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114 Pluripotent Stem Cells

**Chapter 6**

**Human Testis–Derived Pluripotent Cells and Induced**

Pregnancy rates achieved by intercourse in normal human couples are 20-25% per month, 75% by six months, and 90% by one year [1]. However, 15% of couples of unknown fertility status are unable to conceive a baby after one year of intercourse without contraception. For 30% of these couples, their infertility can be attributed to a male factor alone; in an additional 20%, failure to conceive is explained by the presence of both male and female factors [2,3,4]. Among couples known to be infertile, a male factor is involved in 50% of the cases. The most common causes of male infertility include abnormal sperm production or function, impaired delivery of sperm, and overexposure to certain gonadotoxins in the environment. The pathogenesis of male infertility can be attributed to a disorder of germ-cell proliferation and differentiation or

The induction of spermatogenesis depends on the complementary actions of FSH and testosterone. FSH establishes the requisite Sertoli cell population. In the prepubertal primate, FSH alone can induce proliferation of Sertoli cells and spermatogonia, but this does not result in qualitatively and quantitatively normal spermatogenesis unless testosterone is simultane‐ ously present [6] [7]. Testosterone affects the functional completion of meiosis and post-meiotic sperm differentiation and maturation. LH stimulates Leydig cells to produce testosterone. Although FSH appears to play a more dominant role in the maintenance of primate sperma‐ togenesis than in its initiation, normal spermatogenesis is best maintained by the combined

The most severe form of male infertility is nonobstructive azoospermia, which is typically characterized by small-volume testes and elevated FSH. Patients with this disorder cannot

> © 2013 Kobayashi 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 Kobayashi 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.

**Pluripotent Stem Cells**

Koichi Nakajima

**1. Introduction**

http://dx.doi.org/10.5772/55570

to somatic cell dysfunction [5].

effects of FSH and LH [6].

Hideyuki Kobayashi, Koichi Nagao and

Additional information is available at the end of the chapter

## **Human Testis–Derived Pluripotent Cells and Induced Pluripotent Stem Cells**

Hideyuki Kobayashi, Koichi Nagao and Koichi Nakajima

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55570

## **1. Introduction**

Pregnancy rates achieved by intercourse in normal human couples are 20-25% per month, 75% by six months, and 90% by one year [1]. However, 15% of couples of unknown fertility status are unable to conceive a baby after one year of intercourse without contraception. For 30% of these couples, their infertility can be attributed to a male factor alone; in an additional 20%, failure to conceive is explained by the presence of both male and female factors [2,3,4]. Among couples known to be infertile, a male factor is involved in 50% of the cases. The most common causes of male infertility include abnormal sperm production or function, impaired delivery of sperm, and overexposure to certain gonadotoxins in the environment. The pathogenesis of male infertility can be attributed to a disorder of germ-cell proliferation and differentiation or to somatic cell dysfunction [5].

The induction of spermatogenesis depends on the complementary actions of FSH and testosterone. FSH establishes the requisite Sertoli cell population. In the prepubertal primate, FSH alone can induce proliferation of Sertoli cells and spermatogonia, but this does not result in qualitatively and quantitatively normal spermatogenesis unless testosterone is simultane‐ ously present [6] [7]. Testosterone affects the functional completion of meiosis and post-meiotic sperm differentiation and maturation. LH stimulates Leydig cells to produce testosterone. Although FSH appears to play a more dominant role in the maintenance of primate sperma‐ togenesis than in its initiation, normal spermatogenesis is best maintained by the combined effects of FSH and LH [6].

The most severe form of male infertility is nonobstructive azoospermia, which is typically characterized by small-volume testes and elevated FSH. Patients with this disorder cannot

© 2013 Kobayashi 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 Kobayashi 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.

produce biological children. Although microdissection testicular sperm extraction (micro-TESE) is used to treat patients with nonobstructive azoospermia [8], this technique does not have a good success rate. Therefore, new approaches are needed to develop treatments for male infertility.

Stem cells have the potential to differentiate into a variety of functional cell types in the body, and their discovery has given rise to the fields of regenerative medicine and cloning. Stem cells are regulated by the particular microenvironment in which they reside; these microenviron‐ ments are referred to as niches. Male germline stem cells can continuously produce sperm throughout adulthood, and investigators have sought to develop methods using stem cells to improve or restore fertility.

Embryonic stem cells (ESCs) have the potential to differentiate into nearly every cell type in the body. As the cells differentiate, they lose the ability to develop into different tissues. In contrast, specific tissues (gastrointestinal, integumentary, spermatogenic, and hematopoietic systems) maintain their regenerative capacity *in vivo*, and in fact, stem cells have been functionally identified in a wide range of adult tissues. These adult stem cells are believed to hold great promise for tissue generation in clinical settings. Here, we provide a summary of the therapeutic potential of stem cells for the rejuvenation of fertility in infertile males. Our hope is that future research will provide a range of options for the preservation of male fertility or the reversal of infertility.

the stem cell pool, while their differentiation generates a large number of germ cells. Therefore, a balance between SSC self-renewal and differentiation in the adult testis is essential to maintain normal spermatogenesis and fertility throughout life. SSCs need to reside in a unique environment, or niche, that provides the factors necessary for their survival and potency. In mice, Sertoli cells in the testis are a crucial component of the spermatogonial stem cell niche. They produce glial cell line-derived neurotrophic factor (GDNF), a distant member of the TGFβ family, which controls SSC self-renewal [16]. Several groups have reported that adding GDNF to freshly isolated germ cells in culture results in the proliferation of SSCs [17,18]. Other factors within the niche influence the fate of SSCs. One example is colony-stimulating factor 1 (CSF1), which is produced by Leydig cells and some peritubular myoid cells [19], and plays a

Human Testis–Derived Pluripotent Cells and Induced Pluripotent Stem Cells

http://dx.doi.org/10.5772/55570

119

The existence of SSCs was postulated almost 40 years ago on the basis of morphological studies [20] [21] [22] and observations of toxin-induced spermatogenic damage. The early studies of Clermont [23] [24] on human spermatogenesis revealed two types of spermatogonia, the Adark and Apale spermatogonia, which were differentiated by the staining pattern of their nucleus. Both cell types are generally considered stem cells [24,25]. Adark spermatogonia function as reverse stem cells that rarely divide, but can be triggered to self-renew in the case of injury or disease, while Apale spermatogonia are self-renewing stem cells [23,24,25,26]; they

In the last decade, molecular markers that can be used to identify and characterize human SSCs have been sought. A recent study reported that the expression of surface marker G protein coupled receptor 125 (GPR125) can be used in the isolation, characterization, and culture of putative human SSCs [27]. GPR125-positive spermatogonia are very rare, possibly limited to Adark spermatogonia or a sub-population of Apale spermatogonia. Human SSCs are also positive

also divide into B spermatogonia, which further divide into spermatocytes [24].

role in SSC self-renewal (Figure 1).

**Table 1.** Markers of human pluripotent stem cells and germ cells.

## **2. Differentiation and characterization of human primordial germ cells**

Human primordial germ cells (PGCs) can be isolated from tissues and their identity confirmed by observing their migratory activity in vitro [9]. Cultured human PGCs become human embryonic germ cells (hEGCs) in vitro, in the presence of feeder cells, leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) [10]. hEGCs express alkaline phospha‐ tase (AP), OCT4, SOX2, NANOG, stage specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81, which are pluripotent stem cell markers. In vivo, human PGCs do not express FGF4, SOX2 [11] [12], TRA-1-60, or TRA-1-81 [13] [14], which are expressed by hESCs or hEGCs in vitro. The molecular signature of human PGCs in vivo can be characterized as C-KIT+ , SOX2- , TRA1-60- , TRA1-81- , and FGF4- , in contrast with human pluripotent stem cell lines in vitro. (This information is summarized in Table 1.) However, the full complement of genes that are expressed specifically in human PGCs and their functions remain unclear.

## **3. Spermatogonial stem cells**

Spermatogenesis is a complex and tightly regulated process in which a small pool of germline stem cells ultimately gives rise to spermatozoa [15]. These stem cells, called spermatogo‐ nial stem cells (SSCs) are found in the basal compartment of the seminiferous epithelium, where they adhere to the basement membrane. SSC self-renewal ensures the maintenance of

**Table 1.** Markers of human pluripotent stem cells and germ cells.

produce biological children. Although microdissection testicular sperm extraction (micro-TESE) is used to treat patients with nonobstructive azoospermia [8], this technique does not have a good success rate. Therefore, new approaches are needed to develop treatments for

Stem cells have the potential to differentiate into a variety of functional cell types in the body, and their discovery has given rise to the fields of regenerative medicine and cloning. Stem cells are regulated by the particular microenvironment in which they reside; these microenviron‐ ments are referred to as niches. Male germline stem cells can continuously produce sperm throughout adulthood, and investigators have sought to develop methods using stem cells to

Embryonic stem cells (ESCs) have the potential to differentiate into nearly every cell type in the body. As the cells differentiate, they lose the ability to develop into different tissues. In contrast, specific tissues (gastrointestinal, integumentary, spermatogenic, and hematopoietic systems) maintain their regenerative capacity *in vivo*, and in fact, stem cells have been functionally identified in a wide range of adult tissues. These adult stem cells are believed to hold great promise for tissue generation in clinical settings. Here, we provide a summary of the therapeutic potential of stem cells for the rejuvenation of fertility in infertile males. Our hope is that future research will provide a range of options for the preservation of male fertility

**2. Differentiation and characterization of human primordial germ cells**

, and FGF4-

cell lines in vitro. (This information is summarized in Table 1.) However, the full complement of genes that are expressed specifically in human PGCs and their functions remain unclear.

Spermatogenesis is a complex and tightly regulated process in which a small pool of germline stem cells ultimately gives rise to spermatozoa [15]. These stem cells, called spermatogo‐ nial stem cells (SSCs) are found in the basal compartment of the seminiferous epithelium, where they adhere to the basement membrane. SSC self-renewal ensures the maintenance of

, in contrast with human pluripotent stem

Human primordial germ cells (PGCs) can be isolated from tissues and their identity confirmed by observing their migratory activity in vitro [9]. Cultured human PGCs become human embryonic germ cells (hEGCs) in vitro, in the presence of feeder cells, leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) [10]. hEGCs express alkaline phospha‐ tase (AP), OCT4, SOX2, NANOG, stage specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81, which are pluripotent stem cell markers. In vivo, human PGCs do not express FGF4, SOX2 [11] [12], TRA-1-60, or TRA-1-81 [13] [14], which are expressed by hESCs or hEGCs in vitro. The molecular signature of human PGCs in vivo can be characterized

male infertility.

118 Pluripotent Stem Cells

improve or restore fertility.

or the reversal of infertility.

as C-KIT+

, SOX2-

, TRA1-60-

**3. Spermatogonial stem cells**

, TRA1-81-

the stem cell pool, while their differentiation generates a large number of germ cells. Therefore, a balance between SSC self-renewal and differentiation in the adult testis is essential to maintain normal spermatogenesis and fertility throughout life. SSCs need to reside in a unique environment, or niche, that provides the factors necessary for their survival and potency. In mice, Sertoli cells in the testis are a crucial component of the spermatogonial stem cell niche. They produce glial cell line-derived neurotrophic factor (GDNF), a distant member of the TGFβ family, which controls SSC self-renewal [16]. Several groups have reported that adding GDNF to freshly isolated germ cells in culture results in the proliferation of SSCs [17,18]. Other factors within the niche influence the fate of SSCs. One example is colony-stimulating factor 1 (CSF1), which is produced by Leydig cells and some peritubular myoid cells [19], and plays a role in SSC self-renewal (Figure 1).

