Lately, by utilizing the whole-exome sequencing approach, our laboratory has identified a novel *DES* mutation in a patient with left ventricular dilation and impaired left ventricular ejection function [49]. In this *DES* mutation, we recognized a change of the alanine (A) residue to valine (V) at the 285th amino acid position. In the transgenic mouse model with complete desmin deficiency, phenotypes such as hypertrophic and dilated cardiomyopathy [67] were observed. Surprisingly, the patient with *DES*A285V mutation produced a mutant desmin possessing molecular weight and immunoreactivity comparable to the wild type desmin protein. To investigate the pathological significance of this novel *DES* mutation, we have generated skin fibroblasts-derived iPSCs from this *DES*A285V patient. These *DES*A285V iPSCs were subsequently differentiated into cardiomyocytes for structural and functional studies. When compared to the normal cardiomyocytes, the ones carrying the *DES* mutation exhibited abnormal protein aggregations in sarcomere and Z-disc streaming. In addition, contraction arrest was observed in the mutant cardiomyocytes upon isoproterenol stimulation. These observations not only provided an explanation to the pathogenic mechanism underlying the *DES*A285V mutation, but also validated the causal ion relationship between the *DES* mutation and the DCM phenotype observed in that patient [49].

#### *5.4. Modeling Hypertrophic Cardiomyopathy Associated with MYH7 Mutation*

Hypertrophic cardiomyopathy (HCM) is a heritable cardiac disorder characterized by the abnormal left ventricular thickening and diastolic dysfunction in the absence of an identifiable hemodynamic cause [68]. About 13 HCM-associated genes have been identified to date and most of them encode sarcomeric proteins [69]. Transgenic mouse and rabbit models have been established for studying the pathological mechanisms of HCM [70–72]; however, the mechanistic roles of altered contractile function in the development of HCM remain inconclusive. Very recently, Lan and colleagues generated an iPSCs-line from patients carrying one HCM-associated mutation in the *MYH7* gene. In their case, the 663rd residue of the ȕ-myosin heavy chain is changed from an arginine to a histidine as a result of a missense mutation. The patient-specific iPSCs were differentiated into cardiomyocytes for functional analyses. The mutant-containing cardiomyocytes recapitulated the key features of HCM, including increased cell size and arrhythmia. The intracellular calcium transient profile indicated that the diseased cardiomyocytes showed a significant increase in the resting intracellular calcium level when comparing to the normal cardiac muscle fibers. Interestingly, pharmaceutical inhibition of calcium entry helped to prevent the development of HCM phenotypes in the mutant cardiomyocytes suggesting the *MYH7* mutation altered the calcium homeostasis dysfunction [50].

#### *5.5. Modeling Friedreich Ataxia Associated Cardiomyopathy*

Apart from sarcomeric proteins, abnormality in the mitochondrial proteins may also contribute to HCM development. For example, deficiency in the mitochondrial protein frataxin may lead to Friederich ataxia (FRDA), in which patients usually develop with HCM phenotype to varying degrees [73]. In FRDA, abnormal expansions of the GAA repeat within the first intron of the *FXN* gene may result in the silencing of the gene, which in turn reduces or completely abolishes the production of the frataxin protein.

Frataxin has been implicated in the mechanism of iron-sulfur cluster biosynthesis; however, the contribution of frataxin-deficiency to cardiomyopathy development has yet to be elucidated.

To test whether iron homeostasis deregulation accelerates the reduction in energy synthesis dynamics that contributes to impaired cardiac calcium homeostasis and contractile force, we have recently generated skin fibroblasts-derived iPSCs from a FRDA patient [51]. The *FXN* gene expression in that patient was endogenously silenced. Phenotypically, the FRDA iPSCs*-*derived cardiomyocytes exhibited a disorganization of the mitochondrial network complemented with mitochondrial DNA depletion. Consistent with the mitochondrial disorganization, the energy synthesis dynamics, in terms of ATP production rate, in the diseased cardiomyocytes was impaired. Interestingly, when the diseased cardiomyocytes were subjected to iron overloading, a significant impairment in the calcium handling property was observed. These results indicated that patient-specific iPSCs are useful tools for studying FRDA-associated cardiac defects.

#### **6. Application of Patient-Specific iPSCs-Derived Cardiomyocytes in Efficacy Testing and Drug Screening**

Owing to the limited sources of human cardiomyocytes for *in vitro* analyses, the effects of a putative cardiac drug have to be conventionally tested in the well-established rabbit or canine Purkinje fiber model before proceeding to clinical trials. Nevertheless, as the non-human based cellular models often give false-positive or inconsistent results [74–76], many drugs that have passed the animal tests ended up with failure in the clinical trials. Recent reports demonstrated that human embryonic stem cells-derived cardiomyocytes exhibited excellent pharmacological response to various known antiarrhythmic agents; thus they may be a potential alternative to animal cardiomyocytes [77,78]. However, due to the difference in genetic background, individuals with similar cardiac disorders could show quite different responses towards a particular drug. In this regard, the patient-specific iPSC-derived cardiomyocytes offer an exclusive platform for evaluating the efficacy of a particular drug or treatment strategy on a personal basis.

Based on the latest breakthrough in the cardiac differentiation protocol, a yield of more than 80% in cardiomyocyte differentiation has been achieved [27]. When these patient-specific iPSCs-derived cardiomyocytes are applied to a high throughput assay platform, such as multielectrode arrays analysis, the effects of a testing drug on cellular electrophysiology can be evaluated in a short period of time. Emerging evidences from our group and other investigators have pointed out that altered calcium handling could be an important pathogenic mechanism underlying cardiomyopathies [48,49,79,80]; drugs that affect calcium homeostasis should be of great therapeutic potential. Mercola and colleagues have recently developed a high throughput automated kinetic image cytometry system for the measurement of calcium ion dynamics. This system enabled the authors to simultaneously measure individual calcium transients from 100 human iPSCs-derived cardiomyocytes [81]. Taking advantage of such system, high throughput screenings of calcium handling-enhancing properties of known or novel drugs can be performed on patient-specific iPSCs-derived cardiomyocytes.

#### **7. Application of Patient-Specific iPSCs-Derived Cardiomyocytes in Toxicology Test**

In addition to pharmacological studies, the cardiomyocytes derived from iPSCs are of great potential in the toxicology tests. So far, isolated canine cardiomyocytes are the most popular *pre*-clinical model for cardiac safety testing of a developing drug. However, as mentioned in the last section, the reliability of such model remains questionable. As a matter of fact, many drugs that have passed the animal tests turned out to show unanticipated cardiac toxicity when administered to patients [82], thus, a more predictive and reliable human cardiomyocyte-based model for toxicology test is of immediate demand. Increasing evidences suggested that the pharmacological sensitivities of human ESCs and iPSCs-derived cardiomyocytes are much more advanced than any animal models [77,78], and they should be good detectors for any undesired proarrhythmic side effects of a developing drug.

Recently, Mendenius and colleagues proposed the possibility of using human ESCs and iPSCs-derived cardiomyocytes in the evaluation of drug-induced cardiac injury [83,84]. In their studies, the human ESCs- and iPSCs-derived cardiomyocytes were treated with doxorubicin, and the release of cardiac troponin T in culture medium was measured utilizing a Biocore-based system for the degree of cell injury. Compared to the conventional ELISA based assay, the surface plasmon resonance-based method not only provides superior sensitivity and specificity, but also allows simultaneous analysis of multiple samples. Consequently, the use of iPSCs-derived cardiomyocytes in toxicity predication appears to be feasible.

#### **8. Limitations of iPSCs**

The recent achievement in the patient-specific iPSC technology has created a new platform for regenerative medicine, disease modeling and personalized medication development. Yet, like many other technologies, the clinical applications of patient-specific iPSCs-derived cardiomyocytes are also hindered by various limitations. Though the latest advancement in cardiac differentiation protocol allows efficient generation of cardiomyocytes in a high yield, the human iPSCs-derived cardiomyocytes are actually less mature in terms of calcium homeostasis when compared to the human ESCs-derived cardiomyocytes as demonstrated earlier by our laboratory [85]. In other words, the patient-specific iPSCs-derived cardiomyocytes may not be suitable for modeling cardiac defects resulted from mutations of genes that regulate calcium transients, such as the mutations in the gene encoding the phospholamban.

Furthermore, it should be noted that a high yield of cardiac differentiation is not equivalent to high purity. In fact, iPSCs-derived cardiomyocytes are always grown in a mixed population of atrial, ventricular and nodal subtypes. These subtypes do possess different electrophysiology properties. So far, most transplantation studies were performed in rodent models [28,29]. As rodents have a much faster heart rate compared to humans, the injection of human cardiomyocytes into rodent hearts may not create significant arrhythmia problems. However, the injection of mismatched subtypes of cardiomyocytes into a patient's heart may lead to a medical emergency. Unfortunately, no efficient way is available to sort the subtypes of iPSCs-derived cardiomyocytes into pure populations. The direct application of patient-specific iPSCs cardiomyocytes in regenerative medicine, therefore, remains a theoretic foundation. Besides the issue of mixed subtypes, the immature phenotype of iPSCs-derived cardiomyocytes also limits its application in drug screening experiments. To this end, it is important to verify and validate the results obtained in the initial screening steps.

#### **9. Conclusions**

The cardiomyocytes derived from patients-specific iPSCs are of great potential in many clinical applications. This authentic human cardiomyocyte-based system is expected to compensate for the limitations of the current experimental animal models. This review provides detailed descriptions in the strategies and workflow of using the patient-specific iPSCs-derived cardiomyocytes in regenerative medicine, disease modeling and pharmacological applications. The examples illustrated in this review clearly evidenced the practical values of this novel technology. However, various limitations, such as the immaturities of iPSCs-derived cardiomyocytes, still need to be addressed, and future studies resolving these issues would be beneficial to the use of patient-specific iPSCs in clinical applications.

#### **Acknowledgments**

This work was supported by the Hong Kong Research Grant Council: General Research Fund (HKU 775613).

#### **Author Contributions**

Kwong-Man Ng and Cheuk-Yiu Law were responsible for the literature review and the writing of the manuscript. While Hung-Fat Tse was responsible for providing suggestions on the organization of the article and the editing of the final draft.

#### **Conflicts of Interest**

The authors declare no conflict of interest.

#### **References**

Chapter 3: Eye

### **iPS Cells for Modelling and Treatment of Retinal Diseases**

#### **Fred K. Chen, Samuel McLenachan, Michael Edel, Lyndon Da Cruz, Peter J. Coffey and David A. Mackey**

**Abstract:** For many decades, we have relied on immortalised retinal cell lines, histology of enucleated human eyes, animal models, clinical observation, genetic studies and human clinical trials to learn more about the pathogenesis of retinal diseases and explore treatment options. The recent availability of patient-specific induced pluripotent stem cells (iPSC) for deriving retinal lineages has added a powerful alternative tool for discovering new disease-causing mutations, studying genotype-phenotype relationships, performing therapeutics-toxicity screening and developing personalised cell therapy. This review article provides a clinical perspective on the current and potential benefits of iPSC for managing the most common blinding diseases of the eye: inherited retinal diseases and age-related macular degeneration.

Reprinted from *J. Clin. Med*. Cite as: Chen, F.K.; McLenachan, S.; Edel, M.; Cruz, L.D.; Coffey, P.J.; Mackey, D.A. iPS Cells for Modelling and Treatment of Retinal Diseases. *J. Clin. Med.* **2014**, *3*, 1511–1541.

#### **1. Introduction**

The ability to convert a differentiated somatic cell from a patient into a pluripotent stem cell has provided new tools for studying organ development and genotype-phenotype relationships. Three-dimensional tissue structures and cells derived from these induced pluripotent stem cells (iPSCs) are now being used to screen and test the therapeutic and toxic effects of potential pharmacologic agents and gene therapies. More importantly, iPSCs could also be used to provide an easily accessible source of tissue for autologous cellular therapy. To date, the greatest potential benefit of iPSC technology is in the treatment of retinal diseases.

The retina is a complex neurovascular tissue within the eye. It contains a network of neurons nourished by the retinal and choroidal circulations. Specialised neuronal cells, called rod and cone photoreceptors, capture light that enters into the eye. Through phototransduction within the photoreceptors and downstream neural processing by the bipolar, amacrine, horizontal and ganglion cells within the retina, light signals are transmitted to the primary and secondary visual cortex of the brain to enable visual sensation. The functions of these specialised neuronal cells are supported by the Muller glial cells and the retinal pigment epithelium (RPE). The ease of visualising retinal neurons and assessing the structure-function correlation in detail using readily available imaging devices will facilitate the *in vivo* clinical translation of iPSC technology in the diagnosis and treatment of retinal diseases (Figure 1).

Among hundreds of human retinal diseases, the most significant are age-related macular degeneration (AMD) and the inherited retinal diseases (IRDs). Both AMD and IRDs are neither preventable nor curable, and they remain the most significant causes of irreversible blindness. The underlying processes leading to retinal cell death range from cell-autonomous mechanisms related to single gene mutations to complex gene-metabolic-environment interaction, resulting in extracellular remodelling, abnormal angiogenesis, chronic inflammation, defective lipid metabolism and oxidative injury, as proposed in AMD [1]. The discovery of the pathological basis of these diseases was made possible through clinical observation using detailed retinal imaging techniques, human genetic studies, histology of post-mortem, enucleated or aborted foetal eyes, immortalised cell line culture systems and animal models of retinal diseases. However, in routine clinical practice, retinal diagnosis is rarely based on retinal histology because of the significant morbidity associated with retinal biopsy and the ease in making a diagnosis, because the retina is easily visualised. The availability of iPSC technology provides an opportunity to obtain retinal tissue without retinal biopsy. There are now several examples in which iPSC-derived retinal cells are used to confirm the clinical and genetic diagnosis of IRDs [2,3], understand the molecular mechanisms of developmental anomalies of the eye [4] and explore the cellular mechanisms of specific genetic mutations [5–8]. In addition to improving diagnostic capability, the use of iPSCs in clinical practice could also lead to new treatments for retinal diseases (Figure 2).

