Induced pluripotent stem cells (iPSCs): challenges and promises for the future

Reprogramming, the conversion of adult somatic cells to the pluripotent ground state, can be achieved by three approaches: nuclear transfer, cell fusion, and generation of induced pluripotent stem cells (iPSCs) (reviewed in Yamanaka and Blau 2010). The later approach was introduced by the seminal work of Takahashi and Yamanaka 2006, demonstrating that the retroviral transduction of four transcription factors, Oct4, Klf4, Sox2, and c-Myc (known as the Yamanaka’s factors), is sufficient for the conversion of adult cells into ESC-like cells (Takahashi and Yamanaka 2006). Since then, various somatic cells and patient-derived tissues were successfully reprogrammed. Importantly, mouse iPSCs can pass the most stringent test of pluripotency, the tetraploid complementation assay, by giving rise to live, fertile mice (Boland et al. 2009; Kang et al. 2009; Zhao et al. 2009). Thus, the pluripotent potential of iPSCs is probably very similar to that of ESCs and nuclear transfer-derived embryos, supporting that iPSCs may resolve the ethical concerns due to the initial need for embryos in ESC research and therapy.

The discovery of iPSCs has the potential to revolutionize the field of regenerative medicine (Abramovich et al. 2008). In the past few years, iPSCs have been the subject of intensive research towards their application in disease modeling and drug screening (Deshmukh et al. 2012; Bellin et al. 2012). In the future, the cells may be applied in cell therapy to replace or regenerate tissues by autologous transplantation. However, there are currently many barriers to this goal (Grafi 2013), ranging from safety and regulatory issues, financial viability, and lack of knowledge of the underlying biology and genetics. For example, the use of iPSCs in autologous transplantation was recently challenged by a report by Zhao et al. (2011), demonstrating that reprogrammed MEFs were immune-rejected following isograft transplantation in mouse hosts. This immunological incompatibility problem was reconciled by the understanding that immunogenicity occurs only due to the undifferentiated cells. No immunogenicity was observed following isograft transplantation of differentiated cells derived from the iPSCs (Guha et al. 2013), and only minimal immunogenicity was observed when the bone marrow and skin cells isolated from iPSC-derived chimera were transplanted into syngeneic mice (Araki et al. 2013). These studies raise the hope for the clinical use of iPSCs, such as neural cell replacement therapy for Parkinson’s disease patients (Wernig et al. 2008), assuming that these applications will prove to be safe.

To apply iPSCs in regenerative medicine, several major technical and biological issues need to be resolved (Grafi 2013), including the low reprogramming efficiency, the risk of integrating foreign DNA into the genome of the cells, inherited risks due to the presence of tumorigenic cells, and undesired genetic rearrangements in the genome of cultured cells. This notion led to the development of various reprogramming methods categorized into two types (reviewed in Gonzalez et al. 2011): (1) genome-integrating methods, which involve the integration of the reprogramming into the host genome, such as retroviral and lentiviral transduction, transposon-based transfection, and phage integrase-mediated site-specific recombination (Takahashi et al. 2007; Yu et al. 2007; Woltjen et al. 2009; Muenthaisong et al. 2012; Ye et al. 2010; Karow et al. 2011; Davis et al. 2013; Klincumhom et al. 2012); and (2) integration-free or “scar-free” methods, which include transient expression without a genomic insertion (repeated transfections, adenoviral transduction, episomal vector transfection, cell penetrating recombinant protein transductions, Sendai virus transduction, synthetic modified mRNA transfections, and artificial chromosome vector transfection) (Okita et al. 2008; Stadtfeld et al. 2008b; Zhou and Freed 2009; Yu et al. 2009; Zhou et al. 2009; Kim et al. 2009; Zhang et al. 2012a; Fusaki et al. 2009; Ban et al. 2011; Warren et al. 2010; Hiratsuka et al. 2011; Gonzalez et al. 2011).

Factors affecting the efficiency of iPSC generation

Given the therapeutic potential of iPSCs, major efforts have been dedicated to identify the barriers to reprogramming and the ways to overcome the low reprogramming efficiency. It is now clear that reprogramming efficiency is sensitive to various factors, among them the genetic background of the source of cells, the tissue source, the reprogramming method, and the combination of the reprogramming factors. In addition, recent high impact studies suggested that aging is a direct barrier to the generation of iPSCs (Banito et al. 2009; Mahmoudi and Brunet 2012).

