STATE-OF-THE-ART REVIEW Dedifferentiation, transdifferentiation and cell fusion: in vivo reprogramming strategies for regenerative medicine Martina Pesaresi1, Ruben Sebastian-Perez1 and Maria Pia Cosma1,2,3

1 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Spain 2 Universitat Pompeu Fabra (UPF), Barcelona, Spain 3 Institucio Catalana de Recerca i Estudis Avancßats (ICREA), Barcelona, Spain

Keywords Regenerative capacities vary enormously across the animal kingdom. In cell fusion; dedifferentiation; endogenous contrast to most cold-blooded vertebrates, mammals, including humans, regeneration; in vivo reprogramming; have very limited regenerative capacity when it comes to repairing dam- regenerative medicine aged or degenerating tissues. Here, we review the main mechanisms of tis- Correspondence sue regeneration, underlying the importance of cell dedifferentiation and M. P. Cosma, Centre for Genomic reprogramming. We discuss the significance of cell fate and identity Regulation (CRG), C/Dr. Aiguader, 88 08003 changes in the context of regenerative medicine, with a particular focus on Barcelona, Spain strategies aiming at the promotion of the body’s self-repairing mechanisms. Fax: +34 9 33160099 We also introduce some of the most recent advances that have resulted in Tel: +34 9 33160370 complete reprogramming of cell identity in vivo. Lastly, we discuss the E-mail: [email protected] main challenges that need to be addressed in the near future to develop (Received 29 March 2018, revised 1 July in vivo reprogramming approaches with therapeutic potential. 2018, accepted 10 August 2018) doi:10.1111/febs.14633

Introduction Some organisms are able to successfully regenerate cells to the damage site, where they differentiate into entire parts of their bodies, while others can suffer per- tissue-specific cell types; this process is employed, for manent damage following even a mild injury (Fig. 1). instance, by hydra, and does not involve a proliferative Generally speaking, the term regeneration defines a stage. On the other hand, proliferation is crucially process finalised at the re-establishment of the physio- important in epimorphic regeneration, which can logical and morphological functions of damaged tis- depend on either pre-existing stem/progenitor cells sues and/or organs. Regenerative processes can be (e.g. planaria) or on dedifferentiation of resident tissue classified into two broad classes of events, namely cells (e.g. salamanders). morphallatic and epimorphic [1,2]. On the one hand, Some mammalian organs undergo a constitutive morphallatic regeneration involves migration of stem regeneration process that is dependent on the presence

Abbreviations ARF, alternate reading frame; Ascl1, aschaete-scute homolog 1; ASCs, adult stem cells; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; CNS, central nervous system; CTNF, ciliary neurotrophic factor; Dlx2, distal-less homeobox 2; EGF, epidermal growth factor; ESCs, embryonic stem cells; Fah, fumarylacetoacetate hydrolase; FGF1, fibroblast growth factor 1; HSPCs, haematopoietic stem cells; IL6, interleukin-6; iPSCs, induced pluripotent stem cells; Klf4, Kruppel-like factor 4; Mafa, V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog A; MGCs, Mu¨ ller glia Cells; NeuroD1, neurogenic differentiation factor 1; Neurog2, neurogenin 2; Neurog3, neurogenin 3; Oct3/4, octamer-binding transcription factor 3/4; OSKM, Oct4 Sox2 Klf4 and c-Myc; Pdx1, pancreatic and duodenal homeobox 1; pRB, retinoblastoma protein; Sox2, SRY-box 2; Wnt1, Wnt family member 1; Wnt3a, Wnt family member 3a.

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Fig. 1. Spectrum of regenerative potential in the animal kingdom. Platyhelminthes (e.g. planarians) are found on the left-end of the regeneration spectrum, as they are characterised by outstanding regenerative capacities. For instance, an entire planaria can be formed starting from a tissue fragment that is only 1/279th of the original body size [151,152]. Amphibians also possess remarkable regenerative capacities [153]. These observations date back more than 200 years, when Lazarro Spallanzani reported for the first time that salamanders were able to replace not only damaged organs, but also entire limbs and tails [146]. Similarly, newts can fully regenerate their lenses following complete lentectomy [147]. Regenerative potential is also present in animals from other phyla, including Chordates such as ascidians and zebrafish. Zebrafish, in particular, can completely regenerate their heart following amputation of up to 20% of the ventricle [148,149]. It is also able to completely regenerate damaged retinal tissue [12]. Potential for retinal regeneration is also maintained in birds, even if only partially. In fact, the avian retina can be efficiently regenerated in the early postnatal period; this capacity, however, is lost as the animal ages [154]. Mammals are found on the right-(and lower)-end of the regeneration potential spectrum. In fact, the liver is the only mammalian organ that can be endogenously repaired following injury, regaining sufficient function to lead a normal life. Most of the other organs, instead, are endowed with very limited regenerative capacity.

