Two patients enrolled in the clinical trial in order to test the safety of such cell trans‐ plantations. 50 000 viable RPE cells differentiated from the hESC line MA09 (Kliman‐ skaya *et al.*, 2006) by embryoid body formation were injected into the subretinal space of each patient's eye (see Fig. 1 for schematic overview). The cells were resuspended in phosphate buffered saline (PBS) and delivered in a region of pericentral macula that was not completely lost to the disease. The authors thought that engraftment of the cells into a completely atrophic macula was unlikely due to the loss of Bruch's membrane. The primary outcome was positive: none of the concerns related to stem cell transplantations as teratomas, rejection, or inflammation were observed. The transplanted cells attached to Bruch's membrane and persisted for the duration of the observation period. This was however possible only in one of the two patients. Moreover, clear functional visual im‐ provement was noted in the patient with Stargardt's macular dystrophy.

There are many repair strategies in spinal cord injury, as prevention of cell death by antiglutamatergic drugs, promotion of axonal regeneration, compensation of the lost myelina‐ tion or cell replacement therapy (McDonald *et al.*, 2002; McDonald & Sadowsky, 2002). Different sources and types of cells, including stem/progenitor cells (embryonic stem cells, neural progenitor cells, bone marrow mesenchymal cells) and non-stem cells (olfactory en‐ sheathing cells [OECs] and Schwann cells) have been, and/or are being tested in clinical tri‐

Embryonic Stem Cell Therapy – From Bench to Bed

http://dx.doi.org/10.5772/54368

403

As mentioned before in the case of spinal cord injury, diseases of the nervous system in‐ volve proliferation of astrocytes and loss of oligodendrocytes (OLN) and the protective myelin sheath they produce. Transplantation of oligodendrocyte precursors in different animals systems show that these precursors can myelinate axons (Groves *et al.*, 1993). Thus, derivation of oligodendrocytes from ESCs has been an important goal for cell re‐ placement therapy. The most common protocols involve an initial differentiation step to neural progenitors (Reubinoff *et al.*, 2001), followed by expansion, further differentiation, and selection. These protocols follow the differentiation steps that take place *in vivo*. Dur‐ ing development, oligodendrocytes differentiate from precursors, which migrate and pro‐ liferate, through immature oligodendrocytes, which send out processes seeking axons to myelinate, to mature myelinating oligodendrocytes that form myelin sheaths. The precur‐ sor cells are morphologically bipolar (when migrating) or stellate (after migration). These initially differentiate into immature cells that put out processes seeking axons to myeli‐ nate, and eventually form mature cells with parallel processes myelinating up to 30 dif‐

Oligodendrocytes were first efficiently derived from mouse ESCs (Brustle *et al.*, 1999), where ESCs were aggregated to embryoid bodies and plated in a defined medium that favors the survival of ES cell–derived neural precursors, followed by the expansion of progenitors in culture medium containing FGF2 and EGF, and a switch to FGF2 and PDGF to yield bipo‐ tential glial progenitors (Brustle *et al.*, 1999). These glial progenitors were transplanted into the spinal cords of rats with a genetic deficiency in myelin production, yielding myelinated fibers in the majority of animals (Learish *et al.*, 1999). Human ESCs were first shown to dif‐ ferentiate into oligodendrocytes by Zhang et al., 2001, who used a similar strategy involving FGF treatment followed by growth as neurospheres (Zhang *et al.*, 2001). They reported the differentiation of neural precursors into neurons, astrocytes and oligodendrocytes. Howev‐ er, no human oligodendrocytes were detected after transplantation of neural precursors into the brains of newborn mice, although human neurons and some astrocytes were found to

The first detailed protocol for directed differentiation of oligodendrocytes from human ESCs was published in 2005 and involved the induction of neural lineage by retinoic acid treat‐ ment, followed by expansion and selection in various media containing the differentiation factors triiodothyroidin hormone, FGF2, EGF, and insulin (Nistor *et al.*, 2005). After 42 days of culture, the desired cells were found in yellow spheroids, which upon differentiation as

als for spinal cord injury (Fehlings & Vawda, 2011).

**3.1. Differentiation to oligodendrocytes**

ferent axons (Karadottir & Attwell, 2007).

have engrafted (Zhang *et al.*, 2001).

This is the first peer reviewed study that uses human ESCs for cell therapy. Although their report is preliminary, in only two patients, and with a short-term follow-up, the results are impressive - especially considering the progressive nature of both diseases (Atala, 2012).

## **3. Treatment of spinal cord injury**

More than a decade ago, spinal-cord injury meant confinement to a wheelchair and a lifetime of medical care. Published incidence rates for traumatic spinal-cord injury in the USA range between 28 and 55 per million people, with about 10 000 new cases reported every year. Causes include motor vehicle accidents (36–48%), violence (5–29%), falls (17– 21%), and recreational activities (7–16%) (McDonald & Sadowsky, 2002). The primary in‐ jury (the initial insult) is usually due to the mechanical trauma and includes traction and compression forces. Neural elements are compressed by fractured and displaced bone fragments, disc material, and ligaments and leads to injuries on both the central and pe‐ ripheral nervous systems. Blood vessels are damaged, axons disrupted and cell mem‐ branes broken. Micro-haemorrhages occur within minutes in the central grey matter and spread out over the next few hours. Within minutes, the spinal cord swells to occupy the entire diameter of the spinal canal at the injury level. Secondary ischaemia results when cord swelling exceeds venous blood pressure. The more destructive phase of secondary injury is, however, more responsible for cell death and functional deficits. Hemorrhage, edema, ischaemia, release of toxic chemicals from disrupted neural membranes, and elec‐ trolyte shifts trigger a secondary injury cascade that substantially compounds initial me‐ chanical damage by harming or killing neighbouring cells (McDonald & Sadowsky, 2002). Glutamate plays a key part in a highly disruptive process known as excitotoxicity. It was demonstrated that glutamate, released during injury, damages oligodendocytes (Domercq *et al.*, 2005). Oligodendrocytes express glutamate receptors as NMDA (Karadot‐ tir *et al.*, 2005) and AMPA/kainate receptors (Domercq *et al.*, 1999). Up to now, the pri‐ mary approach in treatment is limitation of secondary injury by removal of damaging bone, disc, and ligament fragments to decompress the swollen cord, followed by the ad‐ ministration of the steroid methyl-prednisolone (Bracken *et al.*, 1990).

There are many repair strategies in spinal cord injury, as prevention of cell death by antiglutamatergic drugs, promotion of axonal regeneration, compensation of the lost myelina‐ tion or cell replacement therapy (McDonald *et al.*, 2002; McDonald & Sadowsky, 2002). Different sources and types of cells, including stem/progenitor cells (embryonic stem cells, neural progenitor cells, bone marrow mesenchymal cells) and non-stem cells (olfactory en‐ sheathing cells [OECs] and Schwann cells) have been, and/or are being tested in clinical tri‐ als for spinal cord injury (Fehlings & Vawda, 2011).

#### **3.1. Differentiation to oligodendrocytes**

Two patients enrolled in the clinical trial in order to test the safety of such cell trans‐ plantations. 50 000 viable RPE cells differentiated from the hESC line MA09 (Kliman‐ skaya *et al.*, 2006) by embryoid body formation were injected into the subretinal space of each patient's eye (see Fig. 1 for schematic overview). The cells were resuspended in phosphate buffered saline (PBS) and delivered in a region of pericentral macula that was not completely lost to the disease. The authors thought that engraftment of the cells into a completely atrophic macula was unlikely due to the loss of Bruch's membrane. The primary outcome was positive: none of the concerns related to stem cell transplantations as teratomas, rejection, or inflammation were observed. The transplanted cells attached to Bruch's membrane and persisted for the duration of the observation period. This was however possible only in one of the two patients. Moreover, clear functional visual im‐

This is the first peer reviewed study that uses human ESCs for cell therapy. Although their report is preliminary, in only two patients, and with a short-term follow-up, the results are impressive - especially considering the progressive nature of both diseases (Atala, 2012).

More than a decade ago, spinal-cord injury meant confinement to a wheelchair and a lifetime of medical care. Published incidence rates for traumatic spinal-cord injury in the USA range between 28 and 55 per million people, with about 10 000 new cases reported every year. Causes include motor vehicle accidents (36–48%), violence (5–29%), falls (17– 21%), and recreational activities (7–16%) (McDonald & Sadowsky, 2002). The primary in‐ jury (the initial insult) is usually due to the mechanical trauma and includes traction and compression forces. Neural elements are compressed by fractured and displaced bone fragments, disc material, and ligaments and leads to injuries on both the central and pe‐ ripheral nervous systems. Blood vessels are damaged, axons disrupted and cell mem‐ branes broken. Micro-haemorrhages occur within minutes in the central grey matter and spread out over the next few hours. Within minutes, the spinal cord swells to occupy the entire diameter of the spinal canal at the injury level. Secondary ischaemia results when cord swelling exceeds venous blood pressure. The more destructive phase of secondary injury is, however, more responsible for cell death and functional deficits. Hemorrhage, edema, ischaemia, release of toxic chemicals from disrupted neural membranes, and elec‐ trolyte shifts trigger a secondary injury cascade that substantially compounds initial me‐ chanical damage by harming or killing neighbouring cells (McDonald & Sadowsky, 2002). Glutamate plays a key part in a highly disruptive process known as excitotoxicity. It was demonstrated that glutamate, released during injury, damages oligodendocytes (Domercq *et al.*, 2005). Oligodendrocytes express glutamate receptors as NMDA (Karadot‐ tir *et al.*, 2005) and AMPA/kainate receptors (Domercq *et al.*, 1999). Up to now, the pri‐ mary approach in treatment is limitation of secondary injury by removal of damaging bone, disc, and ligament fragments to decompress the swollen cord, followed by the ad‐

provement was noted in the patient with Stargardt's macular dystrophy.

ministration of the steroid methyl-prednisolone (Bracken *et al.*, 1990).

**3. Treatment of spinal cord injury**

402 Pluripotent Stem Cells

As mentioned before in the case of spinal cord injury, diseases of the nervous system in‐ volve proliferation of astrocytes and loss of oligodendrocytes (OLN) and the protective myelin sheath they produce. Transplantation of oligodendrocyte precursors in different animals systems show that these precursors can myelinate axons (Groves *et al.*, 1993). Thus, derivation of oligodendrocytes from ESCs has been an important goal for cell re‐ placement therapy. The most common protocols involve an initial differentiation step to neural progenitors (Reubinoff *et al.*, 2001), followed by expansion, further differentiation, and selection. These protocols follow the differentiation steps that take place *in vivo*. Dur‐ ing development, oligodendrocytes differentiate from precursors, which migrate and pro‐ liferate, through immature oligodendrocytes, which send out processes seeking axons to myelinate, to mature myelinating oligodendrocytes that form myelin sheaths. The precur‐ sor cells are morphologically bipolar (when migrating) or stellate (after migration). These initially differentiate into immature cells that put out processes seeking axons to myeli‐ nate, and eventually form mature cells with parallel processes myelinating up to 30 dif‐ ferent axons (Karadottir & Attwell, 2007).

Oligodendrocytes were first efficiently derived from mouse ESCs (Brustle *et al.*, 1999), where ESCs were aggregated to embryoid bodies and plated in a defined medium that favors the survival of ES cell–derived neural precursors, followed by the expansion of progenitors in culture medium containing FGF2 and EGF, and a switch to FGF2 and PDGF to yield bipo‐ tential glial progenitors (Brustle *et al.*, 1999). These glial progenitors were transplanted into the spinal cords of rats with a genetic deficiency in myelin production, yielding myelinated fibers in the majority of animals (Learish *et al.*, 1999). Human ESCs were first shown to dif‐ ferentiate into oligodendrocytes by Zhang et al., 2001, who used a similar strategy involving FGF treatment followed by growth as neurospheres (Zhang *et al.*, 2001). They reported the differentiation of neural precursors into neurons, astrocytes and oligodendrocytes. Howev‐ er, no human oligodendrocytes were detected after transplantation of neural precursors into the brains of newborn mice, although human neurons and some astrocytes were found to have engrafted (Zhang *et al.*, 2001).