**Figure 1.** An example of high-resolution retinal images from a patient with hydroxychloroquine toxicity. (**A**) Wide-field colour photography; (**B**) Zoomed-in colour image highlighted by the yellow box in (**A**) of the macular region showing no obvious abnormality; (**C**) Near-infrared reflectance image of the macula showing no obvious abnormality; (**D**) Adaptive optics retinal image highlighted by the yellow box in (**C**) showing the loss of wave-guiding cone outer segments in the perifoveal region; (**E**) Microperimetry showing reduced sensitivity to light in the macular region; (**F**) Zoomed-in image of the perifoveal region showing reduced sensitivity (<25 dB is abnormal); (**G**) Corresponding optical coherence tomography through the fovea showing no obvious loss of the ellipsoid zone of the photoreceptors (yellow arrow).

Central to most blinding retinal diseases is the loss of cone photoreceptors. Strategies to preserve or replace cone cells are under intense investigation. Cones can be preserved by: (1) anti-oxidant therapy; (2) pharmacological therapy that provides neuroprotection; (3) gene correction therapy; and (4) cell-based therapy to provide support to cone cells (e.g., RPE or rod cell transplantation). Lost cone cells can be replaced by: (1) transplantation of patient-specific or allogeneic photoreceptor precursors (along with supporting cells); (2) recruitment of endogenous cells to differentiate into new photoreceptor or to become light-responsive cells (optogenetics); or (3) implantation of *epi*-retinal, *sub*-retinal, suprachoroidal or optic nerve visual prostheses [9–11]. Some of these treatment modalities have been investigated in cell culture systems and animal models, and many of these have also been tested in phase I/II clinical trials [12–15]. A major limitation of clinical therapeutics trial in IRDs is the vast heterogeneity of the underlying genetic mutation. Many of the approaches to preserve cones may only be suitable for one genetic variant, but not another, despite a similar clinical phenotype. Given the rarity of many IRDs, randomised clinical trials are not feasible. As an alternative, iPSC-derived retinal tissue from many patients with IRDs can now be tested *in vitro*, simultaneously, in a pre-clinical study, for the potential dose-therapeutic effect response and toxicity of various pharmacologic agents or gene therapies. As genomic editing techniques are emerging and iPSCs are being used as a cell source for replacing lost retinal cells, we now also have the capability of eliminating specific mutations prior to retinal differentiation, thus providing the option of autologous transplantation even to patients with IRDs [16].

**Figure 2.** A somatic cell from the patient is used to derive induced pluripotent stem cells (iPSCs). The iPSC colonies are characterised to ensure pluripotency markers are present, they form teratoma or embryoid body and they have stable chromosomes. It may take up to three months to derive and validate iPSC lines. The validated iPSC colonies are differentiated to form optic vesicle structures, which contain retinal pigment epithelium and neural retinal cells. Mature retinal cells can be used for confirming the pathogenicity of newly-discovered genetic variants, modelling of developmental or degenerative retinal disease, testing of pharmacologic agents or gene therapy and autologous cellular therapy.

There are several excellent reviews on the use of human iPSCs in the study of retinogenesis, modelling retinal disease, screening of therapeutics and cell replacement therapy in both AMD and IRDs [17–22]. The purpose of this review is to provide an update, from a clinical perspective, on the potential for using iPSC technology in routine clinical care of patients with retinal diseases. It will expand on clinically relevant issues related to laboratory techniques to derive clinical grade iPSC-retina and illustrate examples in which iPSC technology has translated into patient care.

#### **2. Derivation of Patient-Specific Retinal Cells from iPSC for Clinical Use**

The availability of human retinal tissue and pure populations of specific types of retinal cells is critical to our ability to diagnose and treat retinal diseases. Allogeneic sources of retinal tissue and cells can be obtained from donor eyes or cell lines. However, these are not clinically useful for confirming genetic diagnosis of a patient or for autologous cellular therapy. Access to patient-specific retinal tissue requires an intraocular procedure, called a vitrectomy, followed by detachment of the retina, retinectomy, laser retinopexy and a vitreous substitute to provide a temporary tamponade. Although this type of procedure is rarely performed for obtaining retinal tissue for the diagnosis of vitreoretinal lymphoma, there are significant blinding complications, such as retinal detachment, and the harvested retinal tissue will not be of adequate quantity or quality for disease modelling, retinal regenerative therapy or screening new therapeutics. Therefore, there is a clinical need for obtaining patient-specific retinal cells without the need to perform retinal biopsy.

#### *2.1. Creating iPSC from Patients*

#### 2.1.1. Using Pluripotent Stem Cells

An alternative method to obtain patient-specific retinal cells is to use patient-derived adult stem cells for differentiation into retinal lineages. Retinal neural and pigment epithelial progenitor cells [23,24] have been found in the adult retina, but access to these cells is also limited, as they will require vitrectomy surgery, making them equally unsuitable for clinical use in testing therapeutics and administering personalised cell therapy. Multipotent neural stem cells capable of generating retinal lineages have also been found in the ciliary margin zone and corneoscleral limbus [25–28]. The former source is located adjacent to the lens within the eye, and it is even more difficult to access than the retina. In contrast, limbal tissue is routinely harvested by corneal surgeons for autologous limbal transplantation. Despite the ease of limbal cell harvesting and the long-term safety of the limbal graft donor site [29,30], its use for retinal regeneration and disease modelling has not yet been explored due to limited data on the ability for *in vitro* expansion and the potential for differentiation into all retinal cell types.

Unlike adult stem cells that are multipotent or unipotent, *i.e.*, committed to specific cell types, pluripotent stem cells (PSCs) have a capacity for unlimited self-renewal (hence, large quantities of cells) and differentiation into any somatic cell type, including all classes of retinal cells. One source of PSC is the embryonic stem cells (ESCs), harvested from the inner cell mass of the blastocyst, from which each of the three germ layers—the endoderm, ectoderm and mesoderm—can be derived. However, ESCs are derived from discarded surplus embryos, and this is not patient-specific. Human ESC-derived RPE is currently being used in several clinical trials, but recipients are being immunosuppressed, because of the potential risk of graft rejection [12].

More recently, PSCs can also be generated by dedifferentiating a terminally differentiated patient-specific adult somatic cell, such as a fibroblast, into a pluripotent state by nuclear reprogramming. There are three established methods to induce pluripotency: (1) transfer of the nucleus of a differentiated cell into an enucleated oocyte (nucleus removed), so that pluripotency genes within the somatic cell genome are activated by the regulators within the oocyte cytoplasm (nuclear transfer) [31]; (2) fusion of a somatic cell with an ESC to create a hybrid or heterokaryon in which pluripotency regulators override cell differentiation regulators (cell fusion) [32]; and (3) induced overexpression of specific pluripotency transcriptional factors through transfection of an adult somatic cell with integrating virus, non-integrating virus, plasmids, mRNA or even exposure to protein or small molecules (induced pluripotent stem cell (iPSC) reprogramming) [33].

#### 2.1.2. Induced Pluripotent Stem Cells

Since the original description of the iPSC protocol by Yamanaka [34], there has been significant development in the reprogramming approach, and many types of somatic cells have been successfully induced into a pluripotent state. Conceptually, this is a two-step process of (1) nuclear reprogramming of a chosen somatic cell into several clones of iPSC and (2) validation of the pluripotency of the various clones of iPSC to select the most suitable clone for the specific purpose that iPSCs will be used, for diagnostics or therapeutics. The time lag from biopsy to obtain patient's somatic cells to full validation of the best iPSC clone may take 2–3 months. Several factors will influence the choice of somatic cell for deriving iPSC.

For therapeutic purposes, such as autologous cell replacement therapy, the ideal iPSC clone should be derived from an easily accessible somatic cell type in facilities that comply with good manufacturing practice guidelines related to cell therapy. Both adult stem cells and differentiated cells have been used to derive good quality iPSC lines. Although adult stem cells may already express some of the pluripotency-related genes, their expression is significantly lower than that seen in ESC or iPSC. Therefore, the same protocol for deriving iPSC is generally required for adult stem cells as for differentiated cells. Cells that proliferate well also reprogram well. However, there is significant variability in genetic and epigenetic patterns and the degree of reprogramming, even between iPSC clones from the same cell source. Hu *et al.* showed that iPSCs derived from RPE retain a "memory" of cellular origin with respect to the propensity for differentiation back to RPE [35]. However, it will not be feasible to use patients' RPE as a source for deriving iPSC, due to surgical complications associated with tissue harvest. Furthermore, even without "memory" in source cells, RPE and neuroretinal cells have been generated readily from iPSC derived from cells of diverse background, such as cord blood cell, lymphocyte, keratinocyte, adipocyte and fibroblast [2,4,36–38]. Another easily accessible source of somatic cells is the ocular surface. The potential to generate iPSC from cells on the ocular surface (corneal epithelium and limbal niche) warrants further investigation, as they can potentially be reprogrammed to pluripotency without the introduction of transcriptional factors, as shown in rodent limbal-derived neurospheres [39,40]. In contrast to autologous transplantation of iPSC-derived retinal cells, special consideration needs to be given to the ease of transport and storage of somatic cells for deriving iPSC for the purpose of genetic diagnosis, disease modelling and high throughput drug screening. In this situation, blood-derived cells (activated T lymphocytes and endothelial progenitor cells) may be preferable, as they are easily collected, transported, isolated and stored [41–43].

The reprogramming protocol should preferably avoid the use of viruses, such as retroviruses, that were used to create the first human iPSC [34,44]. Non-integrating viral vectors, DNA plasmids, modified RNA, protein and small molecules have all been reported to induce a pluripotent state in a somatic cell [45–50]. There is no one perfect methodology for creating iPSC for all types of clinical use. The main trade-off for the potential mutagenesis by integrating virus is the lower efficiency and higher cost associated with non-integrating methods. There have also been variations on the transcriptional factors used for reprogramming since the original description by Yamanaka (OCT4, SOX2, KLF4 and c-MYC) and Thomson (OCT4, SOX2, NANOG and LIN28) (Figure 3) [34,44]. Some protocols also use additional small molecules, such as 5-aza-deoxycytidine, valproic acid or ascorbic acid, to modify the epigenetic environment and enhance the efficiency and accuracy of nuclear reprogramming. Ultimately, these protocol modifications will also have an impact on the cost and quality of the human iPSC line and the suitability for clinical application, such as autologous transplantation. Regardless of the cell source and reprogramming protocol, successful generation of retinal tissue from iPSCs will depend on the skills of the operator in identifying the "right" iPSC clones for retinal differentiation.

**Figure 3.** Retrovirus vector for induced pluripotent stem cell (iPSC) reprogramming. (**A**) Map of polycistronic retroviral vector. Human fibroblasts two days after infection with polycistronic GFP Oct4/Sox2/Klf4/cMyc; (**B**) iPSC after four weeks post infection negative for GFP indicating that the transgene is silenced in iPSC clone.

#### 2.1.3. Validation of Human iPSC Lines

The key defining features of iPSC are the self-renewal capacity and the ability to produce all three germ layers. Not all iPSC clones generated from the same somatic cell line from the same

(**B**)

patient will be fully reprogrammed or truly pluripotent, and the efficiency of various protocols in generating iPSC clones can vary from 1:100 to 1:10,000. Screening to distinguish partially and fully reprogrammed colonies may add further delay and cost to the generation of patient-specific iPSC lines, and the thoroughness of this process depends on the clinical reasons for deriving the iPSC. It has been suggested that between five and 10 clones may need to be isolated for characterisation and future differentiation, because not all clones will have the same propensity for retinal lineage derivation, despite their potential [6,7,51,52].

Four techniques are used for characterising and subsequently selecting iPSC clones: cellular, molecular, functional and genetic (Table 1). The extent of characterisation required will again depend on the purpose of generating iPSC. Less rigorous criteria may be sufficient for genetic diagnosis and disease modelling compared to drug screening and cellular therapy. However, a minimum set of criteria for establishing putative iPSC has been recommended by the European Consortium of stem cell research (the ESTOOLS project).

The unpredictable variability between clones may be related to the somatic origin of iPSC, the reprogramming technique or the intrinsic clonal variability within the individual. Furthermore, equal performance of iPSC clones against the same "pluripotency" tests does not translate to equal propensity for retinal lineage derivation. Further investigation is required to establish a selection screen and criteria for reducing clonal variation and identifying iPSC clones that have optimal retinal differentiation propensity. It can be envisaged that different criteria for establishing pluripotency may emerge for diagnostic and therapeutic use of iPSC.