Cell source

The cell origin markedly affects the efficiency and the kinetics of iPSC generation. So far, iPSCs have been successfully derived from a variety of primary tissues (Takahashi et al. 2007; Aoi et al. 2008; Tsai et al. 2010; Tsai et al. 2011; Stadtfeld et al. 2008a), patient-derived tissues (reviewed in Unternaehrer and Daley 2011), adult stem cells (Eminli et al. 2009; Sun et al. 2009; Eminli et al. 2008; Kim et al. 2008), and cancer cells (Sun and Liu 2011; Mahalingam et al. 2012). Yet, differences in reprogramming efficiency were observed. For example, the numbers of alkaline phosphatase-positive colonies derived from keratinocytes were shown to be 100-fold higher than from fibroblasts taken from the same skin biopsy. Moreover, iPSC colonies derived from keratinocytes emerged earlier than colonies derived from fibroblasts following retroviral transduction using the same reprogramming factors (Aasen et al. 2008). Interestingly, iPSCs derived from hepatocytes or gastric epithelial cells exhibited less transgene integration events than iPSCs derived from fibroblasts (Aoi et al. 2008). The cell type or donor may also impact the differentiation propensities into a specific lineage due to sustained epigenetic memory, especially during the early passages of iPSCs (Polo et al. 2010; Kim et al. 2010; Bar-Nur et al. 2011; Kajiwara et al. 2012). These observations are consistent with the diverse differentiation potentials observed between hESC lines (Osafune et al. 2008). Furthermore, the cell source may influence the tumorigenic potential, as shown by the differences in teratoma formation of secondary neurospheres versus other adult tissues (Miura et al. 2009). Finally, reprogramming (Schnabel et al. 2012; Vallier et al. 2013) and differentiation propensities (Muenthaisong et al. 2012) are also affected by genetic background variances.

Reprogramming methods and factors

The efficiency of iPSC generation highly depends on the reprogramming method used. The “classical” retroviral-driven reprogramming is still one of the most powerful methods (Gonzalez et al. 2011), especially when compared to the integration-free methods that exhibit markedly reduced efficiency. Of note, the Sendai viral transduction and the synthetic mRNA delivery may be more suitable for future clinical applications, considering that these methods may provide integration-free reprogramming with similar, or even higher, efficiency as retroviral transduction according to some reports (Warren et al. 2010; Ban et al. 2011). For example, Warren et al. (2010) observed a 36-fold increase in reprogramming efficiency determined by Tra1-60 positive colonies and earlier colonies appearance using the synthetic mRNA method than the retrovirus method (Warren et al. 2010). Episomal vector based methods are also gaining momentum and they are used by many teams (Okita et al. 2013; Kondo et al. 2013). Overall, the fast development of the reprogramming methods is expected to result in reliable and efficient “scar-free” methods. After all, the ultimate goal is not to produce a large quantity of colonies from a tissue sample, but rather increasing the rate of successful derivation of high quality, fully reprogrammed iPSC clones from the majority of the samples. Interestingly, in some cases, the genetic (or epigenetic) background has a high impact on the reprogramming efficiency regardless of the method used. In other cases, it may also restrict the differentiation potential of the iPSC clones towards the endoderm or the ectoderm lineages (Vallier et al. 2013).

Not only the four Yamanaka factors, but also other combinations of factors were used in reprogramming. Most notably, Nanog and Lin28 were used either instead of c-Myc and Klf4 (the Thomson factors) (Yu et al. 2007) or in addition (the six factor cocktail) (Lowry et al. 2008; Lapasset et al. 2011; review in Maherali and Hochedlinger 2008; Okita and Yamanaka 2011; Gonzalez et al. 2011). In addition, the expression of the microRNA cluster miR302/367 was suggested to efficiently replace these factors in the reprogramming of mouse and human cells (Anokye-Danso et al. 2011). Small molecules that modulate the cell epigenetics or metabolic pathways have been used in combination with the reprogramming factors in attempts to improve reprogramming efficiency, as well (Okita and Yamanaka 2011; review in Zhang et al. 2012b). An increased understanding of the pluripotency regulatory networks in human, mouse and other species will likely result in the identification of new combinations of factors, leading to enhanced reprogramming efficiency.

