Induced Pluripotent Stem Cells

Induced Pluripotent Stem Cells

In 2006 it was discovered that stem cells could be generated from adult somatic cells through a process of cellular reprogramming (Takahashi and Yamanaka 2006). Stem cells generated by this new technology were termed induced pluripotent stem cells (iPSCs). Since their discovery, there has been substantial interest in iPSCs, as this technology facilitates the generation of adult human pluripotent stem cells without the need for human embryos, thereby bypassing a number of the ethical and legal concerns that have hindered embryonic stem cell research to date (Takahashi and Yamanaka 2006; Maherali et al. 2007; Okita et al. 2007; Takahashi et al. 2007; Wernig et al. 2007). iPSCs appear to have a number of advantages over other dental-derived stem cell populations, such as periodontal ligament (PDL) and dental pulp. Specifically, iPSCs can be generated from readily accessible tissue sources, including oral mucosa (Miyoshi et al. 2010) and gingival tissue (Egusa et al. 2010). More important, iPSCs are highly proliferative, making it possible to obtain the large numbers of stem cells that would be required for use in regenerative therapies in the clinic.

Limitations of iPSCs

Before we outline the potential of iPSCs in dentistry, it is important to note that there are significant safety concerns regarding iPSCs that need to be addressed before they can be considered for use in mainstream treatment approaches in dentistry. The major drawbacks of iPSCs include their genomic instability and their propensity to form tumors in vivo (Ben-David and Benvenisty 2011; Gore et al. 2011). While there are shortcomings associated with iPSCs, the potential that iPSCs have demonstrated in the treatment of multiple disorders demands further research to investigate and minimize the associated therapeutic risks.

The use of viral integrating vectors in the generation of iPSCs is a contributing factor to genomic instability and tumorigenic potential of iPSCs. Consequently, various groups have attempted to utilize nonintegrating vectors to reduce the genomic instability of iPSCs. Zou et al. (2012) successfully utilized a single lentiviral stem cell cassette to generate iPSCs from human stem cells of apical papilla. Stem cell cassette is a single lentiviral cassette flanked by a lox-p site, which allows for its controlled excision with cre-recombinase (Somers et al. 2010). The ability to remove the transgene/vector from the generated population of iPSCs provides a platform for their potential medical therapeutic application.

Furthermore, the tumorigenicity of iPSCs can be minimized by differentiating iPSCs into lineage-specific progenitor cells or mature populations of cells prior to use in regenerative therapies. Assessment of the tumorgenicity of iPSC-derived mesenchymal stem cells (iPSC-MSCs) in NOD/SCID mice failed to identify the formation of any tumors (Zhao et al. 2015). Additionally, iPSC-MSCs have less potential to promote tumors than do bone marrow–derived mesenchymal stem cells (BMMSCs; Zhao et al. 2015). Combining the use of nonintegrating vectors in iPSC generation and the differentiation of iPSCs into lineage-restricted progenitor cells such as mesenchymal stem cells (MSCs) will help to overcome the major safety concerns currently associated with the use of iPSCs in the clinic. As discussed in a recent review, in spite of their limitations, iPSCs still possess vast potential for use in clinical settings (Sanchez Alvarado and Yamanaka 2014).

Generation of iPSCs from Dental Tissues

While iPSCs can be generated from a range of adult tissues, dental tissues present an attractive source of precursors due to tissue accessibility and ease of attainment. Furthermore, for dental applications, it may prove beneficial to utilize iPSCs generated from dental tissues. It has been suggested that iPSCs may maintain epigenetic memory of the source tissue that they were generated from and that the resulting iPSCs will preferentially differentiate back to their original cell type, so using iPSCs derived from dental tissues could enhance their capacity to differentiate into dental tissues. Whether iPSCs do retain epigenetic memory for their original cell type is still a point of contention in the literature, with some studies indicating that iPSCs do maintain a memory of their original cell type (Bar-Nur et al. 2011; Hu et al. 2010; Kim et al. 2011; Ohi et al. 2011), while other studies have shown that this memory is lost during in vitro culture (Kim et al. 2010; Lee et al. 2012; Polo et al. 2010).