The first detailed protocol for directed differentiation of oligodendrocytes from human ESCs was published in 2005 and involved the induction of neural lineage by retinoic acid treat‐ ment, followed by expansion and selection in various media containing the differentiation factors triiodothyroidin hormone, FGF2, EGF, and insulin (Nistor *et al.*, 2005). After 42 days of culture, the desired cells were found in yellow spheroids, which upon differentiation as low-density monolayers formed 85%–95% oligodendrocytes expressing typical markers as GalC, RIP, and O4. Human embryonic stem cell (hESC)-derived oligodendrocytes were able to integrate, differentiate and display a functional myelinating phenotype following trans‐ plantation into the shiverer mutant mouse (Nistor *et al.*, 2005). Recently, other protocols were developed for generation of oligodendrocytes from ESCs. The Neman and de Vellis (Neman & de Vellis, 2012) laboratory has reported usage of defined serum-free media to‐ gether with morphogens, as retinoic acid and sonic hedgehog, to devise a new method to derive a pure population of OLN from ESCs. These experiments show that human oligoden‐ drocytes can be generated in large numbers and used to restore myelination under some cir‐ cumstances in mice.

#### **3.2. Clinical trial**

In October 2010 the world's first clinical trial using human embryonic stem cells began, using ESCs converted into OLN precursor cells. The feasibility of the treatment was proofed by a wide range of pre-clinical studies that have shown that human oligoden‐ drocyte progenitor cells implanted after spinal cord injury in rodent models show func‐ tional improvement (Keirstead, 2005; Keirstead *et al.*, 2005; Sharp *et al.*, 2010). Geron of Menlo Park, California, is the biotech company that received FDA approval to proceed with clinical trials that transplant cells derived from embryonic stem cells into the spinal cord (Alper, 2009). This company has pioneered translational research into human ESC therapies. The Geron trial (trial identification number NCT01217008), which was original‐ ly approved by the FDA, but then halted due to concerns of abnormal cyst formation, was reinitiated and approved for phase I clinical trials in the U.S. in October 2010. The trial was suspended following news that animals in a dose-escalation study developed microscopic cysts in regenerating tissue sites. In november 2011 Geron announced that it is dropping its entire program owing to financial concerns and started looking for part‐ ners for stem cell treatments and decided to not further invest in the clinical trials in‐ volving treatments with ESCs.

**Figure 2.** Scheme of procedure for treating spinal cord injury with human ESCs derived oligodendrocyte precursor cells.

Embryonic Stem Cell Therapy – From Bench to Bed

http://dx.doi.org/10.5772/54368

405

The major safety concerns for the use of hESCs are related to the achievement of xenobi‐ otic-free culture conditions, avoidance of genetic abnormalities, development of good dif‐ ferentiation and selection protocols, and the avoidance of the immune rejection. Moreover, the unlimited proliferative capacity of ESCs is a disadvantage in clinical appli‐ cations because this could cause tumor formation upon transplantation. When implanted in an undifferentiated state, ESCs cause teratoma, a tumor type that consists of different kinds of differentiated cells. Teratomas are encapsulated, usually benign tumors that can occur naturally, but there is the fear, based on some animal studies, that some propor‐ tion of the cells derived from ESCs injected into the body could drift from their intended developmental pathway. Teratoma formation was reported in various cases when mouse

**4. Embryonic stem cells and tumorigenesis**

The trial was planned to involve treating ten patients who have suffered a complete thora‐ cic-level spinal cord injury in a phase 1 multicenter trial. The pioneering therapy is Geron's 'GRNOPC1 product', which contains hES cell–derived oligodendrocyte progenitor cells that have demonstrated remyelinating and nerve growth–stimulating properties. In the human SCI lesion site, it is hoped that OLN precursors will work as a "combination therapy" - phe‐ notypically replacing lost oligodendrocytes and hence remyelinating axons that have be‐ come demyelinated during injury, as well as secreting neurotrophic factors to establish a repair environment in the lesion (Hatch *et al.*, 2009). The ESCs were differentiated into OLN precursors (Hatch *et al.*, 2009) and one injection of 2 million GRNOPC1 cells was adminis‐ tered within 2 weeks in patients with thoracic spinal cord injury (Fig. 2). No serious adverse effects were observed in the 2 patients enrolled, only one of the patients experienced some side effects due to the immunosupression (Watson & Yeung, 2011). However, the data gen‐ erated by Geron for the FDA are not published and no preliminary report on the safety of their product is available up to now.

**Figure 2.** Scheme of procedure for treating spinal cord injury with human ESCs derived oligodendrocyte precursor cells.

## **4. Embryonic stem cells and tumorigenesis**

low-density monolayers formed 85%–95% oligodendrocytes expressing typical markers as GalC, RIP, and O4. Human embryonic stem cell (hESC)-derived oligodendrocytes were able to integrate, differentiate and display a functional myelinating phenotype following trans‐ plantation into the shiverer mutant mouse (Nistor *et al.*, 2005). Recently, other protocols were developed for generation of oligodendrocytes from ESCs. The Neman and de Vellis (Neman & de Vellis, 2012) laboratory has reported usage of defined serum-free media to‐ gether with morphogens, as retinoic acid and sonic hedgehog, to devise a new method to derive a pure population of OLN from ESCs. These experiments show that human oligoden‐ drocytes can be generated in large numbers and used to restore myelination under some cir‐

In October 2010 the world's first clinical trial using human embryonic stem cells began, using ESCs converted into OLN precursor cells. The feasibility of the treatment was proofed by a wide range of pre-clinical studies that have shown that human oligoden‐ drocyte progenitor cells implanted after spinal cord injury in rodent models show func‐ tional improvement (Keirstead, 2005; Keirstead *et al.*, 2005; Sharp *et al.*, 2010). Geron of Menlo Park, California, is the biotech company that received FDA approval to proceed with clinical trials that transplant cells derived from embryonic stem cells into the spinal cord (Alper, 2009). This company has pioneered translational research into human ESC therapies. The Geron trial (trial identification number NCT01217008), which was original‐ ly approved by the FDA, but then halted due to concerns of abnormal cyst formation, was reinitiated and approved for phase I clinical trials in the U.S. in October 2010. The trial was suspended following news that animals in a dose-escalation study developed microscopic cysts in regenerating tissue sites. In november 2011 Geron announced that it is dropping its entire program owing to financial concerns and started looking for part‐ ners for stem cell treatments and decided to not further invest in the clinical trials in‐

The trial was planned to involve treating ten patients who have suffered a complete thora‐ cic-level spinal cord injury in a phase 1 multicenter trial. The pioneering therapy is Geron's 'GRNOPC1 product', which contains hES cell–derived oligodendrocyte progenitor cells that have demonstrated remyelinating and nerve growth–stimulating properties. In the human SCI lesion site, it is hoped that OLN precursors will work as a "combination therapy" - phe‐ notypically replacing lost oligodendrocytes and hence remyelinating axons that have be‐ come demyelinated during injury, as well as secreting neurotrophic factors to establish a repair environment in the lesion (Hatch *et al.*, 2009). The ESCs were differentiated into OLN precursors (Hatch *et al.*, 2009) and one injection of 2 million GRNOPC1 cells was adminis‐ tered within 2 weeks in patients with thoracic spinal cord injury (Fig. 2). No serious adverse effects were observed in the 2 patients enrolled, only one of the patients experienced some side effects due to the immunosupression (Watson & Yeung, 2011). However, the data gen‐ erated by Geron for the FDA are not published and no preliminary report on the safety of

cumstances in mice.

volving treatments with ESCs.

their product is available up to now.

**3.2. Clinical trial**

404 Pluripotent Stem Cells

The major safety concerns for the use of hESCs are related to the achievement of xenobi‐ otic-free culture conditions, avoidance of genetic abnormalities, development of good dif‐ ferentiation and selection protocols, and the avoidance of the immune rejection. Moreover, the unlimited proliferative capacity of ESCs is a disadvantage in clinical appli‐ cations because this could cause tumor formation upon transplantation. When implanted in an undifferentiated state, ESCs cause teratoma, a tumor type that consists of different kinds of differentiated cells. Teratomas are encapsulated, usually benign tumors that can occur naturally, but there is the fear, based on some animal studies, that some propor‐ tion of the cells derived from ESCs injected into the body could drift from their intended developmental pathway. Teratoma formation was reported in various cases when mouse ESCs-derived cells like insulin producing islets (Fujikawa *et al.*, 2005), ESC-derived cardi‐ omyocytes (Cao *et al.*, 2006), and ESC-derived neurons (Schuldiner *et al.*, 2001) were transplanted into immunosuppressed mice even though there was successful engraftment and functional improvement. When undifferentiated human ESCs were injected into the hind limb muscles or under the kidney capsule of SCID mice, teratomas were readily formed after 8–12 weeks (Richards *et al.*, 2002). Evidence of tumor formation has also been observed in differentiated hESC derivatives transplanted *in vivo* (Roy *et al.,* 2006). In another study, successful hESC-derived neuronal engraftment in a Parkinsonian rat model did not yield teratomas after 12 weeks (Ben-Hur *et al.*, 2004). When hESC-derived osteocytes or cardiomyocytes were transplanted into the bone or heart of severe com‐ bined immunodeficient mice (SCID), there was also no teratoma production within 1 month after injection (Bielby *et al.*, 2004; Laflamme *et al.*, 2007). It seems that the longer hESCs are differentiated *in vitro*, the risk of teratoma formation appears to be reduced. Certain sites appear to favor the growth of teratomas, while others do not, confirming a phenomenon already described that tumorigenesis of ESCs is site dependent. For exam‐ ple the rate of teratoma formation with hESCs in immunodeficient mice was subcutane‐ ously 25–100%, intratesticularly 60%, intramuscularly 12.5% and under the kidney capsule 100% (Prokhorova *et al.*, 2009). Furthermore, tumor formation in the lung and thymus had the highest probability of teratoma formation while the pancreas was parti‐ ally site-privileged (Shih *et al.*, 2007). Shih et al. observed an aggressive growth of tu‐ mors when human ESCs were injected into engrafted human fetal tissues in SCID mice (Shih *et al.*, 2007).

hESCs (Bongso *et al.*, 2008). It is also very important to develop very good and reliable methods to detect residual ESCs contamination in ESCs derived cells prior to clinical ap‐ plication. In their review, Fong et al. (Fong *et al.*, 2010) presented some available meth‐ ods for the elimination of undifferentiated ESCs. These included single cell propagation with encapsulation, usage of density gradients, MACS and FACS, usage of tumor privi‐ leged sites, usage of antibodies against undifferentiated ESCs, prolonged differentiation in vitro before transplantation or destruction of teratoma after engraftment. However, be‐ cause differentiation is not an on/off process, it is probably the best to use a combination

Embryonic Stem Cell Therapy – From Bench to Bed

http://dx.doi.org/10.5772/54368

407

**5. Embryonic stem cells versus induced pluripotent stem cells in clinics**

Induced pluripotent cells (iPS) are generated by re-engineering mature, fully differentiat‐ ed cells (e.g. human skin fibroblasts) by modifying the cells with a set of transgenes (Ta‐ kahashi & Yamanaka, 2006; Takahashi *et al.*, 2007). Induced pluripotent stem cells, created by turning back the developmental clock on adult tissues, display similar geneexpression patterns to ESCs, and can produce various tissues in the human body. How‐ ever, iPS cells have a major advantage over ESCs; they can be obtained directly from the individual that has to be treated. Thus, as a source of cells for therapy, they are able to avoid the immunocompatibility issues. Furthermore, the utilization of these stem cells in both clinical and basic research studies does not face ethical and political issues that oth‐

During the last years various studies reported the differentiation of iPS cells to various types of cells in vitro and these cells were used for cellular therapy in various mouse models (Wer‐

However, before bringing these cells into the clinics, their safety should be tested. For example, the initial enthusiasm related to bringing iPS cells into clinics dampened when it was shown that these cells develop teratoma more efficiently than ESCs (Gutierrez-Aranda *et al.*, 2010). It was also shown that iPS retain the epigenetic memory of the cells from which they are derived; this fact makes them to preferentially differentiate into the cell lineage from which they came from. Future clinical applications will demand new techniques for generating factor-free iPS cells such as virus-free or DNA-free approaches at acceptable efficiencies. There are also other disadvantages in using iPS cells in the clinics. Usually, they are made by integrating retroviruses into the cells as shuttle for the reprogramming factors. This problem may be solved by transient gene transfer or by de‐ livering the pluripotency factors in protein form (Murry & Keller, 2008). The second is that iPS cells are not an ''off-the-shelf'' product and would likely only be produced after the patient becomes ill, precluding their use in the acute phase of the disease (Murry & Keller, 2008). Quality control is will also be difficult and expensive, because a separate

of these methods in order to do safe cell therapy.

erwise surround the use of embryonic stem cells.

batch of iPS cells would have to be made for each patient.

nig *et al.*, 2008; Saha & Jaenisch, 2009).