A crossroad between induced pluripotency and aging

In addition to the above mentioned variables (cell type, method and reprogramming factors), culture conditions have been shown to affect iPSC generation. For example, incubation of hESCs under 5 % oxygen supports their maintenance and increases the somatic reprogramming efficiency (Ezashi et al. 2005; Yoshida et al. 2009). Though in some studies these oxygen levels are considered hypoxic, it should be emphasized that they are similar to the physiological levels in embryo tissues. Another important observation is that some of the molecules (see previous section) are longevity-promoting compounds (Table 1), thus suggesting that aging networks and reprogramming are well connected. Further complicating the relationships between induced pluripotency and aging is the observation of Damri et al. (2009) that plant cells committed to senescence first acquire a stem cell-like phenotype before obtaining a new cell fate (Damri et al. 2009).

Table 1 Selected longevity pathway-related compounds that promote reprogramming efficiency
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On the whole, accumulating body of data is in favor of the notion that aging is a barrier to iPSC generation (Fig. 1). Given that the generation of iPSCs is time-consuming and rather costly, understanding the barriers to reprogramming is highly desirable. Moreover, understanding the effects of aging on reprogramming is especially important for therapeutic purposes in order to improve reprogramming success from elderly patients’ tissues, who often suffer from various ARDs (Liu et al. 2012).

Fig. 1
figure 1

The impact of aging on somatic cell reprogramming and regenerative medicine. Cells from young and old individuals can be successfully reprogrammed to the pluripotent ground state, however, not all aging-related marks can be rejuvenated. Aging of cells, characterized by shortened telomeres, accumulated DNA damage, and altered signaling pathways and epigenetics, may impact reprogramming efficiency. Accordingly, reprogramming can be enhanced by the addition of longevity-promoting compounds or by the inhibition of age-related pathways. Prior to the use of iPSCs in regenerative therapy, major issues, including safety and protocol standardization, will need to be resolved. Similar differentiation potential of iPSCs from young and aged tissues provides encouraging results towards the application of iPSCs in regenerative medicine for the elderly

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Is aging a barrier to reprogramming?

Understanding the importance of a particular factor in iPSC generation is not a trivial task due to the sensitivity of the process to numerous technical aspects. Thus, careful considerations need to be taken into account when reprogramming efficiencies are compared, for example, by keeping other variables constant and working with large data sets to eliminate artifacts arising from differences between clones.

Age-related aspects of induced pluripotency have been studied in murine and human models. Murine models are highly advantageous because they allow the examination of reprogramming from an identical genetic background and a controlled environment. Data on induced pluripotency of human somatic cells are more difficult to interpret and thus they are also less conclusive.

The studies on induced pluripotency of isogenic murine fibroblasts suggest that reprogramming efficiency declines with age. The first study that directly examined the effect of aging on iPSCs came from the reprogramming of old and young murine dermal fibroblasts, demonstrating a two-fold reduction in reprogramming efficiency of old versus young mouse cells (Li et al. 2009). Similar studies in mice showed consistent results of either lower efficiency or slower reprogramming of bone marrow cells or fibroblasts (Wang et al. 2011; Kim et al. 2010; Cheng et al. 2011). Yet, in these studies, the differentiation potential of the iPSCs has not been addressed and remains to be examined.

