**2. Clinical trials with iPSC**

After over a decade of research on iPSC, and due to fast-track facilitating procedure in Japan, several clinical studies were launched. While the first clinical trial based on the human ESC started in 2010, taking advantage of the acquired extensive knowledge of ESC biology, despite their relatively recent discovery, the first clinical study based on the iPSC-derived retinal pigmented epithelium was authorized and conducted at the RIKEN Institute in Japan in 2014 [2]. A sheet of autologous iPSC-derived retinal cells were transplanted in a patient with eye-related macular degeneration (AMD). In 2015, the RIKEN Institute decided to suspend the study due to safety concerns on the cells of the second recruited patient [3]. Nonetheless, regarding the first transplanted patient, a 25-month follow-up revealed neither serious events, nor clinical signs of rejection. Moreover, the macular degeneration progress was delayed in the treated eye compared to the untreated eye. This result corroborated all the results obtained previously in the course of the ESC-based clinical studies, where no adverse events related to transplanted cells were observed. Still this problem induced a shift in the approach from patient-specific autologous to highly securized allogeneic iPSC lines. This study was resumed in 2017 and until now five patients with AMD have been treated with allogeneic iPSC-derived cells.

Since then, several clinical studies based on allogeneic iPSCs have been developed and approved. Until mid-2019, there have been nine ongoing clinical studies based on iPSC, mostly nationally approved in Japan, with four of them being approved in the first months of 2019, with indications including Parkinson's disease, AMD, severe cardiac failure, aplastic anemia, spinal cord injury and corneal stem cell deficiency. Furthermore, two private companies—Cynata Therapeutics, an Australian stem cell and regenerative medicine company, and Fate Therapeutics, an American clinical-stage biopharmaceutical company—have developed a line of products based on allogeneic human iPSC-derived cells. In Australia and United Kingdom, Cynata Therapeutics just concluded a phase I study using CYP-001, an iPSC-derived mesenchymoangioblast precursor administered intravenously in 15 patients with graft-versus-host disease (GVHD) occurring after an allogeneic hematopoietic stem cell transplant [4]. Currently, all patients treated so far have demonstrated at least a partial response, while no treatment-related serious adverse events or safety concerns have been observed. The product development activities of CYP-001 will be done in a phase II study in 2019 by Fujifilm in collaboration with Cynata Therapeutics. On its part, Fate Therapeutics received a first approval from Food and Drug Administration (FDA) in November 2018 to transplant an off-the-shelf iPSC-derived Natural Killer cell, FT-500, as cancer immunotherapy to treat solid tumors and for a second cell product derived from a genetically engineered iPSC, FT-516, in February 2019, for the treatment of relapsed/refractory hematologic malignancies. For the first product FT-500, all the three patients with advanced solid tumors have been treated with multiple doses of FT-500, 100 million cells per dose, and it has been well tolerated with no dose-limiting toxicities or adverse events [5].

Even though the first clinical studies have already been started, technical advances in iPSC biology have revealed that several factors could affect their safety for a larger range of medical applications, and should be taken into account for short- and long-term follow-up of patients. Two of the major concerns related to iPSC-based products are their potential tumorigenicity and immunogenicity. The scientific community is still continuing to elucidate the biological mechanisms underlying iPSC's immunogenicity and tumorigenicity and how to manage or overcome them.

**17**

*Induced Pluripotent Stem Cells for Clinical Use DOI: http://dx.doi.org/10.5772/intechopen.88878*

unstable during the *in vitro* production steps.

The potential risk of tumorigenicity to patients from both teratomas and malignant tumors could arise if transplanted cells are contaminated with undifferentiated iPSC, or if transplanted cells have been genetically modified and become

The major concern related to iPSC-based tumorigenicity is the reprogramming method. In the original cocktail of transcription factors developed by Yamanaka, somatic cells are transduced by retroviral vectors that become integrated into the genome of the host cells. Two of these factors—*c-Myc* and *klf4*—are potent oncogenes [6]. Subsequently, reports of tumorigenicity after transplantation of iPSC or iPSC-derived cells are not surprising. Thereby, teratoma formation could be induced by the undesired activation/suppression of essential host genes proximal to integration sites or by residual expression of reprogramming factors in the derived cells in animal model [7, 8]. With hindsight, there is evidence for the necessity to select a non-integrative method for reprogramming, a higher rate of genomic alterations occurring when human iPSCs are generated with viral vectors, compared to mRNA [7, 9]. Numerous studies, focused on the choice of reprogramming factors and methods of delivery, have developed various novel strategies to enhance the efficiency of reprogramming and reduce the potential risk of tumorigenicity. To circumvent this risk, human iPSCs have been generated by several "integration-free" methods, based on the use of viral vectors (adenoviral vectors and Sendai virus-based vectors) or non-viral vectors (piggyBac system, minicircle vector, and episomal vectors). Originally, the four transcription factors needed for complete cell reprograming were *c-myc*, *klf4*, *oct4* and *sox2* [1]. The protumorigenic transcription factor *c-myc* has been found to be unnecessary for the reprogramming process, but the overall efficiency is decreased without it. Several strategies have been developed with the use of different transcription factors and/ or replacement of *c-myc*, or the use of direct protein delivery and synthesized

Furthermore, the tumorigenicity risk is often linked to the genetic instability of iPSC. Random genomic alterations are frequently observed in human iPSCs showing their intrinsic instability, essentially due to the massive genome remodeling, and probably also resulting from various mechanisms such as replicative stress, reactivation of the telomerase and metabolism modification from the oxidative to the glycolytic state. Epigenetic modifications may also contribute to iPSC variation due to residual epigenetic memories of the starting cell type [13]. The incomplete resetting of the non-CpG methylation patterns during reprogramming leads to a biased differential potential in certain cell types depending on the donor cell source [14, 15]. However, it has been shown that their residual epigenetic memory diminishes with the *in vitro* expansion over a period of time [16, 17]. As just mentioned, the selection of the donor cell type is of importance. Many human somatic cell types have been successfully reprogrammed. However, even if the use of different transcription factors, delivery methods and culture conditions does not facilitate any comparison, it is well known that reprogramming efficiencies, kinetics and tumorigenicity vary between somatic cell types. Firstly, cell sources have to be permissive to avoid to turn to integrative methods and to the use of oncogenes. Some human, adult somatic cells, such as melanocytes, are known to naturally express endogenously reprogramming factors, for instance Sox 2, at sufficiently high levels [18, 19]. Moreover, some types of donor cells such as dermal fibroblasts and blood cells are easily accessible, but they might carry more mutational burdens

**3. iPSC safety**

mRNA [10–12].

**3.1 Tumorigenicity**