The simplest way to slow or even eliminate the tumorigenicity of normal stem cells prior to transplantation may be to take advantage of pluripotency by partially differentiating them into progenitors. Therefore, a promising proposed method for making stem cell-based re‐ generative medicine therapies safer may seem paradoxical: to not transplant stem cells at all into patients. The idea is to use the stem cells to produce progenitor or precursor cells of the desired lineage and then transplant progenitors purified by sorting (Knoepfler, 2009). This approach was presented in this chapter and is actually used in the clinical trial with oligo‐ dendrocyte progenitor cells. However, not only the embryonic stem cells, but also the im‐ planted precursor cells seem to form teratoma in some cases. A group of Israeli researchers reported that a boy with ataxia telangiectasia who had received several fetal neural stem cell transplants developed teratomas in his brain and spinal cord four years after treatment (Amariglio *et al.*, 2009). For this reason is very important to achieve a 100% pure population of differentiated cells when using ESCs for cell therapy.

Currently, the only way to ensure that teratomas do not form is to differentiate the ESCs in advance, enrich for the desired cell type, and screen for the presence of undifferentiat‐ ed cells. The elimination of undifferentiated hESCs may best be achieved by (1) destroy‐ ing the remaining undifferentiated hESCs in the differentiated tissue population with specific agents or antibodies, (2) separating or removing the undifferentiated hESCs from the differentiated cell population, (3) eliminating pluripotent cells during the differentia‐ tion process, and (4) inducing further differentiation of left-over rogue undifferentiated hESCs (Bongso *et al.*, 2008). It is also very important to develop very good and reliable methods to detect residual ESCs contamination in ESCs derived cells prior to clinical ap‐ plication. In their review, Fong et al. (Fong *et al.*, 2010) presented some available meth‐ ods for the elimination of undifferentiated ESCs. These included single cell propagation with encapsulation, usage of density gradients, MACS and FACS, usage of tumor privi‐ leged sites, usage of antibodies against undifferentiated ESCs, prolonged differentiation in vitro before transplantation or destruction of teratoma after engraftment. However, be‐ cause differentiation is not an on/off process, it is probably the best to use a combination of these methods in order to do safe cell therapy.

ESCs-derived cells like insulin producing islets (Fujikawa *et al.*, 2005), ESC-derived cardi‐ omyocytes (Cao *et al.*, 2006), and ESC-derived neurons (Schuldiner *et al.*, 2001) were transplanted into immunosuppressed mice even though there was successful engraftment and functional improvement. When undifferentiated human ESCs were injected into the hind limb muscles or under the kidney capsule of SCID mice, teratomas were readily formed after 8–12 weeks (Richards *et al.*, 2002). Evidence of tumor formation has also been observed in differentiated hESC derivatives transplanted *in vivo* (Roy *et al.,* 2006). In another study, successful hESC-derived neuronal engraftment in a Parkinsonian rat model did not yield teratomas after 12 weeks (Ben-Hur *et al.*, 2004). When hESC-derived osteocytes or cardiomyocytes were transplanted into the bone or heart of severe com‐ bined immunodeficient mice (SCID), there was also no teratoma production within 1 month after injection (Bielby *et al.*, 2004; Laflamme *et al.*, 2007). It seems that the longer hESCs are differentiated *in vitro*, the risk of teratoma formation appears to be reduced. Certain sites appear to favor the growth of teratomas, while others do not, confirming a phenomenon already described that tumorigenesis of ESCs is site dependent. For exam‐ ple the rate of teratoma formation with hESCs in immunodeficient mice was subcutane‐ ously 25–100%, intratesticularly 60%, intramuscularly 12.5% and under the kidney capsule 100% (Prokhorova *et al.*, 2009). Furthermore, tumor formation in the lung and thymus had the highest probability of teratoma formation while the pancreas was parti‐ ally site-privileged (Shih *et al.*, 2007). Shih et al. observed an aggressive growth of tu‐ mors when human ESCs were injected into engrafted human fetal tissues in SCID mice

The simplest way to slow or even eliminate the tumorigenicity of normal stem cells prior to transplantation may be to take advantage of pluripotency by partially differentiating them into progenitors. Therefore, a promising proposed method for making stem cell-based re‐ generative medicine therapies safer may seem paradoxical: to not transplant stem cells at all into patients. The idea is to use the stem cells to produce progenitor or precursor cells of the desired lineage and then transplant progenitors purified by sorting (Knoepfler, 2009). This approach was presented in this chapter and is actually used in the clinical trial with oligo‐ dendrocyte progenitor cells. However, not only the embryonic stem cells, but also the im‐ planted precursor cells seem to form teratoma in some cases. A group of Israeli researchers reported that a boy with ataxia telangiectasia who had received several fetal neural stem cell transplants developed teratomas in his brain and spinal cord four years after treatment (Amariglio *et al.*, 2009). For this reason is very important to achieve a 100% pure population

Currently, the only way to ensure that teratomas do not form is to differentiate the ESCs in advance, enrich for the desired cell type, and screen for the presence of undifferentiat‐ ed cells. The elimination of undifferentiated hESCs may best be achieved by (1) destroy‐ ing the remaining undifferentiated hESCs in the differentiated tissue population with specific agents or antibodies, (2) separating or removing the undifferentiated hESCs from the differentiated cell population, (3) eliminating pluripotent cells during the differentia‐ tion process, and (4) inducing further differentiation of left-over rogue undifferentiated

of differentiated cells when using ESCs for cell therapy.

(Shih *et al.*, 2007).

406 Pluripotent Stem Cells

## **5. Embryonic stem cells versus induced pluripotent stem cells in clinics**

Induced pluripotent cells (iPS) are generated by re-engineering mature, fully differentiat‐ ed cells (e.g. human skin fibroblasts) by modifying the cells with a set of transgenes (Ta‐ kahashi & Yamanaka, 2006; Takahashi *et al.*, 2007). Induced pluripotent stem cells, created by turning back the developmental clock on adult tissues, display similar geneexpression patterns to ESCs, and can produce various tissues in the human body. How‐ ever, iPS cells have a major advantage over ESCs; they can be obtained directly from the individual that has to be treated. Thus, as a source of cells for therapy, they are able to avoid the immunocompatibility issues. Furthermore, the utilization of these stem cells in both clinical and basic research studies does not face ethical and political issues that oth‐ erwise surround the use of embryonic stem cells.

During the last years various studies reported the differentiation of iPS cells to various types of cells in vitro and these cells were used for cellular therapy in various mouse models (Wer‐ nig *et al.*, 2008; Saha & Jaenisch, 2009).

However, before bringing these cells into the clinics, their safety should be tested. For example, the initial enthusiasm related to bringing iPS cells into clinics dampened when it was shown that these cells develop teratoma more efficiently than ESCs (Gutierrez-Aranda *et al.*, 2010). It was also shown that iPS retain the epigenetic memory of the cells from which they are derived; this fact makes them to preferentially differentiate into the cell lineage from which they came from. Future clinical applications will demand new techniques for generating factor-free iPS cells such as virus-free or DNA-free approaches at acceptable efficiencies. There are also other disadvantages in using iPS cells in the clinics. Usually, they are made by integrating retroviruses into the cells as shuttle for the reprogramming factors. This problem may be solved by transient gene transfer or by de‐ livering the pluripotency factors in protein form (Murry & Keller, 2008). The second is that iPS cells are not an ''off-the-shelf'' product and would likely only be produced after the patient becomes ill, precluding their use in the acute phase of the disease (Murry & Keller, 2008). Quality control is will also be difficult and expensive, because a separate batch of iPS cells would have to be made for each patient.

## **6. Conclusion**

There is no doubt that after the hurdles are overcome, hESC-derived cells have a promising future for transplantation therapy given the versatility of these cells. It is very encouraging to see that clinical trials involving the use of hESCs have begun, and that extensive efforts are underway to efficiently, and safely differentiate hESCs into specific cell types.

[6] Ben-Hur, T., Idelson, M., Khaner, H., Pera, M., Reinhartz, E., Itzik, A. & Reubinoff, B.E. (2004) Transplantation of human embryonic stem cell-derived neural progeni‐ tors improves behavioral deficit in Parkinsonian rats. Stem Cells, 22, 1246-1255.

Embryonic Stem Cell Therapy – From Bench to Bed

http://dx.doi.org/10.5772/54368

409

[7] Bielby, R.C., Boccaccini, A.R., Polak, J.M. & Buttery, L.D. (2004) In vitro differentia‐ tion and in vivo mineralization of osteogenic cells derived from human embryonic

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## **Acknowledgements**

I would like to thank Paul G. Layer for carefully reading the manuscript.

## **Author details**

Laura E. Sperling

Technische Universität Darmstadt, Fachbereich Biologie, Entwicklungsbiologie & Neuroge‐ netik, Darmstadt, Germany

### **References**

[6] Ben-Hur, T., Idelson, M., Khaner, H., Pera, M., Reinhartz, E., Itzik, A. & Reubinoff, B.E. (2004) Transplantation of human embryonic stem cell-derived neural progeni‐ tors improves behavioral deficit in Parkinsonian rats. Stem Cells, 22, 1246-1255.

**6. Conclusion**

408 Pluripotent Stem Cells

**Acknowledgements**

**Author details**

Laura E. Sperling

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netik, Darmstadt, Germany

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Technische Universität Darmstadt, Fachbereich Biologie, Entwicklungsbiologie & Neuroge‐

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**Chapter 19**

**Disease Models for the Genetic Cardiac Diseases**

The ability to reprogram somatic cells into pluripotent stem cells has presented a significant advancement in stem cell research. This technique enables derivation of induced pluripotent stem (iPS) cells from any individual having a unique genotype. iPS cells can be derived from human somatic cells such as fibroblasts, keratinocytes or blood cells. Since, the production of iPS cell lines does not require the destruction of human embryos as in the production of the human embryonic stem cells (hESCs), the legal and ethical issues associated with hESCs can be at least partly avoided. The characteristics of iPS cells are very similar to those of pluripotent hESCs in many respects, including cell morphology, immortal growth characteristics in culture, expression of pluripotent markers, and differentiation potential. The iPS cells com‐ bined with the various differentiation protocols developed enable the production of genotype specific cell types. This feature enables also to produce disease-specific iPS cell lines from patients bearing defined genetic mutations. Traditionally, it has been challenging to study genetic cardiac diseases because cardiomyocytes from the heart biopsies of patients are difficult to obtain and the procedure carries a high risk. Additionally these cardiomyocytes do not survive long in culture. Animal models, mostly developed in rodent, have aided in elucidating the basic mechanisms of several genetic cardiac diseases. The disadvantages of small animal models are marked differences in anatomy and physiology of the cardiovascular system in comparison to humans. Rodent models are far from ideal when used in the identi‐ fication of contractile deficits and signals that initiate pathological growth [1]. Furthermore, the results obtained from neonatal rat cell experiments can be problematic because these cells possess different relative receptor subtypes and cell-signaling mechanisms. It will thus be especially important to investigate functional consequences of genetic cardiac diseases in human cardiomyocytes in which the functional effects of specific proteins have been adjusted to optimize electrical properties, contractile efficiency and power output of larger hearts [2].