#### *2.2. Creating Retinal Tissue from iPSC*

#### 2.2.1. Derivation of Retina Lineages

The fundamental principles for differentiating iPSC into retinal progeny have been laid down by previous work on mouse and human ESCs. However, the different propensity between iPSC and ESC for retinal differentiation brings into question the validity of the various protocols proposed. Most of these protocols rely on the initial spontaneous induction of retinal differentiation, but there is variability between cell lines. The lack of reproducibility by other laboratories also raises concern regarding their utility in the clinical setting. Nevertheless, there are two broad approaches: one by default differentiation of iPSC into neuroectodermal lineages (upon withdrawal of FGF2) and the other through directed differentiation by the addition of extrinsic molecules, such as growth and transcription factors.

A common approach in deriving retinal cells is to allow human iPSCs to overgrow as adherent layers. With the use of specific extracellular matrix in addition to certain inducing factors and proteins, iPSCs may be preferentially differentiated into RPE or photoreceptor phenotypes. For example, Tucker *et al.* described the formation of two-dimensional eyecup-like structures in a synthetic xeno-free culture substrate when skin keratinocyte-derived iPSCs were used [2]. After formation of small pigmented foci at around 45 days, these clumps expanded over 150 days. In some of these clumps, neural cells fill the centre, whilst in other colonies, pigmented cells wraps around in a C-shape around neural rosettes resembling a cross-section of an optic cup [2]. Similar two-dimensional eye-cup structures have also been reported by Jin *et al.* [53] and Reichman *et al.* [54].

An alternative approach is to culture iPSCs as suspended aggregates to enable the formation of three-dimensional cellular structures. Recapitulation of ocular organogenesis through the formation of an optic cup structure using a serum-free suspension culture system was first demonstrated using murine ESC by Eiraku *et al.* and then human ESC by Nakano *et al.* [55,56]. More recently, Meyer *et al.* described human iPSC-derived cell aggregates with vesicle-like and non-vesicular configurations after 20 days of culture using successive media changes from embryoid body medium (four days) to neural induction medium (seven days) and, finally, to retinal differentiation medium [6,51]. The vesicle-like structures expressed CHX10, a marker of retinal progenitor cells, whereas the non-vesicular spheres expressed ISLET-1, a homeodomain protein involved in early forebrain development. Upon further differentiation, photoreceptor-like cells and RPE were derived from the vesicle-like structures. Similar optic vesicle-like structures have also been generated from lymphocyte-iPSC [4,36]. Zhong *et al.* recently reported three-dimensional laminated retinal cups generated from human iPSC with distinct populations of neural retinal cells interacting through synaptic junctions and photoreceptor cells capable of forming outer segment discs and responding to light [57]. However, it is important to note that efficiency in generating PAX6+ neuroectodermal cells amongst different iPSC clones can vary from 5% to 56% of the total cell population using the same protocol, highlighting the need for further investigation into the methods and screening criteria to identify the most suitable iPSC clone for retinal differentiation [51].

Irrespective of the protocol used for inducing retinal differentiation, the timing of the derivation of specific retinal cell types generally reflects the timeline of embryological development. This temporal recapitulation of embryogenesis by iPSC differentiation supports the notion that derivation of retinal cells is not directed, but rather the outcome of subcloning and culture in a permissive microenvironment. During the first month of embryonic development, the forebrain portion of the primitive anterior neuroepithelium gives rise to cells expressing markers specific for the eye field. Optic vesicles then develop from the eye fields at the end of the first month with cells expressing PAX6 and MITF. Retinal progenitors destined to become RPE preferentially express MITF, whereas those becoming neuronal cells downregulate MITF in response to increased CHX10 expression. Then, there is a 1–2 month(s) lag in the expression of *CRX* and opsin genes in neural retinal cells after the formation of RPE. Hence, the time to generate RPE from iPSC is typically around 4–6 weeks, whereas differentiation of photoreceptor precursors occurs at 2–3 months after retinal induction. Formation of outer segments and the development of light response was reported by Zhong *et al.* at six months after retinal induction [57]. Recently, Reichman *et al.* described a floating culture system for generating neuroretinal-RPE containing retinal progenitor cells within two weeks, which bypassed embryoid body formation and obviated the need for exogenous molecules, coating or Matrigel [54].

The ability to recapitulate retinogenesis using iPSC has tremendous potential for studying diseases that interfere with retinal development and non-cell autonomous mechanisms, in addition to those that cause post-natal cell autonomous retinal degeneration. On a practical level, there are still significant barriers to routine clinical use of this technology, since the generation of patient-specific retinal cells may take 4–6 months from the time of biopsy, and there is significant overhead infrastructure cost to maintain an iPSC laboratory. Future advancement in three-dimensional culture and differentiation techniques may one day enable iPSCs to differentiate into other structures of the eye, such as the choroid and sclera, thus expanding the use of iPSC in understanding complex retinal diseases, such as AMD and myopia.

#### 2.2.2. iPSC to Photoreceptor Cells

Hirami *et al.* described deriving photoreceptor cells using human iPSC from dermal fibroblast, serum-free embryoid body culture system, defined factors (Wnt and Nodal inhibitors) and subsequent plating of aggregates onto poly-D-lysine, laminin and fibronectin to generate retinal progenitors expressing *RX*, *PAX6* and *MITF* [58]. From Day 90, the application of retinoic acid and taurine to the culture system induced the expression of the photoreceptor marker, recoverin, in a quarter of the colonies by Day 120 (four months). Half of these recoverin-positive cells were also immune-positive for rhodopsin. Notably, only two of three iPSC lines could be differentiated into the retinal lineage, and functional assays of putative photoreceptor cells were not performed. Osakada *et al.* from the same group, at the RikenCenter for Developmental Biology, also reported a modified protocol using small molecules (casein kinase I inhibitor CKI-7, Rho-associated kinase inhibitor Y-27632 and ALK4 inhibitor SB-431542) to block Wnt and Nodal pathways to induce retinal progenitors [59]. This method has been used in generating photoreceptor cells from patients with *RP1*, *RP9*, *PRPH-2* and *RHO* mutations [7,53].

Meyer *et al.* [51] described a different culture system using embryonic stem cell medium without FGF2, then chemically-defined neural induction medium with N2 supplement followed by another chemically-defined retinal differentiation medium supplemented with B27. Rosettes were picked and selected for neurosphere culture and generation of optic vesicle-like structures. By Day 80, 14% of the neurospheres expressed rod- and cone-specific transcription factor Crx, within which 65% of the cells were expressing Crx. However, only 8% of the cells within Crx+ spheres expressed recoverin and/or opsin. This protocol was modified by Zhong *et al.* to generate photoreceptor cells (within laminated retinal cup structure) that express synaptic junction proteins, phototransduction molecules, to form outer segments and to respond to light stimulus [57]. The optic vesicle-like system was used to study the effect of *CHX10* mutation [4].

Several other groups have also described the derivation of photoreceptor cells from human iPSC (Table 2). There are many morphological, cellular, molecular, functional and genetic assays for the characterisation of iPSC-derived photoreceptors and their precursors, but there is no consensus on the minimum criteria (Table 1). Lamba *et al.* used their protocol for ESC [60] to derive photoreceptor cells from human iPSCs [52]. Although, they did not test the function of these cells, they demonstrated integration into mouse retina following sub-retinal transplantation. Mellough *et al.* combined the techniques described by Lamba *et al.* and Osakada *et al.* for deriving retinal cells from ESC and added activin A, Shh and T3 to enhance photoreceptor differentiation from human iPSCs [60–62]. Their three-step differentiation protocol involved inducing a neural lineage, then retinal progenitors and, finally, photoreceptor cells expressing blue, red and green opsin. For iPSC-derived photoreceptor cells to be used in human transplantation, animal-derived products should be avoided where possible. Tucker *et al.* and Sridhar *et al.* recently reported the generation of photoreceptor cells from iPSCs using a xeno-free system, where a synthetic culture surface (Synthemax cell culture surface) is used for iPSC derivation and retinal differentiation [37,63].

#### 2.2.3. iPSC to Retinal Pigmented Epithelial Cells

Although the embryoid body culture system can generate RPE from iPSC, adherent culture has been favoured if RPE is the only cell that is required. Hirami *et al.* and Meyer *et al.* showed RPE differentiation occurs earlier than neural retinal progeny derivation [51,58]. Carr *et al.* and Buchholz *et al.* demonstrated that RPE differentiation from human iPSC can be achieved within four weeks, and these cells demonstrated morphological and molecular signatures of RPE, as well as *in vitro* and *in vivo* functional characteristics [65,66]. Morphologically, RPE derived from iPSCs is indistinguishable from RPE in post-mortem eye or human ESC-derived RPE (Figure 4).

Characterisation of iPSC-RPE involves morphological, cellular, molecular, functional and genetic assays (Table 1). Key morphological features include pigmentation, monolayer of hexagonal cells and electron microscopic features of apical microvilli, tight junctions, basal infoldings and cytoplasmic melanosomes. The molecular signature of RPE cells reflects their eye field origin (PAX6 and MITF) and function: RPE65 and CRALBP (retinoid cycle), MERTK (phagocytosis), bestrophin (modulating calcium flux in endoplasmic reticulum) and ZO-1 (tight junctions). *In vitro* functional assessment includes transepithelial resistance measurement, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and pigment epithelium-derived factor (PEDF) secretion, extracellular matrix production (laminin and type IV collagen) and phagocytosis assay using photoreceptor outer segments. *In vivo* functional assessment requires subretinal transplantation in an animal model of RPE or retinal dystrophy, such as the Royal College of Surgeons (RCS) rat, to assess the rescue of visual function [65,67–69]. Gene expression comparing human iPSC-RPE to adult and foetal RPE and other controls through microarray and hierarchical clustering analysis needs to be performed to verify similarity to target tissue [70].

**Figure 4.** Morphology of the retinal pigment epithelium monolayer. (**A**) Hexagonal pigmented monolayer of retinal pigment epithelium derived from induced pluripotent stem cells; (**B**) Comparison of the morphology of retinal pigment epithelial stem cells derived from human embryonic stem cells (HESC), post-mortem (PM) eyes and induced pluripotent stem (iPS) cells.

For autologous transplantation of iPSC-RPE, immunogenicity, cell survival and tumourigenicity studies are also required. These have been addressed for iPSC-RPE specifically by Kamao *et al.* and Kanemura *et al.* [70,71] as part of a pre-clinical study in preparation for human iPSC-RPE autologous transplantation. The generation of patient-specific iPSC-RPE has also been performed in gyrate atrophy and Best disease (BD) [5,6]. The following section will illustrate clinical examples of the use of iPSC-derived retinal cells in genetic diagnosis, discovery of genotype/phenotype relationship, screening of pharmaco- and gene therapies and as a source of autologous cell therapy.

#### **3. Clinical Use of Patient-Specific iPSC-Derived Retinal Cells**

The ability to generate patient-specific retinal tissue and cells offers the opportunity to study the relationship between genetic variants and disease phenotypes. This technology is particularly useful in modelling IRDs, as there are around 200 genes with over 4200 known and many other unknown mutations causing disease phenotype in IRDs [72]. One in 2000–3000 individual are affected by IRDs, and these, collectively, are the most common cause of blindness in children and young adults. Given that emerging therapies for IRDs are likely to be mutation specific, it is important to identify pathogenic mutation(s) in every affected individual [73–75].

Many IRDs have a poor genotype-phenotype correlation; defects in a single gene may lead to a variety of disease phenotypes, while, on the other hand, a particular disease phenotype may be caused by mutations in a large number of different genes. Adding to the challenge of identifying causative mutations is the relatively common occurrence of X-linked and *de novo* autosomal dominant variants. Although Sanger sequencing of selected genes followed by targeted next-generation sequencing (NGS) can identify known pathogenic mutation in many individuals, exome or whole genome NGS combined with genetic linkage studies are required for the identification of novel mutations. Traditionally, these rare mutations (<1%) have been validated through functional modelling, mouse and zebrafish studies and replication of genotyping in large patient and relevant control cohorts. More recently, iPSC technology has also been used to confirm the pathogenicity of genetic variants and to unravel the molecular mechanism of disease phenotype through the *in vitro* study of cellular function and the histogenesis of iPSC-derived retinal tissue.

#### *3.1. IPSC for Genetic Diagnosis and Modelling*

#### 3.1.1. Confirming Pathogenicity of Mutation

Patient-specific iPSC has been used to confirm the pathogenicity of new rare genetic variants. For example, using NGS, single-strand conformation polymorphism screening and Sanger sequencing of a large validation cohort, Tucker *et al.* [3] identified a new mutation (Alu element insertion) in the male germ cell-associated kinase (*MAK*) gene causing rod-cone dystrophy. This was confirmed by examining and comparing the transcripts of *MAK* between iPSC and iPSC-derived photoreceptors from the patient and his unaffected sibling. The proband had no family history of retinal dystrophy, and the affected individual was heterozygous for pathogenic variants in *ABCA4* and *USH2A*. Using iPSC, they discovered a previously unrecognized exon 12 of the *MAK* gene that is expressed in cells differentiated into retinal precursors, but not in undifferentiated cells. This observation confirmed that the homozygous Alu element insertion in exon 9 is pathogenic by affecting the developmental switch from *MAK* bearing only exon 9 to a retina-specific transcript bearing both exons 9 and 12. The insertion of a 353-bp Alu repeat between codons 428 and 429 in exon 9 results in the insertion of 31 incorrect amino acids followed by a premature termination. In another study, Tucker *et al.* [2] reported the discovery of a new pathogenic variant of *USH2A* in another patient with rod-cone dystrophy who was presumed heterozygous for a pathogenic variant in *ABCA4* and *USH2A*. The second possibly disease-causing variant was found in intron 40 of *USH2A*, and this was confirmed by real-time PCR of patient-specific iPSC-derived photoreceptor precursor cells. A pseudoexon (IVS40) was formed by the intronic splice site mutation in the intervening sequence 40 of *USH2A*, and this caused a translation frameshift and a premature stop codon.