The existence of SSCs was postulated almost 40 years ago on the basis of morphological studies [20] [21] [22] and observations of toxin-induced spermatogenic damage. The early studies of Clermont [23] [24] on human spermatogenesis revealed two types of spermatogonia, the Adark and Apale spermatogonia, which were differentiated by the staining pattern of their nucleus. Both cell types are generally considered stem cells [24,25]. Adark spermatogonia function as reverse stem cells that rarely divide, but can be triggered to self-renew in the case of injury or disease, while Apale spermatogonia are self-renewing stem cells [23,24,25,26]; they also divide into B spermatogonia, which further divide into spermatocytes [24].

In the last decade, molecular markers that can be used to identify and characterize human SSCs have been sought. A recent study reported that the expression of surface marker G protein coupled receptor 125 (GPR125) can be used in the isolation, characterization, and culture of putative human SSCs [27]. GPR125-positive spermatogonia are very rare, possibly limited to Adark spermatogonia or a sub-population of Apale spermatogonia. Human SSCs are also positive

servation. However, most couples would prefer a naturally conceived child. Work has progressed in many laboratories to partially enrich the spermatogonial stem cells of species ranging from mice to primates. Today, many urologists bank a testicular biopsy from patients about to undergo chemotherapy, with the expectation that technology will advance rapidly

Human Testis–Derived Pluripotent Cells and Induced Pluripotent Stem Cells

http://dx.doi.org/10.5772/55570

121

Previous studies have demonstrated that neonatal and adult germline stem cells (GSCs) can be self-reprogrammed into ESC-like cells, called germline-derived pluripotent stem cells [32,33,34,35]. In addition, Conrad et al. [36] reported that pluripotent cells can be derived from human testis, which those authors called human adult GSCs (haGSCs). Other research groups subsequently claimed that ESC-like cells could be obtained from cultures of human testicular cells [37,38,39]. Conrad and colleagues compared the global gene expression profile of hESCs and haGSCs, and concluded that the populations presented a similar gene expression profile, and thus, that the haGSCs were pluripotent. However, Ko et al. claimed that the gene expres‐ sion profile of haGSCs differed substantially from the pluripotent profile of hESCs, determined by a number of laboratories [40]. For example, the haGSCs did not express NANOG, and had low OCT4 and SOX2 levels, but showed high levels of the fibroblast markers SNA12 and ACTA2 [40]. Ko and colleagues therefore suggested that the haGSCs originated from fibroblast cells, rather than from pluripotent tissue. They concluded that haGSCs were very similar to a human testicular fibroblast cell line (hTFCs) [40]. Conrad and colleagues argued that micro‐ array data sets cannot be compared unless they are processed in parallel in the same experi‐ ment, suggesting that the similarity between haGSCs and hTFCs was inconclusive. However, studies on microarray results generated by different laboratories [41,42,43] have shown that findings from microarray analyses are comparable across multiple laboratories [44], particu‐ larly when a common platform and set of procedures are used. These findings justify the utility of microarray repositories, such as the GEO database [45], not only as data warehouses but also as resources for comparative and combinatory analyses of microarray data from different laboratories. In conclusion, the global gene expression analysis of haGSCs demonstrated that

over the next 10 years and allow transplantation in the future.

**4. Pluripotency of human testis–derived ESC–like cells**

these cells resembled fibroblast hTFCs more than pluripotent hESCs.

The year 2006 saw the first description of mouse induced pluripotent stem cells (miPSCs), which were generated by the retrovirus-mediated transduction of four transcription factors (OCT3/4, SOX2, KLF4, and C-MYC) into mouse fibroblasts [46]. Human somatic cells can be reprogrammed to become human iPSCs via the introduction of a small set of genes, either those encoding OCT3/4, SOX2 and KLF4, with or without the addition of C-MYC, or an alternate combination of OCT3/4, SOX2, LIN28, and NANOG [47,48,49,50,51,52,53,54,55]. Human iPSCs (hiPSCs) have remarkable similarity to hESCs in terms of their morphology, in

**5. Induced pluripotent stem (iPS) cells**

**Figure 1.** Diagram of the spermatogonial stem cell (SSC) niche showing that extrinsic factors drive SSC maintenance and self-renewal. SSCs and Sertoli cells are attached to the basement membrane. Sertoli cells produce glial cell linederived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF). Leydig cells and peritubular cells pro‐ duce colony-stimulating factor-1 (CSF-1).

for some markers identified in mouse SSCs and other undifferentiated spermatogonia, including GFRA1, UCHL1 (PGP9.5), ZBTB16 (PLZF), and THY1 (CD90) [27,28]. We also have obtained evidence that THY1 is a potential surface marker for human SSCs [29].

Brinster and colleagues proved the existence of mouse SSCs by using unique approaches [30, 31]. These investigators transplanted cells obtained as testicular homogenates expressing the *LacZ* gene into the seminiferous tubules of otherwise sterile mice with a Sertoli-cell-only pathology. After three months, the transplanted spermatogonial stem cells had engrafted and colonized the seminiferous tubules. Spermatogenesis was restored.

The clinical implications of this work are enormous. The findings suggest that the isolation, enrichment, and cryopreservation of spermatogonial stem cells prior to chemotherapy or radiation therapy, with later autologous transplantation, may offer the potential for the subsequent restoration of fertility. The development of this technique will be especially important for survivors of childhood cancer. Adult patients can also bank sperm for cryopre‐ servation. However, most couples would prefer a naturally conceived child. Work has progressed in many laboratories to partially enrich the spermatogonial stem cells of species ranging from mice to primates. Today, many urologists bank a testicular biopsy from patients about to undergo chemotherapy, with the expectation that technology will advance rapidly over the next 10 years and allow transplantation in the future.

## **4. Pluripotency of human testis–derived ESC–like cells**

Previous studies have demonstrated that neonatal and adult germline stem cells (GSCs) can be self-reprogrammed into ESC-like cells, called germline-derived pluripotent stem cells [32,33,34,35]. In addition, Conrad et al. [36] reported that pluripotent cells can be derived from human testis, which those authors called human adult GSCs (haGSCs). Other research groups subsequently claimed that ESC-like cells could be obtained from cultures of human testicular cells [37,38,39]. Conrad and colleagues compared the global gene expression profile of hESCs and haGSCs, and concluded that the populations presented a similar gene expression profile, and thus, that the haGSCs were pluripotent. However, Ko et al. claimed that the gene expres‐ sion profile of haGSCs differed substantially from the pluripotent profile of hESCs, determined by a number of laboratories [40]. For example, the haGSCs did not express NANOG, and had low OCT4 and SOX2 levels, but showed high levels of the fibroblast markers SNA12 and ACTA2 [40]. Ko and colleagues therefore suggested that the haGSCs originated from fibroblast cells, rather than from pluripotent tissue. They concluded that haGSCs were very similar to a human testicular fibroblast cell line (hTFCs) [40]. Conrad and colleagues argued that micro‐ array data sets cannot be compared unless they are processed in parallel in the same experi‐ ment, suggesting that the similarity between haGSCs and hTFCs was inconclusive. However, studies on microarray results generated by different laboratories [41,42,43] have shown that findings from microarray analyses are comparable across multiple laboratories [44], particu‐ larly when a common platform and set of procedures are used. These findings justify the utility of microarray repositories, such as the GEO database [45], not only as data warehouses but also as resources for comparative and combinatory analyses of microarray data from different laboratories. In conclusion, the global gene expression analysis of haGSCs demonstrated that these cells resembled fibroblast hTFCs more than pluripotent hESCs.

### **5. Induced pluripotent stem (iPS) cells**

for some markers identified in mouse SSCs and other undifferentiated spermatogonia, including GFRA1, UCHL1 (PGP9.5), ZBTB16 (PLZF), and THY1 (CD90) [27,28]. We also have

**Figure 1.** Diagram of the spermatogonial stem cell (SSC) niche showing that extrinsic factors drive SSC maintenance and self-renewal. SSCs and Sertoli cells are attached to the basement membrane. Sertoli cells produce glial cell linederived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF). Leydig cells and peritubular cells pro‐

Brinster and colleagues proved the existence of mouse SSCs by using unique approaches [30, 31]. These investigators transplanted cells obtained as testicular homogenates expressing the *LacZ* gene into the seminiferous tubules of otherwise sterile mice with a Sertoli-cell-only pathology. After three months, the transplanted spermatogonial stem cells had engrafted and

The clinical implications of this work are enormous. The findings suggest that the isolation, enrichment, and cryopreservation of spermatogonial stem cells prior to chemotherapy or radiation therapy, with later autologous transplantation, may offer the potential for the subsequent restoration of fertility. The development of this technique will be especially important for survivors of childhood cancer. Adult patients can also bank sperm for cryopre‐

obtained evidence that THY1 is a potential surface marker for human SSCs [29].

colonized the seminiferous tubules. Spermatogenesis was restored.

duce colony-stimulating factor-1 (CSF-1).

120 Pluripotent Stem Cells

The year 2006 saw the first description of mouse induced pluripotent stem cells (miPSCs), which were generated by the retrovirus-mediated transduction of four transcription factors (OCT3/4, SOX2, KLF4, and C-MYC) into mouse fibroblasts [46]. Human somatic cells can be reprogrammed to become human iPSCs via the introduction of a small set of genes, either those encoding OCT3/4, SOX2 and KLF4, with or without the addition of C-MYC, or an alternate combination of OCT3/4, SOX2, LIN28, and NANOG [47,48,49,50,51,52,53,54,55]. Human iPSCs (hiPSCs) have remarkable similarity to hESCs in terms of their morphology, in vitro characteristics, proliferation rate, gene expression, and ability to differentiate into mesoderm, endoderm, and ectoderm, both in vitro and in vivo, in teratoma assays [56,57].

surface markers are available. To purify PGC-like cells from differentiating human ESCs and iPSCs, cell sorting with specific antibodies for SSEA1 [68], SSEA1 and C-KIT [50] [79], or CXCR4 [69] has been effective. In particular, Eguizabal et al. (2011) published a straightforward protocol for germline cell purification that requires only three steps. First, human iPSCs and hESCs are allowed to differentiate for 3 weeks in a monolayer, in the absence of growth cytokines. Second, the cells are cultured for 3 weeks in the presence of retinoic acid. Finally, after these 6 weeks of differentiation, the cells are sorted for a specific combination of surface markers (CD49f++, CD9+, CD90-, and SSEA4-), and the isolated fraction is cultured in the

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presence of LIF, bFGF, Forskolin, and CYP26 inhibitor for 4 more weeks [76].

germline development from piPSCs have been published to date.

**8. Conclusions**

**Acknowledgements**

Science and Technology of Japan, 2011-2015.

**7. Germline differentiation from porcine iPSCs, non–human iPSCs**

Despite their undoubted promise as sources of cells for tissue transplants, many roadblocks remain against using human ESCs clinically. Particularly troubling is the lack of tests for the efficacy of such therapies and the safety of transferring these cells in animals whose anatomy and physiology resemble those of humans better than mouse models do [80] [81] [82] [83] [84]. The pig is a potentially useful model in this regard, because of its similarities to humans in organ size, immunology, and whole animal physiology [85] [86] [87]. It was reported that porcine somatic cells can be reprogrammed to form piPSCs [88]. However, no reports on

Research on stem cells has shown remarkable progress over the past 5 years. In particular, the development of human iPSCs has opened new avenues into the generation of an *in vitro* disease model of male infertility. However, improvements are still needed before stem cells can be used clinically. For the treatment and diagnosis of male infertility, future advances may enable spermatids to be differentiated from germline stem cells or iPS cells. In addition, by examining patient-specific iPSCs that are defective in their ability to generate germ cells and comparing their differentiation capacity with that of normal human ESCs and iPSCs, researchers can hope

This study was supported in part by a Grant-in-Aid for Young Scientists (B) of the Japan Society for the Promotion of Science (JSPS) and a Grant of the Strategic Research Foundation Grantaided Project for Private schools at Heisei 23th from Ministry of Education, Culture, Sports,

to uncover the nature of male infertility and to design new methods to reverse it.