> © 2013 Pekkanen-Mattila et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Pekkanen-Mattila et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Mari Pekkanen-Mattila, Kristiina Rajala and

Additional information is available at the end of the chapter

Katriina Aalto-Setälä

http://dx.doi.org/10.5772/55773

**1. Introduction**

## **Disease Models for the Genetic Cardiac Diseases**

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37-54.

414 Pluripotent Stem Cells

Mari Pekkanen-Mattila, Kristiina Rajala and Katriina Aalto-Setälä

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55773

## **1. Introduction**

The ability to reprogram somatic cells into pluripotent stem cells has presented a significant advancement in stem cell research. This technique enables derivation of induced pluripotent stem (iPS) cells from any individual having a unique genotype. iPS cells can be derived from human somatic cells such as fibroblasts, keratinocytes or blood cells. Since, the production of iPS cell lines does not require the destruction of human embryos as in the production of the human embryonic stem cells (hESCs), the legal and ethical issues associated with hESCs can be at least partly avoided. The characteristics of iPS cells are very similar to those of pluripotent hESCs in many respects, including cell morphology, immortal growth characteristics in culture, expression of pluripotent markers, and differentiation potential. The iPS cells com‐ bined with the various differentiation protocols developed enable the production of genotype specific cell types. This feature enables also to produce disease-specific iPS cell lines from patients bearing defined genetic mutations. Traditionally, it has been challenging to study genetic cardiac diseases because cardiomyocytes from the heart biopsies of patients are difficult to obtain and the procedure carries a high risk. Additionally these cardiomyocytes do not survive long in culture. Animal models, mostly developed in rodent, have aided in elucidating the basic mechanisms of several genetic cardiac diseases. The disadvantages of small animal models are marked differences in anatomy and physiology of the cardiovascular system in comparison to humans. Rodent models are far from ideal when used in the identi‐ fication of contractile deficits and signals that initiate pathological growth [1]. Furthermore, the results obtained from neonatal rat cell experiments can be problematic because these cells possess different relative receptor subtypes and cell-signaling mechanisms. It will thus be especially important to investigate functional consequences of genetic cardiac diseases in human cardiomyocytes in which the functional effects of specific proteins have been adjusted to optimize electrical properties, contractile efficiency and power output of larger hearts [2].

© 2013 Pekkanen-Mattila et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Pekkanen-Mattila et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genetic cardiac diseases, such as long QT syndrome, belong to a severe class of diseases which are unpredictable, have variable clinical picture ranging from asymptomatic to sudden cardiac death and lack specific medication. These inherited arrhythmic diseases are caused by single mutations which are relatively common in population. Earlier we did not have *in vitro* models for these diseases, but with the aid of iPS cell derived cardiomyocytes genetic cardiac diseases can now be modeled in cell culture. The patient specific iPS cell derived cardiomyocytes have been demonstrated to manifest the disease-associated electrophysiological abnormalities in a dish [3-6]. Therefore, these cells allow researchers to study and understand disease mecha‐ nisms more readily as well as to investigate the effects of different chemical compounds on the electrophysiology of the cardiomyocytes. In addition to basic research, iPS cell derived cardiomyocytes would provide an effective tool for novel drug or treatment discovery. However, before iPS cell derived cardiomyocytes are ready to be considered for use as disease models, the cells produced need to be confirmed to exhibit the essential functional character‐ istics of human cardiomyocytes.

efficiency and kinetics as well as to generate iPS cells without viral integration in the genome (Table 1). The nonintegrating reprogramming methods developed thus far include adeno- and sendai viruses, plasmid- and episomal vector-based approaches, excision systems of integrat‐ ed transgenes such as Cre/loxP recombination or PiggyBac transposition, and delivery of reprogramming factors directly as RNAs, proteins and chemicals. However, most of these nonintegrating approaches are still highly inefficient when compared to the original retro- or lentiviral reprogramming systems with the exception of nowadays widely used sendai virus

**Methods Efficiency % Details References**

tumorigenicity possible

tumorigenicity possible

vector-fragment possible

fragment possible, T sensitive Sendai vector allowing removal of the virus

site excision, screening needed, tumorigenicity possible

treatment required, genetic abnormality

treatment required, abnormal signaling

pathway possible, virus used

[7]

Disease Models for the Genetic Cardiac Diseases

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417

[9]

[14]

[15]

[17]

[18, 19]

[20]

[21, 22]

[23, 24]

[25]

[26]

Retroviral vectors Medium, 0.01-0.5 Multiple integration, incomplete silencing,

Lentiviral vectors Medium, 0.1-1 Multiple integration, incomplete silencing,

Adenoviral vectors Low, 0.001 Non-integrating, however integrated

Sendaiviral vectors Medium, 0.01-1 Non-integrating, integrated vector-

Plasmids Low, 0.001 Occasional integration, simple transfection [16]

Cre/loxP system Medium, 0.01-1 Integration but excisable, inefficient loxP

PiggyBac system Medium, 0.1 Precise excision possible, screening

RNAs High, 1 Non-integrating, DNA-free, multiple

Protein Low, 0.001 Non-integrating, DNA-free, long-term

**Table 1.** Overview of the reprogramming methods for the generation of iPS cells.

Low, 0.0003 Non-integrating, long-term persistent transcription

Low, 0.005 Non-integrating, multiple transductions needed

needed

possible

High, >1 Non-integrating, DNA-free, long-term

transfection needed

reprogramming method.

OriP/EBNA-1 episomal vectors

Minicircle DNA episomal vectors

Factors + small molecules

In this chapter, the production and the characterization of patient specific iPS cell derived cardiomyocytes is described. In addition, we discuss the genetic cardiac disease models so far developed based on iPS technique, their demands, advantages and disadvantages. Further‐ more, the future applications for iPS cell derived cardiomyocytes are discussed.

## **2. Production of disease specific iPS cell lines**

The discovery of cellular reprogramming as a technology to generate iPS cells offers a potential solution to the challenge when studying genetic cardiac diseases. In this approach, human adult somatic cells are reprogrammed into stem cells offering comparable function to human pluripotent ESCs in their ability to develop differentiated progeny from all developmental lineages of the human being. When somatic cells are reprogrammed to iPS cells, they shut down the expression of genes specific for that somatic cell type and activate genes that maintain pluripotency. Once reprogramming has occurred, endogenous counterparts of the exoge‐ nously supplied reprogramming factors are activated, indicating that exogenous factors are only required for the induction, not for the maintenance of pluripotency [7]. Up to date, various human somatic cell types, including fibroblasts, keratinocytes, and different blood cells have been reprogrammed to iPS cells [7-11].

The initial methods used to generate iPS cells involved the retroviral overexpression of four transcription factors Oct4, Sox2, Klf4, and c-myc observed to be essential in maintaining pluripotency of hESCs [7, 12]. Another set of four transcription factors Oct4, Sox2, Nanog, and Lin-28 was also found to induce pluripotency [9]. Efficient retro- and lentiviral vector systems that have been most widely used to generate iPS cells have several drawbacks including the possibility of proviral genomic integration, which may cause both the reactivation of silenced exogenous genes and the alteration of genomic integrity, thereby increasing the risk for tumorigenesis [12, 13]. Since the seminal discovery the development in this field has been rapid and numerous alternative strategies have been applied to improve the reprogramming safety, efficiency and kinetics as well as to generate iPS cells without viral integration in the genome (Table 1). The nonintegrating reprogramming methods developed thus far include adeno- and sendai viruses, plasmid- and episomal vector-based approaches, excision systems of integrat‐ ed transgenes such as Cre/loxP recombination or PiggyBac transposition, and delivery of reprogramming factors directly as RNAs, proteins and chemicals. However, most of these nonintegrating approaches are still highly inefficient when compared to the original retro- or lentiviral reprogramming systems with the exception of nowadays widely used sendai virus reprogramming method.

Genetic cardiac diseases, such as long QT syndrome, belong to a severe class of diseases which are unpredictable, have variable clinical picture ranging from asymptomatic to sudden cardiac death and lack specific medication. These inherited arrhythmic diseases are caused by single mutations which are relatively common in population. Earlier we did not have *in vitro* models for these diseases, but with the aid of iPS cell derived cardiomyocytes genetic cardiac diseases can now be modeled in cell culture. The patient specific iPS cell derived cardiomyocytes have been demonstrated to manifest the disease-associated electrophysiological abnormalities in a dish [3-6]. Therefore, these cells allow researchers to study and understand disease mecha‐ nisms more readily as well as to investigate the effects of different chemical compounds on the electrophysiology of the cardiomyocytes. In addition to basic research, iPS cell derived cardiomyocytes would provide an effective tool for novel drug or treatment discovery. However, before iPS cell derived cardiomyocytes are ready to be considered for use as disease models, the cells produced need to be confirmed to exhibit the essential functional character‐

In this chapter, the production and the characterization of patient specific iPS cell derived cardiomyocytes is described. In addition, we discuss the genetic cardiac disease models so far developed based on iPS technique, their demands, advantages and disadvantages. Further‐

The discovery of cellular reprogramming as a technology to generate iPS cells offers a potential solution to the challenge when studying genetic cardiac diseases. In this approach, human adult somatic cells are reprogrammed into stem cells offering comparable function to human pluripotent ESCs in their ability to develop differentiated progeny from all developmental lineages of the human being. When somatic cells are reprogrammed to iPS cells, they shut down the expression of genes specific for that somatic cell type and activate genes that maintain pluripotency. Once reprogramming has occurred, endogenous counterparts of the exoge‐ nously supplied reprogramming factors are activated, indicating that exogenous factors are only required for the induction, not for the maintenance of pluripotency [7]. Up to date, various human somatic cell types, including fibroblasts, keratinocytes, and different blood cells have

The initial methods used to generate iPS cells involved the retroviral overexpression of four transcription factors Oct4, Sox2, Klf4, and c-myc observed to be essential in maintaining pluripotency of hESCs [7, 12]. Another set of four transcription factors Oct4, Sox2, Nanog, and Lin-28 was also found to induce pluripotency [9]. Efficient retro- and lentiviral vector systems that have been most widely used to generate iPS cells have several drawbacks including the possibility of proviral genomic integration, which may cause both the reactivation of silenced exogenous genes and the alteration of genomic integrity, thereby increasing the risk for tumorigenesis [12, 13]. Since the seminal discovery the development in this field has been rapid and numerous alternative strategies have been applied to improve the reprogramming safety,

more, the future applications for iPS cell derived cardiomyocytes are discussed.

**2. Production of disease specific iPS cell lines**

istics of human cardiomyocytes.

416 Pluripotent Stem Cells

been reprogrammed to iPS cells [7-11].