Considering the vast potential of iPSCs in dental research and the prospective benefits of using dental-derived iPSCs, numerous groups have reprogrammed dental tissue–derived cell populations into pluripotent stem cells. Figure 1 summarizes the dental tissues that have been successfully reprogrammed into iPSCs.

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Figure 1.

The dental-derived tissues from which induced pluripotent stem cells have successfully been generated.

iPSCs have been generated successfully from dental pulp cells derived from exfoliated deciduous teeth, stem cells from apical papilla, and dental pulp stem cells (Yan et al. 2010); cells from human extracted wisdom teeth (Oda et al. 2010; Tamaoki et al. 2010); immature human dental pulp stem cells (Beltrao-Braga et al. 2011); dental pulp cells from naturally lost deciduous teeth (Dambrot et al. 2013); oral mucosa fibroblasts (Miyoshi et al. 2010); gingival tissue (Egusa et al. 2010; Wada et al. 2011); and PDL stem cells (Wada et al. 2011). A comparison performed by Yan et al. (2010) identified that the reprogramming efficiency appeared to be greater from dental-derived tissues than human fibroblast cells.

Collectively, the above studies demonstrate the potential and advantages that different dental-derived tissues have for iPSC generation.

Dental Applications of iPSCs

To date, 4 studies have investigated the potential utility of iPSCs in various dental applications, including periodontal regeneration (Duan et al. 2011) and tooth development and regeneration (Otsu et al. 2012; Wen et al. 2012; Cai et al. 2013).

Duan et al. (2011) investigated the potential of human iPSCs to regenerate periodontal tissues when implanted into a surgically created periodontal fenestration defect. The addition of iPSCs resulted in significantly more alveolar bone formation, cementum, and PDL regeneration in mice that received iPSCs in combination with a silk scaffold and enamel matrix derivatives (EMDs), as compared with control mice that received either the silk scaffold and EMDs or the silk scaffold alone. This study identified the ability of iPSCs to contribute to periodontal repair without any noticeable adverse reactions, although it remains to be determined if iPSCs alone without the scaffold or EMDs could achieve the same results.

The ability of iPSCs to differentiate into cells that could be used to aid dental development and regeneration has also been assessed (Otsu et al. 2012; Wen et al. 2012; Cai et al. 2013). Otsu et al. (2012) initially demonstrated that mouse embryonic fibroblast iPSCs can differentiate into neural crest–like cells that, when cocultured with dental epithelium for 2 wk in conditioned media, could differentiate into dental mesenchymal cells. This research was subsequently extended by Wen et al. (2012), who assessed the potential of iPSCs to contribute to regeneration of the tooth. The experimental model used in this study was based on reconstruction of the tooth germ, using fibroblast-derived mouse iPSCs mixed with mouse MSCs and placed in direct contact with epithelial cells derived from mouse oral tissue. The cells were combined in a collagen hemisphere and placed under odontogenic induction conditions for 5 d, to mimic the tooth germ microenvironment and stimulate molecular signaling and communication between the mesenchymal and epithelial populations. The recombinant tooth germ was implanted into subrenal capsules in mice and assessed 4 wk postimplantation. Histomorphometric and immunohistochemical analyses revealed that the engineered construct of combined cells resulted in formation of bone-, dentin-, and pulp-like structures and confirmed direct contribution of the iPSCs to tooth regeneration (Wen et al. 2012).

More recently, Cai et al. (2013) investigated the capacity of human iPSC lines, derived from urine cells, to differentiate into epithelial cells and contribute to the formation of tooth-like structures. Upon differentiation, iPSC-derived epithelial cells were harvested as an intact epithelial sheet and cultured ex vivo for 2 d on top of dental mesenchyme of molar tooth germs extracted from mice at E14.5. The resulting recombinant structure was implanted into the subcapsular renal layer of nude mice and assessed 3 wk postimplantation. Their findings demonstrated that of the 8 iPSC lines assessed, 3 exhibited the capacity to form tooth-like structures containing dental pulp, dentin, enamel space, and enamel organ (Cai et al. 2013). The finding that human iPSCs have the capacity to form tooth-like structures demonstrates that iPSCs are a realistic cell source for future investigations into the in vitro generation of implantable teeth and/or tooth germs.

