**2. General approaches to induce** *in vitro* **differentiation of pluripotent stem cells (PSCs)**

Both mouse and human PSCs are routinely cultivated in the presence of feeder layers. PSCs grow on the feeder layers as colonies (Figure 2). Generally, human and mouse PSCs are enzymatically dissociated with trypsin, acutase, or dispase to obtain a suspension of single cells, which is then transferred for subculture and expansion or differentiation purposes. For mouse PSCs, LIF can substitute for feeder layers. However, since LIF is not effective for human PSCs, in the last years different chemically defined media have been generated in order to sustain human PSCs culture and expansion in feeder-free substrates.

**Figure 2.** PSCs are typically maintained in mitotically inactivated supportive cells. A) Mouse iPSCs cultured on top of irradiated mouse embryonic fibroblasts grow in tight colonies that are further trypsinized for subculture or differentia‐ tion purposes. B) Human iPSCs cultured on top of human irradiated dermal fibroblasts grow as colonies with defined borders.

As an option for culturing human PSCs without feeder cells, Matrigel™ has proven to be a useful alternative enabling the stable culture of human PSCs. Moreover, we have also shown that Matrigel™ allows the generation of hiPSCs without animal-derived feeder cells [19]. Since Matrigel™ was derived from Engelbreth-Holm-Swarm mouse sarcoma cells [20], other types of matrices which do not contain animal-derived agents have been tested and used as feedercell substitutes for the successful maintenance and generation of human PSCs; such as CellStart [21,22], recombinant proteins [23–25], and synthetic polymers [26,27].

The culture media used in the early generation of hESCs contained fetal bovine serum [1]. In order to remove unspecific agents that might cause the differentiation of hESCs, knockout serum replacement (KSR) has now been established as a defined material for maintaining hESCs [28] and is also traditionally used for hiPSC generation [9,12,29,30]. In this regard, mTeSR1 medium was developed as a chemically defined medium for maintaining human PSCs [31]. Importantly, in the last years several authors have reported the generation of commercially developed *xeno-*free media for maintaining hiPSCs, and such media have already been used successfully for iPSCs generation. These media include: TeSR2 [32], NutriStem [33], Essential E8 [24], and StemFit [34].

When factors that sustain PSCs stemness are deprived from the media, PSCs spontaneously differentiate into derivatives of the three embryonic germ layers. This capacity has been profited for more than 30 years in order to direct PSCs to the desired cell product. In this regard, up to day, an infinite number of protocols have been established to promote the development of the cell type of interest.

The following are basic strategies to induce *in vitro* differentiation of PSCs cells:

enzymatically dissociated with trypsin, acutase, or dispase to obtain a suspension of single cells, which is then transferred for subculture and expansion or differentiation purposes. For mouse PSCs, LIF can substitute for feeder layers. However, since LIF is not effective for human PSCs, in the last years different chemically defined media have been generated in order to

**Figure 2.** PSCs are typically maintained in mitotically inactivated supportive cells. A) Mouse iPSCs cultured on top of irradiated mouse embryonic fibroblasts grow in tight colonies that are further trypsinized for subculture or differentia‐ tion purposes. B) Human iPSCs cultured on top of human irradiated dermal fibroblasts grow as colonies with defined

As an option for culturing human PSCs without feeder cells, Matrigel™ has proven to be a useful alternative enabling the stable culture of human PSCs. Moreover, we have also shown that Matrigel™ allows the generation of hiPSCs without animal-derived feeder cells [19]. Since Matrigel™ was derived from Engelbreth-Holm-Swarm mouse sarcoma cells [20], other types of matrices which do not contain animal-derived agents have been tested and used as feedercell substitutes for the successful maintenance and generation of human PSCs; such as CellStart

The culture media used in the early generation of hESCs contained fetal bovine serum [1]. In order to remove unspecific agents that might cause the differentiation of hESCs, knockout serum replacement (KSR) has now been established as a defined material for maintaining hESCs [28] and is also traditionally used for hiPSC generation [9,12,29,30]. In this regard, mTeSR1 medium was developed as a chemically defined medium for maintaining human PSCs [31]. Importantly, in the last years several authors have reported the generation of commercially developed *xeno-*free media for maintaining hiPSCs, and such media have already been used successfully for iPSCs generation. These media include: TeSR2 [32],

When factors that sustain PSCs stemness are deprived from the media, PSCs spontaneously differentiate into derivatives of the three embryonic germ layers. This capacity has been

[21,22], recombinant proteins [23–25], and synthetic polymers [26,27].