**Table 1.** Overview of the reprogramming methods for the generation of iPS cells.

## **3. Cardiomyocyte differentiation**

Cardiomyocytes have been differentiated from the hESCs over a decade [27, 28] and multiple cardiac differentiation methods have been developed. The differentiation methods developed for hESC derived cardiomyocytes have been proven to be applicable also for cardiac differ‐ entiation of iPS cells.

Overall the differentiation event of hESC and iPS cell derived cardiomyocytes is quite rapid, 10-20 days regardless of the differentiation method used. However, all the differentiation methods share common problems, including uncontrolled differentiation and low differen‐ tiation rates. With common differentiation methods the cardiomyocyte yield is between ~1-25 % of the total cell number [28-30]. In addition, the cardiomyocyte differentiation efficiency has been shown to vary markedly between different stem cell lines [31].

All differentiation methods end up with a heterogeneous cell population. In addition to the other cell types, the differentiated population includes all cardiomyocyte subtypes; ventricular, atrial and nodal –like cells [32]. The ventricular cells form usually the majori‐ ty of differentiated cells (60-80%), atrial cells form usually 10-40 % of the population and only <5% of cells are nodal-like cells [32, 33]. However, these numbers can differ depend‐ ing on the cell line used [34].

The cardiac differentiation methods are lately reviewed [35] and described in Figure 1.

## **4. Transdifferentiation of fibroblasts into cardiac cells**

Murine fibroblasts can be reprogrammed directly into cardiomyocytes by overexpression of Gata4, Mef2c and Tbx5 (GMT) [36]. This combination of factors has been reported to convert murine cardiac fibroblasts and tail tip fibroblasts into spontaneous beating cells having cardiomyocyte expression profiles. In addition, epigenetic status is typical for cardiomyocytes in these cells. However, Chen and co-workers have shown this method to be inefficient [37]. Overexpression of GMT factors resulted in an increase in cardiac troponin expression but spontaneous action potentials were lacking even though 22% of the cells exhibited voltagedependent calcium currents.

**Figure 1.** Generation of disease –specific iPS cell lines and cardiomyocytes. Cardiac differentiation methods can be div‐ ided into three classes; (1) Embryoid body (EB) based, (2) END-2 coculture based or (3) directed differentiation meth‐ ods. Traditionally EB method has been based on spontaneous aggregation of EBs and spontaneous differentiation [28]. However, lately multiple methods controlling the EB formation has been developed [41] enhancing the reprodu‐ cibility and productivity of the cardiac differentiation. END-2 method can be performed in two ways, either co-cultur‐ ing the hESC or iPS cells in contact with END-2 cells [42] or by using END-2 conditioned media [43]. A lot of effort has been made in enhancing and defining the cardiac differentiation and this has led to the development of directed dif‐ ferentiation methods with growth factors or small molecules. Activin A and BMP-4 has been used in combination with monolayer cultures on matrigel to differentiate cardiomyocytes [44]. A temporal modulation of Wnt signaling by us‐ ing small molecules has been proven to an even more robust and, in addition, rather inexpensive method for cardio‐ myocyte differentiation [45]. Directed reprogramming of fibroblasts to cardiomyocytes has been successful with

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The first characterization step for the differentiated hESC or iPS cell derived cardiomyocytes is the observation of spontaneously beating cells. In addition, cardiomyocyte phenotype can

mouse cells. However, this method has not yet been proven to work with human cells.

**5. The assesment of the cardiomyocyte functionality**

**5.1. Cardiomyocyte characterization at gene and protein level**

A lot of effort has been done to transdifferentiate human fibroblasts into cardiomyocytes. So far spontaneously beating human cells have not been obtained. However, with transcription factors mesoderm posterior (MESP) homolog and mammalian v-ets erythoblastosis virus E26 oncogene homolog ETS2 cardiomyocyte progenitors expressing cardiac mesoderm marker KDR have been obtained [38]. It seems that the GMT method alone is not robust enough for direct reprogramming of human cardiomyocytes. Therefore it has been suggested that combination of GMT with other transcription factors, mRNAs or small molecules could provide more efficient reprogramming procedure [39]. In addition, based on animal experi‐ ments it can be concluded that cardiac microenvironment has also important role in reprog‐ ramming [40].

**3. Cardiomyocyte differentiation**

entiation of iPS cells.

418 Pluripotent Stem Cells

ing on the cell line used [34].

dependent calcium currents.

ramming [40].

Cardiomyocytes have been differentiated from the hESCs over a decade [27, 28] and multiple cardiac differentiation methods have been developed. The differentiation methods developed for hESC derived cardiomyocytes have been proven to be applicable also for cardiac differ‐

Overall the differentiation event of hESC and iPS cell derived cardiomyocytes is quite rapid, 10-20 days regardless of the differentiation method used. However, all the differentiation methods share common problems, including uncontrolled differentiation and low differen‐ tiation rates. With common differentiation methods the cardiomyocyte yield is between ~1-25 % of the total cell number [28-30]. In addition, the cardiomyocyte differentiation efficiency has

All differentiation methods end up with a heterogeneous cell population. In addition to the other cell types, the differentiated population includes all cardiomyocyte subtypes; ventricular, atrial and nodal –like cells [32]. The ventricular cells form usually the majori‐ ty of differentiated cells (60-80%), atrial cells form usually 10-40 % of the population and only <5% of cells are nodal-like cells [32, 33]. However, these numbers can differ depend‐

The cardiac differentiation methods are lately reviewed [35] and described in Figure 1.

Murine fibroblasts can be reprogrammed directly into cardiomyocytes by overexpression of Gata4, Mef2c and Tbx5 (GMT) [36]. This combination of factors has been reported to convert murine cardiac fibroblasts and tail tip fibroblasts into spontaneous beating cells having cardiomyocyte expression profiles. In addition, epigenetic status is typical for cardiomyocytes in these cells. However, Chen and co-workers have shown this method to be inefficient [37]. Overexpression of GMT factors resulted in an increase in cardiac troponin expression but spontaneous action potentials were lacking even though 22% of the cells exhibited voltage-

A lot of effort has been done to transdifferentiate human fibroblasts into cardiomyocytes. So far spontaneously beating human cells have not been obtained. However, with transcription factors mesoderm posterior (MESP) homolog and mammalian v-ets erythoblastosis virus E26 oncogene homolog ETS2 cardiomyocyte progenitors expressing cardiac mesoderm marker KDR have been obtained [38]. It seems that the GMT method alone is not robust enough for direct reprogramming of human cardiomyocytes. Therefore it has been suggested that combination of GMT with other transcription factors, mRNAs or small molecules could provide more efficient reprogramming procedure [39]. In addition, based on animal experi‐ ments it can be concluded that cardiac microenvironment has also important role in reprog‐

been shown to vary markedly between different stem cell lines [31].

**4. Transdifferentiation of fibroblasts into cardiac cells**

**Figure 1.** Generation of disease –specific iPS cell lines and cardiomyocytes. Cardiac differentiation methods can be div‐ ided into three classes; (1) Embryoid body (EB) based, (2) END-2 coculture based or (3) directed differentiation meth‐ ods. Traditionally EB method has been based on spontaneous aggregation of EBs and spontaneous differentiation [28]. However, lately multiple methods controlling the EB formation has been developed [41] enhancing the reprodu‐ cibility and productivity of the cardiac differentiation. END-2 method can be performed in two ways, either co-cultur‐ ing the hESC or iPS cells in contact with END-2 cells [42] or by using END-2 conditioned media [43]. A lot of effort has been made in enhancing and defining the cardiac differentiation and this has led to the development of directed dif‐ ferentiation methods with growth factors or small molecules. Activin A and BMP-4 has been used in combination with monolayer cultures on matrigel to differentiate cardiomyocytes [44]. A temporal modulation of Wnt signaling by us‐ ing small molecules has been proven to an even more robust and, in addition, rather inexpensive method for cardio‐ myocyte differentiation [45]. Directed reprogramming of fibroblasts to cardiomyocytes has been successful with mouse cells. However, this method has not yet been proven to work with human cells.

## **5. The assesment of the cardiomyocyte functionality**

#### **5.1. Cardiomyocyte characterization at gene and protein level**

The first characterization step for the differentiated hESC or iPS cell derived cardiomyocytes is the observation of spontaneously beating cells. In addition, cardiomyocyte phenotype can be assessed at the gene or protein level with cardiomyocyte specific markers such as structural proteins troponin, alpha-actinin or myosins. The commonly used markers in monitoring the cardiac differentiation are listed in Table 2.

cardiac action potential is composed of co-operation of these channels and the action potential curve can be divided into five different phases (Figure 2). Phase 0 of the action potential is the depolarization phase of the cardiomyocytes from the negative membrane potential to positive, called the upstroke. This is followed by phase 1, the short transient repolarization that is followed by the plateau phase 2. Phase 2 is followed by phase 3, which is the repolarization back to the resting membrane potential. The resting state of the membrane potential is called

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As mentioned, cardiac action potential results from the chain reaction of multiple ion channels. Therefore a malfunction of a single ion channel can be observed from the action potential curve. Figure 2 presents the parameters which are used in analyzing the action potential. In regard to analyzing cardiac disease specific cells, the action potential duration plays an important role

As a method, patch clamp is very informative and provides invaluable data for example for pharmacological and safety pharmacological studies. However, it is very laborious, needs highly specialized machinery and, most importantly, dedicated and specialized users. For these reasons, semi-automated and automated patch clamp machinery are being developed

**Figure 2.** The phases of the cardiac action potential. ADP50 and ADP90 represent the action potential duration at 50% and 90% of the repolarization and these parameters are used in determining the duration of the action potential. The dV/dtmax represents the maximal upstroke velocity and can be used in assessing the electrophysiological phenotype

because the lengthening of the action potential may lead to severe arrhythmias.

and would be valuable for cardiomyocyte applications [50, 51].

as the phase 4 [49].

and maturity stage of the cardiomyocytes.

**Table 2.** Markers used in monitoring the cardiac differentiation.

#### **5.2. Electrophysiological methods**

#### *5.2.1. Patch clamp*

Traditional way to study the functionality and the electrical activity of the cardiomyocytes is the patch clamp technique [46]. Originally patch clamp method has been developed to study ion channels in excitable membranes [47]. In this technique micropipette is attached to the cell membrane by a giga seal and this can be exploited to measure current changes and voltage across the membrane. Due to the unique nature of the cardiomyocyte action potential curve, the ion channel composition and the maturation stage of the cardiomyocyte can be assessed and therefore the method has been widely used with stem cell derived cardiomyocyte studies.

Key cardiac ion channels (and respective current) involved in the human action potential are NaV1.5 (INa), KV4.3 (Ito), CaV1.2 (ICa,L) KV11.1 (IKr), KV7.1 (IKs), and Kir2.X (IK1) [48]. The cardiac action potential is composed of co-operation of these channels and the action potential curve can be divided into five different phases (Figure 2). Phase 0 of the action potential is the depolarization phase of the cardiomyocytes from the negative membrane potential to positive, called the upstroke. This is followed by phase 1, the short transient repolarization that is followed by the plateau phase 2. Phase 2 is followed by phase 3, which is the repolarization back to the resting membrane potential. The resting state of the membrane potential is called as the phase 4 [49].

be assessed at the gene or protein level with cardiomyocyte specific markers such as structural proteins troponin, alpha-actinin or myosins. The commonly used markers in monitoring the

> Nanog SOX2 Tra-1-60 SSEA-4

FoxC1 Dkk-1 Mesp1 Flk-1

Nkx2.5 GATA4 Tbx5 Isl-1 Mef2c Hand1/2

Sarcomeric α-actinin Myosin heavy- and lightchain (MHC and MLC)

cardiac differentiation are listed in Table 2.

