representation of the extracellular matrix of stem cells. Stem cells are surrounded by fibers and adhesion proteins which recruit integrins. Their fate is directed by aspects of the ECM like stiffness, cell-cell interactions, and composition with respect to solubility factors, adhesionproteins, and glycosaminoglycans. Topological signals can be epitopes presented by the latter to direct cell behavior. According to the biomaterial design principles discussed, peptide materials can be designed to comply with the requirements of the natural ECM. A stiff ECM leads to differentiation toward stiff tissues, i.e., osteogenesis. Depending on the specific lineage of stiffness and elastic moduli, for example, MSCs can differentiate tissues according to stiffness of tissues the cells are specializing in. Brain tissue has elastic moduli that range from 0.1 to 1 kPa and can entrap neurocytes; elastic moduli of pancreatic tissue are about 1.2 kPa; cartilage tissue has a typical elastic modulus of 3 kPa and entraps chondrocytes; muscle tissues has elastic moduli between 8 and 17 kPa and entraps myoblasts; and the strongest is osteoblast entrapping bone tissue with elastic moduli from 25 to 40 kPa.
A prominent example of conjugation of adhesion proteins with different biologically relevant polymers was shown by Zhang et al. (2016), who functionalized hyaluronic acid (HA) with methacrylate to achieve photo-crosslinking on glass-slides and conjugation of a synthetic peptide derived from vitronectin (VN) to the glycosaminoglycan. The growth rate of hiPSCs on these VN-MeHA surfaces was compared to that on Madrigal, a mixture of ECM proteins, and similar results were obtained.
Another example of a 3D nanofibrous scaffold supporting cell differentiation is the RAD16 peptide family These two ionic peptides, named RAD16-I and RAD16-II, are able to form stable β-sheets in water and self-assemble spontaneously into nanofiber scaffolds, which could mimic the ECM of tissue cells. Furthermore, three different self-assembling peptide hydrogels were developed to mimic a 3D cell environment for human adipose stem cells by functionalization of RAD16-I with three biologically active peptide motifs (see Table 1) at its C-terminus. The functionalized RAD16-I peptide-based hydrogels were used for cell culture and showed that the peptide motifs increased the viability of human adipose stem cells compared to RAD16-I. The largest number of human adipo
