**3. Strategies to induce** *in vitro* **differentiation of mouse and human PSCs towards myogenic cells**

#### **3.1. Generation of myogenic precursors and/or terminally differentiated multinucleated myogenic cells by exogenous expression of transcription factors in PSCs**

During the last 30 years successful generation of myogenic precursors from mouse and human PSCs has been achieved by exogenous expression of transcription factors crucial for myogenic differentiation. PSCs are especially amenable for genome editing because they can undergo extensive tissue culture manipulations, such as drug selection and clonal expansion, while still maintaining their pluripotency signature and genome stability. In his regard, different authors have explored the possibility to generate PSCs stable cell lines that express the myogenic transcription factor of interest under the control of specific drugs (i.e., antibiotics). The myogenic transcription factor of interest is normally subcloned into a viral vector, which possesses high infection efficiency when transducing PSCs. Other methods involve the use of non-integrative vectors such as transposons or excisable lentiviral vectors. Following these strategies different authors have shown that PSCs monolayers or PSCs-derived EBs can be converted into myogenic cells (see below).

#### *3.1.1. Early studies of myogenic differentiation from mESCs by exogenous expression of transcription factors*

Already in 1992 Dekel and colleagues showed that, when mESCs were electroporated with MyoD1 cDNA driven by the Βeta-actin promoter, some cells could be converted to skeletal muscle cells [69]. Moreover, authors showed that contracting skeletal muscle fibers could be generated when the transfected cells were allowed to differentiate *in vitro*, via EBs, in lowmitogen-containing medium. Although those studies failed to develop efficient protocols for the generation of high yields of myogenic cells, they helped to understand that environmental factors should control MyoD expression and its myogenic differentiation function, and more importantly, that MyoD was required for the establishment of the myogenic program but not for its maintenance.

Thus, those first observations served as a starting point for the definition of enriched cell culture media for mESCs differentiation towards myogenic cells, and more importantly, for the generation of fine-tuned systems in order to control the expression of the desired myogenic factor at a precise moment during the onset of differentiation. Alongside this line, Ozasa and colleagues [70] established a mESC line by introducing a MyoD transgene controlled by a Tet-Off system (ZHTc6-MyoD). The possibility to induce MyoD expression during the time course of differentiation, allowed mESCs to differentiate almost exclusively into the myogenic lineage in the absence of doxycycline, and without pre-differentiation into EBs. To start the differen‐ tiation process, Ozasa and colleagues removed doxycycline and used a differentiation medium containing 4% fetal bovine serum (FBS). Under those conditions and only after 7 days, primed cells started to fuse into myotubes, and occasionally light muscle contractions were observed. In that study the potential of the generated cells to differentiate into myofibers *in vivo* was also investigated by intramuscular injections into *mdx* mice and clusters of dystrophin-positive myofibers were detected in the injected area.

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

**3. Strategies to induce** *in vitro* **differentiation of mouse and human PSCs**

**3.1. Generation of myogenic precursors and/or terminally differentiated multinucleated**

During the last 30 years successful generation of myogenic precursors from mouse and human PSCs has been achieved by exogenous expression of transcription factors crucial for myogenic differentiation. PSCs are especially amenable for genome editing because they can undergo extensive tissue culture manipulations, such as drug selection and clonal expansion, while still maintaining their pluripotency signature and genome stability. In his regard, different authors have explored the possibility to generate PSCs stable cell lines that express the myogenic transcription factor of interest under the control of specific drugs (i.e., antibiotics). The myogenic transcription factor of interest is normally subcloned into a viral vector, which possesses high infection efficiency when transducing PSCs. Other methods involve the use of non-integrative vectors such as transposons or excisable lentiviral vectors. Following these strategies different authors have shown that PSCs monolayers or PSCs-derived EBs can be

*3.1.1. Early studies of myogenic differentiation from mESCs by exogenous expression of transcription*

Already in 1992 Dekel and colleagues showed that, when mESCs were electroporated with MyoD1 cDNA driven by the Βeta-actin promoter, some cells could be converted to skeletal muscle cells [69]. Moreover, authors showed that contracting skeletal muscle fibers could be generated when the transfected cells were allowed to differentiate *in vitro*, via EBs, in lowmitogen-containing medium. Although those studies failed to develop efficient protocols for the generation of high yields of myogenic cells, they helped to understand that environmental factors should control MyoD expression and its myogenic differentiation function, and more importantly, that MyoD was required for the establishment of the myogenic program but not

Thus, those first observations served as a starting point for the definition of enriched cell culture media for mESCs differentiation towards myogenic cells, and more importantly, for the generation of fine-tuned systems in order to control the expression of the desired myogenic factor at a precise moment during the onset of differentiation. Alongside this line, Ozasa and

**myogenic cells by exogenous expression of transcription factors in PSCs**

surface of the host muscle.

converted into myogenic cells (see below).

