Research of in vivo reprogramming toward clinical applications in regenerative medicine: A concise review

Abstract

The successful generation of induced pluripotent stem cells (iPSCs) has significantly impacted many scientific fields. In the field of regenerative medicine, iPSC-derived somatic cells are expected to recover impaired organ functions through cell transplantation therapy. Subsequent studies using genetically engineered mouse models showed that somatic cells are also reprogrammable in vivo. Notably, cyclic expression of reprogramming factors, so-called partial reprogramming in vivo ameliorates cellular and physiological hallmarks of aging without inducing teratoma formation or premature death of animals. Subsequent studies provided evidence supporting the beneficial effects of partial reprogramming in various organs. Although in vivo reprogramming appears to be a promising strategy for tissue regeneration and rejuvenation, there remain unsolved issues that hinder its clinical application, including concerns regarding its safety, controllability, and unexpected detrimental effects. Here, we review the pathway that research of in vivo reprogramming has followed and discuss the future perspective as we look toward its clinical application in regenerative medicine.

1. The discovery of induced pluripotent stem cell (iPSC) generation

All cells that constitute an individual body are derived from pluripotent cells located in the inner cell mass (ICM) of blastocysts originating from a fertilized egg. Embryonic stem cells (ESCs), which maintain pluripotency to give rise to all cell types, can be established from ICM cells in vitro. The cellular differentiation process during organismal development is unidirectional and often compared with a ball rolling down a slope into a landscape with peaks and valleys, which is depicted in Waddington's epigenetic landscape. In this model, a totipotent zygote is located at the top of the hill and each differentiated cell lies at the bottom of each distinct valley. Once the development process proceeds, cells do not climb back up the slope and thus cannot cross the ridges separating individual valleys, representing stable maintenance of somatic cells.

However, cell fate can be altered by artificial reprogramming technologies. This was demonstrated in seminal studies with nuclear transfer, which showed that a somatic nucleus can be reprogrammed to become pluripotent after transplantation into an enucleated egg (Fig. 1) [1,2]. The decisive breakthrough in cell fate control came when Takahashi and Yamanaka successfully generated iPSCs from somatic cells. Forced induction of four transcription factors, namely, Oct3/4 (Pou5f1), Sox2, Klf4, and Myc (OSKM or Yamanaka factors), all of which are highly expressed and associated with maintenance of ESC identity, resulted in generation of iPSCs in both mice (in 2006) and humans (in 2007) [[3], [4], [5]].

Fig. 1.

Advances in cellular reprogramming. The first demonstration that somatic cells can be reprogrammed into pluripotent stem cells was made with nuclear transfer (Gurdon). The decisive breakthrough came when induced pluripotent stem cells were generated by forced induction of four transcription factors (Takahashi and Yamanaka). Subsequent studies using genetically engineered mouse models showed that somatic cells are also reprogrammable in vivo (Abad et al. and Ohnishi et al.). After that, cyclic expression of reprogramming factors, so-called partial reprogramming in vivo, was found to ameliorate cellular and physiological hallmarks of aging (Ocampo et al.). Later, many studies provided evidence supporting the beneficial effects of partial reprogramming in various organs.

These experiments suggest that cell identity is controllable, at least in part, by activating a transcriptional network of the desired cell type; therefore, forced expression of transcription factors that enable establishment of the targeted transcriptional network induces cell fate conversion. It is also important to note that epigenetic regulation involving DNA methylation and histone modifications safeguards the stable transcriptional network in each cell type [6], which contributes to maintenance of somatic cell fate. Given that the DNA methylation pattern in each cell type is generally preserved across the lifespan, the idea that biological age can be predicted by altered DNA methylation patterns, referred to as the epigenetic clock, is gaining attention [7,8].

In addition to the biological impact, the technology of iPSC generation has been applied to medical research fields, such as regenerative medicine, disease modeling, and drug discovery [9]. Prototypically, pluripotent stem cells (PSCs) generated in vitro can be differentiated into a desired cell type, which is applicable to cell transplantation therapy. One pioneering study transplanted a sheet of iPSC-derived retinal pigment epithelial cells into patients with age-related macular degeneration [10]. Additionally, other studies and clinical trials have been conducted aiming to treat several diseases with ESC- and iPSC-derived somatic cells [11,12].

