Abstract
Tissue-resident stem cells are surrounded by a microenvironment known as ‘stem cell niche’ which is specific for each stem cell type. This niche comprises of cell-intrinsic and -extrinsic factors like biochemical and biophysical signals, which regulate stem cell characteristics and differentiation. Biochemical signals have been thoroughly studied however, the effect of biophysical signals on stem cell regulation is yet to be completely understood. Biomaterials have aided in addressing this issue since they can provide a defined and tuneable microenvironment resembling in vivo conditions. We review various biomaterials used in many studies which have shown a connection between biomaterial-generated mechanical signals and alteration in stem cell behaviour. Researchers probed to understand the mechanism of mechanotransduction and reported that the signals from the extracellular matrix regulate a transcription factor yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), which is a downstream-regulator of the Hippo pathway and it transduces the mechanical signals inside the nucleus. We highlight the role of the YAP/TAZ as mechanotransducers in stem cell self-renewal and differentiation in response to substrate stiffness, also the possibility of mechanobiology as the emerging field of regenerative medicines and three-dimensional tissue printing.
Keywords: Mechanobiology, Human pluripotent stem cells, YAP/TAZ, Biomaterials
Introduction
Stem cells are unique cells that can self-renew and differentiate into specialized cells. Stem cell research has opened a new field of regenerative medicine, it has modernized the fields of drug discovery and our understanding of the physiological processes associated with disease or injury [1, 2]. Even though adult human stem cells have been used in clinical settings, their use is impeded because of their limited expansion capabilities, differentiation potential, and availability [3–5]. Human pluripotent stem cells (hPSCs) on the other hand exhibit unlimited expansion potential and unique property to differentiate into three germ layer cells, which makes them an ideal cell source for basic and clinical research [6, 7]. The microenvironment surrounding the stem cells, also known as ‘niche’, maintains a balance between self-renewal and differentiation and is specific for each type of stem cell population. It is mainly composed of tissue-specific extracellular matrix (ECM) proteins, other cells, and soluble factors such as Wingless-Type MMTV integration site family member (WNT), bone morphogenetic proteins (BMPs), ACTIVIN/NODAL, fibroblast growth factor (FGF) among others, all of which regulate cellular functions [8–10].
Signalling pathways originating from soluble factors are well known in regulating stem cell proliferation and differentiation. A number of studies have demonstrated that the physical properties of ECM and the mechanical forces generated from the surrounding niche also play an integral part in defining the stem cell behaviour, inducing specific differentiation pathways. The mechanical forces such as stress, tension, pressure, and stretch can be generated internally or externally in response to fluid flow, substrate stiffness, gas partial pressures or from adjacent cells [11, 12]. The mechanism by which the mechanical signals originating from the ECM are transduced into biological signals is collectively known as mechanotransduction and has been of great interest to the researchers. The knowledge gained so far has been invaluable in understanding metastatic cancers, neuronal regeneration, wound healing, liver regeneration, bone repair, and several other cellular phenomena. Mechanotransduction involves sensing of mechanical signals by cell surface receptors and translating it into biochemical signals by initiating a signalling cascade and eventually regulate the activity of specific genes [13]. Among all the mechanical forces, the role of ECM has been extensively researched; as the ECM plays important role in determining cellular functions [12, 14].
There has been a lot of excitement around organoids, generated from pluripotent and adult stem cells. Advances in the field of organoid research have shown the significance of cell–matrix interactions on stem cell self-renewal and differentiation [15]. For organoid formation, stem cells are embedded in complex ECM matrix which creates small, three-dimensional (3D) and self-organized tissue-like cell clusters. Organoids resembling complex tissues of intestine [16], retina [17], thyroid [18], brain [19], inner ear [20], kidney [21], liver [22], lungs [23], gastrointestinal tract [24] and blood vessels [25] have been generated. Mechanobiology behind organoid formation can shed some light upon the self-organization behaviour of stem cells in organogenesis.
Fundamental understanding of mechanical forces on stem cell behaviour can provide insights for developing an artificial niche, to support stem cells for regenerative medicine as well as it will add to our knowledge of developmental biology. In this review, we have tried to give the reader comprehensive information about the studies that have demonstrated the effect of mechanical signals mediated through hydrogels and artificial substrates on stem cell behaviour. We focus particularly on transcriptional activators yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), their regulation by mechanical signals and their critical role in stem cell proliferation and differentiation.
Extracellular matrix regulated stem cell differentiation
The extracellular matrix is an essential component of the stem cell microenvironment and is indispensable for proliferation and differentiation. Apart from providing physical support to the cells, ECM regulates cell proliferation, growth, differentiation, cell shape, and migration. It is a complex assembly of proteins (mainly composed of collagen, fibronectin, elastin, laminin, and vitronectin), proteoglycans and small integrin-binding glycoproteins secreted by the cells; however, the precise composition of the matrix is different between tissues [26]. For instance, the extracellular matrix of bone is composed of highly insoluble collagen type I fibres, with traces of collagen III, V, XI, and XIII, with many intra- and inter-molecular crosslinks. Based on the composition and structure of the collagen fibres, the estimated stiffness; represented as Young’s modulus (E) expressed in Pascals (Pa); for bone is approximately 100 kPa–1GPa [27]. Whereas, the brain extracellular matrix primarily consists of lecticans, a proteoglycans family, crosslinked by tenascin (a glycoprotein), and hyaluronic acid (a glycosaminoglycan). ECM of the brain has a low content of fibrous proteins such as collagen, fibronectin, and vitronectin, making it soft compared to bone ECM with E of approximately 1 kPa [28–30]. Several experimental studies have shown that changing the biochemical composition of ECM, changes the mechanical properties (stiffness and resulting stress) which in turn regulates cell morphology, growth, differentiation, migration, and gene expression [14, 31, 32].
