**1. Introduction**

Regenerative Medicine aims to restore the loss of function in tissues and organs due to any cause (trauma, stress, aging, or disease) by the replacement of dysfunctional structures with competent cells, tissues, or organs. In order to achieve this goal Regenerative Medicine takes advantage of different forefront methodologies, such the use of stem cells, gene therapy, and tissue engineering among others.

#### **1.1. Human embryonic stem cells (hESCs)**

The isolation and derivation of hESCs by Thompson and colleagues in 1998 attracted signifi‐ cant attention in the Regenerative Medicine field [1]. Indeed, regenerative cell transplantation therapies have been expected to treat incurable diseases, such as spinal cord injury [2], neurodegenerative disease [3], heart failure [4,5], diabetes [6], and retinal disease [7].

Nowadays, clinical application of hESCs still shows many concerns regarding the use of human embryos, tissue rejection after transplantation, and tumour formation. However, hESCs possess the dual ability to proliferate indefinitely without phenotypic alterations, and more importantly, to differentiate, theoretically, into all cell types in the human body. These qualities suggest extensive utility of hESCs in applications varying from the definition of differentiation protocols, to the generation of drug screening platforms for disease treatment. Thus, hESCs represent an ideal source for understanding skeletal muscle development and disease, such skeletal muscle.

#### **1.2. Induced pluripotent stem cells (iPSCs)**

In 2006 Professor Shinya Yamanaka and colleagues [8] showed for the very first time, that by introducing different transcription factors the epigenetic status of somatic cells could be reverted to pluripotency. In particular, the Japanese team ectopically induced the expression of specific transcription factors related with embryonic stem cells (ESCs) biology, generating in a period of only 30 days, cells that were identical to mouse ESCs (mESCs) in terms of selfrenewal capacity, expression of endogenous pluripotency-related factors, and *in vivo* and *in vitro* differentiation potential to give rise to cells belonging to the three germ layers of the embryo (ectoderm, mesoderm, endoderm). This discovery was awarded with the Nobel Price of Medicine in 2012 to Professor Shinya Yamanaka.

While, at first, somatic reprogramming was described using mouse embryonic fibroblasts, the Japanese team could show that also a reduced formula of the original "Yamanaka cocktail" could be used to reprogram human somatic cells towards human iPSCs (hiPSCs) [9]. Since 2007 different research groups, including us, have shown that iPSC technology can be applied to reprogram a huge variety of human somatic cells, independently of their embryonic origin [10–13]. Interestingly, during the last years the generation of protocols avoiding the use of lentiviral or retroviral vectors for the expression of Yamanaka factors has involved the definition of novel strategies for hiPSCs generation, including the use of recombinant proteins [14,15], episomal vectors [16], or mRNAs [17,18], among others [13]. Thus, the generation of hiPSCs, especially the generation of patient-derived iPSCs suitable for disease modelling *in vitro*, opens the door for the potential translation of patient-derived iPSCs into the clinic. Successful replacement or augmentation of the function of damaged cells by patient-derived differentiated stem cells would provide a novel cell-based therapy for skeletal muscle-related diseases.

**1. Introduction**

334 Muscle Cell and Tissue

tissue engineering among others.

disease, such skeletal muscle.

**1.2. Induced pluripotent stem cells (iPSCs)**

of Medicine in 2012 to Professor Shinya Yamanaka.

**1.1. Human embryonic stem cells (hESCs)**

Regenerative Medicine aims to restore the loss of function in tissues and organs due to any cause (trauma, stress, aging, or disease) by the replacement of dysfunctional structures with competent cells, tissues, or organs. In order to achieve this goal Regenerative Medicine takes advantage of different forefront methodologies, such the use of stem cells, gene therapy, and

The isolation and derivation of hESCs by Thompson and colleagues in 1998 attracted signifi‐ cant attention in the Regenerative Medicine field [1]. Indeed, regenerative cell transplantation therapies have been expected to treat incurable diseases, such as spinal cord injury [2],

Nowadays, clinical application of hESCs still shows many concerns regarding the use of human embryos, tissue rejection after transplantation, and tumour formation. However, hESCs possess the dual ability to proliferate indefinitely without phenotypic alterations, and more importantly, to differentiate, theoretically, into all cell types in the human body. These qualities suggest extensive utility of hESCs in applications varying from the definition of differentiation protocols, to the generation of drug screening platforms for disease treatment. Thus, hESCs represent an ideal source for understanding skeletal muscle development and

In 2006 Professor Shinya Yamanaka and colleagues [8] showed for the very first time, that by introducing different transcription factors the epigenetic status of somatic cells could be reverted to pluripotency. In particular, the Japanese team ectopically induced the expression of specific transcription factors related with embryonic stem cells (ESCs) biology, generating in a period of only 30 days, cells that were identical to mouse ESCs (mESCs) in terms of selfrenewal capacity, expression of endogenous pluripotency-related factors, and *in vivo* and *in vitro* differentiation potential to give rise to cells belonging to the three germ layers of the embryo (ectoderm, mesoderm, endoderm). This discovery was awarded with the Nobel Price

While, at first, somatic reprogramming was described using mouse embryonic fibroblasts, the Japanese team could show that also a reduced formula of the original "Yamanaka cocktail" could be used to reprogram human somatic cells towards human iPSCs (hiPSCs) [9]. Since 2007 different research groups, including us, have shown that iPSC technology can be applied to reprogram a huge variety of human somatic cells, independently of their embryonic origin [10–13]. Interestingly, during the last years the generation of protocols avoiding the use of lentiviral or retroviral vectors for the expression of Yamanaka factors has involved the definition of novel strategies for hiPSCs generation, including the use of recombinant proteins [14,15], episomal vectors [16], or mRNAs [17,18], among others [13]. Thus, the generation of

neurodegenerative disease [3], heart failure [4,5], diabetes [6], and retinal disease [7].

Satellite cells (SCs), the adult stem cell pool in skeletal muscle, are often compromised in patients with muscle dystrophies (MDs). Over the last decades the understanding of the transcription factors and intrinsic and extrinsic signals that govern SCs or terminally differ‐ entiated myogenic cells have represented a good starting point for the definition of protocols for the generation of myogenic cells from PSCs (both from mouse and human ESCs and iPSCs). In the same manner, the generation of patient-derived cell platforms can help us to develop experimental strategies toward generating muscle stem cells, either by differentiating patientspecific iPSCs or by converting patient's somatic cells towards myogenic cells (transdifferen‐ tiation). Overall, the possibility to generate disease-free patient iPSCs can help us to identify which are the mechanisms driving muscle disease, and more importantly, to develop new compounds for treating MDs (Figure 1).

**Figure 1.** Patient iPSCs represent an unprecedented tool for the generation of i*n vitro* platforms for disease modelling and the definition of protocols for PSCs differentiation. The correction of the genetic defect(s) leading to disease may help to understand the molecular and cellular mechanisms driving disease gestation and progression, and more im‐ portantly, to identify novel mechanisms leading to muscle regeneration.