So far, various groups succeeded in generating iPSC lines from tissues taken from elderly individuals or patients suffering from ARDs [e.g. (Ohmine et al. 2012; Yagi et al. 2012)]. For example, Yagi et al. (2012) showed that iPSCs can be derived from centenarians’ dermal fibroblasts by the conventional reprogramming method using the four Yamanaka factors and differentiated into neural cells (Yagi et al. 2012). Ohmine et al. (2012) derived iPSCs from keratinocytes of individuals from 56 to 78 years old and further differentiated the cells into insulin-producing cells (Ohmine et al. 2012). Altogether, studies on iPSCs showed that they expressed pluripotent markers, gave rise to the three germ layers, and formed teratomas, demonstrating that fully reprogrammed clones can be derived regardless of the donor’s age. However, it is still unclear whether reprogramming efficiency and differentiation capacity of the iPSCs vary in old versus young patients. In human studies, examining the influence of donor’s age on reprogramming efficiency brought about conflicting results. A large-scale study of iPSC generation from dermal fibroblasts using three or four reprogramming factors did not reveal a significant correlation between reprogramming efficiency and age of donors (Somers et al. 2010). Similar results were also obtained in our lab (unpublished data), when fibroblasts from patients at different ages were reprogrammed by lentiviral transduction using the four Yamanaka factors. It should, however, be noted that in these two studies, the tissues were obtained from patients with various pathologies and, therefore, it is difficult to draw clear-cut conclusions. Recently, reprogramming of vaginal wall fibroblasts from a 78-year-old woman with pelvic organ prolapse was shown to be less efficient than that from a 47-year-old healthy control, since less bona fide reprogrammed colonies emerged 31 days following transduction (Wen et al. 2013). The small number of clones tested in this study further hampers the examination of the impact of age on reprogramming. Notably, the reprogramming of samples derived from old donors involved only highly efficient methods including retroviral (Yagi et al. 2012, 2011; Israel et al. 2012), lentiviral (Lapasset et al. 2011; Ohmine et al. 2012), Sendai viral (Kudva et al. 2012), and episomal vector transfections (Okita et al. 2013; Kondo et al. 2013). In one study, six (but not four) reprogramming factors were required to establish iPSCs from centenarians (Lapasset et al. 2011). Collectively, the data obtained on human cells suggest that reprogramming efficiency is not significantly affected by the donor’s age, but may be masked by other factors, such as pathological conditions, number of passages prior to reprogramming and the individual’s genetic background. Yet, reprogramming of tissues from elderly patients may be slower and less efficient and thus require more robust methods. Recent studies suggest that the differentiation potential of iPSCs and their functionality are not significantly affected by the donor’s age. For example, reprogramming of dermal fibroblasts using three or four factors under a standardized protocol gave rise to functional motor neurons, and no correlation was detected between the differentiation capacity and the donor’s age (29–82 years) (Boulting et al. 2011). Importantly, the differentiation capacity of the obtained iPSCs was similar to that of hESCs. Similar results were obtained when iPSCs produced from young and old donors were differentiated into fibroblasts (Wen et al. 2013) or islet-like cells (Ohmine et al. 2012). Although direct evidence is sparse, current results of the human studies support that the quality of differentiated cells derived from iPSCs may be suitable for regenerative therapy regardless of the donor’s age.

Senescence meets reprogramming: the INK4/ARF locus

Fundamental insights into the relationship between aging and reprogramming arise from studies of the INK4/ARF tumor suppressor locus (Banito et al. 2009; Hong et al. 2009; Kawamura et al. 2009; Li et al. 2009; Marion et al. 2009a) (Fig. 2). This locus encodes three tumor suppressor genes (p16Ink4a, p15Ink4b and p19Arf) that activate the p53 and pRb pathways, leading to apoptosis and senescence (Sharpless 2005). Briefly, p16Ink4a and p15Ink4b activate Rb by relieving the inhibition of the cyclin-dependent kinases, Cdk4 and Cdk6; while p19Arf activates the p53–p21 axis by inhibiting the p53 destabilizing enzyme MDM2. Importantly, during reprogramming, some of the cells enter the apoptosis (Marion et al. 2009a; Kawamura et al. 2009) or the senescence (Banito et al. 2009) pathways, which hampers iPSC generation and accounts, at least in part, for the low reprogramming efficiency. In fact, Banito et al. (2009) demonstrated that the DNA damage response and the induced demethylation on H3K27me3 at the INK4/ARF locus are responsible for the activation of senescence during reprogramming, and termed this process reprogramming-induced senescence (RIS). Senescence directly hampers reprogramming since inhibition of senescence by knocking-down p16, p21, or p53 improves the reprogramming efficiency of human fibroblasts (Banito et al. 2009; Li et al. 2009). These studies are in line with additional reports demonstrating that inhibition or knock-down of p53 and abrogating anti-proliferative pathways significantly improve reprogramming efficiency (Hong et al. 2009; Kawamura et al. 2009). Interestingly, the main barrier to reprogramming differs between humans and mice: while p19Arf dominates in humans, p16Ink4a dominates in mice (Li et al. 2009), consistent with reports in oncogene-induced growth arrested cells (Evan and d’Adda di Fagagna 2009). Finally, the impact of senescence on reprogramming is further supported by the enhancement of iPSC generation by longevity-promoting compounds (Table 1) (Chen et al. 2011; Damri et al. 2009).