The substrate stiffness has been shown to change cellular morphology that leads to the reorganization of the intracellular cytoskeletal network [14, 31]. Human mesenchymal stem cells (hMSCs) develop broader and flattened morphology on the stiff substrate (E ~ 25–40 kPa) and show high expression of osteoblast-specific genes such as SRY-box transcriptional factor 9 (SOX9) and core-binding factor subunit alpha-1 (CBFA1) [33, 34], whereas, on the softer substrate (E ~ 0.1–1 kPa) hMSCs have shown upregulation of neuronal-specific protein markers such as Nestin, Tubulin beta 3 (TUBB3), Neuro Filament Light (NFL) chain, and Neural Cell Adhesion Molecule (NCAM) [33]. Mouse primary spinal cord neural cells grow and extend to neurite on the soft substrate but not on the stiff substrates [35]. On the soft substrate, cells show reduced spreading, reduced organization of actin into stress fibres and morphologically they appear round [36, 37]. Moreover, muscle stem cells maintain stemness and self-renew when cultured on soft synthetic hydrogel which mimics the elasticity of muscle (E ~ 12 kPa) [38]. Such diverse responses of stem cells to substrate stiffness represent a fundamental role of matrix in regulating cell behaviour. This shows that actin filaments regulate cell morphology in response to matrix stiffness. These biosystems help us in understanding the basic principles of tissue and organ functions.
Tools to engineer stem cell niche ex vivo and study ECM-cell interactions
In vitro studies are the ideal way to investigate the ECM-cell interactions; however, the complexity of the niche and variations in ECM composition makes it challenging and difficult. Traditional in vitro culturing methods involves the use of glass and plastic culture dishes for cell culture with E ~ 1GPa, which is very stiff compared to in vivo conditions [39]. Researchers have been using various biocompatible materials to engineer stem cell niches in vitro which mimic the stiffness of biological tissues. Natural polymers such as collagen, agarose, collagen, chitin [40], alginate, and hyaluronic acid or its conjugated hydrogel [32] are used to synthesize scaffolds due to their similarity with native ECM. However, given their limited mechanical properties focus has now shifted to synthetic hydrogels such as polyacrylamide (PA) gels [37], poly-dimethyl-siloxane (PDMS) [41], polyethylene glycol hydrogel [38], polyvinyl alcohol [42] to name a few. These synthetic hydrogels provide a wide array of stiffness range similar to physiological tissue stiffness but synthetic polymers provide limited cellular interactions due to lack of functional group present. To overcome the disadvantage of natural and synthetic polymers, semi-synthetic hydrogels such as gelatin methyl acrylate (GelMa) [43] was synthesized which combines the biocompatibility of natural hydrogel polymer and mechanical properties of synthetic biomaterials. Other than the hydrogels, artificial micropatterned and nanopatterned substrates [44], flexible micropillars [45], and electrospun nanofiber [46] have also been used as scaffolds to understand the effect of different substrate and their stiffness on cell migration, growth, and differentiation. In the following section, we have briefly discussed few natural and synthetic substrates and their effect on stem cells. Table 1 gives an overview of the various biomaterials discussed below and their influence on cell behaviour.
Table 1.
Biomaterial | Stiffness | Cell type cultured | Cell response | References |
---|---|---|---|---|
Natural polymer | ||||
Matrigel (3D culture) |
34 Pa–480 Pa (of 3D Matrigel) |
Primary extravillous trophoblast | Cells showed migration towards stiff region and differentiated into endometrium and placental cells | [47] |
Vitronectin | As coating on tissue culture plate | hESCs | Cell attachment, growth and proliferation | [48] |
Synthetic substrates | ||||
Polyacrylamide (PA) gel substrate | ||||
PA gel substrate coupled with type I collagen |
10 Pa (soft) 90 Pa (stiff) |
Kidney epithelial cells and 3T3 fibroblasts |
Less spreading of cells on soft substrate compared to stiff substrate Cells migrate from soft substrate towards stiff substrate |
[14, 31] |
PA gel substrate coupled with type I collagen |
0.1–1.0 kPa (soft) 8–17 kPa (intermediate stiff) 25–40 kPa (stiff) |
hMSCs |
Differentiated into neural lineage Differentiated into myogenic lineage Differentiated into osteogenic lineage |
[33] |
PA gel functionalized with GAG peptides |
0.7 kPa (soft) 10 kPa (stiff) |
hESCs and hiPSCs |
Better attachment, self-renewal and maintains pluripotency Cells adopted neural morphology and after addition of neuronal maintaining media developed into mature neurons |
[49] [50] |
PA gel substrate coupled with Matrigel |
3 kPa (soft) 165 kPa (stiff) |
hESCs and hiPSCs | On soft substrate which showed stiffness similar to liver tissue, the cells differentiated into endoderm lineage whereas not on stiff substrate | [51] |
Poly (dimethyl siloxane) (PDMS) gel substrate | ||||
PDMS coated with polydopamine | Not determined | Bone-marrow stromal cells | Promoted differentiation into osteogenic lineage in presence of osteogenic differentiation media | [52] |
PDMS coupled with type I collagen | 1.9 MPa–2.7 MPa (stiff) | mESCs | Cells expressed pro-osteogenic gene markers | [53] |
PDMS printed with Fibronectin or Laminin | 5 kPa (soft) |
PC12 (rat adrenal gland derived cell line), C2C12 (mouse muscle derived cell line) |