While these studies have demonstrated the potential that iPSCs have for enhancing periodontal regeneration and generating tooth-like structures, the tumorigenic potential of iPSCs is inhibitory to their use in the clinic. Therefore, numerous groups have attempted to differentiate iPSCs into lineage-restricted progenitor cells that are significantly less tumorigenic than iPSCs. In this review, we focus on 2 differentiation approaches that are particularly relevant to the dental field—the differentiation of iPSCs into MSCs and the differentiation of iPSCs into osteoprogenitor cells. Figure 2 outlines the potential uses of differentiated iPSCs in dentistry.

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Figure 2.

The potential uses of differentiated induced pluripotent stem cells (iPSCs) in dentistry. This figure summarizes the potential clinical dental applications of iPSCs. Given the tumorigenic potential that iPSCs possess, it is highly unlikely that undifferentiated iPSCs will be safe for clinical use; however, the differentiation of iPSCs into more lineage-restricted cell populations has been shown to be a much safer option. To date, 3 main approaches for the use of iPSCs and their derivatives in dentistry have been proposed and/or investigated. First, iPSCs have shown potential for use in tooth generation (Otsu et al. 2012; Wen et al. 2012; Cai et al. 2013). Multiple cell populations are involved in the formation of teeth—namely, dental epithelium, dental mesenchyme, and neural crest–like cells—and iPSCs could be used to generate these cell populations, which, when combined, facilitate tooth generation. A second approach for the use of iPSCs involves their differentiation into mesenchymal stem cells (i.e., iPSC-MSCs), which can then be used in dental applications directly or in the generation of osteoprogenitors. To date, iPSCs-MSCs have demonstrated their immune modulation (Yang et al. 2014) and tissue and periodontal regeneration (Hynes et al. 2013) potential in dental settings. Finally, iPSCs and iPSCs-MSCs have successfully been differentiated into osteoprogenitor cells, and while these cells have yet to be assessed in a dental setting, it is likely only a matter of time until their utility in dental applications is demonstrated.

iPSC-derived MSCs

Numerous groups have successfully differentiated iPSCs into MSCs (i.e., iPSC-MSCs). Not only does this approach yield cells that are less tumorigenic and therefore safer for clinical use, but it also facilitates the generation of unlimited quantities of high-quality, early-passage MSCs for clinical use. MSCs have been shown, in multiple animal models, to have the capacity to enhance dental regeneration (reviewed in Hynes et al. 2012); however, translation of MSCs as a novel cell-based treatment approach for dental regeneration into the clinic has been limited by issues pertaining to access to sufficient quantities of MSCs. While MSCs can be sourced from numerous tissues, including multiple dental tissues, only a limited number of cells can be obtained from a single donor, and these cells have a limited capacity to expand in culture. Therefore, novel sources of MSCs are required before they can be used routinely in the clinic.

A range of methods have been used successfully to generate MSCs from human iPSCs (Lian et al. 2010; Giuliani et al. 2011; Fu et al. 2012; Hynes et al. 2012; Sun et al. 2012; Villa-Diaz et al. 2012; Zhang et al. 2012; Hynes et al. 2013; Liu et al. 2013; Moslem et al. 2013; Diederichs and Tuan 2014; Frobel et al. 2014; Tang et al. 2014; Cheng et al. 2015; Ishiy et al. 2015; Zhang et al. 2015). The approaches used involve spontaneous differentiation of iPSCs, followed by selection of MSC-like cells. The spontaneous differentiation step is typically performed through 1 of 2 approaches. The first is spontaneous differentiation of the iPSCs in flat culture conditions (Lian et al. 2010; Giuliani et al. 2011; Fu et al. 2012; Hynes et al. 2012; Sun et al. 2012; Zhang et al. 2012; Hynes et al. 2013; Moslem et al. 2013; Diederichs and Tuan 2014; Frobel et al. 2014; Tang et al. 2014; Cheng et al. 2015; Ishiy et al. 2015; Zhang et al. 2015). The second approach uses suspension cultures to promote iPSCs to differentiate and form embryoid bodies (Villa-Diaz et al. 2012; Liu et al. 2013; Diederichs and Tuan 2014; Frobel et al. 2014). The subsequent selection of MSCs cells is typically achieved through 1 of 2 approaches. One approach utilizes culture and passaging of the differentiated iPSCs on tissue culture flasks to select for plastic-adherent MSCs and select against nonadherent differentiated cell types and undifferentiated iPSCs (Giuliani et al. 2011; Hynes et al. 2012; Villa-Diaz et al. 2012; Hynes et al. 2013; Liu et al. 2013; Moslem et al. 2013; Diederichs and Tuan 2014; Frobel et al. 2014; Tang et al. 2014; Ishiy et al. 2015; Zhang et al. 2015). The alternative approach uses cell sorting to select for cells that express the mesenchymal cell surface marker CD105 and do not express CD24 (Lian et al. 2010; Fu et al. 2012; Sun et al. 2012; Zhang et al. 2012; Cheng et al. 2015). Inhibition of the transforming growth factor β pathway using the inhibitor SB43152 has also been identified as a rapid and reliable method for achieving MSC conversion from iPSCs (Chen et al. 2012).