In our laboratory, we induced iPS cells from adult human testicular tissue by introducing four transcription factors, OCT4, SOX2, KLF4, and C-MYC, using lentiviral vectors [58]. We also generated ES-like cells from 293FT cells by using OCT4, SOX2, NANOG, and LIN28 [59]. Finally, we generated iPS cells derived from the human testicular tissue of individuals with Klinefelter syndrome (KS, also called 47, XXY) [60].

## **6. Germline differentiation from ESCs and iPSCs in humans**

Recent studies indicate that mouse [61,62,63,64,65] and human [66,67] [50,68,69,70,71] ESCs can differentiate in vitro into oocyte- or sperm-like cells. In particular, Clark et al. first reported the spontaneous differentiation of germ cells in embryoid bodies derived from human ESCs [66]. Male germline cells express specific RNA and protein markers, such as VASA. In 2009, Park et al. demonstrated that PGC-like cells can be differentiated from human iPSCs [50]. Subsequent reports on male germline differentiation from stem cells have used one of three approaches: (1) specific culture conditions, (2) manipulation of gene expression, and (3) purification of germ cells.

**Culture conditions supporting differentiation into germline cells.** Bucay et al. observed that as hESCs differentiate into putative germline cells, they also produce Sertoli-like support cells [69]. In addition, co-cultures of hESCs and hiPSCs with human fetal gonadal stromal cells [50], mouse Sertoli cells [72], or mouse embryonic fibroblasts [67] resulted in the increased efficiency of germ cell-like differentiation. Co-culture systems are used to mimic a suitable microenvir‐ onment for the growing germ cells. For the differentiation of germline-like cells from hESCs and hiPSCs, cytokines and other cell-signaling molecules are often added to the cultures. For example, BMP4 and other BMPs are added to promote PGC-like differentiation from hESCs and hiPSCs [73,74,75]. In addition, retinoic acid has been used to stimulate meiosis [75] [76]. Panula and colleagues reported the differentiation of fetal- and adult-derived iPSCs into germ cells, and showed that ~5% of human iPSCs differentiated into PGCs following induction with BMPs [77].

**Manipulation of gene expression.** By manipulating gene expression, researchers can regulate the cell lineage decisions of differentiating pluripotent stem cells. Overexpression of DAZL and VASA promotes PGC formation in differentiating human ESCs and iPSCs [78]. In addition, Kee and colleagues (2009) reported that hESCs differentiate into germline cells that initiate meiosis and progress to form haploid germ cells. These authors indicated that the overexpres‐ sion of members of the DAZ gene family, DAZ, DAZL, and BOULE, promoted the progression of PGCs to meiosis and the production of haploid cells, a process that is unique to germ cell development [71].

**Purification of germline cells.** The isolation and purification of germline cells from stem cell cultures (ESCs and iPSCs) can be performed efficiently when specific antibodies for germ cell surface markers are available. To purify PGC-like cells from differentiating human ESCs and iPSCs, cell sorting with specific antibodies for SSEA1 [68], SSEA1 and C-KIT [50] [79], or CXCR4 [69] has been effective. In particular, Eguizabal et al. (2011) published a straightforward protocol for germline cell purification that requires only three steps. First, human iPSCs and hESCs are allowed to differentiate for 3 weeks in a monolayer, in the absence of growth cytokines. Second, the cells are cultured for 3 weeks in the presence of retinoic acid. Finally, after these 6 weeks of differentiation, the cells are sorted for a specific combination of surface markers (CD49f++, CD9+, CD90-, and SSEA4-), and the isolated fraction is cultured in the presence of LIF, bFGF, Forskolin, and CYP26 inhibitor for 4 more weeks [76].

## **7. Germline differentiation from porcine iPSCs, non–human iPSCs**

Despite their undoubted promise as sources of cells for tissue transplants, many roadblocks remain against using human ESCs clinically. Particularly troubling is the lack of tests for the efficacy of such therapies and the safety of transferring these cells in animals whose anatomy and physiology resemble those of humans better than mouse models do [80] [81] [82] [83] [84]. The pig is a potentially useful model in this regard, because of its similarities to humans in organ size, immunology, and whole animal physiology [85] [86] [87]. It was reported that porcine somatic cells can be reprogrammed to form piPSCs [88]. However, no reports on germline development from piPSCs have been published to date.

## **8. Conclusions**

vitro characteristics, proliferation rate, gene expression, and ability to differentiate into mesoderm, endoderm, and ectoderm, both in vitro and in vivo, in teratoma assays [56,57].

In our laboratory, we induced iPS cells from adult human testicular tissue by introducing four transcription factors, OCT4, SOX2, KLF4, and C-MYC, using lentiviral vectors [58]. We also generated ES-like cells from 293FT cells by using OCT4, SOX2, NANOG, and LIN28 [59]. Finally, we generated iPS cells derived from the human testicular tissue of individuals with

Recent studies indicate that mouse [61,62,63,64,65] and human [66,67] [50,68,69,70,71] ESCs can differentiate in vitro into oocyte- or sperm-like cells. In particular, Clark et al. first reported the spontaneous differentiation of germ cells in embryoid bodies derived from human ESCs [66]. Male germline cells express specific RNA and protein markers, such as VASA. In 2009, Park et al. demonstrated that PGC-like cells can be differentiated from human iPSCs [50]. Subsequent reports on male germline differentiation from stem cells have used one of three approaches: (1) specific culture conditions, (2) manipulation of gene expression, and (3)

**Culture conditions supporting differentiation into germline cells.** Bucay et al. observed that as hESCs differentiate into putative germline cells, they also produce Sertoli-like support cells [69]. In addition, co-cultures of hESCs and hiPSCs with human fetal gonadal stromal cells [50], mouse Sertoli cells [72], or mouse embryonic fibroblasts [67] resulted in the increased efficiency of germ cell-like differentiation. Co-culture systems are used to mimic a suitable microenvir‐ onment for the growing germ cells. For the differentiation of germline-like cells from hESCs and hiPSCs, cytokines and other cell-signaling molecules are often added to the cultures. For example, BMP4 and other BMPs are added to promote PGC-like differentiation from hESCs and hiPSCs [73,74,75]. In addition, retinoic acid has been used to stimulate meiosis [75] [76]. Panula and colleagues reported the differentiation of fetal- and adult-derived iPSCs into germ cells, and showed that ~5% of human iPSCs differentiated into PGCs following induction with

**Manipulation of gene expression.** By manipulating gene expression, researchers can regulate the cell lineage decisions of differentiating pluripotent stem cells. Overexpression of DAZL and VASA promotes PGC formation in differentiating human ESCs and iPSCs [78]. In addition, Kee and colleagues (2009) reported that hESCs differentiate into germline cells that initiate meiosis and progress to form haploid germ cells. These authors indicated that the overexpres‐ sion of members of the DAZ gene family, DAZ, DAZL, and BOULE, promoted the progression of PGCs to meiosis and the production of haploid cells, a process that is unique to germ cell

**Purification of germline cells.** The isolation and purification of germline cells from stem cell cultures (ESCs and iPSCs) can be performed efficiently when specific antibodies for germ cell

**6. Germline differentiation from ESCs and iPSCs in humans**

Klinefelter syndrome (KS, also called 47, XXY) [60].

purification of germ cells.

122 Pluripotent Stem Cells

BMPs [77].

development [71].

Research on stem cells has shown remarkable progress over the past 5 years. In particular, the development of human iPSCs has opened new avenues into the generation of an *in vitro* disease model of male infertility. However, improvements are still needed before stem cells can be used clinically. For the treatment and diagnosis of male infertility, future advances may enable spermatids to be differentiated from germline stem cells or iPS cells. In addition, by examining patient-specific iPSCs that are defective in their ability to generate germ cells and comparing their differentiation capacity with that of normal human ESCs and iPSCs, researchers can hope to uncover the nature of male infertility and to design new methods to reverse it.

## **Acknowledgements**

This study was supported in part by a Grant-in-Aid for Young Scientists (B) of the Japan Society for the Promotion of Science (JSPS) and a Grant of the Strategic Research Foundation Grantaided Project for Private schools at Heisei 23th from Ministry of Education, Culture, Sports, Science and Technology of Japan, 2011-2015.

## **Author details**

Hideyuki Kobayashi\* , Koichi Nagao and Koichi Nakajima

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Urology, Toho University School of Medicine, Tokyo, Japan

#### **References**

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125

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Hideyuki Kobayashi\*

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[56] Yamanaka, S. Strategies and new developments in the generation of patient-specific

[57] Yamanaka, S. Pluripotency and nuclear reprogramming. Philos Trans R Soc Lond B

[58] Kobayashi, H, Nakajima, K, Oka, Y, Tai, T, Nagao, K, & Ishii, N. Reprogramming of adult human testicular cells by our transcription factors (OCT4, SOX2, KLF4, and C-

[59] Oka, Y, Nakajima, K, Nagao, K, Miura, K, Ishii, N, & Kobayashi, H. FT cells trans‐ duced with four transcription actors (OCT4, SOX2, NANOG, and LIN28) generate aberrant ES-like cells. J. Stem cell and Regenerative Medicine (2010). , 3, 149-156.

[60] Kobayashi, H. Pluripotent stem cells induced from testicular tissue with Klinefelter syndrome (47, XXY) by four ranscription factors (OCT4, SOX2, KLF4, and C-MYC). Methodological Advances in the Culture, Manipulation and Utilization of Embryonic

[61] Hubner, K, Fuhrmann, G, Christenson, L. K, Kehler, J, Reinbold, R, De La Fuente, R, et al. Derivation of oocytes from mouse embryonic stem cells. Science (2003). , 300,

[62] Toyooka, Y, Tsunekawa, N, Akasu, R, & Noce, T. Embryonic stem cells can form

[63] Geijsen, N, Horoschak, M, Kim, K, Gribnau, J, Eggan, K, & Daley, G. Q. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature (2004). ,

[64] Qing, T, Shi, Y, Qin, H, Ye, X, Wei, W, Liu, H, et al. Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differ‐

[65] Nagano, M. C. In vitro gamete derivation from pluripotent stem cells: progress and

[66] Clark, A. T, Bodnar, M. S, Fox, M, Rodriquez, R. T, Abeyta, M. J, Firpo, M. T, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro.

[67] West, F. D, Machacek, D. W, Boyd, N. L, Pandiyan, K, Robbins, K. R, & Stice, S. L. Enrichment and differentiation of human germ-like cells mediated by feeder cells

[68] Tilgner, K, Atkinson, S. P, Golebiewska, A, Stojkovic, M, Lako, M, & Armstrong, L. Isolation of primordial germ cells from differentiating human embryonic stem cells.

and basic fibroblast growth factor signaling. Stem Cells (2008). , 26, 2768-76.

Stem Cells for Basic and Practical Applications (2011). , 295-306.

germ cells in vitro. Proc Natl Acad Sci U S A (2003). , 100, 11457-62.

pluripotent stem cells. Cell Stem Cell (2007). , 1, 39-49.