PC12 cells differentiated into neurons on soft substrate, whereas C2C12 formed myotubes when cultured stiff substrate | [54] |
PDMS coated with type I collagen |
3 kPa (soft) 37 kPa (stiff) |
Cardiac fibroblasts, 3T3 fibroblast, hMSCs |
Cells of all three-cell line showed increased cell spreading on stiff substrate whereas on soft substrate these cells manifest small spread area Stiff substrate promoted myofibroblast activation of cardiac fibroblast |
[55] |
Electrospun nanofibrous substrate | ||||
PCL fibrous substrate PET fibrous substrate PEKK fibrous substrate PCU fibrous substrate (all plasma treated or collagen-conjugated) |
19 kPa (softest) 39 kPa (soft) 74 kPa (stiff) 193 kPa (stiffest) |
hiPSCs |
hiPSCs exhibit round 3D colony morphology on softest/soft substrate, on stiff/stiffest substrate cells spread and have flattened morphology Long-term culture on soft(est) substrate led to ectodermal differentiation, no change in cells on other substrates After adding defined growth factors, stiff substrate promoted motor neurons and soft substrate enhanced posterior foregut specification |
[46] [56] [57] |
Semi-synthetic substrates | ||||
Gelatin methyl acrylate (GelMa) | ||||
5% GelMA 10% GelMA 20% GelMA 30% GelMA |
3.08 kPa (softest) 34.9 kPa (stiffer) ~75 kPa (data not provided, estimated from the graph) 184.52 kPa (stiffest) |
PC12 (rat adrenal gland derived cell line) |
Cells show highest adhesion rate on 5% GelMa compared to 20% and 30% Cells on 10% GelMA have optimum spreading rate and longest neurite length |
[58] |
5% GelMa, 10% Gelma |
Not determined | Rat MSCs | Cells differentiated into osteoblast when supplemented with osteogenic media | [59] |
3D GELMA-PEGDA 3D PEGDA-GELMA |
30 Pa–150 Pa (soft) 1 kPa–8 kPa (stiff) |
MDA-MB-231 (breast cancer cell line) |
Soft substrate support spindle-like morphology Stiff substrate showed tumor-like spheroidal morphology, cells from these spheroids showed epithelial to mesenchymal transition and drug resistance |
[60] [61] |
Mineralised GelMa with functionalized PEGDA | Not determined | hiPSCs | Cells underwent osteogenic differentiation devoid of biochemical signals compared with non-mineralized GelMA | [62] |
Natural polymers
Conventional protocols for growing pluripotent stem cells use mouse embryonic fibroblast cells (MEFs) as a natural substrate. MEFs is known as ‘feeder cells’ are maintained in metabolically active but non-proliferating state, thus allowing them to express soluble, membrane-bound growth factors and extracellular matrix proteins, which controls cell growth and acts as a substrate [63, 64]. In 1998, the first derivation of human embryonic stem cells (hESC) line described the importance of inactivated MEFs feeder layer for maintaining their undifferentiated state [6]. However, owing to the risk of xeno-contamination and variability, a feeder-free culture system was introduced for the maintenance and proliferation of hESCs. The feeder-free culture system uses ECM protein namely laminin [65], collagen [66], fibronectin [67], vitronectin [48, 68], Matrigel [69–71].
Collagen is the abundant ECM protein, which is widely used as a natural substrate by many researchers. Since the majority of the native ECM is composed of collagen, collagen-based biomaterials are highly biocompatible and biodegradable. Collagen alone has very low mechanical property since the mechanical properties depend on the composition of the scaffold, collagen along with co-polymer has shown to support self-renewal and differentiation of hPSCs [66] and regeneration of various tissues [72–75] in several individual studies. Fibronectin is the second most abundant ECM protein after collagen and is often used for increasing cell adhesion [67]. Several studies have shown that fibronectin supports long-term self-renewal of hPSCs [76, 77]. Fibronectin along with another polymer, for example, silk fibroin, have been used to develop a hybrid fibre, which mimics the mechanical properties of desired tissue and sustains cell growth and proliferation [78].
Matrigel is a commercially available basement membrane-like protein matrix made up of laminin, collagen IV, heparan sulphate proteoglycan and a number of growth factors in a non-defined proportion and has been reported in several studies to support the growth of undifferentiated hESCs [79, 80]. By changing the composition of the biopolymer and protein growth factors, the elastic modulus of Matrigel varies from 34 Pa–480 Pa [81, 82]. Given such low elastic moduli, Matrigel has been an ideal substrate to study cell migration in 3D culture and for the generation of many 3D organoid culture models, for example, the organoid culture of endometrium and placenta has been well established [47]. Since Matrigel is not well-defined, it may not be suitable for large-scale manufacturing for therapeutic purposes. It is generated from mouse sarcoma cells and may contain carcinogenic or xenogenic factors; thus, it is not suitable for human clinical trials [83]. Alternatively, other ECM components have been coupled with synthetic polymer matrices with specific compositions. For example, hyaluronic acid (HA), glycosaminoglycans are critical in modulating neural and hematopoietic stem cell behaviour [84, 85]. Also, HA in conjugation with tyramine can form a tuneable 3D microenvironment that modulates chondrogenesis and may impact the spatial organization of cells [32].