– of resident stem cell pools [3 5]. However, this is lim- Mechanisms of tissue regeneration – ited to a few organs with physiological cell turnover, the importance of cell such as the skin, the blood system and the gut. In the dedifferentiation context of tissue injury and degeneration, most mam- malian tissues are endowed with very limited regenera- The broad regenerative potential that characterises tive capacity. Indeed, mammals seem to have “lost” amphibians and other cold-blooded vertebrates can be the potential for extensive organ repair, and their abil- ascribed to the dedifferentiation of resident cells ity to self-regenerate following injury and disease is located in close proximity of the injury site. In the case extremely restricted. of damage to newts’ lenses, for instance, pigmented Despite the collective effort of the scientific commu- epithelial cells of the dorsal iris dedifferentiate back to nity, we still need to satisfactorily explain why mam- a progenitor state, temporarily re-enter the cell cycle mals are unable to regenerate a missing limb or repair and eventually redifferentiate to generate a new, func- a damage organ. Therefore, before proceeding with tional lens [6–8]. Importantly, the newly generated tis- further discussions, it is important to understand what sue is indistinguishable from the lenses of younger are the mechanisms that underlie the ability to exten- animals. This regenerative capacity remains unchanged sively repair damaged tissues in early vertebrates. throughout the entire lifespan of the organism,

2 The FEBS Journal (2018) ª 2018 Federation of European Biochemical Societies M. Pesaresi et al. regardless of the number of injuries suffered [9]. The advantageous for the overall survival of the organism. occurrence of dedifferentiation has also been described Despite promising and intellectually satisfying, how- and characterised for zebrafish cardiomyocytes and ever, these explanations await further confirmation, Muller€ glia cells (MGCs), in the context of heart and and currently remain on a rather speculative level. retina repair respectively [10–12]. Several other questions also remain to be addressed. Following tissue injury in mammals, however, dedif- For instance, it has not been clearly elucidated yet ferentiation does not frequently occur. Indeed, years of why exposure to an extract derived from regenerating intense investigations and debates have culminated in newt limbs can induce proliferation and a certain level the wide acceptance of the hypothesis that the dediffer- of dedifferentiation in mammalian myotubes [19]. entiation step might represent one of the major differ- Indeed, this observation introduces the possibility that ences between the regeneration that occurs in some extrinsic signals might also be released following salamander and that of other warm-blooded verte- injury in amphibians, but not in mammals. Such sig- brates, including mammals. nals could be context-specific, meaning that the local Mechanistically, cell dedifferentiation is dependent microenvironment could also play a crucially impor- on the occurrence of numerous distinct events. In par- tant role. This could explain why local factors (such as ticular, inactivation of the tumour suppressor neurotrophic factors, ECM proteins and hormones) retinoblastoma protein (pRB) plays a crucially impor- allow partial dedifferentiation of Schwann cells and tant role [13] (Fig. 2). In fact, in addition to regulating not, for instance, of cardiomyocytes [20]. Indeed, an exit from quiescence, pRB is also implicated in the increasing number of reports are now showing that maintenance of cell identity. Indeed, its inactivation some mammalian cell types can be induced to dediffer- can render mammalian cells prone to dedifferentiation, entiate upon stimulation with specific signals and/or even when proliferation is impeded [14,15]. It is, how- treatments. As an example from our field of interest, ever, important to specify that proliferation and dedif- damage-induced dedifferentiation and proliferation of ferentiation are independent processes, and re-entry MGCs in the mammalian retina can be promoted via into the cell cycle is not necessarily associated with loss treatment with mitogenic factors, such as insulin, epi- or redefinition of cell identity. dermal growth factor (EGF) and Wnt3a [21–24]. It is also interesting to note that, in mammals, pRB Collectively, these observations are opening up new loss alone is not sufficient to induce dedifferentiation and provocative questions regarding the potential and/or re-entry into the cell cycle [16]. This is most applicability of therapeutic methods that can promote likely due to the INK4b-ARF-INK4a locus, which, in changes in cell identity and enhance endogenous regen- addition to p15INK4b and p16INK4a CDK inhibitors, eration. encodes a protein called ARF (or p14ARF, from alter- nate reading frame). In the absence of pRB, ARF can Cell fate and reprogramming activate compensatory mechanisms that lead to p53- mediated growth arrest [17] (Fig. 2). In support of this In the classical view of development, the differentiated hypothesis, combined inactivation of both pRB and state of a cell was believed to be terminal and irre- ARF has been shown to stimulate dedifferentiation versible. This concept has been best exemplified by and proliferations of mammalian muscle cells [18]. Conrad H. Waddington’s description of the epigenetic Indeed, ARF is not present in many regenerating ver- landscape (Fig. 3) [25]. Building upon Waddington’s tebrates, which could make them intrinsically better at original idea, during the 1960s, John Gurdon success- responding to dedifferentiation signals. This could fully cloned a frog via transplantation of a somatic explain, at least partially, why mammalian tissues have nucleus from the animal’s intestine into an oocyte [26]. lost their self-repairing capabilities [18]. It could even Gurdon showed for the first time that the identity of be speculated that evolution has strongly disfavoured differentiated cells could be reversed. His work was fate changes in mammalian cells. From an evolution- followed by the generation of Dolly the sheep [27], ary point of view, this could be explained by the need which confirmed that the epigenetic state of somatic for a trade-off between maximising tissue repair and cells could be reprogrammed to an embryonic pluripo- minimising the risks of malignant cellular transforma- tent state. tion. In fact, pRB has been implicated uncountable Nowadays, in addition to nuclear transfer, differen- times in the development and progression of various tiated cells can be artificially induced to revert to a types of tumours. In other words, ensuring mainte- pluripotent state via one of two mechanisms: (a) cell nance of the quiescent state (even if at the expense of fusion with embryonic stem cells (ESCs) [28]; (b) trans- regeneration) is probably more “valuable” and duction with specific cocktails of transcription factors,