420 Pluripotent Stem Cells

**Cell stage Markers** Pluripotent cells OCT4

Precardiac/cardiac mesodermal cells Brachyury T

Cardiac precursor cells KDR

**Table 2.** Markers used in monitoring the cardiac differentiation.

**5.2. Electrophysiological methods**

*5.2.1. Patch clamp*

Cardiac cells Troponin I and T

Traditional way to study the functionality and the electrical activity of the cardiomyocytes is the patch clamp technique [46]. Originally patch clamp method has been developed to study ion channels in excitable membranes [47]. In this technique micropipette is attached to the cell membrane by a giga seal and this can be exploited to measure current changes and voltage across the membrane. Due to the unique nature of the cardiomyocyte action potential curve, the ion channel composition and the maturation stage of the cardiomyocyte can be assessed and therefore the method has been widely used with stem cell derived cardiomyocyte studies.

Key cardiac ion channels (and respective current) involved in the human action potential are NaV1.5 (INa), KV4.3 (Ito), CaV1.2 (ICa,L) KV11.1 (IKr), KV7.1 (IKs), and Kir2.X (IK1) [48]. The

As mentioned, cardiac action potential results from the chain reaction of multiple ion channels. Therefore a malfunction of a single ion channel can be observed from the action potential curve. Figure 2 presents the parameters which are used in analyzing the action potential. In regard to analyzing cardiac disease specific cells, the action potential duration plays an important role because the lengthening of the action potential may lead to severe arrhythmias.

As a method, patch clamp is very informative and provides invaluable data for example for pharmacological and safety pharmacological studies. However, it is very laborious, needs highly specialized machinery and, most importantly, dedicated and specialized users. For these reasons, semi-automated and automated patch clamp machinery are being developed and would be valuable for cardiomyocyte applications [50, 51].

**Figure 2.** The phases of the cardiac action potential. ADP50 and ADP90 represent the action potential duration at 50% and 90% of the repolarization and these parameters are used in determining the duration of the action potential. The dV/dtmax represents the maximal upstroke velocity and can be used in assessing the electrophysiological phenotype and maturity stage of the cardiomyocytes.

#### *5.2.2. Micro electrode array*

In addition to the traditional patch clamp technique [46] the micro electrode array (MEA) – platform [52] offers practical, relative easy and non-invasive technique to assess the electrical properties of the differentiated cardiomyocytes [53]. Contrary to the patch clamp, the MEA system measures the electrical activity of a cell population. Therefore the signal resembles electrocardiogram (ECG) and is called field potential instead of action potential. Even though the ion channel function cannot be studied in the similar accuracy as with patch clamp, it allows examination field potential properties, such as cardiac repolarization, and therefore enables drug effect investigation [53]. During the last years, MEA has been widely used in character‐ ization of hESC- and iPS cell derived cardiomyocytes [31, 54]. MEA has been become a basic electrophysiological tool and in addition to cardiomyocytes, it has been successfully used also with other cell types, such as neurons [55].

*5.2.4. Force measurement*

with custom made platforms [61, 62].

**6.1. Long QT-syndrome**

seizures and even sudden cardiac death.

**6. Diseases modeled with iPS cell technique**

edly provide groundbreaking innovations in the future.

Recently a lot of effort has been applied to develop measuring systems to understand the mechanobiology of cardiomyocytes. Force measurement technique can be applied to measure isometric cardiomyocyte force contraction. A number of parameters can be determined by using the cardiomyocyte force measurement such as determination of Ca-sensitivity, cooper‐ ativity of force production and maximal Ca-activated force. Kinetics of the contractile re‐ sponses can also be measured such as the actin-myosin turnover kinetics. These parameters can be useful in the characterization of myofibrillar pathologies of various origin and drug effects. Most of the currently existing systems are only suitable for the study of cardiac tissue slices and therefore inappropriate to be used for iPS cell derived cardiomyocytes. Recently, however, cardiomyocyte force measurement system based on atomic force microscopy (AFM) was developed which can also be used to study single cardiomyocytes and small clusters of cardiomyocytes [60]. With the AFM system they were able to measure contractile forces, beat frequencies and durations of single cardiomyocytes and small cardiomyocyte clusters. The AFM-based method is also applicable for the screening of cardiac-active pharmacological agents. Cardiac microtissues have also been constructed using human pluripotent stem cell derived cardiomyocytes and the contraction force of the beating tissues has been analyzed

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Since the revolutionary discovery of iPS cells, multiple genetic diseases including cardiac and neuronal diseases have been modeled with patient specific iPS cell derived cells. Since primary human cardiomyocytes are not available for research in vitro, iPS cell derived cardiomyocytes are invaluable tool to study the pathophysiology of severe cardiac diseases and will undoubt‐

Long QT-syndrome (LQTS) appears as a genetic or a drug-induced form. It is characterized by a prolonged cardiac repolarization phase resulting in a prolonged QT interval in the surface electrocardiogram (ECG). The clinical symptoms of LQTS include palpitations, syncope and

More than 700 mutations in 12 different genes (LQT1–12) have been found to affect genetic forms of LQTS [63]. However, two of these subtypes account the majority (>90%) of all the genetically identified LQTSs. Both of these mutations affect potassium channels altering their proper function. LQTS type 1 (LQT1) is the most common LQTS subtype, resulting from mutations in the KCNQ1 gene. This gene encodes the α-subunit of the slow component of the delayed rectifier potassium current (IKs) channel [64]. Individuals with LQT1 typically have

LQTS type 2 (LQT2) is due to non-proper functioning of the α-subunit of the rapid delayed potassium channel (IKr), which is encoded by the human ether-a-go-go-related gene (HERG),

symptoms when the heart rate is elevated e.g. during exercise [65, 66].

The MEA system is also applicable in studying cardiac cell responses to pharmaceutical agents [54]. It also enables cells to be measured repeatedly for longer periods of times e.g. multiple days or weeks. However, the analysis of MEA measurement data is laborious. Therefore, semiautomated and automated systems for data analysis have been developed, which makes MEA system more reliable and efficient tool in research [56].

#### *5.2.3. The assessment of calcium homeostasis*

In addition to the unique co-operation of cardiac ion channels, the interaction of calcium-ions with cardiac structure proteins is another crucial feature in cardiomyocytes that is essential for the proper function of the heart. In human cardiomyocytes, calcium ion (Ca2+) influx through L-type calcium channels during the plateau phase triggers the Ca2+-release from the sarcoplasmic reticulum (SR) which is mediated by the ryanodine receptors (RyR2). The Ca2+ influx together with the release raises the free calcium concentration inside the cardiomyo‐ cytes. In sytosol, free calcium binds to troponin C in the myofilaments and triggers the machinery which induces the cell contraction. For the cell relaxation to occur, the calcium has to be rapidly removed from the cytosol. The removal is efficient with the aid of four separate pathways; sarcoplasmic reticulum Ca2+-ATPase (Serca2a), sarcolemmal Ca2+-ATPase, sarcolemmal Na+/Ca2+ exchanger and mitochondrial Ca2+ uniport [57].

Similarly as the regular and synchronous chain of action potentials, calcium concentration fluctuates in the cardiomyocytes. Therefore, with the aid of calcium binding dyes and modern fluorescence microscope systems, the function and response to pharmaceutical agents of cardiomyocytes can be monitored. This method is called calcium imaging [58, 59]. The calcium binding dyes, such as Fura-2 and Fluo-4, can be loaded inside the cardiomyocyte cytosol and when the calcium ions are released to the cytosol, the ions bind to the dyes and a fluorescence signal can be detected. When the fluorescence intensity is measured from the single cell, the calcium handling of the single cardiomyocyte can be monitored and analyzed. From the calcium imaging data, the beating rate and the function of the calcium handling machinery in the cardiomyocytes can be assessed. If the calcium is not released or withdrawn from the cardiomyocyte cytosol in a proper way, irregularity or multiple peaks can be seen in the calcium imaging curve.

#### *5.2.4. Force measurement*

*5.2.2. Micro electrode array*

422 Pluripotent Stem Cells

with other cell types, such as neurons [55].

*5.2.3. The assessment of calcium homeostasis*

calcium imaging curve.

system more reliable and efficient tool in research [56].

In addition to the traditional patch clamp technique [46] the micro electrode array (MEA) – platform [52] offers practical, relative easy and non-invasive technique to assess the electrical properties of the differentiated cardiomyocytes [53]. Contrary to the patch clamp, the MEA system measures the electrical activity of a cell population. Therefore the signal resembles electrocardiogram (ECG) and is called field potential instead of action potential. Even though the ion channel function cannot be studied in the similar accuracy as with patch clamp, it allows examination field potential properties, such as cardiac repolarization, and therefore enables drug effect investigation [53]. During the last years, MEA has been widely used in character‐ ization of hESC- and iPS cell derived cardiomyocytes [31, 54]. MEA has been become a basic electrophysiological tool and in addition to cardiomyocytes, it has been successfully used also

The MEA system is also applicable in studying cardiac cell responses to pharmaceutical agents [54]. It also enables cells to be measured repeatedly for longer periods of times e.g. multiple days or weeks. However, the analysis of MEA measurement data is laborious. Therefore, semiautomated and automated systems for data analysis have been developed, which makes MEA

In addition to the unique co-operation of cardiac ion channels, the interaction of calcium-ions with cardiac structure proteins is another crucial feature in cardiomyocytes that is essential for the proper function of the heart. In human cardiomyocytes, calcium ion (Ca2+) influx through L-type calcium channels during the plateau phase triggers the Ca2+-release from the sarcoplasmic reticulum (SR) which is mediated by the ryanodine receptors (RyR2). The Ca2+ influx together with the release raises the free calcium concentration inside the cardiomyo‐ cytes. In sytosol, free calcium binds to troponin C in the myofilaments and triggers the machinery which induces the cell contraction. For the cell relaxation to occur, the calcium has to be rapidly removed from the cytosol. The removal is efficient with the aid of four separate pathways; sarcoplasmic reticulum Ca2+-ATPase (Serca2a), sarcolemmal Ca2+-ATPase,

Similarly as the regular and synchronous chain of action potentials, calcium concentration fluctuates in the cardiomyocytes. Therefore, with the aid of calcium binding dyes and modern fluorescence microscope systems, the function and response to pharmaceutical agents of cardiomyocytes can be monitored. This method is called calcium imaging [58, 59]. The calcium binding dyes, such as Fura-2 and Fluo-4, can be loaded inside the cardiomyocyte cytosol and when the calcium ions are released to the cytosol, the ions bind to the dyes and a fluorescence signal can be detected. When the fluorescence intensity is measured from the single cell, the calcium handling of the single cardiomyocyte can be monitored and analyzed. From the calcium imaging data, the beating rate and the function of the calcium handling machinery in the cardiomyocytes can be assessed. If the calcium is not released or withdrawn from the cardiomyocyte cytosol in a proper way, irregularity or multiple peaks can be seen in the

sarcolemmal Na+/Ca2+ exchanger and mitochondrial Ca2+ uniport [57].

Recently a lot of effort has been applied to develop measuring systems to understand the mechanobiology of cardiomyocytes. Force measurement technique can be applied to measure isometric cardiomyocyte force contraction. A number of parameters can be determined by using the cardiomyocyte force measurement such as determination of Ca-sensitivity, cooper‐ ativity of force production and maximal Ca-activated force. Kinetics of the contractile re‐ sponses can also be measured such as the actin-myosin turnover kinetics. These parameters can be useful in the characterization of myofibrillar pathologies of various origin and drug effects. Most of the currently existing systems are only suitable for the study of cardiac tissue slices and therefore inappropriate to be used for iPS cell derived cardiomyocytes. Recently, however, cardiomyocyte force measurement system based on atomic force microscopy (AFM) was developed which can also be used to study single cardiomyocytes and small clusters of cardiomyocytes [60]. With the AFM system they were able to measure contractile forces, beat frequencies and durations of single cardiomyocytes and small cardiomyocyte clusters. The AFM-based method is also applicable for the screening of cardiac-active pharmacological agents. Cardiac microtissues have also been constructed using human pluripotent stem cell derived cardiomyocytes and the contraction force of the beating tissues has been analyzed with custom made platforms [61, 62].