Synthetic polymers
Synthetic substrates are promising alternative to natural substrates. Some advantages of synthetic polymers over natural polymers are their tuneable mechanical properties, ease of synthesis, available and low cost.
Polyacrylamide gel
Polyacrylamide gel consists of a linear monomer of acrylamide and a crosslinker bisacrylamide which links the linear acrylamide monomers. PA-gels have been used as a substrate for a diverse range of cell cultures for a long time because of its ideal physical and mechanical properties. By changing the ratio of acrylamide and bisacrylamide, PA-gel substrates can be generated having elastic modulus ranging from 0.1 kPa to 100 kPa, which is equivalent to the physiological elastic moduli of soft tissues namely adipose tissue, brain tissues, endothelial tissue or lung tissue [86–88]. Polyacrylamide is inert in nature, therefore, does not support cell growth and proliferation. For it to be used as a cell culture substrate, PA-gels are first treated with UV-activated cross-linker which couples’ extracellular matrix proteins to the substrate. A number of studies have reported that chemical and physical properties of the various PA substrates have a profound effect on cell locomotion, growth, and differentiation. Early studies with kidney epithelial cells and 3T3 fibroblasts on PA substrates reported that cells on flexible substrates with E between 10 Pa (soft)–90 Pa (stiff) displayed less spreading by limiting the amount of phosphotyrosine at adhesion sites, and more cell death [14]. A similar study with 3T3 fibroblast showed that cells migrate from the soft side towards the stiff side of the PA substrate [31]. Furthermore, PA substrate of varying stiffness has been shown to direct lineage specificity for human mesenchymal stem cells (hMSCs). hMSCs cultured on soft collagen-coated PA substrate (E ~ 0.1–1 kPa), the intermediate stiff substrate (E ~ 8.0–17 kPa) and stiff substrate (E ~ 25–40 kPa) differentiate into neural, myogenic and osteogenic lineages respectively [33]. Musah et al. [49] reported that stiff PA substrate (E ~ 10 kPa), when functionalized with glycosaminoglycans (GAG) peptides, offer better hESCs and human induced pluripotent stem cells (hiPSCs) attachment, self-renewal and promotes pluripotency evident from the expression of octamer binding transcriptional factor-4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and other pluripotency specific markers. In a different study, conducted by the same research group, they reported that PA substrate (E ~ 0.7 kPa) functionalized with GAG-binding peptide selectively differentiates hESCs. After several weeks of culture on these compliant substrates and in absence of neuronal inducing factors, hPSCs adopted neuronal morphology and expressed neuronal-specific tubulin beta 3 chain (TUJ1) protein. When the media was switched to neuronal maintaining media, cells showed expressions of microtubule-associated protein 2 (MAP2), a mature neuronal marker [50]. These results indicate that substrate alone can direct differentiation of hESCs, independent of soluble factors. A similar conclusion was drawn by Chen et al. [51], where they demonstrated that hPSCs cultured on soft PA-substrate with E ~ 3 kPa expresses high levels of the anterior primitive streak and definitive-endoderm-specific gene expression such as Eomesodermin (EOMES), Brachyury (T), Forkhead box protein A2 (FOXA2), and SRY-box transcription factor 17 (SOX17) compared to hPSCs on a stiff substrate with E ~ 165 kPa and tissue culture-treated plastic plates. Another study by Maldonado et al. [56] in hiPSCs cultured on electrospun nanofibrous scaffolds reported a similar observation, which we have discussed under subsection Electrospun Nanofibrous substrate.
Poly (dimethyl siloxane) (PDMS)
PDMS is a silicon-based flexible elastomer having a wide range of applications in medicine and cosmetics, soft lithography even as anti-foaming agents and surfactants [52]. PDMS has been extensively used to understand cell behaviour in the field of mechanobiology. These rubber-like elastomers have E up to 2 MPa [89], thus making them suitable for investigating cellular responses to changing substrate stiffness. Stiff PDMS substrate (E ~ 1.9 MPa-2.7 MPa) has been reported to support proliferation and cell spreading of mouse embryonic stem cells (mESCs). These cells showed significant upregulation of pro-osteogenic transcription factors runt-related transcription factor (Runx2) and secreted phosphoprotein 1 (Spp1), compared to mESCs cultured on soft PDMS [53]. PC12 (rat adrenal pheochromocytoma cell line) differentiate into neurons and develop longer neurites on soft PDMS substrate with elastic modulus similar to brain tissue (E ~ 5 kPa) compared to the cells on stiffer PDMS substrate. The same study also showed that C2C12 (mouse skeletal muscle cell line) when cultured and differentiated on stiff PDMS substrates formed longer myotubes compared to cells cultured on a soft substrate [54].
Many a time because of high surface hydrophobicity of PDMS cell attachment to the substrate is poor. For enhanced cell adhesion and proliferation bio-inspired polydopamine-coated PDMS was used instead of protein-coated PDMS. Polydopamine-coated PDMS promoted long term bone-marrow stromal cells culture while maintaining their multipotency [52]. In regard to cell spreading, 3T3 fibroblasts, cardiac fibroblasts and MSCs showed increased spreading on stiffer PDMS substrate coated with collagen compared to soft substrate, an observation similar to the PA substrate study. Interestingly, cells cultured on soft PDMS respond to secondary crosslinking-induced stiffness, representing similar spread area to the cells on stiff PDMS [55]. These reports indicate that PDMS hydrogels influences cell adhesion and spreading, potency and differentiation. It would be interesting to see whether PDMS modified to low stiffness help maintain the pluripotency of hPSCs or direct them towards differentiation.