MYC). Reprod Med Biol (2011). , 10, 105-112.

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1251-6.

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427, 148-54.

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perspective. Biol Reprod (2007). , 76, 546-51.

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[81] Keefer, C. L, Pant, D, Blomberg, L, & Talbot, N. C. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim Reprod Sci (2007). , 98, 147-68.

**Chapter 7**

**Generation of Induced Pluripotent Stem Cells from**

During early development, human dental pulp is originated from neural crest, which is a transient embryonic structure (Fig. 1). According to current knowledge, neural crest stem cells (NCSCs) have the capacity to self-renewal and display a developmental potential almost the same as embryonic stem (ES) cells (Kerkis and Caplan, 2012). These postmigratory NCSCs generate all craniofacial bones, the majority of the peripheral nervous system cells and tissues, as well as several non-neural cell types, such as smooth muscle cells of the cardiovascular system, pigment cells in the skin, cartilage, connective tissue, corneal epithelium and dental pulp among them. Although postmigratory, postnatal NCSCs are of restricted developmental potential they maintain functional characteristics resembling their embryonic counterparts and an ability to differentiate into a broad spectrum of cell types (Le Douarin et al., 2004, 2007,

**Figure 1.** Early development of NCSCs. According to current knowledge, migrating neural crest cells are stem cells that

© 2013 Lizier 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,

© 2013 Lizier 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.

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

**Dental Pulp Somatic Cells**

Nelson F. Lizier, Irina Kerkis and

Additional information is available at the end of the chapter

2008; Dupin et al., 2007; Le Douarin & Dupin, 2003, 2012).

display almost the same potential as ES cells.

Cristiane V. Wenceslau

http://dx.doi.org/10.5772/55856

**1. Introduction**

## **Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells**

Nelson F. Lizier, Irina Kerkis and Cristiane V. Wenceslau

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55856

### **1. Introduction**

[81] Keefer, C. L, Pant, D, Blomberg, L, & Talbot, N. C. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim Reprod

[82] Brevini, T. A, Antonini, S, Cillo, F, Crestan, M, & Gandolfi, F. Porcine embryonic stem cells: Facts, challenges and hopes. Theriogenology (2007). Suppl 1:S, 206-13.

[83] Talbot, N. C. Blomberg Le A. The pursuit of ES cell lines of domesticated ungulates.

[84] Hall, V. Porcine embryonic stem cells: a possible source for cell replacement therapy.

[85] Prather, R. S, Hawley, R. J, Carter, D. B, Lai, L, & Greenstein, J. L. Transgenic swine

[86] Brandl, U, Michel, S, Erhardt, M, Brenner, P, Burdorf, L, Jockle, H, et al. Transgenic animals in experimental xenotransplantation models: orthotopic heart transplanta‐

[87] Piedrahita, J. A, & Mir, B. Cloning and transgenesis in mammals: implications for

[88] Ezashi, T, Telugu, B. P, Alexenko, A. P, Sachdev, S, Sinha, S, & Roberts, R. M. Deriva‐ tion of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S

for biomedicine and agriculture. Theriogenology (2003). , 59, 115-23.

tion in the pig-to-baboon model. Transplant Proc (2007). , 39, 577-8.

xenotransplantation. Am J Transplant (2004). Suppl , 6, 43-50.

Sci (2007). , 98, 147-68.

130 Pluripotent Stem Cells

Stem Cell Rev (2008). , 4, 235-54.

Stem Cell Rev (2008). , 4, 275-82.

A (2009). , 106, 10993-8.

During early development, human dental pulp is originated from neural crest, which is a transient embryonic structure (Fig. 1). According to current knowledge, neural crest stem cells (NCSCs) have the capacity to self-renewal and display a developmental potential almost the same as embryonic stem (ES) cells (Kerkis and Caplan, 2012). These postmigratory NCSCs generate all craniofacial bones, the majority of the peripheral nervous system cells and tissues, as well as several non-neural cell types, such as smooth muscle cells of the cardiovascular system, pigment cells in the skin, cartilage, connective tissue, corneal epithelium and dental pulp among them. Although postmigratory, postnatal NCSCs are of restricted developmental potential they maintain functional characteristics resembling their embryonic counterparts and an ability to differentiate into a broad spectrum of cell types (Le Douarin et al., 2004, 2007, 2008; Dupin et al., 2007; Le Douarin & Dupin, 2003, 2012).

© 2013 Lizier 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 Lizier 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.

The marathon of induced pluripotent stem cells (iPSC) started when Yamanaka in 2006, by forcing the expression of certain pluripotent genes in fibroblasts, reversed them into a pluripotent state similar to ES cells (Takahashi &Yamanaka, 2006). The main goal of iPSC generation is to create patient-specific cells, which would be advantageous for cell therapy due to immune compatibility (Ohnuki et al., 2009). Research involving the production of iPSC is being developed around the world. Production of iPSC opens new avenues for understanding human genetic diseases; embryogenesis and will likely have a great impact in drug screening and toxicological tests. However, fibroblasts, which were firstly used for iPSC production, present low efficiency and slow process of reprogramming. Moreover, these cells throughout all life are exposed to environmental factors, which can compromise their use as genetic models (Liu, 2008; McDevitt & Palecek, 2008; Nishikawa et al., 2008; Yu & Thomson; 2008; Zhao & Daley, 2008; Maherali & Hochedlinger, 2008; Ooi et al., 2012). Indeed, more immature somatic cells such as, postmigratory NCSCs, and adult stem cells isolated from young organism showed high efficiency of reprogramming (Zouboulis et al., 2008; Muchkaeva et al., 2012). Because of the possibility to isolate NCSCs from easily accessible tissue (e.g. baby teeth is discarded), the dental pulp derived somatic cells have become an ideal model system to study stem cell biology in diseases during different stages of the development (childhood, youth, middle-aged and old) with a special focus on non-invasive source of the cells for investigation of pediatric diseases (Kerkis & Caplan, 2012; Lizier et al., 2012).

deciduous teeth (Miura et al., 2003; Kerkis et al., 2006). DPCs (dental pulp cells) and MStCs (mesenchymal/stromal cells) were isolated from human third molars by two independent groups (Takeda et al., 2008; Ikeda et al., 2008). All these cell types present fibroblast-like morphology and however differ in methods of isolation, show significant difference in expression pattern of stem cell markers and in purity of isolated population (Yan et al., 2010;

Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells

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133

**Figure 2.** iPSC technology. The iPSC production can be induced by forcing the expression of certain pluripotent genes.

For reprogramming of SHED/SCAP/DPSCs, heterogeneous primary human dental stem/ progenitor cell population at passages 2 and 3 were used (Yan et al., 2010). These populations were tested for their cell surface marker expression by flow cytometry and they were positive for STRO-1, CD146, CD73, CD90, CD105 and negative for CD14, CD34, and CD45, showing typical immunophenotype of mesenchymal stem cells (MSC) (Friedenstein et al., 1976; Caplan, 1991). The first study used four factor genes for reprogramming, such as c-Myc [Myc protooncogene protein], Klf4 [Krüppel-like factor], Oct4 [octamer-binding transcription factor 4], and Sox2 [(sex determining region Y)-box 2] into pLenti6.2/C-Lumio/V5-DEST vector system. Although the cells started to present morphological changes (fibroblastic to epithelial cell-like transition), the reprogramming process failed. Further, lentiviral vectors pSin-EF2-gene-Pur carrying 1 of the 4 factors Lin28 [Lin-28 homolog A], Nanog (Nanog homeobox), Oct4, and Sox2 were used and first ES-like colonies were obtained. To improve reprogramming efficien‐ cy, human genes c-Myc, Klf4, Oct4, and Sox2 were subcloned into the vector pMXs and

Beltrão-Braga et al., 2011; Tamaoki et al., 2010; Oda et al., 2010).

**2.1.  The hIDPSC present fibroblast-like morphology, retain characteristics of adult multipotent stem cells and express at least one of three transcription factors: Oct4, Nanog and Sox2 (Kerkis et al., 2006; Lizier et al., 2012). We also used these cells as an alternative source for iPSC derivation (Beltrão-Braga et al., 2011) (Fig. 2).Different research groups derived iPSC from dental pulp fibroblasts and stem cells from young, middle aged and old patients. The difference was observed between the protocols and efficiency of iPSC generation in all these studies (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). The present chapter is focused on comparative investigation of the methods and efficiency of iPSC generation from dental pulp stem cells and fibroblasts (control). Differentiation potential, assuredness and the future perspectives of the use of these iPSC derived from dental pulp stem cells in basic research and in biotechnology will also be broadly discussed.

### **2. Dental pulp somatic cells used for iPSC generation**

Human dental tissues are rich in stem cells (Giordano et al., 2011; Kerkis & Caplan, 2012). Different research groups isolated and characterized several types of stem cells used for iPSC generation: (i) from apical papilla (Yan et al., 2010), (ii) from dental pulp of primary exfoliated deciduous teeth (Yan et al., 2010; Beltrão-Braga et al., 2011) and (iii) from wisdom teeth (Tamaoki et al., 2010; Oda et al., 2010). SCAP (stem cells from apical papilla) were obtained from tissue at the apex of a tooth root (Yan et al., 2010). SHED (stem cells from human exfoliated deciduous) and IDPSC (immature dental pulp stem cells) were derived from exfoliated deciduous teeth (Miura et al., 2003; Kerkis et al., 2006). DPCs (dental pulp cells) and MStCs (mesenchymal/stromal cells) were isolated from human third molars by two independent groups (Takeda et al., 2008; Ikeda et al., 2008). All these cell types present fibroblast-like morphology and however differ in methods of isolation, show significant difference in expression pattern of stem cell markers and in purity of isolated population (Yan et al., 2010; Beltrão-Braga et al., 2011; Tamaoki et al., 2010; Oda et al., 2010).

**Figure 2.** iPSC technology. The iPSC production can be induced by forcing the expression of certain pluripotent genes.

#### **2.1. Transduction and reprogramming**

The marathon of induced pluripotent stem cells (iPSC) started when Yamanaka in 2006, by forcing the expression of certain pluripotent genes in fibroblasts, reversed them into a pluripotent state similar to ES cells (Takahashi &Yamanaka, 2006). The main goal of iPSC generation is to create patient-specific cells, which would be advantageous for cell therapy due to immune compatibility (Ohnuki et al., 2009). Research involving the production of iPSC is being developed around the world. Production of iPSC opens new avenues for understanding human genetic diseases; embryogenesis and will likely have a great impact in drug screening and toxicological tests. However, fibroblasts, which were firstly used for iPSC production, present low efficiency and slow process of reprogramming. Moreover, these cells throughout all life are exposed to environmental factors, which can compromise their use as genetic models (Liu, 2008; McDevitt & Palecek, 2008; Nishikawa et al., 2008; Yu & Thomson; 2008; Zhao & Daley, 2008; Maherali & Hochedlinger, 2008; Ooi et al., 2012). Indeed, more immature somatic cells such as, postmigratory NCSCs, and adult stem cells isolated from young organism showed high efficiency of reprogramming (Zouboulis et al., 2008; Muchkaeva et al., 2012). Because of the possibility to isolate NCSCs from easily accessible tissue (e.g. baby teeth is discarded), the dental pulp derived somatic cells have become an ideal model system to study stem cell biology in diseases during different stages of the development (childhood, youth, middle-aged and old) with a special focus on non-invasive source of the cells for investigation

Our group isolated and fully characterized human immature dental pulp stem cells (hIDPSC), which is a very attractive cell type, from deciduous teeth (baby teeth) (Kerkis et al., 2006; Lizier et al., 2012). The hIDPSC present fibroblast-like morphology, retain characteristics of adult multipotent stem cells and express at least one of three transcription factors: Oct4, Nanog and Sox2 (Kerkis et al., 2006; Lizier et al., 2012). We also used these cells as an alternative source for iPSC derivation (Beltrão-Braga et al., 2011) (Fig. 2).Different research groups derived iPSC from dental pulp fibroblasts and stem cells from young, middle aged and old patients. The difference was observed between the protocols and efficiency of iPSC generation in all these studies (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). The present chapter is focused on comparative investigation of the methods and efficiency of iPSC generation from dental pulp stem cells and fibroblasts (control). Differentiation potential, assuredness and the future perspectives of the use of these iPSC derived from dental pulp stem

Human dental tissues are rich in stem cells (Giordano et al., 2011; Kerkis & Caplan, 2012). Different research groups isolated and characterized several types of stem cells used for iPSC generation: (i) from apical papilla (Yan et al., 2010), (ii) from dental pulp of primary exfoliated deciduous teeth (Yan et al., 2010; Beltrão-Braga et al., 2011) and (iii) from wisdom teeth (Tamaoki et al., 2010; Oda et al., 2010). SCAP (stem cells from apical papilla) were obtained from tissue at the apex of a tooth root (Yan et al., 2010). SHED (stem cells from human exfoliated deciduous) and IDPSC (immature dental pulp stem cells) were derived from exfoliated

of pediatric diseases (Kerkis & Caplan, 2012; Lizier et al., 2012).