## **6. Diseases modeled with iPS cell technique**

Since the revolutionary discovery of iPS cells, multiple genetic diseases including cardiac and neuronal diseases have been modeled with patient specific iPS cell derived cells. Since primary human cardiomyocytes are not available for research in vitro, iPS cell derived cardiomyocytes are invaluable tool to study the pathophysiology of severe cardiac diseases and will undoubt‐ edly provide groundbreaking innovations in the future.

#### **6.1. Long QT-syndrome**

Long QT-syndrome (LQTS) appears as a genetic or a drug-induced form. It is characterized by a prolonged cardiac repolarization phase resulting in a prolonged QT interval in the surface electrocardiogram (ECG). The clinical symptoms of LQTS include palpitations, syncope and seizures and even sudden cardiac death.

More than 700 mutations in 12 different genes (LQT1–12) have been found to affect genetic forms of LQTS [63]. However, two of these subtypes account the majority (>90%) of all the genetically identified LQTSs. Both of these mutations affect potassium channels altering their proper function. LQTS type 1 (LQT1) is the most common LQTS subtype, resulting from mutations in the KCNQ1 gene. This gene encodes the α-subunit of the slow component of the delayed rectifier potassium current (IKs) channel [64]. Individuals with LQT1 typically have symptoms when the heart rate is elevated e.g. during exercise [65, 66].

LQTS type 2 (LQT2) is due to non-proper functioning of the α-subunit of the rapid delayed potassium channel (IKr), which is encoded by the human ether-a-go-go-related gene (HERG), also known as KCNH2-gene [67]. Contrary to type 1, individuals with LQT2 have clinical symptoms when the heart rate is slow [65, 66] and syptoms can be triggered e.g. by an alarm clock during sleep. The drug induced form of LQTS is due to altered function of the HERGchannel by the drug, therefore this channel has a significant importance during drug devel‐ opment and in safety studies.

A human cell model for LQT3 has also been produced and its function and characteristics were compared with a mouse models which were based on both mouse ESCs and mouse iPS cells affected with the same disease specific mutation [71]. LQT3 syndrome is due to mutations in the SCN5A gene, which encodes for the α-subunit of the cardiac sodium (Na+) channel. These mutations disrupt the inactivation of the Na+ channel during the action potential plateu phase and this irruption leads to the delay in repolarization and further prolonged QT interval [72]. In addition to LQT3, another kind of cardiac arrhythmia syndromes such as Brugada syndrome and cardiac conduction disease are associated with mutations in the SCN5A gene. In these syndromes the mutations are loss-of-function-type whereas LQT3 syndrome they are gain-offunction-type mutations [72, 73]. The comparison of multiple types of pluripotent stem cell derived cardiomyocytes showed that all of these models manifest the symptoms of the disease and, furthermore, the characteristics are similar within both species [71]. iPS cell models for

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Timothy syndrome is caused by a single mutation in the CACNA1C-gene. This gene encodes the main L-type calcium channel, Cav1.2, in the mammalian heart which is essential for the cardiac action potential and also for cardiomyocyte contraction [74-76]. Timothy sydrome characterized by LQTS, syndactyly (webbing of fingers and toes), immune deficiency and autism [77] iPS cell derived cardiomyocytes originating from Timothy syndrome patients exhibited irregular functional properties typical for the disease [78]. Interestingly, these irregularities were restored by roscovitine, a compound which increases the voltage-depend‐

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disorder characterized by stress-induced polymorphic ventricular tachycardia in a structurally normal heart. CPVT is a very severe disease and 30-35% of mutation carriers have had symptoms (stress-related syncope, seizures or sudden death) by the age of 30. This disease is caused by mutations in the genes of RyR2 or calsequestrin (CASQ2) which is a regulatory

Multiple iPS-based CPVT disease models have been published, most of them having the disease specific mutation in the RyR2 gene while [83-85] and one having the mutation in the CASQ2 gene [86]. The congruent result from these CPVT model studies was the occurrence of delayed after depolarizations (DADs) and arrhythmias which are caused by the aberrant diastolic Ca2+ from the SR. Notably the model with RyR2-P2328S mutation also exhibited early after depolarizations (EADs) in addition to DADs suggesting suggest‐ ing another pathophysiological mechanism for CPVT [85]. Intriguing finding was also the

these loss-of-function diseases have not yet been described.

*6.1.2. Catecholaminergic Polymorphic Ventricular Tachycardia*

calcium-buffering protein associated with RyR2 in the SR [79-82].

effect of dantrolene in rescuing the arrhythmogenic phenotype [84].

*6.1.1. Timothy syndrome*

ent inactivation of Cav1.2 [78].

The prevalence of the genetic form of LQTS is 1:2,000 in the general population [63]. However, the penetrance of the clinical symptoms of LQTS is low and there is considerable variation in phenotypic expression even within families carrying the same mutation [68]. It has also been suggested, that the population prevalence of milder LQTS mutations might be higher. Therefore the prevalence of latent or concealed LQTS, i.e. relatively asymptomatic individuals, would be higher than currently anticipated [69]. Due to this challenging and complex nature of LQTS, in addition, to the great interest of pharmaceutical industry towards this disease, multiple reports of iPS cell- based LQTS cell models have been published since 2007 when the iPS technology was invented.

Moretti and co-workers produced iPS cell derived cardiomycotyes from two patients carrying a KCNQ1 (R190Q) mutation [6]. In this study, the cardiomyocytes possessed the LQT1 genotype and exhibited prolonged action potential duration. The action potential prolongation was determined to be caused by the ion-channel trafficking defect resulting in a 70-80% IKs current density reduction. A β-adrenergic agonist isoproterenol altered the activation and deactivation kinetics of the IKs and this effect was rescued by the β-blockade [6]. Egashira and co-workers also produced a disease model for LQTS type 1 [70]. In their study, the iPS cells were derived from a sporadic patient who did not have a family history of significant QT interval abnormality. The mutation of the patient in the KCNQ1 was novel (1893delC) and the cells exhibited prolonged action potential duration in addition to arrhythmogenity.

Similarly results were found with iPS-CM derived from a patient suffering from the severe LQT type 2 syndrome. The patient had hERG (A614V) mutation and previously presented episodes of torsade de pointes (TdP), a special type of polymorphic ventricular tachycardia which is associated with LQTS [4]. The LQT2-cardiomyocytes derived from the patient's iPS cells demonstrated increased arrhythmogenicity associated with early after depolarizations (EADs) [5]. In addition, significant APD prolongation due to a reduced IKr current density was observed [4]. Arrhytmia and EADs were also induced by a specific HERG-channel blocker E-4031 to iPS cell derived CM having a hERG (G1681A) mutation. In addition, these cells exhibited EADs caused by the isoproterenol treatment and these EADs were rescued by βblockade [5].

All the aforementioned studies were done with iPS cells derived from the symptomatic LQTS patients. Nevertheless, similar results have been obtained from patients without severe symptoms. In the study made in our institute, iPS cell lines were derived from a patient having a KCNH2 (R176W) mutation and a family history of LQTS. However, this individual was asymptomatic except for occasional palpitations. iPS cell derived cardiomyocytes from this patient manifest the phenotype characteristics to LQT2, such as a prolonged repolarization time and increased arrhythmogenicity [3].

A human cell model for LQT3 has also been produced and its function and characteristics were compared with a mouse models which were based on both mouse ESCs and mouse iPS cells affected with the same disease specific mutation [71]. LQT3 syndrome is due to mutations in the SCN5A gene, which encodes for the α-subunit of the cardiac sodium (Na+) channel. These mutations disrupt the inactivation of the Na+ channel during the action potential plateu phase and this irruption leads to the delay in repolarization and further prolonged QT interval [72]. In addition to LQT3, another kind of cardiac arrhythmia syndromes such as Brugada syndrome and cardiac conduction disease are associated with mutations in the SCN5A gene. In these syndromes the mutations are loss-of-function-type whereas LQT3 syndrome they are gain-offunction-type mutations [72, 73]. The comparison of multiple types of pluripotent stem cell derived cardiomyocytes showed that all of these models manifest the symptoms of the disease and, furthermore, the characteristics are similar within both species [71]. iPS cell models for these loss-of-function diseases have not yet been described.

#### *6.1.1. Timothy syndrome*

also known as KCNH2-gene [67]. Contrary to type 1, individuals with LQT2 have clinical symptoms when the heart rate is slow [65, 66] and syptoms can be triggered e.g. by an alarm clock during sleep. The drug induced form of LQTS is due to altered function of the HERGchannel by the drug, therefore this channel has a significant importance during drug devel‐

The prevalence of the genetic form of LQTS is 1:2,000 in the general population [63]. However, the penetrance of the clinical symptoms of LQTS is low and there is considerable variation in phenotypic expression even within families carrying the same mutation [68]. It has also been suggested, that the population prevalence of milder LQTS mutations might be higher. Therefore the prevalence of latent or concealed LQTS, i.e. relatively asymptomatic individuals, would be higher than currently anticipated [69]. Due to this challenging and complex nature of LQTS, in addition, to the great interest of pharmaceutical industry towards this disease, multiple reports of iPS cell- based LQTS cell models have been published since 2007 when the

Moretti and co-workers produced iPS cell derived cardiomycotyes from two patients carrying a KCNQ1 (R190Q) mutation [6]. In this study, the cardiomyocytes possessed the LQT1 genotype and exhibited prolonged action potential duration. The action potential prolongation was determined to be caused by the ion-channel trafficking defect resulting in a 70-80% IKs current density reduction. A β-adrenergic agonist isoproterenol altered the activation and deactivation kinetics of the IKs and this effect was rescued by the β-blockade [6]. Egashira and co-workers also produced a disease model for LQTS type 1 [70]. In their study, the iPS cells were derived from a sporadic patient who did not have a family history of significant QT interval abnormality. The mutation of the patient in the KCNQ1 was novel (1893delC) and the

cells exhibited prolonged action potential duration in addition to arrhythmogenity.

Similarly results were found with iPS-CM derived from a patient suffering from the severe LQT type 2 syndrome. The patient had hERG (A614V) mutation and previously presented episodes of torsade de pointes (TdP), a special type of polymorphic ventricular tachycardia which is associated with LQTS [4]. The LQT2-cardiomyocytes derived from the patient's iPS cells demonstrated increased arrhythmogenicity associated with early after depolarizations (EADs) [5]. In addition, significant APD prolongation due to a reduced IKr current density was observed [4]. Arrhytmia and EADs were also induced by a specific HERG-channel blocker E-4031 to iPS cell derived CM having a hERG (G1681A) mutation. In addition, these cells exhibited EADs caused by the isoproterenol treatment and these EADs were rescued by β-

All the aforementioned studies were done with iPS cells derived from the symptomatic LQTS patients. Nevertheless, similar results have been obtained from patients without severe symptoms. In the study made in our institute, iPS cell lines were derived from a patient having a KCNH2 (R176W) mutation and a family history of LQTS. However, this individual was asymptomatic except for occasional palpitations. iPS cell derived cardiomyocytes from this patient manifest the phenotype characteristics to LQT2, such as a prolonged repolarization

opment and in safety studies.

424 Pluripotent Stem Cells

iPS technology was invented.

blockade [5].

time and increased arrhythmogenicity [3].