2. The achievement of in vivo reprogramming

After the successful derivation of iPSCs in vitro, researchers investigated the possibility of cellular reprogramming at the organismal level, which has potential biological and clinical relevance. In vivo reprogramming by Yamanaka factors was first achieved in 2013 [13]. This study used transgenic mice that carry the transcriptional activator (rtTA) at the ubiquitously expressed Rosa26 locus and a single copy of a lentiviral doxycycline-inducible polycistronic cassette encoding OSKM (a Tet-On system). After doxycycline administration, these mice developed teratomas in multiple organs, indicating that various somatic cells were reprogrammed into PSCs in vivo. In addition to the demonstration of in vivo reprogramming, the study proposed that in vivo iPSCs might be totipotent based on the findings that they contributed to the trophectoderm after microinjection into morulae and generated embryo-like structures after intraperitoneal injection. Indeed, a subsequent study demonstrated that higher expression levels of OSKM are responsible for the totipotency-like features in the in vivo reprogramming mouse model [14].

In 2014, another study also successfully conducted in vivo reprogramming with similar transgenic mice. The reprogrammable mice carried a rtTA allele at the Rosa26 locus and the doxycycline-inducible OSKM allele within the Col1a1 locus [15]. As in the first in vivo reprogramming study [13], the mice developed teratomas in multiple organs, including the kidneys, pancreas, and liver. Notably, premature termination of in vivo reprogramming (7 days of expression followed by 7 days of withdrawal) resulted in the development of cancer in multiple organs, as represented by kidney tumors that shared characteristics with nephroblastoma, a common pediatric kidney cancer. Of note, the kidney cancer cells did not harbor major oncogenic mutations. Moreover, these cancer cells were readily reprogrammed into PSCs in vitro and contributed to non-neoplastic kidney cells in chimeric mice. Considering that derivation and differentiation of iPSCs do not require alterations in DNA sequences, these results suggest that epigenetic regulations associated with iPSC derivation drive the development of cancer, leading to the concept of epigenetic cancer [[16], [17], [18], [19]]. This concept seems consistent with the fact that non-mutational epigenetic reprogramming is now proposed as a hallmark of cancer [20]. These earlier studies on in vivo reprogramming indicated that the technology can contribute not only to regenerative medicine but also to a deeper understanding of biology including developmental biology and cancer biology. We will not discuss these topics further and will instead focus on the contribution of this technology to regenerative medicine.

Considering that transplantation of in vitro PSC-derived somatic cells is being investigated in clinical trials for regenerative medicine to treat various diseases, it is reasonable to assume that in vivo reprogramming can also be applied in this field. Moreover, in vivo reprogramming might have several advantages over in vitro iPSC-mediated treatment. For instance, it could avoid unintended aberrations related to long-term culture in vitro. Furthermore, given that transplantation is unnecessary, there is no need to consider rejection, which is a common and serious concern following allogeneic transplantations, including those with PSC-derived cells. In addition, in vivo reprogrammed cells might have the advantage of forming functional three-dimensional structures in the physiological tissue environment, which is challenging to achieve with in vitro iPSCs. However, in vivo reprogramming studies, especially those employing systemic induction, have often been hindered by the early death of animals related to tumor formation and toxic effects in the intestines or liver [13,15,21].

3. Modification of the in vivo reprogramming regimen

To avoid detrimental effects of in vivo reprogramming, including the premature death of animals, researchers have mainly adopted two strategies: (1) partial reprogramming with a shortened period of reprogramming factor expression and (2) cell type-specific reprogramming, which can be performed in combination. The breakthrough study of partial in vivo reprogramming, conducted by Ocampo et al. [22], laid the foundation for the application of this strategy for tissue regeneration and rejuvenation. This study, as well as most subsequent studies, used an in vivo reprogrammable system similar to that used in previous studies (i.e., in vivo Tet-ON systems) [13,15,23]. These studies demonstrated that cyclic short-term expression of OSKM (2 days of induction followed by 5 days of withdrawal) ameliorated the cellular and physiological hallmarks of aging and prolonged lifespan in progeroid mouse models. Notably, these mice did not form teratomas, suggesting that acquisition of pluripotency is not required for “rejuvenation” phenotypes. Considering that epigenetic remodeling plays a central role in reprogramming processes, modulation of epigenetic regulation may be responsible for such phenotypes. A subsequent study observed the long-term effect (up to 10 months) of cyclic partial reprogramming and concluded that this regimen effectively delays aging-related phenotypes [24]. The authors also showed that cyclic induction of OSKM (for 7 months) prevented aging-associated alterations of DNA methylation in the kidneys and skin. A recent study also reported that OSK induction extended the lifespan by 109 % in 124-week-old mice using adeno-associated virus (AAV) vectors, which may provide safer methods for clinical applications because of the absence of genome integration [25].