132 Pluripotent Stem Cells

cells in basic research and in biotechnology will also be broadly discussed.

**2. Dental pulp somatic cells used for iPSC generation**

For reprogramming of SHED/SCAP/DPSCs, heterogeneous primary human dental stem/ progenitor cell population at passages 2 and 3 were used (Yan et al., 2010). These populations were tested for their cell surface marker expression by flow cytometry and they were positive for STRO-1, CD146, CD73, CD90, CD105 and negative for CD14, CD34, and CD45, showing typical immunophenotype of mesenchymal stem cells (MSC) (Friedenstein et al., 1976; Caplan, 1991). The first study used four factor genes for reprogramming, such as c-Myc [Myc protooncogene protein], Klf4 [Krüppel-like factor], Oct4 [octamer-binding transcription factor 4], and Sox2 [(sex determining region Y)-box 2] into pLenti6.2/C-Lumio/V5-DEST vector system. Although the cells started to present morphological changes (fibroblastic to epithelial cell-like transition), the reprogramming process failed. Further, lentiviral vectors pSin-EF2-gene-Pur carrying 1 of the 4 factors Lin28 [Lin-28 homolog A], Nanog (Nanog homeobox), Oct4, and Sox2 were used and first ES-like colonies were obtained. To improve reprogramming efficien‐ cy, human genes c-Myc, Klf4, Oct4, and Sox2 were subcloned into the vector pMXs and produced retrovirus was used for second round of transduction. Human fibroblasts, used as a control in this study, were not able to undergo reprogramming under proposed conditions.

efficiency in clonally expanded MStCs was higher and it correlates with cell proliferative ability. The clones, which showed higher proliferative ability, demonstrated a rate of reprog‐ ramming ~ 30–100-fold higher than HDFs and ~ 7-fold higher than clones with lower prolif‐ erative ability. The IDPSC also present high reprogramming efficiency and no difference was observed between the cells from both donors. Therefore, difficulties in reprogramming SHED/ SCAP/DPSCs can be related with cell heterogeneity of original populations (Yan et al., 2010).

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**Figure 3.** hIDPSC-derived iPSC. (A) Representative figure of morphological characteristics of hIDPSC *in vitro* culturing (light microscopy). (B-D) iPSC derivations were shown to be obtained under both conditions: feeder-free, on matrigel-

Furthermore, the aging process influences all organs, tissues and cells of organism. The studies showed that this factor is also important for cells reprogramming (Zouboulis et al., 2008; Banito et al., 2009). SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC were isolated from young donors of variable ages 7, 10, 12, 13, 14 16, 19, 20 and 24 years old. The difference in efficiency of iPSC generation was observed between MStCs isolated from third molars of 10-, 13-, and 16-yearold donors. More efficient reprogramming was observed when MStCs from the 10-year-old donor were used. Similar observation was made by Tamaoki and co-wokers (2010). In our study we used IDPSC from 7 years old donors and we observed rapid and efficient reprog‐

coated dishes (B and C) and on MEF (D).

ramming in both cell populations.

Retroviruses expressing four Oct3/4, Sox2, Klf4, and c-Myc or three (without c-Myc) factors were used for reprogramming DPCs from wisdom teeth and from human dermal fibroblasts (HDFs), which was performed according to the methods previously described (Takahashi et al., 2007). Another group, which used MStCs from wisdom teeth, also demonstrated successful reprogramming of these cells with pMXs retrovirus vectors containing three human Oct3/4, Sox2, and Klf4 factor genes (Oda et al., 2010).

IDPSC is a homogeneous population in respect of the expression of MSCs (Friedenstein et al., 1976; Caplan, 1991) markers, such as CD73, CD105, nestin and vimentin. Within IDPSC population, several cells also express Oct3/4 and Nanog (Kerkis et al., 2006; Lizier et al., 2012). To reprogram IDPSC, our group used four Yamanaka's factors (Klf4, Oct4, c-Myc and Sox2) and previously established protocol (Takahashi et al., 2007; Beltrão-Braga et al., 2011).

#### **2.2. Formation of ES cell-like colonies and expansion**

There are several important points that should be considered when iPSC are isolated and expanded: (i) the use of mouse embryonic fibroblasts (MEF) as a feeder layer, (ii) the efficiency of reprogramming and (iii) the efficiency of expansion (Takahashi & Yamanaka, 2006; Lewitzky & Yamanaka, 2007; Bilic & Belmonte, 2012). Isolation of iPSC on MEF limits the manipulation and further clinical application of these cells. Thus, isolation and expansion of iPSC without MEF is an important step, which avoids contamination of human cells with animal products. Efficiency of reprogramming depends on different factor, such as gene expression profile of cells, which were used in experiments. It has been shown that more immature cells undergo this process more efficiently, then committed or terminally differen‐ tiated cells (Zouboulis et al., 2008; Muchkaeva et al., 2012). And finally, during reprogramming, the cells receive different number of reprogramming factors and/or they did not respond equally to this process, therefore multiple ES-like did not complete reprogramming or non-ES cell-like colonies raised (Aasen et al., 2008; Marchetto et al., 2009). SHED/SCAP/DPSCs/DPCs/ MStCs-derived iPSC were obtained using MEF as a feeder layer (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). IDPSC-derived iPSC were shown to be obtained under both conditions: feeder-free on matrigel-coated dishes and on MEF (Fig. 3) (Beltrão-Braga et al., 2011). It seems that time-course of reprogramming of different cell types varied in accordance with age of cell donor, cell type and number of factors used. Thus, SHED/SCAP/DPSCsderived iPSC, showed the formation of the first colonies ~2-3 weeks after gene transduction. DPCs-derived iPSC were reprogrammed in ~14 days, when 4 factors were used and in ~ 20-25 days, when reprogramming was performed with only 3 factors. MStCs-derived iPSC were reprogrammed in ~25 days, while IDPSC-derived iPSC demonstrated the formation of first colonies at ~ day 5-11. All studies demonstrated that efficiency of iPSC derivation from dental pulp tissues is higher than that from human dermal fibroblast and primary gingival fibroblasts (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). Because the MStCs were a heterogeneous cell population, Oda and co-authors (2010) additionally used clonally expanded MStCs in reprogramming experiments. They observed that reprogramming efficiency in clonally expanded MStCs was higher and it correlates with cell proliferative ability. The clones, which showed higher proliferative ability, demonstrated a rate of reprog‐ ramming ~ 30–100-fold higher than HDFs and ~ 7-fold higher than clones with lower prolif‐ erative ability. The IDPSC also present high reprogramming efficiency and no difference was observed between the cells from both donors. Therefore, difficulties in reprogramming SHED/ SCAP/DPSCs can be related with cell heterogeneity of original populations (Yan et al., 2010).

produced retrovirus was used for second round of transduction. Human fibroblasts, used as a control in this study, were not able to undergo reprogramming under proposed conditions.

Retroviruses expressing four Oct3/4, Sox2, Klf4, and c-Myc or three (without c-Myc) factors were used for reprogramming DPCs from wisdom teeth and from human dermal fibroblasts (HDFs), which was performed according to the methods previously described (Takahashi et al., 2007). Another group, which used MStCs from wisdom teeth, also demonstrated successful reprogramming of these cells with pMXs retrovirus vectors containing three human Oct3/4,

IDPSC is a homogeneous population in respect of the expression of MSCs (Friedenstein et al., 1976; Caplan, 1991) markers, such as CD73, CD105, nestin and vimentin. Within IDPSC population, several cells also express Oct3/4 and Nanog (Kerkis et al., 2006; Lizier et al., 2012). To reprogram IDPSC, our group used four Yamanaka's factors (Klf4, Oct4, c-Myc and Sox2)

There are several important points that should be considered when iPSC are isolated and expanded: (i) the use of mouse embryonic fibroblasts (MEF) as a feeder layer, (ii) the efficiency of reprogramming and (iii) the efficiency of expansion (Takahashi & Yamanaka, 2006; Lewitzky & Yamanaka, 2007; Bilic & Belmonte, 2012). Isolation of iPSC on MEF limits the manipulation and further clinical application of these cells. Thus, isolation and expansion of iPSC without MEF is an important step, which avoids contamination of human cells with animal products. Efficiency of reprogramming depends on different factor, such as gene expression profile of cells, which were used in experiments. It has been shown that more immature cells undergo this process more efficiently, then committed or terminally differen‐ tiated cells (Zouboulis et al., 2008; Muchkaeva et al., 2012). And finally, during reprogramming, the cells receive different number of reprogramming factors and/or they did not respond equally to this process, therefore multiple ES-like did not complete reprogramming or non-ES cell-like colonies raised (Aasen et al., 2008; Marchetto et al., 2009). SHED/SCAP/DPSCs/DPCs/ MStCs-derived iPSC were obtained using MEF as a feeder layer (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). IDPSC-derived iPSC were shown to be obtained under both conditions: feeder-free on matrigel-coated dishes and on MEF (Fig. 3) (Beltrão-Braga et al., 2011). It seems that time-course of reprogramming of different cell types varied in accordance with age of cell donor, cell type and number of factors used. Thus, SHED/SCAP/DPSCsderived iPSC, showed the formation of the first colonies ~2-3 weeks after gene transduction. DPCs-derived iPSC were reprogrammed in ~14 days, when 4 factors were used and in ~ 20-25 days, when reprogramming was performed with only 3 factors. MStCs-derived iPSC were reprogrammed in ~25 days, while IDPSC-derived iPSC demonstrated the formation of first colonies at ~ day 5-11. All studies demonstrated that efficiency of iPSC derivation from dental pulp tissues is higher than that from human dermal fibroblast and primary gingival fibroblasts (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). Because the MStCs were a heterogeneous cell population, Oda and co-authors (2010) additionally used clonally expanded MStCs in reprogramming experiments. They observed that reprogramming

and previously established protocol (Takahashi et al., 2007; Beltrão-Braga et al., 2011).

Sox2, and Klf4 factor genes (Oda et al., 2010).

