**2. Generation of hiPSC**

#### **2.1. Protocols for generating hiPSCs**

Dr. Yamanaka first reported the generation of hiPSCs from fibroblasts using four transcriptional factors (POU5F1, SOX2, KLF4, and MYC) [6]. There are many protocols to further improve the original method. The first improvement was to minimizing the integration risks such as using non-integrating adenoviral vectors, transfection of mRNA, and using cell-penetrating peptide-tagged reprogramming factors [7]. Transgene-free hiPSC generation protocols have been published by multiple groups [8]. Using small molecules such as valproic acid, sodium butyrate, PD0325901, and others to create iPSCs has been reported [9–11]. Haase et al. reported a new non-transgenic protocol to generate hiPSCs from patient cord blood CD34+ cells using CytoTune™ Sendai reprogramming vectors under the exclusive usage of animalderived component-free (ADCF) materials and components [12]. Recently, non-integrative and non-viral mRNA reprogramming technology has been reported for hiPSC generation [13]. Rapid, efficient, and safe strategies which are compliant with standard Good Manufacturing Practice (GMP) regulations pave the way for hiPSC clinical applications.

#### **2.2. Genome editing of hiPSCs**

Genome editing in hiPSCs provides a valuable tool for disease modeling, mechanism study, and gene therapy. A line of technology utilizing engineered nucleases consisting of sequencespecific DNA-binding domains attached to a non-specific DNA nuclease have been developed. These cutting-edge technologies allow researchers to manipulate entire genomes, including specific genes, intergenic regions, promoters, enhancers, silencers, and insulators. After zinc finger nucleases (ZFNs, first-generation) and transcription activator-like effector nucleases (TALENs, second-generation), the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) technology is the third-generation editing tool. Despite the difference in the nucleases, the common mechanisms involve inducing DNA double-strand breaks (DSBs) in targeted DNA. Compared to TALEN and ZFN, CRISPR/Cas9 has become the system of choice because of its features such as high feasibility, high affordability, and precise targeting.

(the brain and spinal cord), the peripheral nervous system, the sensory epithelia of the eye, ear, and nose, the epidermis and its appendages (the nails and hair), the mammary glands,

**Organoids Applications Refs.**

Brain organoids Modeling autism disorder [14]

Brain-region specific organoids Modeling Zika virus infection and human brain development disease [19] Breast organoid Breast cancer research [20]

Fallopian tube organoids Ovarian cancer research [22] Liver bud Organ-bud transplantation for regenerative medicine [23] Lung organoids Lung development and lung disease modeling [22, 25] Pancreas Pancreatic disease model [26] Retinal organoids Modeling glaucoma [27]

Cystic organoids Modeling Alagille syndrome, polycyctic liver disease and cystic

fibrosis

**Table 1.** Summary of hiPSC- and ESC-derived organoids, adapted from Shi et al. [1].

**Figure 1.** Summary of the organs originated from ectoderm.

Modeling ALS disease [15] Modeling Parkinson's disease [16] Modeling Zika virus infection [17] Modeling Seckel syndrome [18]

[21]

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hiPSC-Based Tissue Organoid Regeneration http://dx.doi.org/10.5772/intechopen.76997

iPSC derived organoid model

the hypophysis, the subcutaneous glands, and the enamel of the teeth (**Figure 1**).