Lustremant *et al.* [76] examined the transcriptomics of human iPSC-derived neural stem cells and RPE from two patients with Leber congenital amaurosis (LCA). Although the pathogenic mutation was not known, they showed changes in the expression of 21 genes when compared to wild-type controls. Amongst these, three downregulated genes—*TRIM61*, *ZNF558* and *GSSTT1*—were related to the LCA disease process through protein degradation, altered transcription regulation and oxidation. With better understanding of the interactions between molecular pathways, detection of altered transcriptomics may help to narrow down candidate genes in this patient with LCA.

#### 3.1.2. Modelling Developmental Diseases of the Retina

The impact of known mutations on retinogenesis and cellular function has also been explored. The transcription factor CHX10 (*Caenorhabditis elegans* Ceh-10 homeo-domain-containing homolog 10), also known as VSX2 (visual system homeobox 2), has a critical role in the development of the retina. The very rare mutation in *VSX2* leads to severe malformation of the eye. Although animal models of this disease (the *VSX2*<sup>í</sup>/<sup>í</sup> and (R200Q) *VSX2* mice) have contributed greatly to our understanding of the importance of *VSX2* in repressing MITF, production of the bipolar cell and maturation of the photoreceptors, it was not possible to confirm that the same mechanism occurs in humans. Phillips *et al.* [4] used iPSCs from a patient with a (R200Q) *VSX2* mutation to generate an embryoid body and then vesicles that recapitulated retinogenesis [6,36]. They confirmed previous observations in animal models and went a step further, using lentiviral *VSX2* overexpression to examine the reversibility of the developmental defect *in vitro*. Although suppression of MITF and enhanced photoreceptor maturation was achieved, bipolar cell markers were not restored by wild-type lenti-*VSX2*. Transcriptome analysis at Day 20 and 30 cells demonstrated overall upregulation of genes. Most of these were related to the WNT and TGFb signalling pathways that promote RPE differentiation. In contrast, the FGF pathway, which promotes neuroretinal differentiation, was downregulated.

The process of deriving retinal cells from iPSCs provides an opportunity for studying retinal development and developmental anomalies due to specific mutations that cause ocular and retinal dysgenesis (e.g., *MITF*, *PAX6*, *VSX2*, *CRB1*, *etc.*). However, terminally differentiated retinal cells from iPSCs can also be tested for altered cell function to understand degenerative diseases of the retina (see below). IRDs that have an earlier onset may be expected to demonstrate abnormality earlier in the differentiation protocol, whilst late onset IRDs (e.g., BD, pattern dystrophy, Sorsby fundus dystrophy and Doyne honeycomb retinal dystrophy) may not manifest altered cellular physiology unless the cells are aged and stressed *in vitro* to recapitulate senescence.

#### 3.1.3. Modelling Degenerative Diseases of the Retina

Both RPE and photoreceptor disease models have been created using patient-specific iPSCs. AMD (Figure 5) and two types of RPE dystrophies have been modelled: Best disease and gyrate atrophy.

**Figure 5.** Clinical images of early age-related macular degeneration and its variants. (**A**) Colour photograph of the macula of a 72-year-old male showing soft drusen; (**B**) Optical coherence tomography (OCT) shows a sub-retinal pigment epithelial (RPE) deposit, which did not significantly alter fundus autofluorescence (**C**); (**D**) Colour photograph of the macula of a 78-year-old female showing reticular pseudo-drusen; (**E**) OCT shows deposits above the RPE, resulting in subtle hypo autofluorescent lesions (**F**); (**G**) Colour photograph of the macula of a 57-year-old female showing basal laminar drusen; (**H**) OCT shows a compact sub-RPE deposit forming a saw-tooth pattern, and these lesions were mildly hyper autofluorescent (**I**); (**J**) Colour photograph of the macula of an 83-year-old female showing dominant drusen or Doyne honeycomb retinal dystrophy; (**K**) OCT shows outer retinal layer loss; (**L**) The fovea was hypo autofluorescent due to RPE loss, and the linear radial drusen are seen as hyper autofluorescent streaks.

Chang *et al.* reported reduced ZO-1 and RPE65 staining in iPSC-RPE generated from five atrophic AMD patients compared to two controls [38]. There was also increased accumulation of reactive oxygen species following exposure to H2O2 compared to controls. Furthermore, expressions of antioxidant genes (*HO-1*, *SOD2* and *GPX1*) were lower, whilst PDGF, VEGF and IGFBP-2 expressions were higher compared to controls. There were no details regarding the age of the two control subjects, and the AMD risk allele profiles for all seven subjects were not reported. Further studies are needed to replicate these findings by controlling other potential confounders. It is particular important that control subjects are age matched when iPSC is used to model late-onset degeneration as AMD. This is because AMD cannot be diagnosed until drusen is visualised, usually after the age of 50 years. Although the presence of risk alleles and family history increases the risk of developing AMD, these biomarkers are not 100% predictive. Therefore, somatic cells from young healthy individual cannot be used as controls, because it is not possible at this stage to predict if this subject will or will not develop AMD later in life.

Heterozygous, compound heterozygous and homozygous mutation of the *BEST1* gene (bestrophin protein) can cause ocular disease characterised by abnormal RPE function, accumulation of debris between RPE and photoreceptors and a variable amount of retinal degeneration and ocular dysgenesis (Figure 6). There are over 100 mutations in *BEST1*, and the pathogenicity and molecular mechanism of RPE dysfunction arising from these mutations is not well understood. The traditional approach to study the effects of *BEST1* mutation is based on transfection of mutant *BEST1* gene into the human foetal RPE or Madin Darby canine kidney (MDCK II) epithelium cell lines. Sing *et al.* described the use of RPE derived from patient-specific iPSCs to study the impact of two mutations in the *BEST1* gene, (A146K and N296H) on RPE function. They demonstrated increased intracellular accumulation of autofluorescent materials compared to controls after long-term (3.5 months) feeding of the BD hiPSC-RPE with bovine photoreceptor outer segments (POS, 50/cell) and decreased net fluid transport. Conflicting data at 4 and 24 hours were shown regarding delayed degradation of POS when overfed with FITC-POS (50 *vs.* 20 POS per cell). As expected, there was no significant difference in the RPE differentiation potential of BD hiPSC compared to controls. BD hiPSC-RPE also had a similar transepithelial resistance, level of *BEST1* mRNA and localisation pattern of the mutant bestrophin compared to controls. The dysfunction in mutant bestrophin was found to be associated with altered endoplasmic reticulum (ER)-mediated calcium homeostasis. Furthermore, there was increased expression of genes involved in regulating oxidative stress (*GPX1*, *SOD2*) and iron homeostasis (*TRF*, *TRFR*) after long-term (3.5 months) POS feeding [5]. In this study, the genetic background between the cell lines was not controlled by genomic editing of the *BEST1* mutation into the control iPSC or out of the BD hiPSC (see below). Nevertheless, this is a good example where early onset disease with a well-characterised clinical disease phenotype can be recapitulated *in vitro*. It is not known if similar cellular abnormalities can also be detected in iPSC-RPE from patients with the much commoner late-onset vitelliform macular dystrophy due to other types of *BEST1* mutations. Phagocytosis assay may be a better readout for iPSC-RPE generated from patients with a known genetic defect that impairs phagocytosis, such as *MERTK* or *REP-1* mutation (choroideremia).

In a very different RPE dystrophy, gyrate atrophy, homozygous mutation in the ornithine-į-aminotransferase gene (*OAT*) leads to RPE damage and loss, leading to severe peripheral and central vision loss. RPE has been successfully generated from iPSCs derived from the fibroblasts of a patient with *OAT* mutation (A226V). Enzyme activity of OAT within the iPSC-RPE can be measured [6]. Correction of the *OAT* mutation by bacterial artificial chromosome-mediated homologous recombination restored the enzymatic activity [77]. There are other RPE dystrophies resulting from mutations affecting visual cycle enzymes or regulators (e.g., the acyltransferase, LRAT, the isomerohydrolase, RPE65, the dehydrogenase, RDH12, and RPGR and RLBP1). Deriving iPSC-RPE from patients with various genetic mutations in these enzymes or regulators may also provide opportunities to understand genotype-phenotype molecular mechanisms and variability. **Figure 6.** Clinical images of various types of inherited retinal diseases. (**A**) Colour photograph of the macula of a 10-year-old boy showing multifocal vitelliform lesions resulting from homozygous deletion of exon 2–6 of the *BEST1* gene; (**B**) Optical coherence tomography (OCT) shows intraretinal cystic change with sub-retinal fluid and vitelliform deposits; (**C**) Increased fundus autofluorescence was noted in the area of vitelliform deposits; (**D**) Colour photograph of the macula of a 57-year-old male showing yellow deposits due to pattern dystrophy of the retinal pigment epithelium (RPE); (**E**) OCT shows deposits above and below the RPE; (**F**) Multifocal hyper autofluorescent lesions are seen; (**G**) Colour photograph of the macula of a 56-year-old female showing extensive macular atrophy with cone-rod dystrophy due to two missense mutations in the *ABCA4* gene (c.2915 C > A and c.3041 T > G); (**H**) OCT shows severe retinal and choroidal atrophy with pigment migration into the fovea; (**I**) Extensive RPE loss resulting in wide-spread hypo autofluorescent lesions; (**J**) Colour photograph of the macula of a 32-year-old male showing retinal flecks with mild cone dysfunction due to two pathogenic mutations in the *ABCA4* gene (c.4139 C > T and c.6079 C > T); (**K**) OCT shows outer retinal layer loss to retinal atrophy; (**L**) Retinal flecks were hyper autofluorescent.

Many genes are involved in photoreceptor cell degeneration. Clinically, there are two broad classes of disease phenotypes based on electrophysiology: macular dystrophy, which is limited to the macular region, and retinal dystrophy, where the entire population of photoreceptors (central and peripheral) is affected. Generalised retinal dystrophy can affect cones or rods predominantly. Different mutations in one gene, such as *ABCA4*, can have varied disease phenotypes, including macular dystrophy, cone dystrophy, cone-rod dystrophy or rod-cone dystrophy (Figure 6). The overlap between various disease phenotypes and causative mutation reinforces the importance to understand the molecular mechanisms of genotype-phenotype relationships.

Amongst the genes causing rod-cone dystrophy, the molecular effect of mutations in *RHO*, *RP1*, *RP9*, *PRPH-2* and *USH2A* have been studied using iPSCs. Jin *et al.* [7] used iPSCs from patients with *RHO*, *RP1*, *RP9* and *PRPH-2* to generate rod photoreceptor precursors. They demonstrated reduced rod cell number at Day 120 in iPSC-rod precursors derived from patients with *RP9* mutation (early-onset retinal degeneration). This effect was seen in *RHO*, *RP1* and *RHO* mutation at Day 150, whereas no rods were detectable for the iPSC line carrying the *RP9* mutation. They also showed that the *RP9* mutant iPSC-rod precursors had increased oxidative stress. In contrast, the *RHO* mutant iPSC-rod precursors had mislocalisation of the rhodopsin protein and increased expression of ER stress markers, which might be explained by the accumulation of unfolded rhodopsin [7,53]. Tucker *et al.* also examined the impact of *USH2A* mutation on photoreceptor precursor cells. They described increased expression of *GRP78* and *GRP94* in iPSC-derived photoreceptor precursor cells, indicative of ER stress related to protein misfolding. It is important to note that controls used in these reports were not genetically matched, *i.e.*, the mutation was not removed by genomic editing.

A major limitation of these genotype-phenotype studies is the choice of controls. For metabolic syndromes or disease with early manifestation (ocular dysgenesis or early degeneration), healthy related or unrelated controls will be adequate, because of the robust and rapid cellular manifestation of the phenotype. However, controls used for studying diseases with delayed onset will need to be genetically matched to avoid the cofounding effect of the (1) genetic background, (2) retinal cell differentiation process and (3) genetic alteration introduced during the process of iPSC reprograming. Yoshida *et al.* confirmed the effect of *RHO* mutation (E18K) on rod precursor cell ER stress responses, apoptosis markers and autophagy activation by repairing and introducing the mutation in the affected and control (provided by Yamanka laboratory) cell lines, respectively, using a helper-dependent adenoviral vector gene transfer [8]. Similar genetic control was also reported for gyrate atrophy-iPSC-RPE, where restoration of the *OAT* gene in the iPSC using bacterial artificial chromosome-mediated homologous recombination resulted in normalisation of *OAT* enzyme activity in iPSC-RPE [6,77].