134 Pluripotent Stem Cells

**2.2. Formation of ES cell-like colonies and expansion**

**Figure 3.** hIDPSC-derived iPSC. (A) Representative figure of morphological characteristics of hIDPSC *in vitro* culturing (light microscopy). (B-D) iPSC derivations were shown to be obtained under both conditions: feeder-free, on matrigelcoated dishes (B and C) and on MEF (D).

Furthermore, the aging process influences all organs, tissues and cells of organism. The studies showed that this factor is also important for cells reprogramming (Zouboulis et al., 2008; Banito et al., 2009). SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC were isolated from young donors of variable ages 7, 10, 12, 13, 14 16, 19, 20 and 24 years old. The difference in efficiency of iPSC generation was observed between MStCs isolated from third molars of 10-, 13-, and 16-yearold donors. More efficient reprogramming was observed when MStCs from the 10-year-old donor were used. Similar observation was made by Tamaoki and co-wokers (2010). In our study we used IDPSC from 7 years old donors and we observed rapid and efficient reprog‐ ramming in both cell populations.

#### **2.3. Provirus integration**

Viral vectors are commonly used to deliver genetic material into cells, which can be performed *in vivo* (living organism) or *in vitro* (cell culture). Delivery of genes by a virus is efficient, however with respect to safety, it is ideal not to use lentivirus and retrovirus vectors, since they can integrate into the host DNA. As opposed to lentiviruses and retroviruses, adenoviral DNA does not integrate into the genome and are considered to be safer (Tamaoki et al., 2010). All studies performed with stem cells of dental tissue origin used lentivirus and/or retrovirus vectors for reprogramming. Yan and co-workers (2010) examined the presence of transgenes in the genome of iPSC clones isolating genomic DNA and generating primers specific for each transgene. They showed that the 4 factors were all integrated into the genome of the transduced SHED/SCAP/DPSC-iPSC. Other study, which used MStCs from wisdom teeth for reprogram‐ ming, did not provide any records about viral vectors integration (Tamaoki et al., 2010), while another group demonstrated retroviral silencing (Oda et al., 2010). Beltrão-Braga and coauthors (2011), which used retroviral vector for reprogramming, showed the lack of transgene expression by RT-PCR analyses in iPSC-derived from IDPSCs.

higher in iPSC derived from clonally isolated MStCs, when compared with parental cell lines,

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**Figure 4.** Expression of Oct3/4, Nanog, Sox2 and TRA-1-81 proteins in two lineages of hIDPS-IPSC five days after trans‐ duction with four factors. In A-C3,G) hIDPS-IPSC1 and D-F3,H) hIDPS-IPSC2 are presented, both showing multiple small colonies, which already express hallmarks of pluripotent cells, such as, A-A3) and D-D3) Oct3/4; B-B3) and E-E3) Nanog, C-C3) and F-F3) Sox2; G) and H) TRA-1-81, respectively. Nucleus stained with DAPI (blue). Note, that Oct3/4, Nanog, Sox2 present nuclear, while TRA-1-81 presents cytoplasm localization. Several cells, which did not present ex‐ pression of these proteins and served as a control, are indicated by white arrows. Confocal Microscopy: A-F) Differen‐ tial interference contrast (DIC); A1-F1 and A2-F2) Fluorescent microscopy (Fm); A3-F3, G, H) DIC+Fm. Scale Bars: A-D3,

Yan et al., (2010) quantified by real-time PCR the expression levels of endogenous Klf4 and c-Myc. Klf4 showed relative higher expression in DPC lines than in HDFs, however lower than in ES cells. Endogenous c-Myc expression in most DPC lines was also slightly higher than that in HDFs and in a few iPSC clones were close to ES cells. In contrast, Oda et al., (2010) observed low expression of Klf4 in high reprogramming cells, which was unexpected, once Klf4 is a reprogramming factor. Yan et al., (2010) showed that endogenous Klf4 expression level determined by real-time PCR did not completely correlate with the reprogramming efficiency of each DPCs (wisdom teeth) line. It is noteworthy that highly expression of KLF4 was

F-F3, H =50μm; E-E3,G=100μm.

iPSC-derived from these lines and HDFs, used as a control.

#### **2.4. Characterization of SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-derived iPSC**

#### *2.4.1. Expression of pluripotent stem cell markers*

As expected in all studies, the iPSC obtained from tissues of dental origin, which showed ESlike cells morphology, express key markers of pluripotent stem cells in an appropriate manner. Immunofluorescence study demonstrates uniform expression of these antigens in iPSC colonies derived from different types of dental stem cells. Transcription factor proteins as Oct3/4, Nanog, Sox2 demonstrate nuclear localization, while cell surface markers, such as stage specific embryonic antigen (SSEA) 3 and SSEA4, as well as cell surface antigens of human embryonic carcinoma cells (TRA-1-60 and TRA-1-81) show cell surface localization. Appro‐ priate expression of transcription factors Klf4, c-Myc, Lin28, that were part of the transgene used for reprogramming, also was observed (Yan et al. 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011) (Fig. 4).

#### *2.4.2. Expression of molecular markers of pluripotent stem cells*

Only one study performed quantitative PCR analysis before and after reprogramming for endogenous expression of Oct4, Nanog and Sox2 genes and compared the expression level of all these genes with those in pluripotent human ES cells. Albeit we revealed a tendency for increasing of expression of pluripotent factors Oct4, Nanog and Sox2, when compared to nonreprogrammed cells (18%, 1% and 2%, respectively), it was significantly lower 20% (Oct4), 10% (Nanog) and 40% (Sox2) in comparison with human ES cells (100% - Oct4, Nanog, Sox2) (Beltrão-Braga et al., 2011). Other studies did not provide any data about expression of these key markers in SHED/SCAP/DPSCs/DPCs/MStCs – derived iPSC in comparison with ES cells (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). However, Oda and colleagues (2010) demonstrated that expression levels of Oct4, Nanog, Sox2, Klf4, c-Myc, Lin28 and P53 was higher in iPSC derived from clonally isolated MStCs, when compared with parental cell lines, iPSC-derived from these lines and HDFs, used as a control.

**2.3. Provirus integration**

136 Pluripotent Stem Cells

Viral vectors are commonly used to deliver genetic material into cells, which can be performed *in vivo* (living organism) or *in vitro* (cell culture). Delivery of genes by a virus is efficient, however with respect to safety, it is ideal not to use lentivirus and retrovirus vectors, since they can integrate into the host DNA. As opposed to lentiviruses and retroviruses, adenoviral DNA does not integrate into the genome and are considered to be safer (Tamaoki et al., 2010). All studies performed with stem cells of dental tissue origin used lentivirus and/or retrovirus vectors for reprogramming. Yan and co-workers (2010) examined the presence of transgenes in the genome of iPSC clones isolating genomic DNA and generating primers specific for each transgene. They showed that the 4 factors were all integrated into the genome of the transduced SHED/SCAP/DPSC-iPSC. Other study, which used MStCs from wisdom teeth for reprogram‐ ming, did not provide any records about viral vectors integration (Tamaoki et al., 2010), while another group demonstrated retroviral silencing (Oda et al., 2010). Beltrão-Braga and coauthors (2011), which used retroviral vector for reprogramming, showed the lack of transgene

expression by RT-PCR analyses in iPSC-derived from IDPSCs.

*2.4.1. Expression of pluripotent stem cell markers*

2010; Beltrão-Braga et al., 2011) (Fig. 4).

*2.4.2. Expression of molecular markers of pluripotent stem cells*

**2.4. Characterization of SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-derived iPSC**

As expected in all studies, the iPSC obtained from tissues of dental origin, which showed ESlike cells morphology, express key markers of pluripotent stem cells in an appropriate manner. Immunofluorescence study demonstrates uniform expression of these antigens in iPSC colonies derived from different types of dental stem cells. Transcription factor proteins as Oct3/4, Nanog, Sox2 demonstrate nuclear localization, while cell surface markers, such as stage specific embryonic antigen (SSEA) 3 and SSEA4, as well as cell surface antigens of human embryonic carcinoma cells (TRA-1-60 and TRA-1-81) show cell surface localization. Appro‐ priate expression of transcription factors Klf4, c-Myc, Lin28, that were part of the transgene used for reprogramming, also was observed (Yan et al. 2010; Tamaoki et al., 2010; Oda et al.,

Only one study performed quantitative PCR analysis before and after reprogramming for endogenous expression of Oct4, Nanog and Sox2 genes and compared the expression level of all these genes with those in pluripotent human ES cells. Albeit we revealed a tendency for increasing of expression of pluripotent factors Oct4, Nanog and Sox2, when compared to nonreprogrammed cells (18%, 1% and 2%, respectively), it was significantly lower 20% (Oct4), 10% (Nanog) and 40% (Sox2) in comparison with human ES cells (100% - Oct4, Nanog, Sox2) (Beltrão-Braga et al., 2011). Other studies did not provide any data about expression of these key markers in SHED/SCAP/DPSCs/DPCs/MStCs – derived iPSC in comparison with ES cells (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). However, Oda and colleagues (2010) demonstrated that expression levels of Oct4, Nanog, Sox2, Klf4, c-Myc, Lin28 and P53 was

**Figure 4.** Expression of Oct3/4, Nanog, Sox2 and TRA-1-81 proteins in two lineages of hIDPS-IPSC five days after trans‐ duction with four factors. In A-C3,G) hIDPS-IPSC1 and D-F3,H) hIDPS-IPSC2 are presented, both showing multiple small colonies, which already express hallmarks of pluripotent cells, such as, A-A3) and D-D3) Oct3/4; B-B3) and E-E3) Nanog, C-C3) and F-F3) Sox2; G) and H) TRA-1-81, respectively. Nucleus stained with DAPI (blue). Note, that Oct3/4, Nanog, Sox2 present nuclear, while TRA-1-81 presents cytoplasm localization. Several cells, which did not present ex‐ pression of these proteins and served as a control, are indicated by white arrows. Confocal Microscopy: A-F) Differen‐ tial interference contrast (DIC); A1-F1 and A2-F2) Fluorescent microscopy (Fm); A3-F3, G, H) DIC+Fm. Scale Bars: A-D3, F-F3, H =50μm; E-E3,G=100μm.

Yan et al., (2010) quantified by real-time PCR the expression levels of endogenous Klf4 and c-Myc. Klf4 showed relative higher expression in DPC lines than in HDFs, however lower than in ES cells. Endogenous c-Myc expression in most DPC lines was also slightly higher than that in HDFs and in a few iPSC clones were close to ES cells. In contrast, Oda et al., (2010) observed low expression of Klf4 in high reprogramming cells, which was unexpected, once Klf4 is a reprogramming factor. Yan et al., (2010) showed that endogenous Klf4 expression level determined by real-time PCR did not completely correlate with the reprogramming efficiency of each DPCs (wisdom teeth) line. It is noteworthy that highly expression of KLF4 was previously reported in senescent cells and terminally differentiated cells (Shields et al., 1996; Conkright et al., 1999). Taken together, these data suggest that endogenous Klf4 expression may not be the single factor in charge for the reprogramming efficiency to MSCs derived from wisdom teeth.

*2.4.6. Karyotype analysis of dental stem cell-derived iPSC*

Magnification 63X. Differential interference contrast (DIC)

glial fibrillary acidic protein (GFAP) (Fig. 6).

*2.5.1. Embryoid body formation and in vitro differentiation*

Karyotype study has been performed by all authors and demonstrated that karyotype of reprogrammed cells remained unchanged (Fig. 5). Overall, during reprogramming of stem cell from dental pulp, numerical and gross structural chromosomal abnormalities were not detected (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011).