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Fig. 2. Regulation of proliferation and dedifferentiation in mammalian cells. The retinoblastoma protein (pRB) regulates cell exit from quiescence [155]. (A. Quiescent and Differentiated Cells): When the activity of the cyclinD-Cdk4/6 complex is inhibited by cyclin-dependent kinase (CDK) inhibitors (e.g. p27KIP1, p21CIP1, p15INK4b and p16INK4a), pRB is found in its active, hypophosphorylated state. Active pRB blocks proliferation by sequestering members of the E2F family of transcription factors. It can also recruit histone deacetylases (HDAC) to further repress expression of cell cycle genes. Additionally, pRB can enhance recruitment of lineage-specifying transcription factors (e.g. MyoD in skeletal muscle cells), promoting expression of differentiation genes and the stable maintenance of a specific somatic cell identity [156,157]. (B. Proliferating Cells): In proliferating cells, CDK-inhibitors are inactive, which allows the cyclinD-Cdk4/6 complex to phosphorylate and inactivate pRB. pRB inactivation causes exit from quiescence, as E2F transcription factors are available to drive expression of genes involved in DNA replication and entry into S-phase of the cell cycle [155]. Moreover, independently from the induction of proliferation, pRB inactivation can lead to a decrease in the expression of differentiation genes, thereby favouring dedifferentiation. In other words, cellular dedifferentiation and proliferation can both be favoured by pRB inactivation. After passing this checkpoint, a particular cell is committed to a single round of division. (C. Cell Cycle Arrest): However, if DNA damage occurs, ATR and ATM protein kinases are activated [158]. They phosphorylate checkpoint kinases 1 and 2 (Chk1 and Chk2), which, in turn, phosphorylate and activate p53. Active p53 drives expression of p21, which induces cell cycle arrest by blocking activity of Cyclin/Cdk complexes. p53 also activates the DNA repair machinery, and, if DNA damage is too extensive to be repaired, it eventually induces cell apoptosis or senescence [159]. pRb and p53 operate as part of a larger network that is wired for functional redundancy. This means that dedifferentiation and/or re-entry into the cell cycle cannot be induced simply by inactivating pRB. Indeed, in the absence of pRB, oncogenic stimulation can activate ARF, which leads to p53-mediated growth arrest. In fact, active ARF can inactivate mouse double minute 2 homolog (mdm2), a E3 ubiquitin-protein ligase that normally stimulates p53 degradation [160]. resulting in the generation of the so-called induced cells. It is almost unanimously considered a promising pluripotent stem cells or iPSCs [29,30]. Furthermore, strategy for the repair of damaged organs. Indeed, the the establishment of ESCs cultures in the 1980s [31,32] development and optimisation of detailed, fine-tuned has allowed for extensive in vitro investigation of protocols have enabled us to generate iPSCs directly mechanisms controlling pluripotency, contributing to from accessible tissues of human patients. Addition- the unravelling of puzzling concepts such as potency, ally, both ESCs and iPSCs can be differentiated into lineage-commitment and cell fate. Actually, combining organ-specific cells to be transplanted back to the what is being learnt from ESCs/iPSCs-based in vitro patient [30,33,34]. Transplantation of iPSC-derived studies with the investigation of in vivo regenerative cells has been proven beneficial in rodent models of mechanisms could potentially help us gain an in-depth sickle cell anaemia [35], Parkinson’s disease [36], dia- understanding of repair processes, opening up the pos- betes [37] and spinal cord injury [38]. The possibility sibility of designing successful strategies for the of transplanting adult stem cells is also being explored, replacement of lost and/or damaged cells, which is the with bone marrow derived cells being particularly fundamental purpose of regenerative medicine. promising candidates. Regardless of the identity of the transplanted cells, Regenerative medicine cell therapy offers the advantage of an ex vivo expan- sion step, making it possible to obtain sufficient start- Regenerative medicine aims at the restoration of physi- ing material to ensure adequate replacement of lost ological function in organs and tissues not normally tissue. Furthermore, the use of patient-derived iPSCs prone to injury-induced repair. Over the past few dec- and adult stem cells would introduce the possibility of ades, increasing time and effort have been invested in autologous transplantation, reducing (even though not the development of approaches to achieve such goal. completely eliminating, as reviewed by Boyd et al.[39]) In our view, however, two are the main strategies that the chances of immune rejection. could eventually be employed in the clinical setting: However, for the time being, cell therapy is still in cell therapy and stimulation of endogenous regenera- its early stages, and it only works reliably for bone tion. marrow replacement. Undoubtedly, it requires signifi- Here, after briefly discussing some of the key aspects cant further development before being applicable to of cell therapy, we will focus on strategies aimed at the other organs of the human body. Indeed, cell therapy promotion of endogenous repairing mechanisms, with approaches are intrinsically associated with a number particular emphasis on their advantages, their draw- of limitations, including poor engraftment and poor backs and the challenges that need to be addressed. functional integration. Moreover, standardised proto- cols for the derivation of uncontaminated clinical- Cell therapy grade cells need to be established. Actually, ESC/ Cell therapy is based on transplantation into the iPSC-based therapies can raise controversial concerns injured site of either stem/progenitor or differentiated with respect to the risk of tumorigenesis [40]. Many of