To date iPSC-MSCs have demonstrated therapeutic potential in a range of rodent disease models. Specifically, iPSC-MSCs have been shown to promote cutaneous wound healing (Zhang et al. 2015), significantly improve survival and hepatic function in mice with lethal fulminant hepatic failure (Moslem et al. 2013), attenuate limb ischemia (Lian et al. 2010), prevent allergic airway inflammation, and ameliorate diabetic polyneuropathy (Himeno et al. 2013).

iPSC-MSCs in Dental Applications

Two published studies investigated the therapeutic utility of iPSC-MSCs in dental settings, both in rodent models of periodontitis (Hynes et al. 2013; Yang et al. 2014). The first study examined the utility of iPSC-MSCs in periodontal regeneration, while the second investigated the use of iPSC-MSCs in treatment of periodontal disease.

Differentiation of iPSCs into Osteoprogenitor Cells

iPSCs have the capacity to form bone, which is of importance in the context of periodontal regeneration (Duan et al. 2011) and tooth generation (Wen et al. 2012); however, concerns regarding the safety of iPSCs in patients significantly limits their use in the clinical setting, as discussed above. Differentiation of iPSCs into osteoprogenitor cells could overcome the limitations associated with the use of iPSCs by providing large populations of high-quality and safe cells for use in bone regeneration and would have significant implications for promoting bone regeneration in dental applications. Several groups have successfully differentiated human iPSCs into bone-forming osteoprogenitor cells in recent years using a range of methods (Villa-Diaz et al. 2012; Kanke et al. 2014; Ochiai-Shino et al. 2014; Phillips et al. 2014; Tang et al. 2014; Wang, Deng, et al. 2015; Wang, Liu, et al. 2015). The generation of bone progenitor cells from iPSCs has been achieved through 2 approaches, either through direct differentiation of iPSCs into osteoprogenitor cells (Kanke et al. 2014; Ochiai-Shino et al. 2014; Phillips et al. 2014; Wang, Deng, et al. 2015) or through differentiation of iPSCs to iPSC-MSCs and then to osteoprogenitor cells (Villa-Diaz et al. 2012; Tang et al. 2014; Wang, Liu, et al. 2015). The Table summarizes the in vitro and in vivo assessments that were performed in these studies.

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Table.

In Vitro and In Vivo Analysis Performed on Osteoprogenitor Cells Derived from iPSC and iPSC-MSC.