*factors*

for its maintenance.

**towards myogenic cells**

340 Muscle Cell and Tissue

#### *3.1.2. Myogenic differentiation from human PSCs by exogenous expression of transcription factors*

In the same manner, within the last years several studies have demonstrated the possibility to generate myocytes, and even multinuclear myotubes from both hESCs and patient hiPSCs by means of different systems in which the expression of MyoD is driven under the control of soluble factors during the time course of differentiation. In this regard, early in 2012 two different reports indicated that mesodermal [71] or mesenchymal cells [72] could be generated from iPSCs, demonstrating a high potential for myogenic differentiation in response to *MyoD* over-expression.

Also Rao and colleagues (2012) generated a transgenic Tet-inducible MyoD cassette in which all the transgenic elements were inserted in hESCs making use of lentiviral vectors. In that particular study, authors were able to generate multinucleated myotubes with 90% of effi‐ ciency in a period that lasted only 10 days. Later on, Yasuno and colleagues [37] improved a previous protocol [36] for the generation of terminal multinucleated cells from iPSCs derived from patients affected with Carnitine palmitoyltransferase II (CPT II). Their protocol consisted in the transduction of a self-contained Tet-inducible MyoD1 expressing *piggyBac* vector (Tet-MyoD1 vector) and transposase into hiPSCs by lipofection. This system allowed the indirect monitoring of induced *MyoD* expression in response to doxycycline by co-expression of a red fluorescent protein (mCherry). Moreover, in that particular setting authors increased the purity of the generated myocytes by culturing the cells in low glucose conditions, a condition that was also reported to increase differentiated cardiomyocytes out of undifferentiated iPSCs based on the substantial biochemical differences in glucose and lactate metabolism between differentiated cells and undifferentiated iPSCs [73].

Very recently, Abujarour and colleagues [41] found that it is possible to derive myotubes from control iPSC and iPSC lines from patients with either Duchenne or Becker muscular dystro‐ phies. In particular, by using a lentiviral system expressing MyoD under the control of a Tetinducible promoter, and under-optimized culture conditions, the authors achieved an efficient myogenic differentiation setting the bases for the production of scalable sources of normal and dystrophic myoblasts for further use in disease modelling and drug discovery.

MyoD1 has not been the sole transcription factor of choice when differentiating human PSCs towards myogenic cells. Iacovino and colleagues [74] generated an unprecedented system in which it was possible to integrate the gene of interest into the desired cells (mESCs, kidney murine cells and hESCs) by means of a system that authors called inducible cassette exchange (ICE). In that particular setting, authors were able to integrate one single copy of Myf5 into mESCs and hESCs. Overall, Iacovino and colleagues showed that Myf5 expression is sufficient to promote the myogenic commitment of nascent mesoderm thereby establishing a novel and rapid method of differentiating mESCs and hESCs into skeletal muscle tissue.

Taking advantage of Iacovino´s system [74], Darabi and colleagues generated an improved version of ICE system in order to generate mESCs in which Pax7 expression was controlled under the control of doxycycline, and they succeeded in inducing the myogenic program in mouse cultures [75,76]. Later on, the same authors generated inducible Pax7 hESCs and hiPSCs with a doxycycline-inducible lentiviral vector encoding Pax7 (iPax7 and the expression of the Pax 7 transgene was detected by incorporating an IRES-GFP reporter downstream of the Pax7 gene. Next, iPax7 hESCs and hiPSCs were induced to generate EBs and after three days doxycycline was added into the media in order to induce Pax7 expression. Following 4 days of induction, Pax7+GFP+ cells were purified by FACS and expanded in a secondary monolayer culture in a medium containing doxycycline and bFGF. Under those conditions iPax7 hESCs and hiPSCs expressed markers of early muscle differentiation (Pax7 and Pax3), and terminally differentiated when iPax7 hESCs and iPSCs were subjected to differentiation-inducing conditions (culture media with 5% horse serum and withdrawal of doxycycline and bFGF). Finally, Darabi and colleagues demonstrated that transplantation of Pax7-derived myogenic progenitors into dystrophin-deficient mice (mdx) promotes extensive and long-term muscle regeneration accompanied by functional improvement [77].