Timothy syndrome is caused by a single mutation in the CACNA1C-gene. This gene encodes the main L-type calcium channel, Cav1.2, in the mammalian heart which is essential for the cardiac action potential and also for cardiomyocyte contraction [74-76]. Timothy sydrome characterized by LQTS, syndactyly (webbing of fingers and toes), immune deficiency and autism [77] iPS cell derived cardiomyocytes originating from Timothy syndrome patients exhibited irregular functional properties typical for the disease [78]. Interestingly, these irregularities were restored by roscovitine, a compound which increases the voltage-depend‐ ent inactivation of Cav1.2 [78].

#### *6.1.2. Catecholaminergic Polymorphic Ventricular Tachycardia*

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disorder characterized by stress-induced polymorphic ventricular tachycardia in a structurally normal heart. CPVT is a very severe disease and 30-35% of mutation carriers have had symptoms (stress-related syncope, seizures or sudden death) by the age of 30. This disease is caused by mutations in the genes of RyR2 or calsequestrin (CASQ2) which is a regulatory calcium-buffering protein associated with RyR2 in the SR [79-82].

Multiple iPS-based CPVT disease models have been published, most of them having the disease specific mutation in the RyR2 gene while [83-85] and one having the mutation in the CASQ2 gene [86]. The congruent result from these CPVT model studies was the occurrence of delayed after depolarizations (DADs) and arrhythmias which are caused by the aberrant diastolic Ca2+ from the SR. Notably the model with RyR2-P2328S mutation also exhibited early after depolarizations (EADs) in addition to DADs suggesting suggest‐ ing another pathophysiological mechanism for CPVT [85]. Intriguing finding was also the effect of dantrolene in rescuing the arrhythmogenic phenotype [84].

#### *6.1.3. Cardiomyopathies*

Mutations in the genes expressed in the cardiomyocytes can cause heart diseases known as cardiomyopathies. Cardiomyopathies are currently categorized into the following four classes: arrythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy [87]. Cardiomyopathies that are associated with mutations in genes encoding for sarcomeric proteins are a frequent cause of heart failure. changes including increase in protein synthesis, enhanced sarcomere reorganization as well

Disease Models for the Genetic Cardiac Diseases

http://dx.doi.org/10.5772/55773

427

iPS cell technology has not yet been reported to model HCM. However, iPS cells were used to generate cardiomyocytes from two LEOPARD syndrome patients carrying mutation in the PTPN11 gene encoding for the SHP2 phosphatase [101]. LEOPARD syndrome is an autosomaldominant developmental disorder belonging to inherited RAS-mitogen-activated protein kinase signalling diseases. A major disease phenotype of the LEOPARD syndrome patients is HCM [102]. The iPS cell derived cardiomyocytes from LEOPARD syndrome were larger, had a higher degree of sarcomeric organization as well as preferential localization of NFATC4 in the nucleus when compared to iPS cell derived cardiomyocytes from healthy sibling of the LEOPARD syndrome patient thus presenting some indications of hypertrophy in patient

There are still several challenges that need to be carefully considered when designing disease modeling studies with specialized cell types derived from iPS cells. One potential challenge relates to the reactivation of silenced exogenous transgenes in the iPS cells or in their differ‐ entiated derivatives leading to the altered genomic integrity which may have unknown effects on the differentiation potential and characteristics of differentiated cell types. Efforts to improve the reprogramming methods have led to the technical development of nonintegrating approaches for iPS cell generation which will eliminate this risk in the future iPS cell lines and their differentiated derivatives. The nonintegrating sendai virus tehnique is already widely used in the generation of iPS cells. Regular monitoring of exogenous genes in iPS cells lines

Many genetic cardiac diseases are complex demonstrating huge clinical heterogeneity even within families and patients having the same mutation. In addition, reprogrammed cells carry genetic alterations that have accumulated through life, thus there is a risk that the variance overwhelms the ability to detect the authentic mechanisms in the pathophysiology of the disease. Thus, it will be essential to investigate adequate number of iPSC lines and patients to be able to demonstrate the common features of the cardiac disease phenotype. Further, it may be advantageous to initially compare the characteristics of cardiomyocytes from patients

Most likely in many genetic cardiac diseases various cell types in the heart contribute to the pathophysiological responses of the disease, thus there is a risk that it is impossible to recapitulate the features of the disorder by using solely cardiomyocytes. A 3D human heart tissue model with proper composition of cardiomyocytes, endothelial cells, fibroblasts, smooth muscle cells as well as neurons has not been developed but in recent years the advancement in this field of research has been rapid and hopefully in future we have besides cell models

**7. Challenges with iPS cell technology and disease modeling**

generated by using the integrating techniques is advisable.

authentic tissue models to study genetic cardiac diseases.

as activation of specific cardiac genes [98-100].

specific cardiomyocytes.

having severe symptoms.

iPS cells have been used to generate cardiomyocytes from patients in a family with inherited dilated cardiomyopathy (DCM) [88]. The researchers generated a large number of individualspecific cardiomyocytes from a family carrying a deleterious point mutation (R173W) in TNNT2, a gene encoding for a sarcomeric protein cardiac troponin T, which regulates cardiomyocyte contraction. When compared to cardiomyocytes derived from iPS cells of healthy controls within the same family, the researchers showed that cardiomyocytes derived from iPS cells of DCM patients exhibited an increased heterogenous myofilament organization due to abnormal distribution of α-actinin, compromised ability to regulate calcium flux, and decreased contraction force. When DCM specific cardiomyocytes were stimulated with a βadrenergic agonist, the cells showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric αactinin distribution. The authors also showed that the function of DCM-specific cardiomyo‐ cytes was improved with the treatment with β-adrenergic blockers or overexpression of Serca2a.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is another genetic cardiomyop‐ athy characterized by replacement of cardiomyocytes by adipose and fibrous tissue leading to right ventricular failure, arrhythmias and even sudden death [89]. Twelve different genes have been linked to ARVC and all these encode cardiac cell adhesion proteins resulting in dysfunc‐ tional cardiac desmosomes. Cell adhesion proteins resulting in ARVC include plakoglobin (JUP), desmoplakin (DSP) and plakophilin 2 (PKP2). Patient specific iPS cells have been generated from an ARCV patient carrying a PKP2 mutation and having clinical manifestations of the disease [90]. ARVC specific cardiomyocytes revealed reduced amount of desmosomal proteins and more lipid droplets in the cardiomyocytes compared to control cardiomyocytes thus presenting the abnormalities observed in ARCV patients.

The third form of cardiomyopathy, hypertrophic cardiomyopathy (HCM), is a complex autosomal-dominant disease and the affected individuals acquire cardiac hypertrophy without external stimuli. Cardiac hypertrophy can be induced by different exogenous factors such as hypertension and valvular disease and even by severe exercise [91]. Affecting in 1 in 500 individuals within the general population, genetic HCM is the most common inherited cardiovascular disorder and the leading cause of sudden cardiac death in adolescents and young adults, especially in atheletes [92-94]. The majority of gene mutations associated with HCM occur in 13 sarcomere-related genes where several hundred mutations have been identified [94-97]. Typically cardiac hypertrophy affects the left ventricle and the interventric‐ ular septum and may eventually lead to left ventricular outflow tract obstruction, arrythmias, diastolic dysfunction, and sudden death. Other hallmark features are myocyte disarray and fibrosis [94-97]. The hypertrophic process in cardiomyocytes is characterized by morphological changes including increase in protein synthesis, enhanced sarcomere reorganization as well as activation of specific cardiac genes [98-100].

*6.1.3. Cardiomyopathies*

426 Pluripotent Stem Cells

Serca2a.

Mutations in the genes expressed in the cardiomyocytes can cause heart diseases known as cardiomyopathies. Cardiomyopathies are currently categorized into the following four classes: arrythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy [87]. Cardiomyopathies that are associated with mutations in genes encoding for sarcomeric proteins are a frequent cause of heart failure. iPS cells have been used to generate cardiomyocytes from patients in a family with inherited dilated cardiomyopathy (DCM) [88]. The researchers generated a large number of individualspecific cardiomyocytes from a family carrying a deleterious point mutation (R173W) in TNNT2, a gene encoding for a sarcomeric protein cardiac troponin T, which regulates cardiomyocyte contraction. When compared to cardiomyocytes derived from iPS cells of healthy controls within the same family, the researchers showed that cardiomyocytes derived from iPS cells of DCM patients exhibited an increased heterogenous myofilament organization due to abnormal distribution of α-actinin, compromised ability to regulate calcium flux, and decreased contraction force. When DCM specific cardiomyocytes were stimulated with a βadrenergic agonist, the cells showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric αactinin distribution. The authors also showed that the function of DCM-specific cardiomyo‐ cytes was improved with the treatment with β-adrenergic blockers or overexpression of

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is another genetic cardiomyop‐ athy characterized by replacement of cardiomyocytes by adipose and fibrous tissue leading to right ventricular failure, arrhythmias and even sudden death [89]. Twelve different genes have been linked to ARVC and all these encode cardiac cell adhesion proteins resulting in dysfunc‐ tional cardiac desmosomes. Cell adhesion proteins resulting in ARVC include plakoglobin (JUP), desmoplakin (DSP) and plakophilin 2 (PKP2). Patient specific iPS cells have been generated from an ARCV patient carrying a PKP2 mutation and having clinical manifestations of the disease [90]. ARVC specific cardiomyocytes revealed reduced amount of desmosomal proteins and more lipid droplets in the cardiomyocytes compared to control cardiomyocytes

The third form of cardiomyopathy, hypertrophic cardiomyopathy (HCM), is a complex autosomal-dominant disease and the affected individuals acquire cardiac hypertrophy without external stimuli. Cardiac hypertrophy can be induced by different exogenous factors such as hypertension and valvular disease and even by severe exercise [91]. Affecting in 1 in 500 individuals within the general population, genetic HCM is the most common inherited cardiovascular disorder and the leading cause of sudden cardiac death in adolescents and young adults, especially in atheletes [92-94]. The majority of gene mutations associated with HCM occur in 13 sarcomere-related genes where several hundred mutations have been identified [94-97]. Typically cardiac hypertrophy affects the left ventricle and the interventric‐ ular septum and may eventually lead to left ventricular outflow tract obstruction, arrythmias, diastolic dysfunction, and sudden death. Other hallmark features are myocyte disarray and fibrosis [94-97]. The hypertrophic process in cardiomyocytes is characterized by morphological

thus presenting the abnormalities observed in ARCV patients.

iPS cell technology has not yet been reported to model HCM. However, iPS cells were used to generate cardiomyocytes from two LEOPARD syndrome patients carrying mutation in the PTPN11 gene encoding for the SHP2 phosphatase [101]. LEOPARD syndrome is an autosomaldominant developmental disorder belonging to inherited RAS-mitogen-activated protein kinase signalling diseases. A major disease phenotype of the LEOPARD syndrome patients is HCM [102]. The iPS cell derived cardiomyocytes from LEOPARD syndrome were larger, had a higher degree of sarcomeric organization as well as preferential localization of NFATC4 in the nucleus when compared to iPS cell derived cardiomyocytes from healthy sibling of the LEOPARD syndrome patient thus presenting some indications of hypertrophy in patient specific cardiomyocytes.

## **7. Challenges with iPS cell technology and disease modeling**

There are still several challenges that need to be carefully considered when designing disease modeling studies with specialized cell types derived from iPS cells. One potential challenge relates to the reactivation of silenced exogenous transgenes in the iPS cells or in their differ‐ entiated derivatives leading to the altered genomic integrity which may have unknown effects on the differentiation potential and characteristics of differentiated cell types. Efforts to improve the reprogramming methods have led to the technical development of nonintegrating approaches for iPS cell generation which will eliminate this risk in the future iPS cell lines and their differentiated derivatives. The nonintegrating sendai virus tehnique is already widely used in the generation of iPS cells. Regular monitoring of exogenous genes in iPS cells lines generated by using the integrating techniques is advisable.