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Fig. 3. The epigenetic landscape. The epigenetic landscape can be compared to a mountain with various valleys and several possible tracks leading to the bottom. In this analogy, cells are balls rolling down the side of such mountain. Cells at the very top of the mountain are found in a totipotent or pluripotent stage. Every time the ball meets a bifurcation, it has to commit to a choice, which will affect and limit its developmental potential, giving access to a more restricted number of developmental paths. This process continues until the ball reaches the bottom of the mountain, where it will remain in a stable valley, corresponding to its defined differentiated state. In contrast to what originally postulated, the identity of terminally differentiated cells can be induced to change via several processes: dedifferentiation, transdifferentiation, transdetermination and reprogramming. Dedifferentiation: The term dedifferentiation is intrinsically associated with lineage reprogramming, meaning that fully differentiated cells revert back to a less differentiated (and proliferative) stage from within their own lineage, without reaching pluripotency. Reprogramming: Reprogramming refers to the full restoration of pluripotency, up to a stage where cells can switch lineages and differentiate into any cell type; it could be seen as a case of extreme dedifferentiation, involving dramatic changes at the levels of both gene expression and epigenetic signatures. Transdifferentiation: Transdifferentiation describes the conversion of a fully differentiated cell of a given tissue lineage directly to one of a distinct lineage, without passing through a pluripotent intermediate. Transdetermination: Transdetermination describes the conversion of adult multipotent stem cells from their normal lineage to a closely related one [161]. So far, transdetermination has only been observed in the heapato-pancreatic system, most likely owing to the fact that the pancreas and the liver share a common developmental origin, with hepatic and pancreatic progenitors being derived from the same endodermal stem cells [162]. More specifically, upon transduction with the endocrine transcription factor neurogenin3 (Neurog3), hepatic progenitor cells were reported to generate, in the liver, insulin-producing cells that could rescue a mouse model of diabetes [161]. Transdetermination could also explain the reported presence of hepatocytes in the pancreas of rodents [163], and the other cases of pancreatic cells located in the liver, previously thought to be the result of trandifferentiation events [164–166]. these limitations can be overcome by approaches tar- they would most likely derive from the same (or a sim- geting the endogenous regenerative potential of mam- ilar) cell type. This means that they would already be malian tissues and organs. found where needed, and that they would be exposed to a local microenvironment that favours differentia- tion into the appropriate cell type. Furthermore, Endogenous regeneration in vivo reprogramming is associated with lower risks of Stimulation of endogenous regenerative pathways tumorigenesis [41,42], and with very few ethical issues could hold a tremendous potential as a therapeutic and concerns. These kinds of approaches would also tool, offering numerous advantages over transplanta- eliminate the need of strictly assessing the phenotype tion-based strategies. of the cells to be transplanted, with respect not only to To begin with, newly generated functional cells will gene expression, but also to chromatin state and func- be autologous by definition, eliminating the risk of tionality. In fact, reprogramming does not seem to be immune rejection. Additionally, these cells would accompanied by a proper reset of the epigenome to a already be located in the vicinity of the injury, and true ESC-like state. Consequently, some iPSCs retain