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**Figure 5.** Representative figures of karyotype analysis of both hIDPSC and hIDPS-iPSC: Routine Giemsa staining did not reveal any numerical changes in chromosome number (A and B) and any chromosomal structural changes (B).

Similar to human ES cells, iPSC require the formation of embryoid bodies (EB) in order to undergo *in vitro* differentiation into various cell types. All studies confirm successful differentiation of iPSC-derived from different types of dental stem cells into all three germ layers (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). As expected, the majority of iPSC formed EBs with cystic cavities. The histological analysis demonstrates that EBs differentiated into different cell types of ectodermal, mesodermal, or endodermal origin, which was demonstrated using antibodies against specific pro‐ teins, which is expressed in each of three germ layers. It has been shown that after reprogramming, cells tend to maintain their original commitment. Dental pulp stem cells are multipotent stem cells derived from neural crest and they showed strong commit‐ ment into neural lineages. Therefore, neural differentiation is widely presented in all iPSCderived from different types of dental stem cells. Under appropriate neurogenic culture medium, the EBs developed into the cells with neural-like morphology (Fig. 6), which express such markers as nestin, β-tubulin III (TUJ1), neuron-specific Enolase (NSE) and

**2.5. Differentiation of SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-iPSC**

#### *2.4.3. Searching for new factors of reprogramming*

Oda et al., (2010) tried to find the additional unknown factor(s) that could help in the cell reprogramming. They focused their study on practically two genes: PAXIP1 (or PTIP) and PARP. PAXIP1 acts as component of a histone H3 lysine four (H3K4) methyltransferase complex (Cho et al., 2007; Patel et al., 2007) and has a role in DNA double-strand break repair (van Attikum and Gasser, 2009). It was demonstrated that efficient reprogramming of pluripotent gene (Oct3/4, Sox2) expression is associated with H3K4 methylation in mouse somatic cell nuclei transplantation into amphibian oocytes (Murata et al., 2010). The expression of this gene was about 30% more in the high reprogramming cells than in low reprogramming as well as 3–4 times more in iPSC when compared with each parental cell line. PARP-1 belongs to PARP family being the most abundant member and is responsible for > 85% of nuclear PARP activity modifying histone structure through DNA-dependent "PARylation". Higher expres‐ sion of PARP-1 was also seen after induction of reprogramming in cells derived from wisdom teeth. The authors supposed that due to possible conformational change of chromatin by direct/indirect actions of chromatin modification proteins such as PAXIP1 and possibly PARP-1, high iPSC generation clones may be accessible for reprogramming factors. However, further investigation is needed to illuminate the iPSC reprogramming mechanisms using these genes.

#### *2.4.4. Methylation status of cytosine guanine dinucleotides (CpG)*

The methylation status of CpG in the promoter regions of Nanog and Oct4 was examined using bisulfite DNA sequencing method in two studies (Yan et al., 2010; Oda et al., 2010) and of Nanog in one study (Tamaoki et al., 2010). They showed that parental MStCs from wisdom teeth were highly (Oct3/4) or partially (Nanog) methylated and the iPSC-derived from these cells were highly unmethylated, suggesting that these promoters were active after cells reprogramming. In contrast, the analysis of iPSC clones derived from DPSCs (wisdom teeth) and SHED (deciduous teeth) showed that Nanog promoter had similar or slightly higher number of methylated sites, than their non-transduced counterparts. The SHED-/DPSC-iPSC had less methylated sites of Oct3/4 promoter than the non-transduced cells (Yan et al., 2010).

#### *2.4.5. Telomerase activity*

Telomerase activity is known to be highly activated in ES cells in order to maintain the integrity of chromosome structure. After reprogramming, SHED- (deciduous teeth) SCAP-, and DPSC (wisdom teeth) - iPSC showed telomerase activity very close to ES cells and a lot more in comparison to their non-transduced counterparts (Yan et al., 2010). Parental DPSCs (wisdom teeth) showed low telomerase activity whereas in each iPSC telomerase activity was high (Oda et al., 2010).

#### *2.4.6. Karyotype analysis of dental stem cell-derived iPSC*

previously reported in senescent cells and terminally differentiated cells (Shields et al., 1996; Conkright et al., 1999). Taken together, these data suggest that endogenous Klf4 expression may not be the single factor in charge for the reprogramming efficiency to MSCs derived from

Oda et al., (2010) tried to find the additional unknown factor(s) that could help in the cell reprogramming. They focused their study on practically two genes: PAXIP1 (or PTIP) and PARP. PAXIP1 acts as component of a histone H3 lysine four (H3K4) methyltransferase complex (Cho et al., 2007; Patel et al., 2007) and has a role in DNA double-strand break repair (van Attikum and Gasser, 2009). It was demonstrated that efficient reprogramming of pluripotent gene (Oct3/4, Sox2) expression is associated with H3K4 methylation in mouse somatic cell nuclei transplantation into amphibian oocytes (Murata et al., 2010). The expression of this gene was about 30% more in the high reprogramming cells than in low reprogramming as well as 3–4 times more in iPSC when compared with each parental cell line. PARP-1 belongs to PARP family being the most abundant member and is responsible for > 85% of nuclear PARP activity modifying histone structure through DNA-dependent "PARylation". Higher expres‐ sion of PARP-1 was also seen after induction of reprogramming in cells derived from wisdom teeth. The authors supposed that due to possible conformational change of chromatin by direct/indirect actions of chromatin modification proteins such as PAXIP1 and possibly PARP-1, high iPSC generation clones may be accessible for reprogramming factors. However, further investigation is needed to illuminate the iPSC reprogramming mechanisms using these

The methylation status of CpG in the promoter regions of Nanog and Oct4 was examined using bisulfite DNA sequencing method in two studies (Yan et al., 2010; Oda et al., 2010) and of Nanog in one study (Tamaoki et al., 2010). They showed that parental MStCs from wisdom teeth were highly (Oct3/4) or partially (Nanog) methylated and the iPSC-derived from these cells were highly unmethylated, suggesting that these promoters were active after cells reprogramming. In contrast, the analysis of iPSC clones derived from DPSCs (wisdom teeth) and SHED (deciduous teeth) showed that Nanog promoter had similar or slightly higher number of methylated sites, than their non-transduced counterparts. The SHED-/DPSC-iPSC had less methylated sites of Oct3/4 promoter than the non-transduced cells (Yan et al., 2010).

Telomerase activity is known to be highly activated in ES cells in order to maintain the integrity of chromosome structure. After reprogramming, SHED- (deciduous teeth) SCAP-, and DPSC (wisdom teeth) - iPSC showed telomerase activity very close to ES cells and a lot more in comparison to their non-transduced counterparts (Yan et al., 2010). Parental DPSCs (wisdom teeth) showed low telomerase activity whereas in each iPSC telomerase

wisdom teeth.

138 Pluripotent Stem Cells

genes.

*2.4.5. Telomerase activity*

activity was high (Oda et al., 2010).

*2.4.3. Searching for new factors of reprogramming*

*2.4.4. Methylation status of cytosine guanine dinucleotides (CpG)*

Karyotype study has been performed by all authors and demonstrated that karyotype of reprogrammed cells remained unchanged (Fig. 5). Overall, during reprogramming of stem cell from dental pulp, numerical and gross structural chromosomal abnormalities were not detected (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011).

**Figure 5.** Representative figures of karyotype analysis of both hIDPSC and hIDPS-iPSC: Routine Giemsa staining did not reveal any numerical changes in chromosome number (A and B) and any chromosomal structural changes (B). Magnification 63X. Differential interference contrast (DIC)

#### **2.5. Differentiation of SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-iPSC**

#### *2.5.1. Embryoid body formation and in vitro differentiation*

Similar to human ES cells, iPSC require the formation of embryoid bodies (EB) in order to undergo *in vitro* differentiation into various cell types. All studies confirm successful differentiation of iPSC-derived from different types of dental stem cells into all three germ layers (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). As expected, the majority of iPSC formed EBs with cystic cavities. The histological analysis demonstrates that EBs differentiated into different cell types of ectodermal, mesodermal, or endodermal origin, which was demonstrated using antibodies against specific pro‐ teins, which is expressed in each of three germ layers. It has been shown that after reprogramming, cells tend to maintain their original commitment. Dental pulp stem cells are multipotent stem cells derived from neural crest and they showed strong commit‐ ment into neural lineages. Therefore, neural differentiation is widely presented in all iPSCderived from different types of dental stem cells. Under appropriate neurogenic culture medium, the EBs developed into the cells with neural-like morphology (Fig. 6), which express such markers as nestin, β-tubulin III (TUJ1), neuron-specific Enolase (NSE) and glial fibrillary acidic protein (GFAP) (Fig. 6).

neurons and peripheral neuroendocrine tissue, and synaptophysin (a synaptic vesicle glycoprotein), which is found in neuroendocrine cells as well as virtually in all neurons that participate in synaptic transmission in the brain and spinal cord are expressed in

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**Figure 7.** HE stained differentiated tissues from hIDPS-iPSC teratoma seven weeks after transplantation into nude mice right limb. A) Morphogenesis observed during differentiation of hIDPS-iPSC: glomerulus-like structure formation, with Bowman's capsule and convoluted tubule–like structures. B) Cartilage and in (C) Condrocytes-like cells (higher magnification). D) Respiratory-like epithelium. E) Gastrointestinal-like epithelium. F) Neural tubes-like structures. G)

Tamaoki and co-wokers (2010) determined the human leukocyte antigens (HLA) types of 107 dental pulp cells lines in the Japanese population and identified 2 cell lines with homozygous HLA types at all 3 loci (A, B, and DR) examined. They showed that in the Japanese population the frequencies of haplotypes of these 2 homozygous cell lines were estimated to be 8.7% and 1.5%, data provided by the Japanese Red Cross Society (http://www.bmdc.jrc.or.jp/stat.html). Using these frequencies, the coverage rate for a perfect match of iPSC lines, which were established from these 2 lines was calculated. The authors showed that iPSC lines established from these 2 homozygous cell lines would cover 16.6% and 3.0% of the Japanese population,

respectively, which corresponds to approximately 20% of the Japanese population.

One of the major challenge of pluripotent stem cells use in cell therapies is an immunemediated rejection after transplantation. Today, this problem can be overcome by direct reprogramming of patients somatic cells and by creating an iPSC bank consisting of various HLA types thus providing therapeutic tool for the patients, which need cell transplantation free from immune-mediated rejection. Two works reported that the establishment of 50 unique stem cells lines, having homozygous alleles of the 3 HLA loci (A, B, and DR), would cover ~

**2.7. SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-iPSC bank and therapeutic use**

Blood vessel. H) Adipose-like tissue. Magnifications: A, C) 100x, B, D-G) 20x, Scale Bar (H) = 200μm.

**2.6. HLA typing**

neuronal cells produced by IDPSC-iPSC in teratomas (Beltrão-Braga et al., 2011).

**Figure 6.** Representative figure of *in vitro* differentiation of hIDPS-iPSC. A) EBs adherent on Petri dish showing differ‐ entiation into neural-like cells after culturing in neurobasal+B27 medium. B) Same as in (A) showing neural-like cells in high magnification. C) Neural-like cells present positive immunostaining with anti-nestin antibody. D) positive immu‐ nostaining with anti-TUJ1 antibody. E) Neuron-specific enolase positive immunostaining (green) in hIDPS-iPSC derived neuronal cells. F) Morphological presentation of glial-like cells derived from hIDPS-iPSC. Positive immunostaining for (F) anti-GFAP antibody in glial-like cells. Nucleus stained with DAPI (blue). A, B= Differential interference contrast (DIC). C-E=Epi. F= DIC+Epi. Scale Bars: A-F=20μm.