In the next section, we will introduce the application of in vivo reprogramming in regenerative medicine in various organs. Specifically, because cell type-specific in vivo reprogramming is gaining popularity, we will introduce these studies according to the organ system.

4. Partial reprogramming studies according to the organ system

4.1. Central nervous system (CNS)

Neurodegenerative disorders pose significant challenges in medicine, especially in an aging society. Therefore, the CNS is one of the most attractive targets for regeneration/rejuvenation therapies with in vivo reprogramming technologies. Readers may also refer to excellent reviews on this topic for detailed discussion [26]. Direct reprogramming, i.e., cell type conversion without passing through the pluripotent state, is a possible strategy for tissue regeneration in this organ system. Although direct reprogramming is not a topic of this review, we will briefly discuss these studies because SOX2, a Yamanaka factor and the master regulator of neuronal stem cells, was often used to convert glial cells into neurons [[27], [28], [29]]. Subsequently, researchers demonstrated that retroviral-mediated expression of OSKM induces reprogramming of glial cells in the adult neocortex following traumatic brain injury (TBI) in mice [30]. Surprisingly, the reprogrammed cells differentiated into neural stem cells in their tissue environment, which further differentiated into neurons and glial cells that compensated for the tissue defects induced by TBI.

These studies, including those of direct reprogramming, often intended to recover impaired brain function after injury. Recent studies focused more on ameliorating aging phenotypes with partial reprogramming. One study targeted dentate gyrus (DG) cells because these cells play a central role in higher brain functions, such as learning memory and spatial navigation. Importantly, the functional decline upon aging is associated with alterations of epigenetic modifications in DG cells [31]. The authors reported that cyclic induction of Yamanaka factors in the whole body prevented the age-dependent reduction of H3K9 trimethylation, increased the survival of newborn DG neurons during their maturation, and elevated synaptic plasticity of neurons. Notably, the intervention improved mouse performance in object recognition. In the same year, another study focused on the eye as a model of CNS function and showed that AAV2-delivered polycistronic OSK promoted axon regeneration after injury, which was accompanied by restoration of youthful DNA methylation patterns and transcriptomes in retinal ganglion cells. Moreover, the authors successfully reversed vision loss in a mouse model of glaucoma and aged mice [32]. They further proposed that these beneficial effects are associated with the DNA demethylases TET1 and TET2, suggesting that modulation of epigenetic regulation plays a critical role in this regeneration/rejuvenation.

Additionally, a recent study investigated the effects of in vivo partial reprogramming in cells in the subventricular zone, the neurogenic niche, utilizing single-cell transcriptomic analysis [33]. They reported that partial reprogramming targeting the whole body or neurogenic niche elicited beneficial effects for neuroblasts to maintain their functions and improved production of new neurons in the brains of aged mice. Another study, which induced OSKM specifically in CamKII-positive, post-mitotic neurons with a Tet-Off system, demonstrated that cyclic induction of OSKM improved the cognitive function of adult mice [34]. This study also implied that a low expression level of OSKM can reverse age-associated phenotypes because these mice did not develop tumors even with continuous OSKM induction.

4.2. Heart

The number of patients with heart failure continues to increase because of a growing population of aged individuals [35]. Adult mammalian cardiomyocytes (CMs), in contrast to fetal CMs, have little regenerative capability, and it is beneficial to explore methods that stimulate proliferation of adult CMs after cardiac injury or diseased CMs. A previous study generated transgenic mice in which OSKM is specifically expressed in CMs by crossing Xmlc2-Cre mice and reprogrammable mice [36]. The authors demonstrated that transient OSKM expression (6 or 12 days) activated a fetal-like gene expression program in adult CMs. Furthermore, they demonstrated that transient OSKM expression (6 days) before or during myocardial infarction ameliorated myocardial damage and improved cardiac functions in a myocardial infarction model, which raised the possibility that activation of an embryonic transcriptional network through partial reprogramming can recover the impaired function of adult CMs with a minimal regenerative capacity.