Once the clinically-relevant cellular phenotype and readouts can be defined for the specific genetic variant and mutation, high throughput analysis will need to be developed to enable a large number of therapeutics to be screened across the potential thousands of genetic variant cell lines from patients with IRDs. Although this is not yet possible, there are several examples where this has been reported on a smaller scale (see below). Further investigations are needed to determine if late-onset retinal or macular degeneration, such as reticular pseudodrusen (Figure 5), basal laminar drusen (Figure 5), pattern dystrophy of the RPE, vitelliform macular dystrophy and mutations arising from *EFEMP-1* (Doyne honeycomb retinal dystrophy, Figure 5), *TIMP-3* (Sorsby fundus dystrophy) or *CTRP5* (late-onset retinal degeneration), can also be modelled through derivation of retinal cells from iPSCs. The readout or functional assay for each of these diseases may differ significantly because of variable environmental contribution to the disease phenotype and diverse molecular pathogenic mechanisms. For example: Sorsby fundus dystrophy may be caused by deposition of abnormal extracellular protein (TIMP-3); Doyne honeycomb retinal dystrophy may be related to activation of unfolded protein response due to misfolded fibulin-3 (EFEMP-1); and late-onset retina degeneration may be associated with abnormal intracellular protein aggregates, as well as extracellular deposition [78–81]. There are also many IRDs that affect retinal cells downstream from the photoreceptors, such as congenital stationery night blindness and X-linked retinoschisis [82]. Patient-specific iPSC-derived laminated retinal structures may be ideal for the study of pathophysiology of these inner retinal IRDs, since many of the genes involved in these diseases (e.g., *NYX*, *CACNA1F*, *GRM6*, *TRPM1*, *CABP4*, *CACNA2D4* and *RS1*) are involved in the extracellular matrix of neural retina and the synaptic interaction between photoreceptor and bipolar cells [83].

#### *3.2. IPSC for Therapeutics Development and Treatment*

#### 3.2.1. IPSC for Drug Screening

Current therapeutic modalities in IRDs are aiming to preserve residual cells or replace missing cells. Because gene therapies and many pharmacotherapies will need to be tailored for individual genetic variants or mutations, iPSCs provide an ideal platform for pre-clinical therapeutic and toxicology testing. There are several examples in both AMD and IRDs, where pharmaco- and gene therapies are tested using iPSC.

Chang *et al.* tested the protective effect of curcumin on iPSC-RPE derived from AMD patients [38]. They showed that curcumin had a beneficial effect on H2O2-induced cell death and reactive oxygen specifies generation in both control and AMD iPSC-RPE. Exposure of curcumin also increased the expression of *HO1*, *SOD2* and *GPX1* and decreased the expression of *PDGF*, *VEGF* and *IGFBP-2* in AMD iPSC-RPE. Whether this also occurred in control iPSC-RPE was not reported.

*OAT* deficiency in the RPE leads to a buildup of ornithine and a reduction in high-energy creatine phosphate. In some patients, the *OAT* enzymatic activity can be reversed by a high dose pyridoxine (vitamin B6) supplement, because of the effect of *OAT* mutation on the binding affinity to pyridoxine. Clinically, vitamin B6 responsiveness is tested in patient fibroblasts. Although based on the fibroblast assay, A226V *OAT* mutation is not responsive to vitamin B6, Meyer *et al.* has demonstrated that iPSC-RPE from this patient is, in fact, responsive to vitamin B6 based on an *in vitro* dose titration experiment. Therefore, this patient has directly benefited from iPSC disease modelling and pharmacotherapy screening.

Jin *et al.* [7] demonstrated the benefit of Į-tocopherol (vitamin E) on improving the survival of iPSC-rod precursor cells in the *RP9* mutation. They also confirmed no toxic effect from 1.6 ȝM of ascorbic acid (vitamin C) and ȕ-carotene (vitamin A) on iPSC-rod precursors affected by *RP1*, *RP9*, *PRPH-2* and *RHO* mutations. In addition to vitamins, modulators of signal pathways have also been screened using iPSC. Yoshida *et al.* showed that inhibition of mTOR (using rapamycin or PP242), activation of AMP kinase (using AICAR), inhibition of apoptosis signal-regulating kinase (using NQDI-1) and inhibition of protein synthesis (using salubrinal to inhibit eukaryotic translation initiation factor 2 subunit Į phosphatase) can reverse the increased ER stress and apoptosis and autophagy marker expression seen in *RHO* mutant iPSC-rod precursor cells [8].

#### 3.2.2. IPSC for Testing Gene Therapy

In addition to screening prospective pharmacological agents, iPSC has also been used to test the efficacy of gene therapy approaches in LCA and choroideremia, which aim to deliver the *CEP290* and *REP-1* genes, respectively, to retinal cells. CEP290 is a centrosomal protein involved in ciliogenesis and ciliary trafficking. Mutation in CEP290 leads to abnormality of the inner and outer segments of cone cells, resulting in early-onset severe visual loss. Previous ocular gene therapy used adeno-associated viral (AAV) vectors for delivery of the *RPE65* gene. However, the large size of CEP290 precludes the use of AAV, and therefore, lentivirus is necessary for gene delivery. Burnight *et al.* [64] described the method to package full-length human CEP290 into a lentiviral vector and demonstrated restoration of a ciliogenesis defect in LCA patient-derived fibroblasts. Although they also demonstrated expression of wild-type CEP290 after lentiviral transduction of the iPSC-photoreceptor precursor cells, they did not examine the impact of this on cone development and the formation of inner or outer segments. In contrast, Vasireddy *et al.* [84] showed successful transfection of AAV2 carrying full-length human *REP-1* cDNA into iPSCs rather than transfection into differentiated retinal cells, the presumed target cell primarily affected in choroideremia. They used a prenylation assay to confirm restoration of *REP-1* function following AAV2. *REP-1* infection of the iPSC. There was also improved trafficking of RAB27 in iPSCs, because of prenylation by viral-derived REP-1. The efficiency and toxicity of iPSC transduction was compared to patients' fibroblasts, but not patient-derived iPSC-RPE. These two examples demonstrate the potential of iPSC in pre-clinical studies of patient-specific gene therapy.

#### 3.2.3. iPSC for Cellular Therapy

Cell therapy for retinal disease aims to replace (1) photoreceptors and/or (2) supporting cells that provide trophic and metabolic support to prevent further degeneration of remaining photoreceptors. The main challenges in establishing clinically acceptable cell therapy for retinal disease are: patient selection, surgical technique, carrier system and choice of cell source. Each of these questions has its complexity in several dimensions. However, the use of iPSCs as a source for retinal cell transplantation is one of the most exciting, but also complex and challenging, issues facing scientists, clinicians, pharmaceutical companies and regulatory authorities.

In comparison to human ESC, the use of iPSC-derived cells for cell therapy has the additional requirement of quality control associated with the surgical procedure in harvesting patient somatic cells, isolation of a single cell type from the tissue biopsy, reprogramming vectors and techniques, methods of genomic editing in the case of IRDs and techniques of hiPSC clone selection and storage. Some of these steps have been defined in standard operating procedures for the production of clinical-grade iPSCs from retrovirus reprogramming. However, this is yet to be established for the numerous published non-integrating reprogramming methods.

Derivation of clinical-grade retinal cells from hESC has been conducted in GMP facilities, and it is currently being used in phase I/II clinical trials as hESC-RPE suspension for Stargardt disease, geographic atrophy due to AMD and myopic atrophic macular degeneration (Clinical Trial: NCT01469832, NCT01345006, NCT01344993, NCT02122159) and hESC-RPE patch graft for wet AMD (NCT01691261). There is only one human trial using iPSC-RPE, at the RIKEN Center for Developmental Biology (CDB), Japan. In the CDB trial, a monolayer of iPSC-RPE without substrate is used to treat neovascular AMD after a course of ranibizumab injections. One patient, in her 70s with wet AMD, has already received her own iPSC-RPE as a 1.3 × 3.0-mm cell patch at Kobe City Medical Center General Hospital in September [85]. This group has published data to support the *in vitro* and *in vivo* function of the iPSC-RPE in performing the visual cycle [68]. They also demonstrated suppression of the tumour-forming potential of iPSC by iPSC-RPE following subcutaneous transplantation in NOD/Shi-*scid*/IL-2RȖnull (NOG) mice. They postulated that pigment epithelium-derived factor secreted from iPSC-RPE or RPE of host tissue can cause apoptotic cell death of iPSC [86]. Following from this, the tumourigenicity of iPSC-RPE was also tested in nude, severe combined immune deficiency (SCID), non-obese diabetic (NOD)-SCID and NOG mice in the subretinal and subcutaneous location. They observed no tumour formation at 6–12 months following transplant [71]. Immune reaction to autologous iPSCs has not been studied, but Kamao *et al.* [70] demonstrated a lack of immune response after one year when nonhuman primate iPSC-RPE was transplanted as an autograft into the subretinal space. This study also confirmed no evidence of tumour formation following monkey iPSC-RPE autograft [70]. The RIKEN CDB has already enrolled patients with neovascular AMD for iPSC-RPE transplantation after the disease is stabilised with anti-VEGF therapy. Although the functionality of the graft seems to be well characterised, significant work still needs to be done in developing the optimal surgical instrumentation, technique and approach in resurfacing the RPE in the submacular space and patient selection for optimal visual and anatomical outcome. It is not known if iPSC-RPE survive, as multiple small patches of epithelial monolayer are superior to cell suspension and not inferior to a single large sheet of epithelial-substrate complex that can cover the entire macular region. The importance of restoring damaged Bruch's membrane during iPSC-RPE replacement therapy in AMD cannot be underestimated, as this is considered as an important aspect of the pathophysiology of AMD [87].

*In vitro* genomic editing or mutation repair of harvested somatic cells, iPSCs or iPSC-derived retinal cells will provide patients with IRDs the opportunity to receive autologous cell therapy. For some IRDs that develop late-onset degeneration, genetic mutation correction in the patient-specific iPSCs may not be necessary [2]. This is relevant in the situation where the strategy is to transplant (1) iPSC-derived retinal supporting cells that are not affected by the mutation (e.g., RPE cells for ABCA4 retinopathy); or (2) iPSC-derived retinal cells affected by the mutation, but it has delayed non-cell autonomous effects due to reduced trophic factor release (e.g., rod precursor cells for the cone preservation function through the release of rod-derived cone viability factor) [88]. For replacement of cone photoreceptor cells in cone dystrophy or RPE in RPE dystrophy, *in vitro* genomic editing may be required to allow iPSCs to differentiate into mature photoreceptors or the RPE phenotype and to ensure long-term survival of the autograft. Examples of *in vitro* gene therapy testing have been described in the previous section. Future strategies may include site-specific transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing techniques, where the mutation is edited through a double-strand break (DSB) and off-target mutagenesis minimised by single-guide RNA.

Following induction and subsequent homology-directed repair (HDR), the corrected gene will remain under the normal endogenous expression control elements.

#### **4. Conclusions**

Since the description of iPSCs in 2006, there has been an exponential increase in the translation of this technology towards understanding disease mechanisms and the discovery of therapeutics. The full potential is yet to be realised because of the complexity and variations in reprograming technology and retinal differentiation protocols. The relationship between the clinical disease phenotype and the molecular and cellular features of specific genetic variants in iPSC-derived retinal cells is still poorly understood. These issues will need to be resolved for iPSC-derived retinal tissue to become clinically relevant and useful in modelling retinal dysgenesis and degeneration. Standardisation and development of high throughput technology to interrogate specific retinal progeny derived from iPSCs will facilitate screening of genetic mutation and testing of pharmacologic and gene therapy in rare IRDs. Early data from several publications show that this may have a direct benefit to the patient [2,3,64,84]. iPSCs as a source of autologous cells are an attractive option, but there is a significant barrier to overcome for this to become scalable to treat large numbers of patients. However, progress in cell culture automation and refinements of reprogramming methods will undoubtedly facilitate the translation of iPSC-derived tissue into clinically applicable personalized cell therapy. Despite the mountain of challenge, the escalating costs of biologic therapy for treating neovascular and atrophic AMD and the suffering from irreversible childhood blindness due to IRDs, further ophthalmic translational research in iPSCs is worthy of the costly investment.

#### **Acknowledgments**

The authors would like to thank Lori Bonertz for proof reading the manuscript.

#### **Author Contributions**

Fred K. Chen, Samuel McLenachan, Michael Edel, Lyndon Da Cruz, Peter J. Coffey and David A. Mackey contributed reagents/materials/analysis tools; Fred K. Chen wrote the paper; Samuel McLenachan, Michael Edel, Lyndon Da Cruz, Peter J. Coffey and David A. Mackey proof read and made amendments to the paper.

#### **Conflicts of Interest**

The authors declare no conflict of interest.

#### **References**

1. Ding, X.; Patel, M.; Chan, C.C. Molecular pathology of age-related macular degeneration. *Prog. Retin. Eye Res.* **2009**, *28*, 1–18.

### **Patient-Specific iPSC-Derived RPE for Modeling of Retinal Diseases**

#### **Huy V. Nguyen, Yao Li and Stephen H. Tsang**

**Abstract:** Inherited retinal diseases, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world. Currently, treatments for these conditions are limited. Recently, considerable attention has been given to the possibility of using patient-specific induced pluripotent stem cells (iPSCs) as a treatment for these conditions. iPSCs reprogrammed from adult somatic cells offer the possibility of generating patient-specific cell lines *in vitro*. In this review, we will discuss the current literature pertaining to iPSC modeling of retinal disease, gene therapy of iPSC-derived retinal pigmented epithelium (RPE) cells, and retinal transplantation. We will focus on the use of iPSCs created from patients with inherited eye diseases for testing the efficacy of gene or drug-based therapies, elucidating previously unknown mechanisms and pathways of disease, and as a source of autologous cells for cell replacement.