Fig. 2
figure 2

Successful reprogramming of somatic cells to iPSCs is hampered by alternative cellular outcomes. The generation of fully reprogrammed iPSCs is hampered by incomplete reprogramming, cell death (apoptosis or senescence) and transformation to other cell types. Apoptosis and senescence hold a barrier to reprogramming by activating the ARF/INK4 locus, which is silenced in iPSCs and hESCs

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Rejuvenation of the age-related characteristics by reprogramming

A remarkable aspect in reprogramming of somatic cells into iPSCs is the ability to “reset the clock” and rejuvenate the somatic cell, as suggested for the first time by Abramovich et al. (2008), and experimentally evidenced by global lengthening of telomeres (Suhr et al. 2009; Marion et al. 2009b) and mitochondrial rejuvenation equivalent to the ESC state (Prigione et al. 2011a). Along this line, to be able to use iPSCs in regenerative medicine for older individuals including those with ARDs in the future, it is important to understand whether the characteristics associated with age can be fully rejuvenated by reprogramming. Herein, we will address this complex yet important question.

A key study by Lapasset et al. (2011) showed that the age-related cellular physiology can be reversed by reprogramming (Lapasset et al. 2011). Telomere length, gene expression profiles and mitochondrial metabolism of reprogrammed senescent cells and cells from centenarian were shown to be similar to hESCs. These findings are consistent with the observation that elongated telomeres are generally displayed in human reprogrammed cells, although some heterogeneity in telomere length can be observed in different lines (Suhr et al. 2009). While telomere length was maintained in several cell lines, its maintenance over time was challenged by prematurely aged telomeres (Vaziri et al. 2010). Remarkably, telomere length was maintained in all iPSC lines generated by Lapasset et al. (2011) for more than 110 population doublings (Lapasset et al. 2011). Telomere elongation and mitochondrial rejuvenation were also observed in iPSCs derived from tissues of patients with ARDs, for example, in reprogrammed epidermal keratinocytes from type 2 diabetes patients (Ohmine et al. 2012). Another important aspect in the rejuvenation of somatic cells is the enhanced levels of DNA repair capacities observed in iPSCs when compared with the non-pluripotent cells (Fan et al. 2011; Luo et al. 2012), although heterogeneous DNA repair capacities were observed in different iPSC lines (Luo et al. 2012). It remains to be established whether cells derived from tissues of patients with ARDs display an equivalent DNA repair efficacy. Yet, not all age-associated features can be rejuvenated by reprogramming, including the accumulation of mutations with age (Grafi 2013). Moreover, accumulation of mutations proceeds during reprogramming due to random mutational events that occur both in the nuclear (Gore et al. 2011; Pasi et al. 2011) and the mitochondrial DNAs (Prigione et al. 2011b). Altogether, accumulated DNA alterations may cause genomic instability, rendering the cells more prone to tumorogenesis, and thus may hamper the application of iPSC-based therapies. Interestingly, iPSCs with age-related karyotype aberrations demonstrated increased sensitivity to drug-induced apoptosis, similarly to hESCs, indicating that reprogramming may reverse age-related features despite the genomic alterations (Prigione et al. 2011a). In any case, future research is needed to develop safe standardized protocols for the derivation of iPSCs from the elderly and patients suffering from ARDs.

It should be noted that cells obtained from elderly patients are heterogeneous in the degree of accumulated mutations, age-associated phenotype and response to the DNA damage stimuli. During reprogramming, only a small subset of cells are cloned, therefore, the reprogramming and thus rejuvenation of somatic cells may be challenged by the possibility that a subset of cells with a younger phenotype is selected for reprogramming (i.e. a bias occurs in favor of healthy cells). Future research will be needed in order to test this possibility, for example by monitoring the reversion of age-related features of single cells.

Modeling premature-aging syndromes using iPSCs

Using iPSCs derived from tissues of patients with progeroid (premature-aging) syndromes may provide attractive cell-based models to study the mechanisms of aging in general and the pathogenesis of progeria in particular. Human iPSCs were successfully established (albeit at low efficiency) from tissues derived from carriers of several telomere maintenance disorders, including patients with dyskeratosis congenita, aplastic anemia, hypocellular bone marrow carrying mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) genes [e.g. (Winkler et al. 2013; Agarwal et al. 2010)]. The iPSCs from dyskeratosis congenita patients displayed restored telomere elongation, suggesting that reprogramming may provide a beneficial therapeutic strategy in the future (Agarwal et al. 2010). On the other hand, limited telomere elongation and impaired hematopoietic differentiation have been observed in iPSCs harboring TERC and TERT mutations, thus reflecting the clinical phenotypes in patients, and supporting the utility of the iPSCs for disease modeling, at least for dyskeratosis congenita (Winkler et al. 2013).