Another study of CM-specific expression of Yamanaka factors, utilizing the alphaMHC-rtTA allele, also successfully induced dedifferentiation of adult CMs in vivo [37]. Two days after induction, adult CMs exhibited dedifferentiated phenotypes and increased proliferation in vivo. Transcriptomic analyses revealed that upregulation of ketogenesis, for which HMGCS2 is a rate-limiting enzyme, was central to this process. Consistently, AAV-driven HMGCS2 overexpression led to ketogenesis in adult CMs and induced dedifferentiation and proliferation of CMs reminiscent of cellular responses during partial reprogramming. Similar responses occurred after myocardial infarction, specifically in border zone tissue. Moreover, the HMGCS2-knockout model exhibited impaired cardiac function in response to injury. Finally, exogenous HMGCS2 restored cardiac function after ischemic injury, suggesting that OSKM-mediated in vivo partial reprogramming can be substituted by a simple or feasible strategy.

4.3. Intestines

As mentioned above, the intestines are an organ related to the early death of animals when reprogramming factors are systemically induced [21]. However, one study was able to investigate the effect of partial in vivo reprogramming on the intestines while inducing OSKM systemically utilizing single-cell analyses in combination with organoid assays [38]. Notably, intestinal cells after OSKM expression exhibited similar molecular features as those observed during physiological intestinal regeneration, including a transition from homeostatic cell types to “injury response-like” cells consisting of two distinct cell types. Moreover, partial reprogramming promoted intestinal repair through prostaglandin synthesis. The study also showed that a common molecular mechanism is shared between partial reprogramming and injury-induced dedifferentiation [39], suggesting that in vivo reprogramming exerts its effect by activating machinery involved in physiological regeneration (see Section 5).

4.4. Kidneys

To the best of our knowledge, only one study has suggested that kidney cells can be rejuvenated via in vivo partial reprogramming by presenting a reverted epigenetic clock [24], although functional recovery has not been demonstrated. Chronic kidney disease (CKD) affects 8–16 % of the population worldwide, and progressive CKD is associated with adverse outcomes, including end-stage kidney disease, cardiovascular disease, and increased mortality [40]. Given that the kidneys, and particularly renal tubular cells, are one of the organs/cell types in which reprogrammable mice often show phenotypic changes [14,15], future studies of in vivo partial reprogramming focusing on the kidneys are warranted.

4.5. Liver

A previous study investigated the consequence of in vivo reprogramming of mouse hepatocytes by performing partial reprogramming using the Alb-Cre allele for hepatocyte-specific induction of OSKM [41]. Transient OSKM expression reprogrammed mature hepatocytes into a progenitor state in terms of transcriptional and epigenetic signatures, with the emergence of SOX9-positive cells. Functionally, partial reprogramming of hepatocytes enhanced the liver regenerative capacity after acetaminophen-induced liver damage, which provides additional evidence that activation of fetal signatures through epigenetic reorganization is involved in the enhanced regeneration elicited by in vivo reprogramming.

4.6. Muscle

Pioneering studies of partial reprogramming have focused on muscle while expressing OSKM in the whole body [22,24]. These studies showed that partial reprogramming can suppress degeneration of vascular smooth muscle cells, as indicated by increased nuclei in the medial layer of the aortic arch. Moreover, partial reprogramming significantly improved the regenerative capacity of the tibialis anterior muscle, as indicated by an increase of the cross-sectional area of muscle fibers and a reduction in the number of aberrant muscle fibers with central nuclei in the injured area in the cardiotoxin-induced muscular injury model [22].

A recent study induced partial reprogramming of myofibers by utilizing the Acta1-Cre allele [42]. The researchers found that genes related to metabolism and muscle differentiation were suppressed while cytoskeleton organization pathways were upregulated. Of note, regeneration in the extensor digitorum longus muscle was improved after the reprogramming. Although this phenomenon was associated with activation of muscle stem cells or satellite cells (SCs), SC-specific OSKM expression did not improve regeneration, suggesting the indirect effects of OSKM or potential significance of non-reprogrammed cells in terms of the microenvironment of the stem cell niche.

4.7. Pancreas

Ocampo et al. showed the beneficial effects of partial reprogramming in the pancreas [22]. In this study, aged mice were subjected to 3 weeks of partial reprogramming, and then islet beta cells were injured by administration of streptozocin. Histological analysis of the pancreas at 2 weeks after pancreatic injury revealed that islets were larger in partially reprogrammed mice than in control mice. Moreover, partially reprogrammed mice had significantly decreased blood glucose levels in the glucose tolerance test. These results suggest that the regenerative capacity of beta cells is enhanced by in vivo reprogramming. Indeed, a recent study selectively expanded beta cells into immature proliferating islet cells by Mycl-mediated reprogramming [43].