6 The FEBS Journal (2018) ª 2018 Federation of European Biochemical Societies M. Pesaresi et al. memory of their previous epigenetic status, or some specific epigenetic marks, and de novo establishment of residual methylation reflective of the donor cells others. In particular, reprogramming cells undergo [43,44]. This could explain why iPSCs derived from major rearrangements in heterochomatic regions, with different tissues tend to preferentially differentiate an overall loss of centromeric clusters, and of H3K9 along lineages close to the one from which they were and H4K20 trimethylation [55,56]. Profound changes originally derived [44]. in heterochromatin structure are also accompanied by Promotion of the body’s self-repairing mechanisms improvements in the nuclear envelope architecture [57] could be theoretically achieved in a number of ways and by telomere elongation [55,56]. that include: (a) activation of resident stem cells; (b) However, it is important to discuss how most of the proliferation of uninjured residual tissue cells; (c) fate epigenetic changes described to date in the context of conversion of other, healthy resident cells in vivo [45– in vivo reprogramming also occur during cancer devel- 48]. For instance, we have recently shown that, in the opment [58]. Indeed, the very same epigenetic changes context of tissue damage, MGCs of the murine retina that drive reprogramming can increase the likelihood can dedifferentiate and convert into ganglion and ama- that a given cell would undergo tumorigenesis. crine neurons following fusion with endogenous bone Accordingly, sustained OSKM induction prevents the marrow cells [49]. Indeed, in most tissues, endogenous long-term survival of doxycycline-treated animals due repair is dependent on injury-induced progenitor cells to extensive dedifferentiation and malignant transfor- that can undergo dedifferentiation, transdifferentiation mation in vital organs [53,54]. Nonetheless, it is also and reprogramming (Fig. 3). important to note that reducing the temporal extent of Some mammalians tissues, however, also contain a OSKM-induction can induce partial cell reprogram- reservoir of adult stem cells (ASCs) that can contribute ming in vivo, while simultaneously preventing teratoma to the maintenance of tissue homeostasis [50]. ASCs formation [57]. These findings have already been con- are normally quiescent, but they can become active if firmed by various independent studies [59,60], suggest- exposed to appropriate stimuli [51]. Indeed, upon ing that modulation of epigenetic remodelling may injury, they can re-enter the cell cycle and differentiate represent a valid strategy for the promotion of in vivo into tissue-specific cell types [52]. This endogenous regeneration, without induction of tumorigenesis. repair mechanism, however, is not sufficient to main- Further investigation showed that tissue damage is tain organ integrity and ensure adequate functionality an important factor in the promotion of cellular repro- in the face of severe injuries. gramming. In fact, OSKM induction induces extensive In addition to stem cells, dedifferentiation, transdif- damage in most of the cells, which respond by becom- ferentiation and reprogramming events involving tis- ing senescent [58]. Senescence is a protective cellular sue-resident somatic cells can also play a central role response associated with the secretion of a plethora of in endogenous tissue regeneration (Fig. 3). cytokines, which can promote tissue remodelling. These cytokines, and most notably interleukin-6 (IL6), Reprogramming cell identity in vivo can create a more permissive environment for the reprogramming of neighbouring cells [58]. The power of cellular reprogramming and the fact that Importantly, in vivo-generated iPSCs are signifi- dedifferentiation seems to promote successful regenera- cantly different from in vitro-generated ones, showing tion in mammals lead to wonder whether the genera- an expression profile remarkably more similar to that tion of dedifferentiated or pluripotent cells can be of true ESCs. Actually, like ESCs, in vivo-derived achieved directly in vivo. iPSCs can efficiently contribute to the inner cell mass In this direction, it has been shown that cell repro- (ICM) when injected into morulas. In vivo iPSCs can gramming can be achieved within a living organism also undergo trophectoderm lineage differentiation, via induction of the four Yamanaka factors, namely showing some totipotency features that even ESCs do Oct3/4, Sox2, Klf4 and c-Myc (OSKM) [53,54]. These not display [53]. studies were based on the use of ‘reprogrammable These observations have been backed up by various mice’, in which OSKM expression could be induced independent studies, reporting case of in vivo repro- with doxycycline. Doxycycline-mediated induction of gramming in multiple organs, including the liver OSKM resulted in the generation of NANOG-positive [59,61] and skeletal muscle [60]. Transient OSKM clusters of dedifferentiated cells in several tissues [53]. induction can also induce rejuvenation in a mouse Analogously to the in vitro process, in vivo reprogram- model of progeria (i.e. premature ageing) without ming involves substantial remodelling of chromatin tumorigenic side effects [55]. Interestingly, in this organisation. This, in turn, is mediated by erasure of study, full pluripotency was not achieved; however,

The FEBS Journal (2018) ª 2018 Federation of European Biochemical Societies 7 M. Pesaresi et al. pulses of OSKM activity were sufficient to extend ani- astroglia towards a glutamatergic neuronal fate, while mal lifespan and improve multiple hallmarks of age- Ascl1 or Dlx2 push the GABAergic fate instead [63– ing, including: (a) establishment of nuclear envelope 65]. In the context of tissue damage, Ascl1 has also architecture; (b) increase in stem cell populations in been shown to induce a neurogenic state in mam- the muscle and hair follicles; and (c) proliferation of malian MGCs [80,81], especially when combined with beta cells in the pancreas and satellite cells in the inhibition of a histone deacetylase [82]. skeletal muscle [55]. It is interesting to point out that splicing is also Collectively, these studies suggest that in vivo repro- emerging as an important mechanism modulating gramming could be beneficial for the functional main- reprogramming and regeneration. For instance, the tenance of various tissues. splicing regulator poly-pyrimidine-tract-binding (PTB) Importantly, in addition to OSKM induction, repro- protein can play a key role in the regenerative pro- gramming can be accomplished by expression of speci- cess [83]. PTB controls a network of microRNA that fic cocktails of transcription factors, as discusses targets the protein complex built on RE1-silencing below. transcription factor (REST). The REST complex normally acts to repress expression of several neu- ronal-specific genes in non-neuronal cells. PTB Reprogramming in the central nervous system downregulation activates a series of cascades that (CNS) can induce non-neuronal cells (e.g. HeLa, MEFs) to Glial cells are abundant in the CNS, and they possess transdifferentate into neurons in vitro. It would be a remarkable level of plasticity, being able to convert interesting to test whether this holds true for repro- into functional neurons [61–65]. Under normal circum- gramming in vivo. stances, glial cells are generally quiescent; however, they can re-enter the cell cycle following injury or Reprogramming in the heart other pathological conditions like stroke and neurode- generative disease [66]. Indeed, tissue damage and the Mammals possess some potential for heart regenera- associated process of reactive gliosis favour dedifferen- tion. In fact, contrary to normal belief, human car- tiation and identity changes [65–67]. Reactive gliosis, diomyocytes are constantly being renewed, with in fact, is associated with changes in the expression about 50% of the myocardial mass being replaced profiles of resident astrocytes, which acquire some of during a normal lifespan [84]. However, following the hallmarks of neural stem cells, thereby becoming a injury, regeneration is almost absent, and scarring good target for conversion into neurons [65,68–70]. occurs instead. This is due to the inability of car- In the uninjured mouse brain, Sox2 is sufficient to diomyocytes to efficiently dedifferentiate and re-enter induce conversion of NG2 glia into neurons [71], and the cell cycle. conversion of astrocytes into proliferative neuroblasts Nonetheless, cardiomyocytes can be forced to re- [72]. These cells can be generated even in aged brains enter the cell cycle by: (a) overexpression of specific and do not undergo apoptosis. Although they gener- factors, for example FGF1, periostin or neuregulin1 ally fail to become electrophysiologically functional [83,85–87]; (b) removal of different negative regulators, neurons on their own, these cells can be induced to such as cyclin-dependent kinase inhibitor p27Kip1, acquire a more mature neuronal phenotype by various pRB/p130 or p38 MAP kinase [15,85,88]; (c) treatment treatment, including exposure to epigenetic modulators with oncostatin M [87]. Interestingly, oncostatin M is (e.g. valproic acid) or neurogenic factors such as nog- an IL6-related cytokine, and its treatment is associated gin, brain-derived neurotrophic factor (BDNF) and with improved cardiac function in the context of acute bone morphogenetic protein (BMP) [72]. Encourag- myocardial infarction and chronic dilated cardiomy- ingly, newly generated cells can functionally integrate opathy [87]. into neural networks, which is of tremendous impor- In addition to cardiomyocytes, even adult cardiac tance from a clinical and translational point of view fibroblasts have been reprogrammed to cardiac-like [73]. Even transplanted astrocytes can be induced to myocytes [89]. Even though this was originally become neurons in vivo when expression of specific achieved in vitro, the findings could be recapitulated in reprogramming genes is activated [74]. an in vivo model of coronary artery ligation [46,47,90]. Extremely fine-tuned protocols have also been estab- Importantly, newly generated cells were found to be lished to generate specific neuronal subtypes, including functional, showing spontaneous contraction, rhythmic motor neurons [75] and dopaminergic neurons [76–79]. calcium oscillations and evidence of electrical coupling For instance, Neurog2 or NeuroD1 can convert [46,47,90].