	In Vitro Assessment	In Vivo Assessment
iPSC-derived Osteoprogenitors
Phillips et al. (2014)	Mineral synthesis by cells was detected by alizarin red staining.	iPSC-derived osteoprogenitor cells were combined with hydroxyapatite / tricalcium phosphate and transplanted into immunocompromised mice.
	Quantitation of mineral formation was performed with a numeric score system based on the amount of visible alizarin red staining.	The presence of bone was visualized through histologic sections stained with hematoxylin and eosin and the detection of collagen bundles, via polarized light.
	Real-time polymerase chain reaction analysis of expression of bone- and cartilage-associated genes: PDGFR, RUNX2, ALP, COL1, and COL2.
Kanke et al. (2014)	Mineral synthesis by cells was detected by alizarin red staining.	Not assessed.
	Alizarin red staining–based quantification of calcification was performed.
	Assessed gene expression of RUNX2, SP7, COL1A1, and IBSP.
	Assessed protein expression of RUNX2 and SP7.
Ochiai-Shino et al. (2014)	Mineral synthesis by cells was detected by Alizarin red staining and toluidine blue staining.	Not assessed.
	Assessed gene expression of SOST, RELN, and NYP.
Wang, Deng, et al. (2015)	Mineral synthesis by cells was detected by alizarin red staining.	Not assessed.
	Quantitation of the alizarin red staining was performed.
	Assessed gene expression of RUNX2 and COL1A1.
	Assessed protein expression of RUNX2.
iPSC-MSC-derived Osteoprogenitors
Villa-Diaz et al. (2012)	Mineral synthesis by cells was detected by alizarin red staining.	iPSC-derived osteoprogenitor cells were transplanted into calvarial defects generated in immunocompromised mice skulls.
	Assessed gene expression of ALP, BGLAP, BMP3, COL1A1, DLX5, SPARC, and SPP1.	The presence of new bone was identified through histologic sections stained with hematoxylin and eosin.
Tang et al. (2014)	Mineral synthesis by cells was detected by alizarin red staining.	Not assessed.
	Assessed gene expression of ALP, OC, COL1, RUNX2.
	Mineral synthesis quantitated using an in vitro osteogenesis assay kit.
Wang, Liu, et al. (2015)	Mineral synthesis by cells was detected by alizarin red staining.	Bone regeneration was assessed by implantation of differentiated iPSCs cultured on biofunctionalized macroporous calcium phosphate cement into cranial defects in rats.
	Mineral concentration was measured by the osteogenesis assay.	The presence of new bone was identified through the combined use of micro–computed tomography analysis and histologic sections stained with hematoxylin and eosin and Masson’s trichrome stain.
	Assessed gene expression of ALP, OC, COL1, RUNX2.

iPSC, induced pluripotent stem cell; iPSC-MSC, induced pluripotent stem cell–derived mesenchymal stem cells.

Phillips et al. (2014) assessed different methods for generating osteoprogenitors from multiple iPSC lines. Four additives or combinations of additives were investigated through different differentiation protocols and time frames. The additives assessed included 1) a combination of dexamethasone + ascorbic acid, 2) retinoic acid, 3) rapamycin, and 4) a combination of basic fibroblast + bone morphogenetic protein 4. From 15 of the differentiated iPSC populations generated through these methods, only 4 formed true bone in vivo (the gold standard test for bone-forming abilities). While only a limited number of the differentiated iPSC lines generated in this study formed true bone in vivo, the results demonstrate that it is possible to differentiate iPSCs into bone-forming cells using basic fibroblast + bone morphogenetic protein 4 and/or dexamethasone + ascorbic acid (Phillips et al. 2014). Further improvements to the differentiation protocols are, however, required to establish a reliable method for the generation of osteoprogenitors from iPSCs for clinical use.

Kanke et al. (2014) have since reported an effective mass-production strategy for the stepwise differentiation under serum- and feeder-free conditions of iPSCs into osteoblasts. Their approach utilized 4 small molecules—CHIR99021, cyclopamine, smoothened agonist, and helioxanthin derivative—to aid osteogenic differentiation. Their differentiation strategy involved mesoderm induction, followed by osteoblast induction, and finally an osteoblast maturation phase leading to a significant increase in expression of osteoblast-related genes and proteins.

Ochiai-Shino et al. (2014) utilized a different combination of growth factors (transforming growth factor b, insulin-like growth factor 1, and fibroblast growth factor 2) to promote the formation of osteoprogenitor cells from iPSCs. The use of these growth factors enhanced the frequency of osteolineage cell production. The osteolineage cells generated by this method expressed high levels of the osteogenic marker osterix and could terminally differentiate into osteocyte-like cells.

Wang et al. have designed a method for differentiating iPSCs into osteoblastic lineage cells using a biomaterial rather than growth factors (Wang, Deng, et al. 2015). The biomaterial used was a fully defined synthetic peptides-decorated 2-dimensional microenvironment, which was generated through the use of polydopamine chemistry and carboxymethyl chitosan grafting. Culturing iPSCs on the biomaterial enhanced osteogenic differentiation (Wang, Deng, et al. 2015). The generation of osteogenic lineage cells from iPSCs using a synthetic surface and under fully defined conditions represents a major step toward the utilization of iPSCs for bone regeneration in a clinical setting.