Electrospun nanofibrous substrate
These substrates have been around for many years now and researchers have shown keen interest in using these substrates for as biological scaffolds in tissue engineering. They provide a porous mesh of nanoscale and microscale fibrous structures which highly resembles natural ECM [90]. A notable report by Maldonado et al. (2015 and 2016) [46, 56] studied the proliferation and differentiation of hiPSCs on various synthesized electrospun nanofiber substrates exhibiting different chemical and mechanical properties. These substrates were poly(ε-caprolactone) (PCL), polyethylene terephthalate (PET), polycarbonate-urethane (PCU), and poly(etherketoneketone) (PEKK). The measured Young’s modulus of these substrates ranged from ~ 19 kPa to ~ 313 kPa. Short- and long-term culture of hiPSCs showed characteristic pluripotent colony morphology and expressed pluripotency-associated markers OCT4, homeobox protein Nanog (NANOG) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) on all the substrates. However, after 12 days in culture, cells on 19 kPa substrate highly expressed ectodermal markers PAX6 and NEUROD1, whereas these makers showed minimal expression on 193 kPa substrate and no expression on tissue culture-treated plastic dish.
Electrospun fibrous substrates induce substrate-dependent changes in colony morphology and gene expressions in hiPSCs. hiPSCs, when subjected to defined growth factors of specific lineages, tend to differentiate into motor neurons on a soft substrate during early differentiation, conversely, stiff substrates promoted motor neuron specification during late stages. In contrast, hiPSCs differentiated into the mesendodermal lineage on the stiff substrate, but soft substrate enhanced further their specification into the posterior foregut [57]. These observations are different from the studies that have used hydrogels as substrates, suggesting that differentiation is not restricted to single stiffness and dynamic changes in the mechanical microenvironment may help in enhancing the differentiation efficiency of hiPSCs.
Semi-synthetic polymers
Biohybrid or semi-synthetic polymers combine the best of both the natural and synthetic polymers. A number of scaffolds with at least one natural polymer such as collagen, fibrin, laminin in well-defined proportion with synthetic polymer namely polyethylene glycol (PEG), polyglycolic acid (PGA) or polycaprolactone (PCL) have been synthesized for 2D and 3D culture of many cell lines including stem cells [91]. Recently, GelMA has emerged as an attractive candidate for mimicking native ECM conditions in vitro.
Gelatin methyl acrylate (GelMa)
GelMa is a photo-reactive hydrogel made up of gelatin and methacrylic groups. First synthesized in 2000 by Van Den Bulcke et al. [92], GelMa is an inexpensive, natural polymer synthesized from hydrolysis and denaturation of collagen which makes it suitable biomaterial for in vitro studies. GelMa has proven to provide optimal conditions for cell culture due to the presence of arginine-glycine-aspartic acid (RGD) motifs for adhesions and matrix metalloproteinase (MMP) degradation motif for cellular enzymatic degradation. Depending upon the percentage of GelMa (w/v) or the concentration of the other polymers, such as alginate or hyaluronic acid, mixed with it, stiffness of GelMa can range from 1 kPa to 200 kPa [58, 93, 94]. Owing to such a large physiological stiffness range, GelMa hydrogels have been thoroughly investigated for its physical and biochemical properties as 2-D and 3-D scaffolds in tissue engineering [95]. Notable application of GelMa 3-D scaffolds for tissue engineering had been reported for bone [96], skin [97, 98], cardiac tissue [99], endochondral bone [100], skeletal muscles [101], and vascular networks [102].
To determine whether soft GelMa substrate supports neuronal development, PC12 cells were cultured on GelMa substrates having E ~ 3 kPa–184 kPa. The cells showed maximum adhesion on softest substrate of 3 kPa compared to other stiffness, however, the longest neurite length was observed on substrate with intermediate stiffness (E ~ 34.9 kPa) [58]. The authors, however, did not show the gene expression profile of the differentiated cells. It would be interesting to know whether these substrate support neurite maturation for a long duration and generate mature as well as functional neurons. In another study, rat bone-marrow derived MSCs were cultured on 5% (soft) and 10% (stiff) GelMa substrates and when they were supplemented with osteogenic media, MSCs differentiated into osteoblasts. The stiffness of these two GelMa substrates was not determined; hence which stiffness range of GelMa supports osteoblast differentiation still remains a question [59].
For 3D cell culture, a blend of GelMa and polyethylene (glycerol) diacrylate (PEGDA) was developed to study cell metastasis using cancer cells. By increasing the concentration of PEGDA, the elastic moduli of non-degradable 3D PEGDA-GELMA scaffold was between E ~ 1 kPa to 8 kPa whereas soft degradable 3D scaffolds were obtained by increasing GelMa concentration (E ~ 30 Pa to 150 Pa). Encapsulated MDA-MB-231 breast cancer cells in these substrates showed tumor-like spheroidal morphology in stiff PEGDA-GELMA substrate, while they showed spindle-like morphology in soft GELMA-PEGDA substrate. Invasion chick aorta arch assay showed that the softer GELMA-PEGDA substrate supports the invasion of both MDA-MB-231 and endothelial cells, along with sprouting of endothelial cells from chick aortic arches [60]. The gene expression profile of the spheroids derived cell from stiff PEGDA-GELMA showed epithelial-mediated transition (EMT) behaviour and drug resistance [61]. GelMa substrate mineralized with calcium and phosphate minerals induce osteogenic differentiation of hiPSCs in 2D and 3D, evident from gene expression study and immunofluorescent staining for osteogenic specific transcription factors RUNX2, OCN and SPP1. The hiPSCs were cultured devoid of growth factors and the differentiation was achieved through substrate-based biomechanical cues. However, non-mineralized GelMa or gelatin-coated dishes functionalized with PEGDA does not support the growth and differentiation of hiPSCs [62]. Though GelMa has shown great promise in tissue engineering, questions such as—whether GelMa directs differentiation into lineages other than osteogenic differentiation, is GelMa a better substrate than normal culture dishes or other substrates for differentiation, how stem cells differentiated on GelMa functionally differ from differentiated cells generated via the normal stiff culturing conditions, need to be addressed before GelMa could be routinely used for basic and possibly for translational purposes.