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Reprogramming in the pancreas total bone marrow unless otherwise specified). For instance, some transplanted adult mouse BMDCs were Pancreatic exocrine cells have been reprogrammed found to adopt a neural phenotype in the CNS [101]. in vivo to endocrine b-cells in rodents with streptozo- Similarly, lineage-depleted (Lin ) HSPCs transplanted tocin-induced diabetes. This was originally achieved via in rodent models of ischaemia and myocardial infarc- adenoviral-mediated expression of three transcription tion were reported to mobilise to the heart and transd- factors (namely Neurog3, Pdx1 and Mafa), which ifferentiate into cardiomyocytes and endothelial cells, induced direct cell transdifferentiation [91]. Newly gen- thereby promoting functional repair [102,103]. erated cells were able to secrete insulin at levels compa- Even though these events occur with an extremely rable to those of endogenous b-cells, significantly variable and generally low frequency, they have been improving glucose tolerance and ameliorating hypergly- observed in numerous independent studies and in mul- caemia [91]. Interestingly, the identity of the cell of ori- tiple tissues, including: epithelia, skin and lung epithe- gin is extremely important, as the same combination of lium [104,105]; endothelium [102,106]; heart [102,103]; factors is unable to induce conversion into b-cells of cardiomyocytes and skeletal muscle [99,107–110]; liver other cell types (e.g. fibroblasts and skeletal muscle [105,111–114]; kidneys [115,116]; pancreas [117]; CNS cells). Following severe b-cell loss, other endocrine cells neurons [101,118–120]. can also spontaneously change their identity [92,93]. However, it is important to stress that only a handful For instance, some glucagon-producing a-cells were of these studies have characterised the functional rele- reported to transdifferentiate into insulin-secreting cells vance of in vivo cell fate conversion [102,103,112,116]. [92]. Such conversion potential did not seem to drasti- Most of the other investigations reported the cally decrease with ageing [93], and robust transdiffer- observation, but did not provide evidence of functional entiation correlated with improved blood glucose levels readout. and reduced dependence from exogenous insulin [92,94]. Intriguingly, reprogrammed b-cells continued expressing some a-cell specific genes, indicating that Cell fusion-mediated reprogramming functional transdifferentiation does not necessarily and regeneration requires complete repression of the starting transcrip- Cell fusion is a developmentally important process. As tional programme [92]. Moreover, such transdifferentia- a matter of fact, our own life starts with the fusion of tion routes can be bidirectional, as b-cells can also be our mother’s ovum and our father’s spermatozoa dur- converted to a-cells in vivo [95,96]. Similarly, both aci- ing fertilisation. Cell fusion is also involved in the nar cells [97] and somatostatin-producing d-cells [93] development of muscle, bone, and placenta [121,122], can become b-like cells. Acinar cells, in particular, can and in the formation of multinucleated giant cells in be converted into b-like, glucose-responsive cells fol- response to chronic infections and tumours [123]. The lowing exposure to EGF and CNTF, which activates molecular mechanisms involved in membrane fusion the endocrine progenitor transcription factor Neurog3 are still largely unknown [121,122]. However, it is [97]. This opens up the possibility to convert cells via becoming increasingly clearer that cell fusion events transient cytokine exposure rather than viral delivery of can induce cell plasticity, thereby contributing to tissue exogenous transcription factors [97]. regeneration [124,125]. Since the vast majority of reported fusion events Reprogramming mediated by bone in vivo involved BMDCs, we decided to focus on marrow-derived cells (BMDCs) BMDC-mediated in vivo regeneration. Fusion of recruited BMDCs with terminally differentiated tissue Haematopoietic stem cells (HSPCs) are the best char- cells can induce cell dedifferentiation and proliferation, acterised stem cells of the adult organism. They were representing a regenerative mechanism alternative to isolated from the bone marrow in the 1980s [98] and transdifferentiation [126,127]. found to be able to differentiate into all the lineages of Indeed, BMDCs have been reported to fuse with mature blood cell types. Indeed, a single transplanted several types of somatic cells (Fig. 4). HSC can repopulate the entire haematopoietic system of lethally irradiated animals [99,100]. Then, in the late 1990s, some studies started to indicate that BMDCs Cell fusion-mediated regeneration in the central could possess a wider plasticity than previously postu- nervous system (CNS) lated, being able to generate multiple lineages other Cell fusion events have been observed in the CNS. For than blood (hereinafter, the term BMDCs will refer to instance, postmortem biopsies of women who had