Additional data came from the successful establishment of iPSC lines from two rare, premature aging diseases: Hutchinson-Gilford Progeria syndrome (HGPS) and Werner syndrome (WS) (Zhang et al. 2011; Liu et al. 2011; Ho et al. 2011). Fibroblasts of early passages from HGPS and WS patients were successfully reprogrammed using the four Yamanaka factors (Ho et al. 2011; Zhang et al. 2011). Interestingly, these HGPS- and WS-derived iPSCs lacked the disease-associate features such as the expression of progerin (the short and toxic protein produced in diseased cells due to a mutation in the Lamin A gene), the aberrant nuclear envelope and the altered epigenetic marks. Nevertheless, when the iPSCs were differentiated into vascular smooth muscle cells (VSMCs), mesenchymal stem cells (MSCs) or fibroblasts, the differentiated cells displayed premature senescence phenotypes, reflecting the clinical features of vascular aging (Ho et al. 2011; Zhang et al. 2011; Liu et al. 2011). Furthermore, these studies identified DNA-dependent protein kinase catalytic subunit as a downstream target of progerin (Zhang et al. 2011). Taken together, these results support that iPSCs are excellent cell-based models to study the pathogenesis of progeria, as well as physiological vascular aging.

Concluding remarks

Aging and reprogramming are well interconnected processes. While it is clear that the donor’s age of murine somatic cells poses a barrier to reprogramming into iPSCs, the picture is vaguer for human cells. It is conceivable that in the case of human cells, the effect of aging on reprogramming is modest and could be masked by other, more dominant factors such as genetic background, and disease state and progression. Yet, large-scale reprogramming experiments using standardized protocols and quantitative assays are needed to provide clear-cut evidence for this possibility. Importantly, iPSCs have been successfully generated from various cells derived from elderly individuals (including those with ARDs), centenarians, and senescent cells, indicating that reprogramming of these cells is in fact possible.

DNA damage accumulates with age, at least in some cell types, and is considered one of the main inducers of cellular senescence (reviewed in (Moskalev et al. 2012; Moskalev et al. 2013). Recent evidence demonstrated that cellular senescence is induced during reprogramming (reprogramming-induced senescence) and that this process limits the generation of iPSCs (Banito et al. 2009). Abrogating cellular senescence has been used to enhance reprogramming efficiency, however, special care needs to be taken when doing so. For example, p53 inhibition (Marion et al. 2009a) or Ataxia-telangiectasia mutated (ATM) deficiency (Kinoshita et al. 2011) may result in the reprogramming of cells with aberrant DNA integrity and thus pose a safety issue in using the cells for transplantation. This issue was resolved recently when the p53 mediator PUMA was suggested as an alternative target to p53 because its inhibition lead to reduced DNA damage and fewer chromosomal aberrations in iPSCs (Li et al. 2009). In the same line, maintaining genomic integrity by over-expressing Zscan4 increased the efficiency of reprogramming and the quality of iPSCs (Jiang et al. 2013). Altogether, it is clear that future research should be dedicated to better understand the molecular mechanisms underlying reprogramming in general, and more specifically, the age-related effects on reprogramming.

The use of iPSCs in cell-based aging models led to exciting achievements and is expected to remarkably contribute to the study of physiological and premature aging and to the identification of drugs for the treatment of ARDs. As for the use of iPSCs in regenerative therapy, apart from the general efforts required in the field, additional aspects associated with aging call for further research. A necessary step is evaluating to what extent somatic cells can be rejuvenated by reprogramming, for example, by examining if there are age-associated epigenetic marks that are not “erased” during reprogramming.

Finally, another vital aspect highly relevant to the potential use of iPSCs in regenerative medicine for the elderly or patients with ARDs is the effect of the aging microenvironment on the transplanted cells and vice versa. Although this issue is beyond the scope of this review, it should be thoroughly addressed in future investigations. Assuming the safety of iPSCs will be resolved, future successful transplantation will not only require efficient reprogramming and differentiation protocols, but also proof of concepts that the cells can maintain their functionality in an aging microenvironment and presumably even modify it towards a younger phenotype.