4.8. Skin

Skin is visible and can be closely monitored and is therefore an attractive target to analyze the effects of in vivo partial reprogramming. Indeed, the effects of in vivo partial reprogramming have been extensively investigated in skin [22,24]. A previous study highlighted the beneficial effects of long-term partial reprogramming (for 3 months) on wound healing in aged mice [24]. Another study reported that OSKM induction in reprogrammable mice with cutaneous wounds suppressed transdifferentiation of fibroblasts into myofibroblasts and wound contraction, which eventually reduced scar formation [44].

In summary, in vivo reprogramming, especially partial reprogramming, appears to be a promising strategy to recover cell function upon tissue injury or aging in various organs.

5. Molecular mechanisms of in vivo reprogramming

In parallel with investigating the effects of in vivo reprogramming in the whole body or particular organs, researchers have attempted to understand the molecular mechanisms or trajectories of in vivo reprogramming. They recognized differences between in vitro and in vivo reprogramming based on the findings of previous studies. For example, induction of Ngn3, Pax4, and MafA successfully converted exocrine cells into pancreatic beta cells in vivo but not in vitro [45]. However, an overlapping process between in vitro and in vivo reprogramming is expected to exist. Reference to the mechanisms of in vitro reprogramming is helpful to better understand in vivo reprogramming.

The mechanisms of in vitro reprogramming have been intensively studied [46]. Derivation of iPSCs in vitro is generally inefficient and occurs in a stochastic manner [47]. In the reprogramming process, the consensus is that the identity of somatic cells is first effaced and that of pluripotent cells is then acquired, with OSK playing a major role. Consistent with the fact that DNA methylation is a stable epigenetic mark, alterations of DNA methylation occur at the later stage of reprogramming [48,49]. In detail, OSK predominantly binds to active somatic enhancers early in reprogramming and immediately initiates their genome-wide inactivation through the redistribution of somatic transcription factors away from somatic enhancers, which is accompanied by changes of histone modifications [48]. A recent study reported that OCT4 binds to nucleosomes containing binding motifs for LIN28B or nMATN1, which induces changes in the nucleosome structure and facilitates cooperative binding of additional OCT4 and SOX2 to their internal binding sites [50], while KLF4 binds to enhancers independent of OCT4 and SOX2 [48]. It takes approximately 48 h for somatic cells to lose their enhancer landscape, leading to loss of cell identity. These molecular mechanisms of early reprogramming and kinetics are similar to those of in vivo reprogramming [48,51], indicating that mechanisms are shared between in vitro and in vivo reprogramming (Fig 2).

Fig. 2.

A scheme of in vitro and in vivo reprogramming. In the reprogramming process in vitro, the identity of somatic cells is first effaced and that of pluripotent cells is then acquired, with OSK playing a major role. OSK predominantly binds to active somatic enhancers early in reprogramming and immediately initiates their genome-wide inactivation, which is accompanied by changes of histone modifications. It takes approximately 48 h for somatic cells to lose their enhancer landscape, leading to loss of cell identity. Subsequently, OSK binds to pluripotent enhancers and activates a transcription network of pluripotent stem cells, which eventually induce pluripotent stem cells. The molecular mechanisms of early reprogramming and kinetics are similar to those of in vivo reprogramming, and these partially reprogrammed somatic cells in vivo can contribute to restoration from injury and tissue rejuvenation. When somatic cells are completely reprogrammed in vivo, these cells form teratomas, whereas incomplete reprogramming may lead to cancer development.

Conversely, differences between in vivo and in vitro reprogramming have been proposed. For instance, in vivo reprogramming couples with cellular senescence. Senescent cells, which can be induced by OSKM expression, can promote reprogramming of neighboring cells in a non-cell autonomous manner, which is not observed during in vitro reprogramming. Mechanistically, senescence-related cytokines, particularly interleukin-6, promote reprogramming [39]. This study further raised the possibility that similar conceptual interplay may occur in physiological conditions, where damage-triggered senescence could induce dedifferentiation to promote tissue repair or dedifferentiation-associated cancer development.

A subsequent study that performed single-cell analyses confirmed the presence of reprogrammed and non-reprogrammed cells in vivo [52]. In this study, the authors identified markers along the trajectory from pancreatic acinar cells to pluripotency. These markers allowed the direct in situ visualization of cells undergoing dedifferentiation and acquiring features of early and advanced stages of reprogramming. They found that some cells do not exhibit reprogramming upon OSKM expression but are characterized by expression of stress markers induced by REG3 and AP-1 proteins. Another study reported that the initiation phase of in vivo reprogramming ameliorates DNA damage in mice. Notably, this process can be phenocopied by pharmacological inhibition of the ALK5 and ALK2 receptors, suggesting that these signals are involved in the early stage of in vivo reprogramming [53]. By contrast, studies of the late phase and the full span of in vivo reprogramming are relatively rare, and this warrants further investigation.