#### *2.5.2. Teratoma formation*

To test the pluripotency, iPSC were injected into the testis or intramuscularly into the right and/or left hind leg of severe combined immunodeficient (SCID) mouse. Teratomas formation by SHED/SCAP/DPSCs/DPCs/MStCs–iPSC occurs of nine to eleven weeks after injection and histological examination of the tumor shows representative tissues of three embryonic germ layers, such as gut-like epithelium (endoderm), cartilage (mesoderm), and neuroepithelial rosettes (ectoderm) (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). Teratomas obtained from IDPSC-iPSC were formed between 5 and 7 weeks after reprog‐ rammed cells injection. The mice injected with parental IDPSC, as expected, did not form teratomas. We observed that teratomas were composed by tissues originated from three primary germ layers. Histological characterization of tumor masses showed that these teratomas includes ectodermal: primitive neural tissues, including neural tube and neural rosettes and retinal epithelium; mesodermal: muscle-like cells and gromerulus-like structures and endodermal tissues: respiratory or gastro-intestinal-like epithelium and glandular-like tissue formation (Fig. 7). Similar to *in vitro* differentiation, teratomas derived from IDPSC-iPS cells, display strong neuronal commitment forming rosette-, neuro‐ sphere- and neural tube-like structures. Neuron-specific enolase, which is a marker of neurons and peripheral neuroendocrine tissue, and synaptophysin (a synaptic vesicle glycoprotein), which is found in neuroendocrine cells as well as virtually in all neurons that participate in synaptic transmission in the brain and spinal cord are expressed in neuronal cells produced by IDPSC-iPSC in teratomas (Beltrão-Braga et al., 2011).

**Figure 7.** HE stained differentiated tissues from hIDPS-iPSC teratoma seven weeks after transplantation into nude mice right limb. A) Morphogenesis observed during differentiation of hIDPS-iPSC: glomerulus-like structure formation, with Bowman's capsule and convoluted tubule–like structures. B) Cartilage and in (C) Condrocytes-like cells (higher magnification). D) Respiratory-like epithelium. E) Gastrointestinal-like epithelium. F) Neural tubes-like structures. G) Blood vessel. H) Adipose-like tissue. Magnifications: A, C) 100x, B, D-G) 20x, Scale Bar (H) = 200μm.

#### **2.6. HLA typing**

**Figure 6.** Representative figure of *in vitro* differentiation of hIDPS-iPSC. A) EBs adherent on Petri dish showing differ‐ entiation into neural-like cells after culturing in neurobasal+B27 medium. B) Same as in (A) showing neural-like cells in high magnification. C) Neural-like cells present positive immunostaining with anti-nestin antibody. D) positive immu‐ nostaining with anti-TUJ1 antibody. E) Neuron-specific enolase positive immunostaining (green) in hIDPS-iPSC derived neuronal cells. F) Morphological presentation of glial-like cells derived from hIDPS-iPSC. Positive immunostaining for (F) anti-GFAP antibody in glial-like cells. Nucleus stained with DAPI (blue). A, B= Differential interference contrast (DIC).

To test the pluripotency, iPSC were injected into the testis or intramuscularly into the right and/or left hind leg of severe combined immunodeficient (SCID) mouse. Teratomas formation by SHED/SCAP/DPSCs/DPCs/MStCs–iPSC occurs of nine to eleven weeks after injection and histological examination of the tumor shows representative tissues of three embryonic germ layers, such as gut-like epithelium (endoderm), cartilage (mesoderm), and neuroepithelial rosettes (ectoderm) (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). Teratomas obtained from IDPSC-iPSC were formed between 5 and 7 weeks after reprog‐ rammed cells injection. The mice injected with parental IDPSC, as expected, did not form teratomas. We observed that teratomas were composed by tissues originated from three primary germ layers. Histological characterization of tumor masses showed that these teratomas includes ectodermal: primitive neural tissues, including neural tube and neural rosettes and retinal epithelium; mesodermal: muscle-like cells and gromerulus-like structures and endodermal tissues: respiratory or gastro-intestinal-like epithelium and glandular-like tissue formation (Fig. 7). Similar to *in vitro* differentiation, teratomas derived from IDPSC-iPS cells, display strong neuronal commitment forming rosette-, neuro‐ sphere- and neural tube-like structures. Neuron-specific enolase, which is a marker of

C-E=Epi. F= DIC+Epi. Scale Bars: A-F=20μm.

*2.5.2. Teratoma formation*

140 Pluripotent Stem Cells

Tamaoki and co-wokers (2010) determined the human leukocyte antigens (HLA) types of 107 dental pulp cells lines in the Japanese population and identified 2 cell lines with homozygous HLA types at all 3 loci (A, B, and DR) examined. They showed that in the Japanese population the frequencies of haplotypes of these 2 homozygous cell lines were estimated to be 8.7% and 1.5%, data provided by the Japanese Red Cross Society (http://www.bmdc.jrc.or.jp/stat.html). Using these frequencies, the coverage rate for a perfect match of iPSC lines, which were established from these 2 lines was calculated. The authors showed that iPSC lines established from these 2 homozygous cell lines would cover 16.6% and 3.0% of the Japanese population, respectively, which corresponds to approximately 20% of the Japanese population.

#### **2.7. SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-iPSC bank and therapeutic use**

One of the major challenge of pluripotent stem cells use in cell therapies is an immunemediated rejection after transplantation. Today, this problem can be overcome by direct reprogramming of patients somatic cells and by creating an iPSC bank consisting of various HLA types thus providing therapeutic tool for the patients, which need cell transplantation free from immune-mediated rejection. Two works reported that the establishment of 50 unique stem cells lines, having homozygous alleles of the 3 HLA loci (A, B, and DR), would cover ~ 90% of the Japanese population with a faultless match of these loci (Nakajima et al., 2007; Nakatsuji et al., 2008). Considering that iPSC derivation is a time consuming process and of elevated cost, it should be necessary for cell therapies and regenerative medicine to establish iPSC banks with a sufficient collection of HLA types, thus avoiding additional costs which are required for iPSC production for each individual patient.

*2.7.2. Perspectives of iPSC therapeutic use*

**3. Final considerations**

Currently, iPSC are used to understand human diseases, including Alzheimer's disease, Parkinson's disease, cardiovascular disease, diabetes, and amyotrophic lateral sclerosis (ALS), to develop and screen bioactive molecules - candidate to therapeutic drugs and to identify molecules or genes implicated in tissue regeneration. These *in vitro* studies enable researchers to understand fundamental principles of iPSC function and differentiation, which further will provide knowledge, necessary for therapeutic use of iPSC. Based on this knowledge multiple pre-clinical and clinical protocols will be produced optimizing iPSC transplantation in diverse animal and human diseases thus becoming a tool in cellreplacement therapy. Therefore, iPSC in the future may have tremendous clinical poten‐ tial when highly efficient and safe protocols of generation of reprogrammed stem cells will be developed. Increasing our understanding of the molecular mechanisms that underlie reprogramming, we will be able to identify the cell types and methods of reprogram‐ ming, which will minimize DNA alterations, and conditions of iPSC cultivation that will allow widespread use of these cells in clinic. The scientists, however, should answer the question if iPSC are truly equivalent to human ES cells. Although iPSC potential for regenerative medicine is great, our current knowledge about iPSC variability, and utility must also increase greatly before iPSC became a standard tool for regenerative medicine.

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Different dental tissues, which include apical papilla, primary exfoliated deciduous and permanent teeth, as well as wisdom teeth were used to derivate iPSC. The data obtained by different authors indicate that these tissues can be easily isolated and MSCs cells in sufficient quantities can be obtained. MSCs *in vivo* are reversibly arrested cells, which are localized in their niches maintaining their temporarily quiescent state. They differ from terminally differentiated cells by developmental path that involves a set of increasingly committed stages of specialization. The fact that these cells are undifferentiated cells suggests that their reprog‐ ramming will occur more easily than that of terminally differentiated cells. Indeed, all studies demonstrated that in SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC the reprogramming process occurs more easily than in human fibroblasts used as a control, under the similar protocols and vectors used for transduction, which showed to be efficient. It was also possible to reprogram MSCs from wisdom teeth using only three factors (without c-Myc) avoiding future implications with potential risks of oncogene use (Yu et al., 2007). For the clinical applications of stem cells xenogeneic reagents pose the risk of a severe immune response, and the trans‐ mission of viral or bacterial infections, prions, and unidentified zoonosis. We demonstrated that isolation of IDPSC-iPSC can occur under feeder-free conditions on matrigel-coated dishes. Clinical stem cell therapy trials are ongoing, which request a strong focus on the safety and quality of *in vitro* expanded stem cell transplants. By replacing xenogeneic products with a defined xeno-free medium, the safety and quality of the cells with therapeutic potential may be enhanced significantly. Similar to human ES cells, dental MSCs derived iPSC form compact colony and retain immortal growth characteristics in culture. They express markers charac‐

#### *2.7.1. Requirements of iPSC generation*

In spite of optimistic prognosis in respect of how many iPSC should be produced in order to satisfy their immunological matching within definite human population, several requirements must be challenged before establishing iPSC bank. The principal require‐ ment is a method of reprogramming, which should be safe. Therefore, three major concerns exist in the current reprogramming strategies for clinical applications: (i) the low reprog‐ ramming efficiency of human somatic cells makes it difficult to generate patient-specific iPSC, when a small amount of the cells of the patient is used; (ii) carcinogenesis may be caused by genomic integration of retro- or lentiviral fragments into host DNA; and (iii) Myc is an oncogene, which after reactivation might cause malignant tumor formation. Whereas iPSC can be generated by three transcription factors (Oct3/4, Sox2, and Klf4) without Myc, reprogramming efficiency are significantly reduced.  Next important issue is availability of donor cells, which can provide high efficiency in the generation of non-integrated human iPSC. Therefore, source of the cells also makes its own demand, such as, it should be easily accessible with minimum discomfort for the patient, the procedure of stem cell isolation should be non-invasive, the tissue should be easily processed, the cells should be rapidly proliferating and produced in sufficient quantities, these cells should be young and collected from healthy volun‐ teers. Furthermore, the possibility of genetic abnormalities in donor cells due to ultravio‐ let (UV) irradiation should be minimized and finally, these cells would be able to be stored in liquid nitrogen for a long time without the loss of their prime characteristics.

Dental pulp stem cells from deciduous and wisdom teeth are an ideal source that meets the majority of aforementioned requirements. The loss of baby (deciduous) teeth occurs naturally and they can be removed with minimal discomfort to the patient during a routine visit to the dentist, in many clinics, as well as wisdom teeth. We also showed that not only cells, but also dental pulp can be cryopreserved and new cells can be obtained later, after thawing (Lizier et al., 2012). Therefore, frozen dental pulp does not require *in vitro* cultivation in order to produce the cells of donor until he needs these cells for iPSC production and/or clinical treatment. Additionally, several dental pulps from the same individual can be cryopreserved. This elevates a probability of successful MSCs isolation in high quantities. These cells are safer, once they can be used by first and second degree relatives and within all family (Kerkis and Caplan, 2012; Lizier et al., 2012).

#### *2.7.2. Perspectives of iPSC therapeutic use*

90% of the Japanese population with a faultless match of these loci (Nakajima et al., 2007; Nakatsuji et al., 2008). Considering that iPSC derivation is a time consuming process and of elevated cost, it should be necessary for cell therapies and regenerative medicine to establish iPSC banks with a sufficient collection of HLA types, thus avoiding additional costs which are