Mechanobiology: Intracellular sensory system
We have described the effect of various natural and synthetic biomaterials on cell proliferation and differentiation, this implies that cells have a mechanism to sense substrate stiffness and make modifications to generate specific proteins. In order to generate supportive biomaterial, it is essential to understand what changes occur to mechanosensing machinery inside the cells. Cell–matrix interactions are mediated by cell surface receptor integrins, focal adhesion kinases (FAK), and cytoskeletal filaments that connects the extracellular matrix to the intracellular environment. When cells come in contact with mechanical signals from the extracellular region, a multiprotein complex is formed near the cell surface receptor, known as focal adhesions (FA). FA comprises of talin, vinculin, paxillin, alpha-actinin, p-130cas, FAK, SRC, and FAs act as a mediator between integrins and actin filaments (Fig. 1). Other than FA, Rho family small GTPases, its downstream effectors Rho-associated kinases (ROCK), and myosin light chain kinases also relay the mechanical signals to cytoskeletal filaments [103–107]. The cytoskeletal components mainly actin filaments, microtubules, and non-muscle myosin undergo conformational changes (stretching or relaxing, shortening or elongating) [108, 109] which affects gene-specific transcription due to cytoplasmic or nuclear localization of gene-specific transcriptional factors. For example, the formation of stress fibres causes nuclear localization of transcriptional coactivators YAP and TAZ or cytoskeletal remodeling activates and translocates beta-catenin into the nucleus [110, 111], where these two proteins interact with their respective coactivators and activate specific gene expression.
Apart from these, other transcriptional factors which have been reported to respond to different mechanical signals are tight junction protein ZO-1 [112], tyrosine kinase c-Abl [113], myocardial-related transcriptional factor (MRTF) [114], Nuclear Factor-kappa-B (NFκB) [115], nuclear factor erythroid-2-related factor 2 (NRF2) [116], epigenetic regulator HDAC3 in both mouse and human cell lines [117]. An important point to be noted here is that, these transcriptional factors have been known to be regulated by specific signalling pathways and do not solely function as mechanotransducers. The shuttling of these transcriptional factors between cytoplasm and nucleus is gated by linker of nucleoskeleton and cytoskeleton (LINC) complex present on the nucleus envelope. The tensed actin cytoskeleton causes stretching of actin binding LINC complex component Nesprin1, resulting in stiffening of nuclear envelope and localization of transcriptional factors [118, 119]. The mechanical signal-regulated signalling pathways are complex; numerous studies have identified YAP/TAZ proteins as mechano-transmitters that respond to signals from substrate stiffness, substrate topology, surface area, cell density, cell polarity, and cell geometry. Given such a vast array of regulators, YAP/TAZ has garnered the attention of many research groups. Here, we review the data highlighting the functions of YAP/TAZ as transcriptional co-activators and as mechanosensors.
Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)
Yes-associated protein (YAP) was first discovered in Drosophila and was termed Yorkie due to its association with Src family kinase Yes [120] and transcriptional co-activator of PTZ binding motif (TAZ) was first identified as 14-3-3 binding protein as a paralog to YAP [121]. These transcriptional co-activators share 46% sequence homology and display a similar domain organization, except for a proline right domain, one WW domain and SH3 binding region [122]. YAP and TAZ are primary effectors of the Hippo pathway however; these coactivators also interact with TGF β, WNT, BMP, ERK and FGF signalling pathways [123, 124]. Figure 2 shows the core pathway consists of a cascade of kinase activations in which the upstream signals phosphorylates mammalian Ste20-like kinases 1/2 (MST 1/2; Drosophila homologous Hippo [Hpo]) which phosphorylates and binds to SAV1 (Drosophila homologous Salvador [Sav]). This complex then phosphorylates and activates large tumor suppressor kinase 1/2 (LATS1/2; Drosophila homologous Warts [Wts]) and monopolar spindle-one-binder proteins (MOB1; Drosophila homologous Mats) complex. LATS1/2 in turn activates YAP/TAZ by phosphorylating at specific serine residues, thereby causing it to localize in the cytoplasm. Besides LATS1/2 several other proteins—Angiomotin (AMOT), 14-3-3, SMADs, casein kinase 1 isoform epsilon and delta (CSNK1E/D), glycogen synthase kinase 3 beta (GSK3B), catenin beta (CTNNB), CAPZ and beta transducing repeat-containing protein (BTRC) also affect YAP/TAZ localization [121, 125–128] (Fig. 2A). Since YAP/TAZ does not have their own DNA binding motifs, unphosphorylated nuclear YAP/TAZ interacts with various DNA-binding transcription factors regulating targeted gene expression. The function of YAP/TAZ depends upon the upstream signal and its binding partner [129] (Fig. 2B). Their nuclear localization results in cell proliferation, differentiation, migration, epithelial-mediated transition (EMT), cancer development or suppression, and loss of contact inhibition [129–133].