6. Possibilities of environment-induced partial reprogramming

It is ideal to adopt a non-genetic approach, such as a pharmacological intervention, for the clinical application of partial reprogramming. A recent study reported partial chemical reprogramming of fibroblasts in vitro and proposed the underlying mechanisms [54]. When the authors investigated the epigenome and phosphoproteome, they observed genome-wide modulations, with the most notable signature being upregulation of mitochondrial oxidative phosphorylation after partial chemical reprogramming. Furthermore, the partial reprogramming led to reductions of aging-related metabolites, as well as the biological age of mouse fibroblasts, which was revealed by both transcriptomic and epigenetic clock-based analyses.

Other studies suggested that daily activities, such as eating and exercise, may affect natural cellular reprogramming. Kovatcheva et al. claimed that in vivo reprogramming in mice induces global depletion of vitamin B12 and molecular hallmarks of methionine starvation [55]. Notably, supplementation with vitamin B12 increases the reprogramming efficiency in a cell-intrinsic manner. They also showed that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters, was sensitive to vitamin B12 levels, proposing a link between vitamin B12 levels, H3K36 methylation, transcriptional fidelity, and efficient reprogramming. Of note, vitamin B12 supplementation accelerated tissue repair in a model of ulcerative colitis, which raised the possibility that vitamin B12 promotes tissue regeneration by inducing reprogramming. Vitamin D may also be involved in tissue regeneration because a lack of this vitamin was reported to induce aging of the thymus [56].

Another unique study suggested a mechanistic link between in vivo partial reprogramming and exercise [57]. Using genetically modified mouse models, wild-type mice, and human samples, the authors first defined exercise-induced genes that are also induced upon partial reprogramming. They found that Myc is the reprogramming factor most induced by exercise in muscle and is upregulated following exercise training in aged mice. With further experiments and observations, they proposed that exercise induces epigenetic profiles in muscle reminiscent of those observed upon partial reprogramming.

7. Future perspective of in vivo reprogramming in regenerative medicine

The in vivo reprogramming studies with Yamanaka factors not only successfully generated teratomas in animals but also increased the significance of this technology in many fields. The proposal of totipotent cells and non-genetic cancer development is relevant only to in vivo (not in vitro) studies [13,15]. Recent studies highlighted the potential of in vivo reprogramming in regenerative medicine. The first report on cyclic partial reprogramming [22] accelerated and expanded studies in this field, and many subsequent studies of different organs demonstrated the beneficial effects of in vivo reprogramming for recovery of tissue damage and cell rejuvenation. In addition, efficient methods for in vivo reprogramming have been developed, and the mechanisms underlying in vivo reprogramming are gradually being clarified. In particular, the fact that expression of reprogramming factors in vivo initiates not only reprogramming but also senescence in surrounding cells suggests the importance of the cellular microenvironment in reprogramming at the organismal level. Future studies delineating this mechanism might lead to broader application of this technology in the fields of biology and medicine.

However, it is premature to consider the medical use of in vivo reprogramming. The study of in vivo partial reprogramming is in its infancy, and the reproducibility of most results should be carefully investigated. Additionally, non-genetic chemical reprogramming, which is perhaps an ideal strategy, has not yet been achieved in vivo. Considering that AAV-based gene therapies have already been applied in several clinical settings [58], this is another approach. However, even with such a method, critical challenges are how to precisely induce partial reprogramming that leads to rejuvenation of target cells without any detrimental effects, particularly abnormal cell growth. More studies are necessary to precisely understand the mechanisms and kinetics of such a “point of no return”. In addition, most previous studies performed doxycycline-dependent partial reprogramming in mice, and there is a lack of studies about whether this strategy can be applied to humans.

Several studies suggest that some physiological activities, such as eating and exercise, might involve or even induce partial reprogramming of human bodies. Detailed studies investigating the molecular mechanism of such environment-induced partial reprogramming in vivo may further reveal relationships between physiological activities and partial cellular reprogramming and discover new strategies to more efficiently induce these effects. It is hoped that future multifaceted studies will address the remaining challenges, which will eventually demonstrate the benefits of in vivo reprogramming for health promotion.