Reprinted from *J. Clin. Med*. Cite as: Nguyen, H.V.; Li, Y.; Tsang, S.H. Patient-Specific iPSC-Derived RPE for Modeling of Retinal Diseases. *J. Clin. Med*. **2015**, *4*, 567–578.

#### **1. Introduction**

Human vision is vital for nearly every major activity of daily living, and degeneration of one of the responsible cell types, the retinal pigmented epithelium (RPE), leads to severe visual impairment and blindness. RPE cells exist as a monolayer located at the back of the eye between the retina and Bruch's membrane and is essential for photoreceptor function and survival. Retinal diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) result in clinical pathophysiology characterized by progressive loss of RPE. The adult retina does not intrinsically regenerate, so RPE degeneration may ultimately lead to blindness. Anti-vascular endothelial growth factor (VEGF) therapy has been shown to slow the rate of vision loss, but it has no more than a 10% rate of effectiveness in all AMD cases [1]. There are no other treatments currently established for RPE degenerative diseases, so the disease burden of these conditions are expected to continue to rise. AMD and RP are both leading causes of blindness in the developed world, affecting up to one third of people over the age of 75. Among the elderly, blindness is feared more than any other illness outside of cancer. Currently, nine million Americans have been diagnosed with AMD, and its incidence is expected to double within a decade, affecting 20% of Americans between the ages of 65 and 75 years [2].

Cell transplantation into the human retina has the potential to restore vision and provide treatment in diseases like AMD and RP with significant RPE loss. Since these diseases spare the inner retina and optic nerve, retinal transplantation has focused on replacement of the photoreceptors and RPE. Retinal stem cells have been shown to be efficient at integrating into the degenerative host retina [3]. Replacement of damaged RPE in patients with AMD is now being offered [4]. In 2011, the U.S. Food and Drug Administration advanced the treatment of macular degenerations by approving clinical trials using embryonic stem (ES) cell-derived RPE transplants [5].

Induced pluripotent stem (iPS) cells reprogrammed from adult somatic cells offer the possibility of generating patient-specific cell lines *in vitro*. As a platform to study patient-specific targeted disease cells, iPS cells (iPSC) have exciting potential in regenerative medicine and human disease modeling. As one example, after human embryonic stem cells were shown to be able to produce 3-D optic vesicle-like structures displaying a precise apical-basal orientation [6], human iPS cells were used to also create optic vesicle-like structures which self-assembled into rudimentary, multilayered retinal tissue [7]. Similarly, human iPSCs have been used to model primary open-angle glaucoma (POAG). The optineurin E50K mutation is a mutation currently affirmed as causative for POAG, and human iPSCs have been created with the E50K mutation to study the molecular and cellular characterization of POAG onset [8]. hiPSC modeling has also suggested that normal-tension glaucoma via TBK1 gene duplication is due increased levels of LC3-II, a key marker of autophagy [9].

Specifically, iPS-based therapies holds great promise for treating retinal degenerative diseases, given the advantage of ocular immune privilege and the ease of ocular non-invasive imaging. Moreover, iPS cell technology facilitates investigations of pathophysiological mechanisms of genetic mutations and testing of gene therapy vectors on RPE-based disease models. Indeed, iPS-derived RPE (iPS-RPE) can be reproducibly isolated and closely monitored both morphologically and functionally before experiments, effectively minimizing variability in the timing of differentiation. In addition, RPE, unlike many other human cell types, has a well-described culture standard, which ensures proper controls [4,10].

The *in vitro* phenotypes of disease-specific iPS-derived cells can be used to bridge the gap between the clinical phenotype and molecular or cellular mechanisms, creating new strategies for drug screening, and developing novel therapeutic agents [11]. Human iPS cell-based disease models can prove that a disease is caused by a genetic mutation, hypothesize potential treatment options before using more expensive animal models [12], and assist in the development of novel treatments for clinical trials [13–15].

#### **2. iPSC Disease Modeling**

#### *2.1. Use of iPS-Derived RPE Cells for Cell Therapy*

The eye is an ideal site for stem cell therapies. First, it is considered an immune privileged organ since the inflammatory responses of the eye differ significantly from those in other tissues. Second, the eye allows for easy accessibility for monitoring and imaging. Third, in the case of serious complications, the eye as a unit can be removed, due to its relative isolation from other body systems. Stem cells in turn are an appealing option for retinal cell replacement due to their pluripotency and potentially unlimited capacity for self-renewal. Currently, there are two leading options for stem cells in retinal transplantation: (i) embryonic stem cells (ESCs), which can be isolated from developing embryos four to five days after fertilization; and (ii) induced pluripotent stem cells (iPSCs), which can be created from adult cells by the viral transduction of transcription factors [16]. However, due to the ethical and technical concerns with using ESCs, iPSCs have largely been favored for retinal transplantation.

iPSCs in particular offer a compelling alternative approach for stem cell therapy. When derived from the transplant recipient, autologous iPS-derived cells reduce the risk of post-transplant rejection and obviate the need for immunosuppression after transplantation. The well-described iPSC culture standards also aid in the development of functional testing and optimization studies. Likewise, RPE transplantation into the retina poses fewer challenges than other kinds of cell transplantation since routine culture of RPE cells has been well described [17,18]. RPE monolayers exist in an easily identifiable hexagonal structure and can be isolated and transferred to a variety of substrates without the need for synaptic integration. Subsequently, studies on RPE replacement therapies using pluripotent stem cells have progressed rapidly. A multicenter trial focusing on the treatment of dry macular degeneration and Stargardt macular dystrophy showed that purified human ESC-derived RPE can be subretinally injected into patients with good results [5]. This is also possible since the retina normally enjoys relative immune privilege, due to the blood-retinal barrier. This barrier consists of non-fenestrated retinal vasculature ensheathed by pericyte and astrocyte processes on the inner aspect and by tight junctions between RPE on the outer aspect. In a healthy state, this blood-retinal barrier provides protection to transplanted cells beneath the retina from the systemic immune system. However, in a diseased RPE state, the monolayer is disrupted due to faulty tight junctions and the retina may also become much more pro-inflammatory [19,20]. Therefore, cells transplanted into a diseased retina are likely to be at a higher risk for rejection, so autologous iPSC transplantation represents the best stem cell approach for curing degenerative retinal diseases. In fact, hiPSC-derived RPE has recently been approved in Japan for use in patient safety trials for treatment of AMD [21].

Currently, human iPS-derived RPE (iPS-RPE) experiments are largely confined to animal models. In 2009, Carr *et al.* performed subretinal injections of dissociated human iPS-RPE into Royal College of Surgeons (RCS) rats and observed restoration of RPE phagocytotic function, as measured by intracellular RHO staining, and long-term preservation of visual function, as measured by optokinetic head-tracking [22]. Another model is the RPE-specific protein 65 kDA (RPE65) mutant mouse model, which is used to study Leber congenital amaurosis (LCA) and RP since the RPE 65 defect leads to a faulty isomerase which can no longer convert the chromophore necessary for rhodopsin to detect light [23]. In 2012, Li *et al.* injected dissociated human iPS-RPE into the subretinal space of the RPE65 mutant mouse model and showed integration of the transplant with host RPE, as well as a modest improvement of visual function as measured by electroretinogram (ERG) [10]. The *Mfrprd6*/*Mfrprd6* (rd6) mouse, which has a deletion in the Membrane Frizzled-Related Protein (*Mfrp)* gene, is another widely used model. The resulting MFRP protein, an RPE-specific membrane receptor of unknown function, is abnormal and the mice exhibit progressive retinal degeneration, making the model a preclinical and progressive model of RP [24]. In a recent study, subretinal injections of AAV-packaged wild-type *Mfrp* into rd6 mice showed improvement in visual function and RPE cell layer thickness [25].

The most advantageous aspect of iPSC based therapy is the potential of autologous transplantation, which intends to address the problem of immune rejection. Despite the assumption that these autologous cells should not provoke an immune response in the recipient from whom the cells were derived, there have been conflicting reports that raise some concern of the immunogenicity of iPSCs. In a recent study, teratomas originating from subcutaneous injection of murine derived iPSCs were found to have abnormal gene expression in some cells, which elicited a T-cell dependent immune response in syngeneic mice [26]. However, when Guha *et al.* transplanted various types of murine iPS-derived cells to a site under the kidney capsule of B6 mice, they found no evidence of immune response to the iPSCs, no increased T cell proliferation *in vitro*, no rejection of syngeneic iPSC-derived cells after transplantation, and no antigen-specific secondary immune response [27]. Findings by Liu *et al.* in 2013 suggests that iPSC immunogenicity increases with *in vivo* differentiation, as the authors observed immune responses after transplantation of differentiated iPS-derived cardiomyocytes but no response when transplanting undifferentiated iPSCs [28]. In contrast, Morizane *et al.* performed a direct comparison between autologous and allogeneic transplantation of iPS-derived neural cells in brains of non-human primates and found that the autologous transplantation of iPS-derived neurons caused only a minimal immune response in the brain, while the allografts elicited an acquired immune response [29]. Moreover, a higher number of dopaminergic neurons survived in autografted iPS-derived cells, which further support their use. Taken together, these findings reveals that different cell types derived from iPSCs might have distinctive immunogenicities in their syngeneic hosts. For the development of human iPS-based cell therapy, there remains still a challenge to evaluate the immunogenicity of human iPS-derived cells in an autologous human immune system.

#### *2.2. Progress of RPE Disease Modeling Using iPSCs*

Human iPS cells are useful for modeling RPE disorders since they can be isolated, expanded, re-seeded, and closely monitored both morphologically and functionally prior to testing [30]. Phenotypes of patient-specific iPS cells may differ from those from a mouse model with the same mutation [25], underscoring the necessity for multiple models of human genetic diseases. Since differences in phenotypic expression can be observed among species with the same genetic mutation, it is important to study patient-specific cell lines as a complement to mouse models.

The first retinal disease modeled with patient-specific iPS cells is Best vitelliform macular dystrophy (BVMD) [13]. Caused by a defect in the RPE gene BEST1, which results in the subretinal accumulation of photoreceptor waste products, BVMD is characterized by central vision loss due to photoreceptor death. Singh *et al.* created iPS-RPE from affected patients and compared them with those created from unaffected siblings. From their model, they concluded that the pathophysiology of the disease included delayed rhodopsin degradation after photoreceptor outer segment feeding, as evidenced by disrupted fluid flux and increased accumulation of autofluorescent material [13]. This hiPSC model of BVMD possessed functional deficiencies consistent with the clinical features of the disease and was used to characterize clinically relevant disease phenotypes for BVMD.

iPS-derived RPE cells have also recently been used to model and study the pathophysiology of AMD. While genome-wide association studies (GWAS) have identified risk alleles for the disease, such as the ARMS2 and HTRA1 genes, how these alleles lead to pathology is still unclear. There is currently a lack of appropriate models for AMD; autopsy eyes from end-stage patients already possess terminal changes and cannot be used to determine how abnormal gene expression can lead to RPE pathology, and mice do not have maculae. To bypass these obstacles, Yang *et al.* created a model for AMD by obtaining patient-specific iPS-derived RPE and pharmacologically accelerating the aging process with treatment of bisretinoid N-retinylidine-N-ethanolamine (A2E) and blue light [12]. From a proteome screen of multiple A2E-aged patient-specific iPS-RPE lines, impaired superoxide dismutase 2 (SOD2) function was identified as a high risk factor for developing AMD. Using their iPS model, the researchers concluded that the ARMS2/HTRA1 risk alleles decreased SOD2 defense, making RPE more susceptible to oxidative damage and thus contributing to AMD pathogenesis.

#### **3. Personalized Medicine: Patient-Specific iPSC-Based Therapy**

#### *3.1. Development of Gene Therapy on Patient-Specific iPSCs*

Gene-corrected patient specific iPSCs offer a unique approach to autologous therapies, which have the potential to treat a wide range of acquired and inherited diseases. However, gene targeting in human pluripotent stem cells has been exceedingly difficult [31]. One approach is using recombinant adeno-associated virus (AAV) as a gene transfer vector to carry the missing gene into affected cells. Vasireddy *et al.* published the first study which successfully transduced iPSCs developed from a patient with choroideremia with AAV subtype 2 (AAV2) [32]. Choroideremia is an inherited disorder due to loss of the *CHM* gene and the resulting Rab Escort Protein 1 (REP-1), which leading to degeneration of the choroid and retina and blindness by the 2nd decade of life. Research moving towards clinical trials has been stymied due to a lack of an animal model with similar functional and morphological features as the human retina, since the knockout of the murine *Chm* is lethal. The authors developed a preclinical model of choroideremia using iPSCs and successfully transduced wildtype human *Chm* cDNA into these cells using AAV2 mediated therapy. They observed a functional restoration of REP-1 enzymatic activity and protein trafficking, showing that their gene therapy was successful and that iPSCs can be used as a preclinical model for choroideremia [32].

The development of genome editing tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system have facilitated gene targeting in human iPSCs [33]. These tools use double strand break induction and subsequent homology-directed repair to edit the mutations in the patients' genomic DNA, so that the corrected gene will remain under the normal endogenous promoters and enhancers. Thus, compared to conventional viral-mediated gene replacement, gene editing using ZFNs, TALENs, or the CRISPR system can avoid genetic expression in inappropriate cell types as well as incorrect levels of expression [34].