#### **3.2. Generation of myogenic precursors and/or terminally differentiated multinucleated myogenic cells by soluble factors**

Although in the last years different authors have shown the possibility to generate myogenic cells from human PSCs by means of the ectopic expression of specific transcription factors, these methods do not reflect normal development, and most importantly, are not suitable for therapeutic purposes or *in vitro* disease modelling. For this reason, in the last years different groups have investigated the possibility to expose EBs or monolayers of mouse and human PSCs to different culture media mimicking muscle development. In order to monitor and control the myogenic signature of the produced cells, authors have isolated the different potential populations based on the acquisition of surface markers related to myogenic fate (i.e., paraxial mesoderm) by means of FACS. In the same manner, the majority of these studies have relayed in the analysis of the expression of myogenic-related markers by common techniques such as the expression of myogenic-related factors by polymerase chain reaction or immuno‐ histochemistry. In that way, the different protocols evaluate the efficiency of their method quantifying the percentage of cells that are differentiated towards myogenic cells.

#### *3.2.1. Early studies in myogenic differentiation from mESCs by soluble factors*

Although EBs exposed to undefined differentiation cell culture media spontaneously develop skeletal muscle cells and other cells *in vitro*, transplantation of EBs without any induction to direct development along a specific pathway leads to a failure of integration into recipient tissues and often forms teratomas in transplanted tissues. Thus, the definition of the specific conditions able to instruct PSCs towards myogenic cells requires establishment of robust conditions able to guide cells through the different stages of muscle differentiation.

In the first moment authors thought that the co-culture of EBs on top of freshly isolated muscle cells could serve as a novel method for myogenic differentiation. Although authors showed that differentiated cells generated by this method developed vascularized and muscle tissue when transplanted in dystrophic mice (mdx mice), still the number of engrafted cells was too low for potential applications in a clinical setting [68]. Later, Zheng and colleagues [78] showed that human EBs (hEBs) from two different hESCs lines cultured in the presence of differentia‐ tion media with different percentages of animal serum with or without Epidermal growth factor and 5-azacytidine could give rise to myogenic precursors. Interestingly, in that same work authors demonstrated that when those hESC-derived myogenic precursors were transplanted in NOD-SCID mice they could incorporate into the host muscle efficiently and become part of regenerating muscle fibers; giving rise to myocytes, myotubes, and myofibers, as well as satellite cells.

#### *3.2.2. Generation of myogenic cells from human PSCs by soluble factors*

murine cells and hESCs) by means of a system that authors called inducible cassette exchange (ICE). In that particular setting, authors were able to integrate one single copy of Myf5 into mESCs and hESCs. Overall, Iacovino and colleagues showed that Myf5 expression is sufficient to promote the myogenic commitment of nascent mesoderm thereby establishing a novel and

Taking advantage of Iacovino´s system [74], Darabi and colleagues generated an improved version of ICE system in order to generate mESCs in which Pax7 expression was controlled under the control of doxycycline, and they succeeded in inducing the myogenic program in mouse cultures [75,76]. Later on, the same authors generated inducible Pax7 hESCs and hiPSCs with a doxycycline-inducible lentiviral vector encoding Pax7 (iPax7 and the expression of the Pax 7 transgene was detected by incorporating an IRES-GFP reporter downstream of the Pax7 gene. Next, iPax7 hESCs and hiPSCs were induced to generate EBs and after three days doxycycline was added into the media in order to induce Pax7 expression. Following 4 days of induction, Pax7+GFP+ cells were purified by FACS and expanded in a secondary monolayer culture in a medium containing doxycycline and bFGF. Under those conditions iPax7 hESCs and hiPSCs expressed markers of early muscle differentiation (Pax7 and Pax3), and terminally differentiated when iPax7 hESCs and iPSCs were subjected to differentiation-inducing conditions (culture media with 5% horse serum and withdrawal of doxycycline and bFGF). Finally, Darabi and colleagues demonstrated that transplantation of Pax7-derived myogenic progenitors into dystrophin-deficient mice (mdx) promotes extensive and long-term muscle