YAP and TAZ share high protein sequence similarity (Fig. 3) and thereby have similar functions. However, there are few structural differences that suggest non-overlapping functions of YAP and TAZ [134, 135]. YAP regulates TAZ cytoplasmic retention, but the exact mechanism is still not clear, although interestingly, the same cannot be said for TAZ because an increase in TAZ expression does not show any effect on YAP levels [136]. Transgenic expression of only YAP in mouse liver showed a dramatic increase in liver mass [137]. YAP and TAZ exhibit some physiological differences as well, and knockout studies have shown that YAP knockout mice embryo having normal TAZ develop severe developmental defects and subsequent embryonic lethality by embryonic day 8.5 [138]. Conversely, TAZ knockout embryos with normal YAP exhibit partial embryo lethality, where only half of the embryos survive while the other half die [139–141]. From these observations, it can be said that TAZ is unable to compensate for the loss of YAP.
The differentiation of MSCs into adipocytes, osteoblasts or myocytes has been shown to be regulated by YAP/TAZ activation [142, 143], whereas, YAP/TAZ has been associated in maintaining the pluripotent state and self-renewal in mESCs [137]. Qin et al. (2016) [144] reported that YAP overexpressing hPSCs cultured in DMEM F12 supplemented with bFGF, N2B27, ERK, GSK inhibitors, Forskolin and human LIF did not undergo differentiation as opposed to primed hESCs. Even in differentiating media these hESCs overexpressing YAP strongly expressed pluripotency markers such as OCT4, NANOG, SSEA3, SSEA4, alkaline phosphatase (AP), TRA-1-60 and TRA-1-81. The YAP overexpressing cells when cultured for more than 70 passages showed increased growth rate and dome-like colony morphology. They further proved the role of YAP in maintaining the naïve state by generating clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-generated YAP-/-cells. YAP knockout impairs hESCs ability to exhibit naïve-specific colony characteristics. Apart from this, YAP was shown to play a crucial role in anterior–posterior streak specification. YAP1-/- pluripotent stem cells specify into anterior–posterior streak progenitors in the presence of ACTIVIN which further differentiate into cardiac mesoderm and endoderm [145]. Other than stem cells, YAP/TAZ activation is well known in cancer cells where they function as an oncogene and play an important role in cancer initiation, progression, and metastasis [146]. Numerous knockouts, conditional knockout, deletion studies in various cancer models have revealed the potential role of YAP/TAZ in promoting tumorigeneses in skin, liver, breast and ovarian cancer. We would suggest the readers refer to other excellent reviews on the role of YAP/TAZ in cancer [147–150].
YAP and TAZ as mechanosensors in stem cells
Apart from acting as transcriptional activators, YAP/TAZ have emerged as key mechanotransducers, acting as nuclear relays in response to cell polarity, substrate stiffness, topology, surface area, and cell density [151–153]. YAP/TAZ activity as mechanosensors has been widely associated with the focal adhesion (FA) components namely integrins, talins, and FAKs. Indeed, on larger stiff substrates cells are well spread, in these conditions they exhibit high ROCK, non-muscle myosin II and F-actin levels, experience high contractile forces which activate YAP/TAZ. Conversely, cells on the soft or small substrates with reduced adhesive area display round morphology, low F-actin, low contractile forces causing cytoplasmic retention, and inactivation of YAP/TAZ [151, 154]. Additionally, the F-actin-capping and -severing proteins Cap Z, Cofilin and Gelsolin have shown to bind to YAP/TAZ thus, limiting YAP/TAZ nuclear localization in cells experiencing low mechanical stress [153]. hESCs on stiff PA-gel substrate (E ~ 10 kPa); showed high levels of nuclear YAP/TAZ, F-actin and OCT4 which is indicative of pluripotency [49]. Conversely, hESCs on soft PA-substrate (E ~ 0.7 kPa) showed more cytoplasmic YAP and differentiation into post-mitotic neurons [50]. These findings highlight that, substrate stiffness affects the formation of F-actin and further regulates YAP/TAZ activity. Interestingly, a recent study reported that by changing the stiffness of the PDMS substrate, the mesoderm differentiation kinetics of hiPSCs can be modulated and this mechanical change activates YAP during mesoderm induction [155]. This curious observation can be explored further by differentiating hiPSCs into other lineages on PDMS substrate of varying stiffness and studying the YAP/TAZ dynamics.
The nuclear translocation of YAP in response to mechanical stimuli has also been associated with the FAK activity. Computational model studies suggest that substrate rigidity affects the FAK activation levels which mediate YAP nuclear localization, and subsequent activations of downstream proteins and eventually gene regulation [119]. Among all the mechanosensing molecules, Talin directly links Integrins to Actin, thus the ECM-Integrin-Talin-Actin clutch is of particular interest in understanding mechanotransduction by matrix rigidity. Substrate stiffness above 5 kPa triggers conformational changes in Talin, it unfolds, binds to and activates vinculin, leading to an increase in focal adhesions and nuclear localization of YAP/TAZ as illustrated in Fig. 1. This role of Talin was demonstrated by culturing Talin-depleted cells on PA-gel, these cells showed normal spreading however they neither develop focal adhesions nor caused nuclear localization of YAP [153].