The CRISPR-Cas system has several advantages over ZFNs and TALENs for enhancing gene targeting efficiency. Most CRISPR-Cas subtypes target DNA directly, suggesting the possibility of engineered, RNA-directed gene editing systems. This usage of easily generated RNA guides avoid the need for repeated protein design, which sets CRISPR-Cas apart from ZFNs and TALENs, which use protein-based DNA targeting motifs. Using the CRISPR-Cas9 system, Mali *et al.* targeted the endogenous AAVS1 locus in human iPSCs to achieve homology-directed repair of fibroblast-derived iPSCs [35]. Recently, Hou *et al.* developed a CRISPR-Cas system from *N. meningitides* to generate accurately targeted clones in human iPSCs with increased efficiency as compared to TALENs [36]. There several concerns with CRISPR-Cas technology in human genome editing, primarily off-target DNA cleavage [37]. However, recent experiments showed that "nickases", or enzymes that cleave only a single strand of DNA in DNA repair, can increase the specificity and safety of the CRISPR-Cas9 system [38].

#### *3.2. Gene Therapy on Patient-Specific iPSC-Derived RPE Cells*

With the aim of correcting genetic defects, gene therapy has been attempted not only on patient-specific iPS cells, but also RPE cells derived from these cell lines. A proof of concept study was performed by Cereso *et al.* which used a hybrid vector comprised of AAV2 and AAV5 (AAV2/5) to mediate gene therapy to the RPE derived from iPS created from a choroideremia patient [39]. The authors successfully developed a human iPS-derived retinal cell model of choroideremia, performed gene therapy on the iPS-RPE, and showed that AAV2/5-mediated therapy could potentially restore RPE phenotype. Working with MFRP, Li *et al.* also showed that patient-specific iPS-RPE could be a recipient for gene therapy [25]. The researchers applied the AAV8 vector expressing human MFRP to iPS-RPE from patients with MFRP mutations and confirmed that gene therapy led to restoration of RPE phenotype, specifically with regards to actin organization. These studies suggest that gene therapy using AAV vectors can be applied to RPE created from patient-specific iPS for retinal diseases without previous models, and that these diseases may be potential targets for additional gene therapy trials.

#### *3.3. Transplantation of iPSC-Derived RPE Cells*

Considerable attention has been paid to the potential of human iPSCs as a source for regenerative medicine, disease modeling, and drug testing. In particular, the limitations in existing treatments for AMD have led to attention being given to alternative approaches in which damaged RPE is replaced by healthy RPE. In a recent landmark trial in Japan, patient specific iPSC-derived RPE cells were transplanted for the first time into a human patient with AMD. Clearance for a human trial was given after Takahashi *et al.* showed that transplantation of iPSC-derived RPE did not provoke an immune reaction nor lead to tumor growth in monkeys or mice [40]. Autologous iPSCs were created from the patient's skin cells and then differentiated into RPE so that they would grow in a monolayer without the use of synthetic scaffolds or matrices. To achieve this, iPSC-RPE were seeded onto type I collagen gel on a Transwell insert. After the RPE reached confluence, collagenase was applied to dissolve the collagen gel and leave a sheet of RPE. A 1.3 millimeter by 3.0 millimeter cut of this sheet was then grafted into the patient's retina following excision of her existing damaged RPE.

This marks the first clinical trial on humans using iPSCs. The safety and feasibility of using iPSCs from patients to treat their blindness is still being established, but this trial holds great potential for the advancement of translational medicine in retinal disease.

#### **4. Future Directions**

Patient-specific iPSCs have been shown to not only complement animal models of human disease, but also function as an excellent model in their own right. Patient-specific cell lines created from somatic cells from patients with inherited eye diseases can: (i) provide a window for testing the efficacy of gene or drug-based therapies; (ii) elucidate previously unknown mechanisms and pathways of disease; (iii) demonstrate the pathogenicity of unusual mutations in individual patients; and (iv) enable researchers to optimize parameters for successful cell replacement therapy *in vitro*. Skin-derived iPSCs can be used to investigate the function or dysfunction of a mutant gene product in tissues such as retina that are inaccessible to molecular analysis in living patients [41]. Finally, gene therapy tools such as ZFNs, TALENs, and the CRISPR-Cas system are rapidly improving the prospects of restoring the function of diseased RPE from patients with inherited retinal diseases. These patient-specific iPS-RPE, after undergoing gene therapy, can be optimized to become transplantable retinal cells, with the goal of restoring sight to patients with no other therapeutic options.

However, despite these advances, improvements still must be made in reprogramming, differentiation, and cell characterization protocols before employing this technology in clinical transplantation trials. In moving from animal models to human trials, potential safety issues must be carefully addressed. The use of potent oncogenic transgenes such as c-myc and Klf4 in the reprogramming process as outlined by Yamanaka is one area of concern [16]. If these transgenes are not silenced or are reactivated after reprogramming, genomic instability may result and not only confound results of disease modeling studies but also cause tumor formation after transplantation. To this end, iPS reprogramming protocols are still being optimized. An alternative reprogramming protocol by Yu *et al.* obviates the use of oncogenic transgenes by using a combination of Oct4, Sox2, Nanog, and Lin28 [42]. The methodology for generating iPSCs has markedly improved and now integration-free iPSCs, without transgene insertion in the host genome, can be obtained using plasmid vectors, RNA viruses, or mature microRNAs [43–47]. Integration-free iPSCs appear ideal since exogenous genes integrated in the host genome may affect the genetic properties of the iPSCs generated and thus modify the resulting cellular phenotypes of differentiated progeny. Additional studies are also required to ensure that the risk of rejection is significantly reduced in patient-specific iPSCs, given that immune rejection when certain tissues derived from iPSCs were transplanted into syngeneic murine hosts have been reported [26].

ESCs are still the gold standard for *in vitro* pluripotency. A significant concern of using iPSCs in development of therapies is still whether they are truly equivalent to ESCs. For example, key differences between iPSCs and ESCs in transcribed genes, epigenetic landscape, differentiation potential, mutational load, and premature senescence has been described [48]. If iPSCs cannot closely replicate ESCs, the results from studies using iPSCs must be interpreted with this in mind. Significant differences between iPSCs and ESCs may hinder the translation of study results from an *in vitro* iPSC-based disease model to human disease.

A further step likely to accelerate the integration of iPS technology in regenerative medicine is the development of industry and biotechnology collaboration in order to develop large-scale stem cell production [49]. In this way, availability of iPS-based technology will increase, making them more widespread in investigative and translational studies in the future. Patient-specific iPS-derived cells offer the hope of slowing progression or improving visual function for patients with currently untreatable retinal diseases. In addition to curing blindness, stem cell transplantation in the eye can also be seen as a model system for investigating cell-based treatments for other degenerative disorders of the CNS.

#### **5. Conclusions**

Stem cells have revolutionized the field of human cell culture because they provide an immortal population of pluripotent cells which can theoretically differentiate into any cell type in the body. This technology, when applied to retinal cells, has the promise to make significant contributions to our understanding of the most pressing blinding diseases of our time. Stem cells also allow for the development of therapies for exceedingly rare retinal conditions which currently have little to no funding for research. In particular, patient-specific iPSCs represent an excellent tool for modeling retinal disease since they can be generated from adult somatic cells, thus avoiding the ethical considerations involved with using embryonic stem cells. iPSCs will continue to be a sustainable method to model disease as gene therapies, drug therapies, and transplantable retinal cells continue to be developed for inherited retinal disorders.

#### **Acknowledgments**

Stephen H. Tsang is a member of the RD-CURE Consortium and is supported by Tistou and Charlotte Kerstan Foundation, NIH R01EY018213, the Research to Prevent Blindness Physician-Scientist Award, the Schneeweiss Stem Cell Fund, New York State (N09G-302 and N13G-275), and the Foundation Fighting Blindness New York Regional Research Center Grant (C-NY05-0705-0312), the Joel Hoffman Fund, Gale and Richard Siegel Stem Cell Fund, Charles Culpeper Scholarship, Laszlo Bito and Olivia Carino Foundation, Irma T. Hirschl Charitable Trust, Bernard and Anne Spitzer Stem Cell Fund, Gertrude Rothschild Stem Cell Foundation, and Gebroe Family Foundation. Huy V. Nguyen is supported by the RPB Medical Student Fellowship.

#### **Author Contributions**

Huy V. Nguyen, Yao Li and Stephen H. Tsang performed research and wrote the paper.

#### **Conflicts of Interest**

The authors declare no conflict of interest.

#### **References**

1. Rosenfeld, P.J.; Brown, D.M.; Heier, J.S.; Boyer, D.S.; Kaiser, P.K.; Chung, C.Y.; Kim, R.Y*.* Ranibizumab for neovascular age-related macular degeneration. *N. Engl. J. Med.* **2006**, *355*, 1419–1431.

### **Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface**

### **Ricardo P. Casaroli-Marano, Núria Nieto-Nicolau, Eva M. Martínez-Conesa, Michael Edel and Ana B. Álvarez-Palomo**

**Abstract:** The integrity and normal function of the corneal epithelium are crucial for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio replacement—cultured limbal epithelial transplantation (CLET) and cultured oral mucosal epithelial transplantation (COMET)—present very encouraging clinical results for treating limbal stem cell deficiency (LSCD) and restoring vision. Another emerging therapeutic approach consists of obtaining and implementing human progenitor cells of different origins in association with tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal or induced pluripotent stem cells (IPSCs), represent a significant breakthrough in the treatment of certain eye diseases, offering a more rational, less invasive, and better physiological treatment option in regenerative medicine for the ocular surface. This review will focus on the main concepts of cell-based therapies for the ocular surface and the future use of IPSCs to treat LSCD.

Reprinted from *J. Clin. Med*. Cite as: Casaroli-Marano, R.P.; Nieto-Nicolau, N.; Martínez-Conesa, E.M.; Edel, M.; Álvarez-Palomo, A.B. Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface. *J. Clin. Med.* **2015**, *4*, 318–342.

#### **1. Introduction**

The ocular surface is mainly composed of the cornea and the conjunctiva with their epithelia. The cornea is the primary refractive element at the anterior surface of the eye that is responsible for approximately two-thirds of its total optical power. Basically, the cornea is composed of five well-defined layers (Figure 1). It consists of an outermost stratified, squamous and non-keratinized epithelial layer (corneal epithelium) limited posteriorly by Bowman's layer. The underlying stroma, which accounts for about 90% of the middle thickness of the cornea, comprises aligned arrays of collagen fibrils interspersed with cellular components (keratocytes) and it is this highly organized arrangement of lamellae that is responsible for the cornea's transparency. The stroma is separated from the endothelial layer (corneal endothelium) by Descemet's membrane, which acts as a basement membrane for these endothelial cells. The corneal endothelium is a single cuboidal layer of metabolically active cells that are in direct contact with the aqueous humor in the anterior chamber. These cells help to maintain corneal transparency by actively pumping water out of the stroma [1]. The corneal epithelium has a key role in keeping the cornea transparent and free of blood vessels and, to this end, presents permanent repair phenomena essential for the conservation of the cornea's physiology [1–3]. The homeostasis of the corneal epithelium is crucial to maintaining the structural integrity of the ocular surface, the transparency of the cornea and visual function.

#### *1.1. Limbal Stem Cells*

It has been observed that progenitor cells responsible for the continual renewal of the corneal epithelium are located in the basal layers of the sclerocorneal limbus. The human limbus—the circumferential anatomic area (approximately 1.5 mm wide) that separates the clear cornea from the opaque sclera, which is covered by conjunctiva—serves as the "reservoir" for the stem cells and also provides a barrier to the overgrowth of conjunctival epithelial cells and its blood vessels onto the cornea [1–3] (Figure 1). Due to their particularities, the *limbal stem cells* (LSCs) have a crucial role in maintaining the integrity and in the renewal events of corneal epithelium. Their main features are highlighted: it is their behavior as oligopotent progenitor cells, with high nuclear-cytoplasmic ratio a slow cell cycle, and a high proliferative potential that adds its great capacity for self-renewal by asymmetric division [3–5]. In the limbus, it is possible to identify several cell subpopulations of different progenies (typical progenitors and amplifying cells at different stages of differentiation), melanocytes, antigen-presenting and mesenchymal cells, vascular elements and nerve endings that form a specialized and unique environment called *niche*. This particular microenvironment is considered responsible for the proliferative and self-renewal cellular characteristics of the limbal region [2,3,6]. The LSC niche is an anatomically defined area that is thought to provide a variety of factors, such as physical protection, survival factors and cytokines and is deemed essential to the maintenance of the "stemness" of the stem cell population while preventing entry into differentiation [2,6]. Within the niche, LSCs maintenance and function are controlled in a particular environment by several elements, including extracellular matrix components, cell adhesion molecules, and growth and survival factors secreted by stromal fibroblasts, mesenchymal stem cells and blood capillaries [6]. To date, four limbal anatomic structures have been proposed as the corneal stem cell niche [2,6], Palisades of Vogt, limbal epithelial crypts [7], limbal crypts and focal stromal projections [8].