NutriStem [33], Essential E8 [24], and StemFit [34].

sustain human PSCs culture and expansion in feeder-free substrates.

borders.

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**a. Embryoid Bodies' (EBs) formation**: In contrast to monolayer cultures, EBs are spherical structures that allow PSCs culture in suspension (Figure 3). The three-dimensional structure, including the establishment of complex cell-adhesions and paracrine signaling within the EB microenvironment, enables differentiation and morphogenesis. For that reason, the first protocols for muscle differentiation took advantage of EB induction from mESCs, followed by different periods of exposure to specific cell culture media in which serum, mitogenic factors, and essential substrates (such as amino-acids or glutamine) were formulated. In that manner, those first assays proved the feasibility of mESCs to give rise to myogenic cells, setting the bases for the definition of robust protocols for the differen‐ tiation of muscle cells from human PSCs. Up to day, most of the protocols for the gener‐ ation of myogenic cells from PSCs make use of the differentiation of EBs derived from either wild type or transgenic PSCs.

**Figure 3.** PSCs are capable to differentiate into cells belonging to the three somatic germ layers of the embryo. The generation of EBs from PSCs is a common method for producing different cell lineages for further purposes. A) EBs from mouse iPSCs grown in suspension. B) EBs derived from human iPSCs grown in suspension.

**b. Modification of medium composition**: Monolayers of PSCs and also EBs have been traditionally subjected to changes in nutrient composition, (i.e, reduction/increase of serum concentration, addition/removal of a growth factor or addition/removal of cyto‐ kines, among others) in order to induce their differentiation towards the desired cell type. These changes are conducted in order to promote changes on gene expression profiles and cell proliferation rates. In this manner, by means of relatively simple methods, PSCs are artificially guided towards the desired cell type. Although these methodologies have proven low efficiency yields for specific cell types (i.e., motorneurons, hepatic cells; among others), they are extremely valuable when combined together with PSCs in which the expression of master factors critical for differentiation are under the control of hormones (i.e., tamoxifen inducible reporters) or antibiotics (i.e., puromycin, or hygromycin, among others). The control of expression of the specific transcription factor of choice (i.e., MyoD1) together with the addition of specific molecules mimicking tissue development [i.e., insulin like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF)] has demon‐ strated good results when differentiating mouse or human PSCs towards myogenic cells.


and natural scaffolds have been designed to provide support for muscle growth and allow myofibroblasts to rebuild their native ECM. Natural scaffolds based on ECM proteins such as fibrin and collagens have been used to form hydrogels for musculoskeletal tissue engineering [54–56]. Commercially available ECM substitutes such as Matrigel™ hydro‐ gels are also showing promising results in the differentiation of PSCs towards cardio‐ myocytes [57]. However, current ECM protein-based scaffolds are limited by their immune rejection and scaling up technologies. Synthetic scaffolds, which can be fabricated with ideal architectures at the nanoscale, pore sizes and mechanical properties, represent an advantageous solution to mimic the 3D ECM microenvironment (Figure 4). Technol‐ ogies such as electrospinning, which allows organizing the polymers into thin sheets of fibrous meshes, are promising in this field [58,59]. Recently, it has been proved the reprogramming of mouse fibroblasts onto cardiomyocyte-like cells on polyethylene glycol (PEG) hydrogels functionalized with laminin and RGD peptides [60]. This opens new perspectives toward the use of custom-engineered synthetic scaffolds in the differentia‐ tion of PSCs to muscle cells. Finally, the use of acellularized tissue scaffolds is also being explored in muscle regeneration. They offer a native ECM with the optimal biochemical and mechanical properties for cell culture and preserve the architectural features of the tissue. Their use as a matrix supporting the commitment of cardiac muscle cells has been recently reported, thus showing the potential of this approach [61].

others), they are extremely valuable when combined together with PSCs in which the expression of master factors critical for differentiation are under the control of hormones (i.e., tamoxifen inducible reporters) or antibiotics (i.e., puromycin, or hygromycin, among others). The control of expression of the specific transcription factor of choice (i.e., MyoD1) together with the addition of specific molecules mimicking tissue development [i.e., insulin like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF)] has demon‐ strated good results when differentiating mouse or human PSCs towards myogenic cells.