The FEBS Journal (2018) ª 2018 Federation of European Biochemical Societies 9 M. Pesaresi et al.

Fig. 4. Cell fusion-mediated regeneration occurs in multiple tissues. BMDCs (in red) have been reported to fuse with several somatic cell types (in blue), generating hybrid cells that contain both parental DNA. Such events have been reported in multiple tissues, including skeletal muscle, liver, lungs, intestine, skin, heart, brain and the retina. received a sex-mismatched BM transplant revealed the site [118]. Microglial cells, astrocytes and neurons of presence of tetraploid, Y chromosome-positive the CNS constitutively express a variety of chemoki- (XXXY) Purkinje neurons in the brain [119]. These nes, which are involved in migration, differentiation findings were also confirmed in animal models. More and proliferation of glial and neuronal cells. Such con- specifically, transplantation of GFP-expressing stitutive expression is increased in the presence of BMDCs in irradiated mice led to the generation of a inflammatory mediators, released in the context of few GFP-positive BMDC-Purkinje neurons heterokar- acute and chronic conditions [131]. yons, indicating that BMDCs could contribute to adult Indeed, transplanted Lin HSPCs have also been neuronal circuits through cell fusion [120]. shown to fuse with glial cells in two distinct models of The overall frequency of fusion events is quite low, Parkinson’s disease [132]. The resulting hybrids gener- but it seems to be significantly increased during ated new mature astroglial cells, able to secrete neu- chronic inflammation, degeneration and injury rotrophic factors (such as Wnt1), which, in turn, could [49,128,129]. In fact, Johansson and colleagues showed prevent degeneration of some dopaminergic neurons that, in the context of severe dermatitis or autoim- [132]. Both endogenous [49] and locally transplanted mune encephalitis, the frequency of fusion between [129,133] Lin HSPCs can also fuse with resident BMDCs and Purkinje neurons was 10- to 100-fold MGCs of the retina. Resulting hybrids undergo partial higher than previously reported [119,120,130], resulting dedifferentiation, re-enter the cell cycle and then in the generation of hundreds of fused heterokaryons rapidly commit to the neuroectodermal lineage, even- [128]. This could be explained by the fact that neu- tually differentiating into mature retinal neurons ropathology is associated with a significant enhance- [129,133]. Importantly, BMDC–MGC hybrids can ment of the chemokine-mediated recruitment and contribute to the regeneration of the damaged retinal engraftment of BMDCs, especially around the damage tissue [129,133]. Interestingly, retinal injury or