Another approach in which iPSCs have been used to aid bone regeneration is by differentiating iPSC-MSCs into bone-forming osteoprogenitor cells rather than starting with undifferentiated iPSCs. Villa-Diaz et al. (2012) differentiated iPSC-MSCs in osteoblast differentiation media for 4 d, then transplanted the resulting cells into calvaria defects in mice. The transplantation of osteoblast-differentiated iPSC-MSCs led to de novo bone formation in recipients, while no bone formation was evident in control mice. Furthermore, the authors identified transplanted osteoblast-differentiated iPSC-MSCs within the regenerated bone, demonstrating that osteoblast-differentiated iPSC-MSCs have the capacity to aid bone formation in vivo.

Tang et al. (2014) utilized an injectable osteoconductive biomaterial, calcium phosphate cement (CPC), to promote osetogenic differentiation of iPSC-MSCs. CPC has Food and Drug Administration approval for use in repairing craniofacial defects. Osteogenic differentiation of iPSC-MSCs using CPC and osteogenic media resulted in increased expression of key osteogenic markers, including alkaline phosphatase, osteocalcin, collagen type I, and Runt-related transcription factor 2. Osteogenic iPSC-MSCs generated through this method had the capacity to synthesize bone mineral in vitro (Tang et al. 2014). Subsequent in vivo assessment of iPSC-MSCs and CPC constructs also demonstrated that osteogenic iPSC-MSCs could enhance bone formation in critical-sized cranial defects in rats (Wang, Liu, et al. 2015). Implantation of osteogenic-induced iPSC-MSCs led to a significant increase in the extent of de novo bone formation that occurred when compared with CPC alone, demonstrating the robust bone-forming capacity of osteogenic-induced iPSC-MSCs (Wang, Liu, et al. 2015). This investigation also compared the bone-forming capacity of iPSC-MSCs to umbilical cord MSCs and bone marrow MSCs. Implantation of iPSC-MSCs resulted in the highest new bone area fraction; however, the differences between the cell populations were not statistically significant (Wang, Liu, et al. 2015).

The ability of osteoprogenitor cells derived from iPSCs or iPSC-MSCs to enhance bone regeneration in the dental settings has not yet been investigated; however, it is likely to be only a matter of time until their dental utility is demonstrated.

Additional iPSC Differentiation Approaches with Dental Applications

An interesting paper by Ozeki et al. (2013) reported the use of a “hanging drop” method to differentiate mouse iPSCs into odontoblast-like cells. iPSCs were spontaneously differentiated into embryoid bodies, then cultured on a collagen type I scaffold combined with BMP4 without an epithelial-mesenchymal interaction. The cells generated through this method strongly expressed mature odontoblast markers, dentin sialophosphoprotein, and dentin matrix protein 1 and displayed both physiologic and functional characteristics of odontoblasts in vitro. The generation of odontoblast cells from iPSCs may prove highly valuable for the treatment of dentin and/or dental pulp damage (Ozeki et al. 2013).

Concluding Remarks

Despite substantial progress in iPSC research, significant challenges must be addressed for iPSCs to reach their full potential into a clinical translation. It is evident that iPSCs could be of use in preventing bone loss and regenerating different dental structures. However, the generation of functional teeth will almost certainly remain elusive in the foreseeable future due to the complex interactions between numerous cell types and their complex and tightly controlled microenvironments. In closing, this review reconfirms the potential that iPSCs and their derivatives possess for enhancing the regeneration of dental tissues; however, further studies are required to evaluate efficacy and safety of iPSCs prior to human clinical trials.

Author Contributions

K. Hynes, D. Menichanin, contributed to conception, data analysis, and interpretation, drafted and critically revised the manuscript; R. Bright, contributed to data analysis and interpretation, drafted and critically revised the manuscript; S. Ivanovski, D.W. Hutmacher, S. Gronthos, contributed to data interpretation, critically revised the manuscript; P.M. Bartold, contributed to conception, data analysis, and interpretation, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.