**c. Genetic manipulation of PSCs**: Forced expression of transcription factors can direct differentiation of PSCs toward specific lineages. In the last years, the generation of platforms for transgene expression in PSCs has emerged as one of the most potent tools for PSCs differentiation. Whereas the first studies took advantage of exogenous gene expression systems (i.e., lentivirus or retrovirus), nowadays the use of integrative vectors are limited, since they incur uncertain risks for potential cell-based therapeutic applica‐ tions [35]. In this regard, the use of excisable vectors (i.e., transposons; [36,37], or mRNAs [17]) offer an unprecedented opportunity for the derivation of differentiated PSCs suitable

**d. Use of extracellular matrix (ECM) and signaling molecules**: Unlike *de novo* embryonic muscle formation, muscle regeneration in higher vertebrates depends on the injured tissue retaining of an ECM scaffolding that serves as a template for the formation of muscle fibers [38]. In this regard, the interaction between cells and ECM via integrins determines the expression of signaling molecules that affect PSCs differentiation [39]. Of note, when mouse iPSCs have been cultured in the presence of matrigel, myogenesis (this is, prolif‐ eration of myoblasts and further fusion into myotubes) has been positively induced [40]. Similar results have been observed when using collagen-based matrix for the differentia‐ tion of human iPSCs expressing a Dox-inducible expression cassette of MyoD1 [41]. For the organization and alignment of muscle fibers not only the composition of the ECM but also its anisotropic architecture are essential. To address this, a number of strategies have been developed to organize myotubes: topography-based approaches based on the use of nanofibers, [42], microabrated surfaces [43], and microcontact printing of ECM proteins such as fibronectin [44] and vitronectin [45]. In a complementary approach, biochemical cues have also been introduced to promote cell alignment and differentiation. By using inkjet bioprinting, spatially defined patterns of myogenic and osteogenic cells were derived from primary muscle-derived stem cells (MDSCs) as a response to BMP-2 patterning [46]. The combination of topographical and biochemical signaling has also been explored by coating sub-micron polystyrene fibers with either FGF-2 or BMP-2 to provide spatial control of cell fate and alignment in order to mimic native tissue organization [47]. The vast majority of these works present cells to static microenvironments. Latest trends point out the relevance of presenting cells to spatially and temporally dynamic microen‐ vironments [48]. Surfaces with gradient concentrations of growth factors (BMP-2 and BMP-7) have shown to successfully drive cell differentiation [49,50]. Although not yet a reality, these strategies appear a promising way to direct the differentiation of PSCs [51].

**e. Use of biomatrices mimicking skeletal muscle niche:** Tissue engineering approaches have been used to design synthetic and natural 3D scaffold materials to mimic the structural, biochemical, and mechanical properties of the stem cell niche [52,53]. Synthetic

for regenerative medicine.

338 Muscle Cell and Tissue

**Figure 4.** Bio-inert polyethylene glycol (PEG)-based hydrogels have been designed as the scaffold substrate for biomi‐ metic matrices supporting muscle migration in three dimensions. The picture shows PEGDA hydrogel (MW 550 KDa) cross-linked by UV light.

**f. Co-culture with supportive cells (feeder cells)**: The co-culture of mouse and human PSCs (either as monolayers or EBs) with feeder cells has been traditionally used in order to induce PSCs differentiation [39]. The stromal cell line OP9 [62,63], which is derived from newborn mouse calvaria, supports hematogenesis [64,65]. The preadipose cell line PA6 [66] promotes neural differentiation of mouse and human PSCs [10,11,67]. In this regard, Baghavati [68] showed that the co-culture of mESCs together with primary muscle cells suffice for myogenic differentiation, since donor-derived myofibers generated by coculturing mouse EBs on top of primary muscle fibers could be occasionally found on the surface of the host muscle.