**Figure 1.** The corneal limbus is the circumferential anatomic area, approximately 1.5 mm wide, which separates the clear cornea from the opaque sclera (**a**); The limbal region represents the "reservoir" for LSCs in the ocular surface. In a cross-section of the human cornea stained with hematoxylin-eosin, (**b**) to (**d**), details of its main layers can be observed. The cornea is composed of a stratified non-keratinized squamous epithelial layer (epithelium), the stroma and an endothelial cuboidal layer (endothelium) (**b**); The corneal epithelium (48 to 55 μm thick) consists of the outermost layer, which presents five to seven stratified cell layers (**c**), limited posteriorly by Bowman's layer (10 to 12 μm thick; c, asterisk). The stroma (480 to 510 μm thick; b), composed of compacted collagen lamellae and keratocytes (c and d), offers transparency and scaffolding to maintain the shape of the cornea in its middle portion. The stroma is separated from the endothelium (about 5 μm thick; d, large arrows) by Descemet's membrane (8 to 10 μm thick; d, narrow arrows), which acts as a basement membrane for the corneal endothelial cells (**d**). Bar = 150 μm for b; Bar = 25 μm for c and d.

#### *1.2. Renewal of Corneal Epithelium*

It has been shown that cell subpopulations with progenitor features, located in the deeper basal layers of the corneal epithelium, have the capacity to differentiate into post-mitotic cell populations located in the outermost epithelial layers. This continuous centripetal movement (the XYZ hypothesis)—from the peripheral deeper epithelial layers to the more central outermost layers—ensures constant renewal of the corneal epithelium and maintains its integrity [1–6,9]. The *X* component represents the anterior migration from cells of the basal epithelium of the limbal region, the *Y* component represents the centripetal migration of cells from the limbus, and the *Z* component represents the desquamation from the surface of corneal epithelium. However, this XYZ theory has recently been challenged by evidence in the mouse and other mammals suggesting that uninjured cells in the central cornea can generate holoclones with characteristics of stem cells, presenting regenerative epithelial capabilities, which may also be responsible for the maintenance of the corneal epithelium [10]. Also, in support of these controversial findings, the presence of central islands of normal corneal epithelial cells has been described in patients with apparently complete clinical absence of LSCs [11]. These interesting observations may have the following interpretation: the central basal epithelial cells of the surviving corneal epithelium present the capability to regenerate, or some LSCs remain and contribute to the maintenance of the central epithelium.

#### *1.3. Limbal Stem Cell Multipotency*

An *in vitro* study of the clonogenic capacity of epithelial cells located in the ocular limbal region revealed a progenitor cell system stratified into levels (cellular stages or "compartments") [2,6,12]. Undifferentiated small cells presenting progenitor cell features with high self-renewal capacity are found in the first compartment but they lose these characteristics as they migrate through the following compartments. Lastly, the final level contains a cell population with terminal differentiation features associated with little or no self-renewal capability. The latter cells, once their epithelial differentiation events are completed, lose their ability to self-renew and are incorporated as corneal epithelial cells on the surface of the central cornea. In this regard, some studies [12,13] concluded that epithelial cells of the limbal region can form holoclones with higher clonogenic potential, in contrast to epithelial cells from the central cornea. In addition, epithelial cells isolated from basal layers in the limbal region exhibit a high proliferative potential *in vitro* during expansion or in response to corneal injury [14], and show an undifferentiated phenotype lacking the expression of differentiated corneal cell markers such as cytokeratins 3 and 12 [15]. They have also been shown to retain labeled precursors of DNA for an extended time, in contrast to more differentiated cells that quickly lose them due a higher division rate [16]. This lack of differentiation and slow cell cycling are characteristics of the quiescent state of stem cells.

#### **2. Limbal Stem Cell Deficiency (LSCD)**

The disappearance, reduction or functional impairment of LSCs may produce a clinical state (limbal stem cell deficiency, LSCD) that can give rise to significant changes in the ocular surface. These changes include the occurrence of persistent corneal defects, epithelial keratinization, conjunctivalization phenomena with the development of newly formed vessels in the corneal tissue, and scarring. All this compromises the corneal physiology, reducing transparency and decreasing vision [1–5]. The presence of a complete loss of the corneal-limbal epithelium leads to a reactive reepithelialization by conjunctival cells, which have a high proliferative capacity. This event is followed by neovascularization, chronic inflammation with scarring of corneal stroma, causing a pronounced decrease in vision and severe discomfort (Figure 2). Furthermore, the chronic inflammatory condition not only leads to the death of more LSCs but also leaves the surviving epithelial cells unable to function properly, explaining the worsening of clinical symptoms and features over time [17–24]. In patients with severe lacrimal dysfunction syndrome (dry eye) suffering from LSCD, the conjunctival epithelium that replaced the corneal epithelium (conjunctivalization) becomes partially or totally keratinized [18,21,22,24]. Several processes and diseases (Table 1) may lead to unilateral or bilateral LSCD, and depending on its extent, the disorder can be classified as either partial or total. Chemical burns (alkalis and acids) are, however, the most frequent cause of limbal ischemia and epithelial destruction causing the loss and/or impairment of LSCs function, and are the main indication for cell-based therapy approaches [18–24].

**Figure 2.** Clinical findings related to Limbal Stem Cell Deficiency (LSCD). Limbal deficiency secondary to ocular cicatricial pemphigoid with the presence of peripheral newly formed vessels leading to a loss of corneal transparency (**a**); Limbal deficiency, secondary to a chemical burn (bleach) of the ocular surface leads to a corneal conjuntivalization and neovascularization with loss of transparency (**b**). LSCD can be treated with cell therapy techniques such as cultured limbal epithelial transplantation (CLET) or cultured oral mucosal epithelial transplantation (COMET).

**Table 1.** Main etiologies and pathological conditions for primary and secondary Limbal Stem Cell Deficiency (LSCD).

MMC, mitomycin-C; 5FU, 5-fluorouracil; UV, ultra-violet.

#### *2.1. Cell-Based Treatments for LSCD*

The concept of ocular surface reconstruction was introduced with the application of autologous conjunctiva for unilateral ocular chemical alkali burns [25]. Since then, several surgical approaches have been developed with the aim of restoring the corneal epithelium on the diseased ocular surface. In recent decades, limbal transplantation techniques using auto or allografts have been introduced as bio-replacement approaches for limbal tissues to improve and reconstruct the altered ocular surface [5]. Building on previous experience treating patients with large surface areas of burned skin, the epithelial cells of the ocular surface have been obtained by cell culture techniques for *ex vivo* expansion. Subsequently, the ocular surface was successfully reconstructed by using LSCs in patients with severe unilateral ocular surface pathology [17]. Since then, various translational approaches have been developed and optimized, with satisfactory long-term clinical results [18–23].

Treatment approaches for LSCD can be divided into three main categories [4,5,23,24]: (a) transplants and bio-replacement of tissue; (b) cell-based therapy by *ex vivo* cell culture expansion; and (c) symptomatic and alternative treatment: keratoprosthesis implantation, provisional debridement of conjunctival corneal tissue, therapeutic contact lenses and drug (steroids, anti-angiogenic drugs, tear substitutes, autologous serum) therapy [5].

*Ex vivo* expansion of LSCs is the most innovative approach for ocular surface bio-replacement (CLET: cultured limbal epithelial transplantation). From a minimally invasive biopsy (1–2 mm2 ) of the healthy limbal region (the same or the contralateral eye), an explant culture technique can be applied on a suitable substrate (such as the amniotic membrane) or by separating the epithelial layer from the fragment obtained by enzymatic treatment [19,21,22,26–29]. In the latter approach, the cells obtained are *in vitro* co-cultured on feeder-layers (3T3 murine fibroblasts growth arrested by irradiation or mitomycin-C). Once cell growth is achieved, the cell suspensions are transferred to suitable substrates, such as fibrin, collagen or biocompatible polymers. The bio-replacement is carried out after removal of most of the diseased tissue from the ocular surface [17–21]. This methodology has many advantages over the tissue transplantation techniques used to date: essentially, it requires a substantially smaller limbal biopsy, which reduces the risk of limbal deficiency in healthy donor tissue. Its other advantages include a final high cell population that is more efficiently selected, homogeneous and, theoretically, more enriched with progenitor characteristic cells [26–29]. However, enzymatic techniques involve a more complex approach, with additional manipulation of the tissue and the need for xenoproducts at different stages of cell culture production. For its part, the explant technique has certain advantages—among them its technical simplicity, the lack of xenoproducts and its cost-effectiveness, despite the heterogeneity of the cell population cultured (sclera fibroblasts, antigen presenting cells, melanocytes, conjunctival epithelium cells and others) [21,22,26–29]. It is always desirable to use autologous cells for *ex vivo* expansion to avoid the risk of immune response. However, in the presence of severe bilateral ocular pathology, the use of heterologous epithelial cells (from cadaveric or related living donor corneas) is acceptable [18–21]. Autologous oral mucosal epithelia expanded *ex vivo* have also been successfully used as an alternative source of epithelial cells (COMET: cultured oral mucosal epithelial transplantation) [30,31].

The mechanism by which cultured LSCs may restore the ocular surface is still poorly understood. Cells may replace the progenitor population, and/or "reactivate" nonfunctioning host progenitor cells by providing stimuli for growth, or change niche behavior. It has been speculated that there may be "dormant" stem cells despite clinical features of LSCD [11].

Currently, *ex vivo* expansion methods applied in cell-based therapy for clinical application are based mostly on the use of xenogeneic or allogeneic products such as murine cells for feeder layer approaches, fetal calf or bovine serum in culture media, supplements of non-human origin for cell growth and maintenance and the human amniotic membrane as a cell carrier. These products potentially carry a risk for transmitting diseases; they may induce tumorigenesis or precipitate an immunological response in the host [18,20–23,28,29]. They also show idiosyncratic biological variability that may adversely affect the quality of cultured grafts and also the final results after transplantation. Thus, there is currently a special need to investigate options for replacing potentially hazardous xenobiotic materials with others of human origin or xeno-free chemically defined media.

#### *2.2. Alternative Cell Sources for LSCD Treatment*

Corneal transplantation (penetrating keratoplasty) is considered the conventional therapy to restore the corneal tissue. However, this technique is not a viable strategy for patients suffering LSCD because it does not replace the LSCs population [32]. Cell-based therapy is the most rational approach for ocular surface bio-replacement, and the ideal cells for corneal reconstruction are autologous corneal LSCs using CLET approach. Minimally invasive biopsy of the limbal tissue from the same patient's healthy eye (unilateral disease) is the preferred method, although this source of progenitor cells is not always available. If both eyes present serious surface damage, the source of healthy LSCs will be lost; COMET is among the current therapeutic alternatives. In fact, therapy for LSCD is also rapidly evolving to include alternative cell types (of autologous or heterologous origin) and clinical approaches as treatment modalities. As a consequence, other strategies, such as the use of mesenchymal stem cells from adult tissue (bone marrow mesenchymal stromal cells or adipose derived stromal cells, among others) for cell regenerative therapy in corneal injuries, are gaining prominence at present. Other sources of cells or stem cells have been tested with regenerative aims in the ocular surface, and may be useful in situations where both eyes are affected although many of them, still without clinical use at present, but which have great translational potential (Table 2).

**Table 2.** Cell sources for *ex vivo* expansion cell-based therapy to treat Limbal Stem Cell Deficiency (LSCD).

Nevertheless, the application of CLET using human amniotic membrane (hAM) or fibrin gel as a scaffold has been clinically validated and today is the most frequently used cell-based therapy applied at clinical level in ophthalmology [18–21]. Since its introduction [17], it has been used with long-term clinical follow-up periods. Despite many differences between studies regarding inclusion/exclusion criteria, the culture methods applied, transplantation techniques, and clinical outcome measures, the overall success rate of this procedure is around 70% [18,21,23]. On the other hand, oral mucosa has also been shown to be an attractive autologous epithelial cell source for cases of severe bilateral LSCD. COMET has already been used in clinical settings, offering promising long-term results with improved vision in over half of treated patients [30–35]. However, peripheral corneal neovascularization is commonly found with this approach since oral mucosal cells have greater angiogenic potential than limbal epithelial cells [65,66]. It has been suggested that these new-formed vessels may regress following local anti-angiogenic therapy [33–35]. Further studies are needed in this regard to assess the long-term efficacy of COMET technique. In the past five years, several clinical trials have been conducted to test, compare or consolidate the application of other approaches and other sources of progenitor cells for the treatment of LSCD (Table 3).

#### **3. IPSCs and Corneal Epithelial Differentiation**

As discussed above, adult stem cells make it possible to repair and regenerate damaged epithelial tissue. In general, these cells reside in the basal layer of the epithelium, are able to self-renew continuously, and produce transient amplifying cells (TACs) that differentiate terminally after a brief period of proliferation [68–72]. However, there are limitations to LSCs transplantation therapies. On one hand, for unilateral LSCD, taking biopsies from the healthy eye carries along the risk of damaging the donor eye. On the other hand, for bilateral LSCD, allogenic transplantation presents the risk of immune rejection by the patient. In this sense, induced pluripotent stem cells (IPSCs) can be obtained from minimally invasive sources from the patient himself and be differentiated into LSCs, avoiding immune rejection problems and cell availability. The discovery of IPSCs has been one of the most significant advances in regenerative medicine in the last decade. Overexpression of a specific set of transcription factors (e.g., Oct4, Sox2, c-Myc, and Klf4; or Oct4, Sox2, Lin28 and Nanog) in adult differentiated cells can reprogram cell fate and IPSCs [68–70]. These can be differentiated into various cell types, a property that has opened up a wide range of possibilities for the investigation of cell states, the mechanisms of differentiation, pluripotency and other related cellular identities and behaviors. Contrary to embryonic stem cells, IPSCs can be created from easy access differentiated cells, such as fibroblast or keratinocytes, and allow the creation of autologous sources of different cell types for regenerative therapies or disease modeling.