A common observation across current literature shows that hPSCs attach poorly on the soft substrate due to lack of binding sites on substrates, which directly affects their survival. To overcome this, a semi-interpenetrating matrix of PA gel and Matrigel was synthesized ranging from 150 to 12000 Pa in stiffness. hESCs on all the substrates maintain the expression of pluripotency markers even in small colonies. In response to the substrate stiffness, hESCs mimicked colony characteristics of epiblast, inner cell mass, and proamnion. hESCs on soft substrates with E < 450 Pa exhibit decreased nuclear YAP compared to cells on stiff substrates having E > 1000 Pa. Immunofluorescence probing of single cells grown on soft substrate showed a heterogeneous distribution of YAP in peripheral cells and nuclear localization in the colony interior, indicating a significant variation in YAP distribution within a single colony. When the substrate was switched from soft PA to glass coverslips, an increase in nuclear YAP was observed. Further analysis showed that YAP expression drastically decreased on soft substrate (E ~ 450 Pa and 150 Pa) whereas no significant changes in OCT4 expression were seen. This implies that on soft substrate, YAP expression in pluripotent stem cells is sufficient for proliferation and survival but it does not seem to play any role in maintaining pluripotency [156]. Substrate stiffness has a significant effect on cell migration, human adipose-derived stromal/stem cells, human hepatic stellate cells (HHSteC) and MSCs cultured on PA-gel substrate of stiffness spanning the in vivo physiological range, migrate towards the stiff region of the rigidity gradient substrate, a process known as durotaxis. To understand the mechanism behind migration, cells were treated with FAK inhibitor and siRNA YAP. Treated cells showed less motility compared to untreated cells indicating that the directed movement of cells from soft region towards stiff region is controlled by FAK and YAP [157–159].
To summarize, YAP/TAZ regulation is largely controlled by the integrity of the cytoskeleton mainly actin-myosin contractility [151]. Actin-severing proteins and unfolded Talin affects the YAP/TAZ nuclear localization [153, 154], while actin-capping proteins, impairment, and depletion of LINC or Nesprins, and finally stiffness of the substrates cause the cytoplasmic retention of YAP/TAZ in the cytoplasm [119, 154]. Elosegui-Artola et al. [107] have shown that by applying force to the nucleus, nuclear import of the YAP/TAZ and other proteins increases irrespective of their active or inactive state. Whether this passive transport initiates expressions of YAP/TAZ-regulated genes is yet to be shown.
Mechanobiology: future perspectives
Bone marrow transplantation or blood transfusions harbouring stem cells have been used for decades and have been reported to be very successful, however when isolated stem cells that are cultured on stiff substrates are transplanted, the results are variable. Sudden change in substrate stiffness may cause changes at gene-level leading to tumour formation or death of the injected cells. This might be the reason why many of the stem cell-based therapies have failed to show the desired effect. 3D bioprinting is a rapidly developing field, where cells are printed onto synthetic or natural biomaterials to generate tissues that closely resemble natural cell arrangement. Recently many newer biomaterials have been designed [160, 161], these biomaterials need to be stiff enough to support the growth of epithelial cells and pliable enough to support angiogenesis, to be clinically successful. Therefore, it is important that we understand the relationship between substrate stiffness and stem cells, so that cells can be cultured on substrate having similar physiological stiffness in order to achieve better results. We hope that with the knowledge about YAP/TAZ and the mechanism by which they regulate genes in response to varied substrate stiffness, would help us design functional 3D printed tissues. Most of the research work done to understand the effect of substrate stiffness on cell behaviour, has been carried out with MSCs, however not much data is available for primary tissues/cells or human pluripotent stem cells.
On a very fascinating note, researches have been focused on understanding the effect of microgravity on stem cell growth and differentiation, necessitated by the advances in space travel and challenges it poses to human physiology. Microgravity has a reversible effect on cell adhesion and microfilament rearrangements of MSCs as shown by the expression levels of vinculin, integrin, VCM1, and actin filaments [162]. Recently it has been demonstrated that the reduced gravity of the international space station (ISS) altered the functioning of neonatal and adult human cardiac progenitor cells [163]. These newer observations demonstrate that there is a lot to be uncovered about how living cells respond to external cues. Mechanobiology would be important discipline that should be studied if humans have to travel to distant planets or survive in low gravity environments of space stations.
Conclusion
Until recently, most of our understanding of molecular biology was from the cells cultured on very stiff tissue culture-treated plastic dishes. Cellular functions were defined by the signalling molecules used during culturing; however, the overarching function of mechanical signals suggests that mechanotransduction contributes to defining cellular activity. This raises profound questions such as- in the absence of signalling molecules are the external signals such as substrate stiffness or topology sufficient to direct the cellular fate of stem cells, the answers as and when are uncovered will have significant clinical impact.
Acknowledgements
We would to acknowledge Sunandan Divatia School of Science, NMIMS (deemed to-be University) and Symbiosis Centre for Stem Cell Research (SCSCR), Symbiosis International University, for providing the necessary resources and infrastructure. The work was funded by Department of Biotechnology (DBT), Govt of India, sanction order BT/PR28474/MED/31/393/2018. JKV was provided Senior Research Fellowship by Council of Scientific and Industrial Research (CSIR).
Compliance with ethical standards
Conflict of interest
The authors have no financial conflict of interest.
Ethical statement
There are no animal experiments carried out for this article.
Footnotes
Publisher's Note
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