**2.3 iPSC disease modeling**

These groundbreaking discoveries have revolutionized our understanding of stem cell development and created novel opportunities for human disease modeling and drug screening in "disease-in-a-dish" models (**Figure 1**) [15]. To date, significant progress has been made utilizing human iPSCs to model various neurological disorders, inherited heart diseases, and other genetic diseases such as Duchene muscular dystrophy and recessive dystrophic epidermolysis bullosa (RDEB) [16, 17]. Utilizing amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease as an example, ALS is a neurodegenerative disease that primarily affects corticospinal "upper" motor neurons (UMNs) and spinal cord "lower" motor neurons (LMNs), resulting in progressive muscle weakness [18]. In about 10% of patients with ALS, the disease runs in the family (familiar ALS) with mutations in around 20 genes including SOD1, TARDBP, FIS, and C9orf72 identified as common causes [18]. The remaining 90% of the patients are classified as sporadic ALS, with the causative mutations largely unidentified. As the iPSCs generated from ALS patients and differentiated into motor neurons carry the same genetic background as the patients, it represents a novel tool for studying disease pathology of ALS, particularly the sporadic form, which is not possible in the other model systems. A proof-of-principle study on derivation of iPSCs from an ALS patient and differentiation into LMNs was reported in 2008 [19]. Subsequently, Kiskinis et al. and Chen et al., respectively, established in vitro models of ALS by generating iPSC-derived LMNs from patients carrying different SOD1 mutations. Both studies recapitulated the spontaneous and progressive decrease in cell viability and ALS-related morphological changes including reduction in soma size and altered dendrites, which was linked to neurofilament aggregation [20, 21]. Chen et al. further demonstrated the pathological features of mutated SOD1 in patient-derived MNs, but not in non-MNs. Only in MNs, mutated SOD1 bound to the 3′UTR region of neurofilament (NF)-L mRNA resulted in neurofilament aggregation, restoring the expression of NF-L mitigated neurite degeneration of the ALS-iPSC-derived MNs. Meanwhile, Wainger et al. generated iPSC-derived LMNs from patients carrying SOD1, C9orf72, or FUS mutations [22]. All these ALS-iPSC-derived LMNs with distinct genetic mutations have recapitulated essential disease features and discovered common molecular pathways driving ALS pathogenesis, opening the possibility of new and effective drug screening [23]. However, challenges still remain for in vitro modeling for ALS using iPSCs [18]. Different protocols have been reported in deriving LMNs from iPSCs; thus, criteria need to be established to compare the MNs generated using different methods. Moreover, generation of UMNs from PSCs involves a series of steps and

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*Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine*

is more challenging than that of LMNs [18]. The current protocols for deriving UMNs mostly resulted in heterogeneous, neocortical-like neurons that are immature and "stalled" at a stage resembling mid-embryonic differentiation in vivo [24]. Therefore, promoting subtype-specific differentiation and maturation will be crucial to an accurate ALS modeling. Indeed, the abilities of iPSC-derived cells to exhibit maturation and aging are crucial for accurate in vitro modeling of all the

The most significant advantage of iPSCs lies in its application in cell-based therapies. iPSCs can be developed without destroying human embryos, therefore circumventing the ethical obstacles of utilizing and generating human ES cells. Being able to differentiate into all cell types in the body similar to ES cells, iPSCs theoretically provide an unlimited source of cells for autologous transplantation, eliminating the need for immunosuppression. Moreover, scientists have established robust directed differentiation protocols with sequential activation and inhibition of molecular differentiation pathways to generate a wide range of somatic cells from

A challenge for the PSC or iPSC differentiation, as also mentioned above in the iPSC-derived MNs, is that the PSC-derived cells tend to be immature. This is indeed the major limitation for translating iPSC-derived red blood cells into the clinic [25]. In 2008, Lu et al. reported differentiation of human ES cells into functional oxygencarrying erythrocytes on a large scale with up to 60% enucleation rate [26]. In comparison, differentiation of iPSCs along the erythroid lineage generated orthochromatic (nucleated) erythroblasts and reticulocytes. In most reports, the differentiated red blood cells express mainly fetal and embryonic globins, but very little adult-type (β-) globin [27]. This is likely due to the low level of erythroid Kruppellike factor 1 (EKLF1) and absence of BCL11A in these iPSC-derived red blood cells. These two factors have been demonstrated to be essential for the developmental switch from fetal to adult globin expression [28]. Inducible expression of KLF1 during later stages of the differentiation process has been recently demonstrated to enhance differentiation and maturation of red blood cells from both human ES cells

Recent advances in the development of programmable site-specific nucleases, including zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas)9 system, have enabled target-specific introduction of transgene or correction of disease-specific mutations by homologous recombination, creating novel opportunities not only for disease modeling and drug testing but also generation of genetically corrected cells for autologous

The development of iPSC technology has also revolutionized the future treatment for end-stage organ failure. Takebe et al. recently reported vascularized and functional mini-livers or liver buds created in vitro based on human iPSCs [30]. In this proof-of-concept demonstration, the authors first prepared hepatic endoderm cells from human iPSCs by directed differentiation. About 80% of the differentiated cells express liver-specific marker HNF4A. To recapitulate early organogenesis, the investigators next cultured the iPSC-derived hepatic endoderm cells with two stromal cell populations, i.e., human umbilical vein endothelial cells and human mesenchymal stem cells (MSCs), in a traditional two-dimensional culture condition. Intriguingly, the iPSC-derived hepatic cells

*DOI: http://dx.doi.org/10.5772/intechopen.88790*

iPSCs, such as β cells and cardiomyocytes (**Figure 1**).

adult-onset diseases.

and iPSCs [29].

transplantation (**Figure 2**).

**2.4 iPSC-based therapies**

*Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.88790*

is more challenging than that of LMNs [18]. The current protocols for deriving UMNs mostly resulted in heterogeneous, neocortical-like neurons that are immature and "stalled" at a stage resembling mid-embryonic differentiation in vivo [24]. Therefore, promoting subtype-specific differentiation and maturation will be crucial to an accurate ALS modeling. Indeed, the abilities of iPSC-derived cells to exhibit maturation and aging are crucial for accurate in vitro modeling of all the adult-onset diseases.