**3.2. Generation of myogenic precursors and/or terminally differentiated multinucleated**

Although in the last years different authors have shown the possibility to generate myogenic cells from human PSCs by means of the ectopic expression of specific transcription factors, these methods do not reflect normal development, and most importantly, are not suitable for therapeutic purposes or *in vitro* disease modelling. For this reason, in the last years different groups have investigated the possibility to expose EBs or monolayers of mouse and human PSCs to different culture media mimicking muscle development. In order to monitor and control the myogenic signature of the produced cells, authors have isolated the different potential populations based on the acquisition of surface markers related to myogenic fate (i.e., paraxial mesoderm) by means of FACS. In the same manner, the majority of these studies have relayed in the analysis of the expression of myogenic-related markers by common techniques such as the expression of myogenic-related factors by polymerase chain reaction or immuno‐ histochemistry. In that way, the different protocols evaluate the efficiency of their method

quantifying the percentage of cells that are differentiated towards myogenic cells.

Although EBs exposed to undefined differentiation cell culture media spontaneously develop skeletal muscle cells and other cells *in vitro*, transplantation of EBs without any induction to direct development along a specific pathway leads to a failure of integration into recipient

*3.2.1. Early studies in myogenic differentiation from mESCs by soluble factors*

rapid method of differentiating mESCs and hESCs into skeletal muscle tissue.

regeneration accompanied by functional improvement [77].

**myogenic cells by soluble factors**

342 Muscle Cell and Tissue

In the quest for protocols suitable for regenerative purposes, Barberi and colleagues [79,80] developed simple feeder-free-monolayer culture systems in order to generate mesenchymal precursors that could be further differentiated towards myogenic cells from hESCs. In those studies multipotent mesenchymal precursors (MMPs) were purified for the acquisition of CD73 surface marker using FACS technology. First, MMPs were maintained in inactivated foetal serum and in the presence of the mouse skeletal myoblast line C2C12 [79]. Later, Barberi and colleagues could avoid the use of C2C12 cells by using serum-free N2 medium. Moreover, in that work authors further purified skeletal muscle myoblasts by means of a second FACS analysis for the neural cell adhesion molecule (NCAM), a marker of embryonic skeletal muscle. Those changes allowed for the expansion of hESC-derived myoblasts in a serum-free N2 medium in the presence of insulin [80].

Following a similar strategy Sakurai and colleagues [81] differentiated a murine ESC line towards paraxial mesodermal progenitors. Specifically, authors selected paraxial mesodermal progenitors based on the expression of platelet-derived growth factor receptor α (PDGFR-α) and the absence of Flk-1–a lateral mesodermal marker. Later on, the same authors demon‐ strated that mESCs could be directed toward the paraxial mesodermal lineage by a combina‐ tion of bone morphogenetic protein (BMP) and Wnt signaling under chemically-defined conditions [82]. Interestingly, the same group developed a protocol for the generation of paraxial mesoderm progenitors from both miPSCs and hiPSCs. Although some differences in growth factor requirement between mESCs and miPSCs cells were observed, the PDGFR-α+ population derived from miPSCs was almost identical to that of mESCs. Importantly, the work of Sakurai and colleagues showed that, under their specific conditions, two different lines of hiPSCs could be differentiated towards PDGFR-α+/KDR- cells. Those progenitors could be further differentiated into osteocytes, chondrocytes, and skeletal muscle cells, demonstrating the suitability of their procedures for the generation of myogenic cells for regenerative purposes.

Notably, other authors have shown the possibility to generate PDGFR-α+ from hESCs [83]. However, those same authors showed few engraftments of transplanted hESCs-derived myogenic cells into injured skeletal muscle. Interestingly, the same authors have recently demonstrated that, by incorporating Wnt3a in culture medium, myogenic commitment is rapidly achieved from hESCs, and more significantly, that those cells can contribute to finally regenerate cardiotoxin-injured skeletal muscle of NOD/SCID mice [84]. In the same line, other authors have demonstrated that the inhibition of GSK3B and treatment with FGF2 could specifically promote skeletal muscle differentiation. In particular, Xu and colleagues [85] have demonstrated that simultaneous inhibition of GSK3B, activation of adenyl cyclase and stimulation with FGF2 during EBs formation could promote the generation of myogenic precursors that terminally differentiate *in vitro* and act as satellite cells upon transplantation. Also, Borchin and colleagues [86] have shown that human PSCs can be differentiated towards Pax3/Pax7 double positive cells after GSK3B and FGF2 exposure.