10 The FEBS Journal (2018) ª 2018 Federation of European Biochemical Societies M. Pesaresi et al. degeneration (and, most likely, the associated inflam- chromosome correlated with restored dystrophin matory environment) are essential for cell-hybrid for- expression [137]. mation in vivo [49,129,133]. BMDC fusion with the skeletal muscle has been also reported in human patients with Duchenne muscular dystrophy [138]. More specifically, muscle biopsies Cell fusion-mediated regeneration in the liver from a female patient who had received a BM-trans- Cell fusion events have been extensively shown to con- plant from a male donor showed that Y-containing tribute to liver regeneration [113,130,134–136]. Actu- nuclei were present within muscle myofibres, even ally, BM-derived hybrids are essential to trigger 13 years after transplantation [138]. efficient liver regeneration after injury. Accordingly, regeneration is severely impaired when BMDC recruit- in vivo – Challenges for reprogramming ment is impeded, or BMDC hepatocytes hybrids are approaches genetically ablated [136]. Interestingly, BMDC transplant in mice lacking the Recent years have witnessed an explosion of interest in Fah enzyme (Fah/, a model of tyrosinemia type I) regenerative medicine and, more specifically, in resulted in restored expression of the wild type gene in approaches aimed at boosting the endogenous regener- some hepatocytes [112]. More specifically, Lagasse and ative capacities of mammalian organs. However, colleagues isolated and transplanted the so-called numerous open questions and technical challenges KTLS (c-kithigh, Thylow, Lin, Sca-1+) fraction which remain to be addressed. is highly enriched for long-term repopulating HSC In addition to identifying the most appropriate activity. This correlated with a rescue of the biochemi- in vivo cell type sources for lineage reprogramming, it cal function of the liver [112]. Originally, Fah+ hepa- is essential to define the best strategy for the conver- tocytes were proposed to derive from BMDC sion of such cells into the desired cell type. Currently, transdifferentiation. However, convincing evidence has adenoviral transduction of transcription factor cock- now shown that, during hepatic degeneration, cell tails represents the most common approach for cell fusion represents the main source of BM-derived hepa- reprogramming in vivo. This is associated with a num- tocytes [113,114]. In fact, Fah+ cells were found to ber of issues, including the intrinsic risk of insertional express both donor and host genes [113], and southern mutations, which represents a major safety concern blot analysis revealed that hepatocytes in the liver were and severely hinders their clinical applications. More- heterozygous for alleles unique to the donor marrow over, when the reprogramming process is completed, [114]. the endogenous pluripotency programme is normally Importantly, these studies have shown that fusion activated. This means that cells can self-sustain their with transplanted BMDCs could be used to deliver pluripotent state, with expression of exogenous factors wild-type alleles to tissue cells with recessive muta- becoming dispensable after about 10 days [139]. tions, thereby representing a valuable strategy for the Expression of virus-mediated transcription factor, correction of genetic defects. however, is often maintained beyond the reprogram- ming stage, with unknown consequences. In other words, viral-induced reprogramming might Cell fusion-mediated regeneration in the skeletal result in some hurdles for clinical application, and the muscle quest for more appropriate, safer and less-invasive Transplanted BMDCs have been reported to migrate delivery strategies continues. into areas of muscle degeneration in mice lacking dys- In this direction, recent studies have shown that trophin (Mdx/, a model of Duchenne Muscular pluripotent stem cells can be generated in vitro with Dystrophy). BMDCs were found to differentiate into minicircle DNA constructs and in vivo via hydrody- myogenic cells, thereby contributing to the regenera- namic tail-vein injection of DNA [56]. Additionally, tion of damaged fibres either when unfractionated administration of synthetic mRNA could be used to bone marrow cells [109] or lineage-depleted HSPCs induce not only reprogramming, but also redifferentia- were transplanted [110]. Once again, cell fusion rather tion into specific cell types [140]. Sendai virus vectors than transdifferentiation seems to account for the also represent a valid alternative, as they do not inte- observed cell identity changes. In fact, Y chromosome- grate into the genome. They have been used to deliver positive muscle fibres were found in female animals cardiac reprogramming factors to both rodent and that had undergone sex-mismatched BM transplanta- human fibroblasts, efficiently inducing their conversion tion [137]. Importantly, the presence of the Y into cardiomyocytes [141]. Induced cardiomyocytes

The FEBS Journal (2018) ª 2018 Federation of European Biochemical Societies 11 M. Pesaresi et al. generated in this way could improve cardiac function reprogramming in vitro [149]. This opens the possibil- and reduce fibrosis in a mouse model of myocardial ity that the chemical cocktails identified and charac- infarction [141]. terised in vitro could eventually be implemented for Small molecules represent an even more attractive reprogramming in vivo. However, the road to in vivo alternative to transcription factors, as they are gener- applications is still long and tortuous. ally less expensive, more stable and nonimmunogenic. Other additional issues to be overcome include the They can also be more easily synthesised and stan- yield of a number of cells sufficient to mediate func- dardised. Additionally, the administered concentra- tional recovery and, most importantly, the systematic tions and combinations can be fine-tuned and and thorough assessment of the final phenotype optimised, allowing for a higher degree of spatial and obtained. In fact, it is crucial to generate mature cells temporal control. Indeed, the use of small molecules that are fully converted into the desired cell type, with for chemical reprogramming could facilitate the the ability to successfully and functionally integrate removal of the epigenetic barriers that normally act to within the host tissue. Among the various tissues and soundly maintain somatic cell identity. Many interest- organs, the brain microenvironment seems to be par- ing small molecules and chemical compounds able to ticularly permissive for the survival and maturation of modulate the reprogramming process have already induced neurons. Indeed, it allows for a remarkable been identified [142]. Furthermore, reprogramming level of regenerative plasticity [36,75,77]. Notably, mediated by small molecules could potentially (and extracellular cues of the cortical and striatal microenvi- attractively) be cell type-specific, introducing the possi- ronments uniquely instruct non-neuronal cells to adopt bility to target specific cell subpopulations of interest. distinct neuronal subtypes of varying functional matu- Due to all these advantageous properties, chemical rity when transduced with Neurog2 [150]. Other reprogramming could significantly speed up clinical microenvironments, however, might not be as favour- translation of in vivo reprogramming strategies. So far, able. in an attempt to minimise the number of transgenes Overcoming the constraints imposed by the host tis- required, direct conversion of fibroblasts into choliner- sue represents a critical challenge to be addressed in gic neurons has been achieved with the combination of order to promote cellular plasticity. It is therefore a single proneural gene (Neurog2) with the small important to understand what role the local microenvi- molecules forskolin and dorsomorphin [143]. Taking ronment plays, and if there are any specific inhibitory these findings one step further, iPSCs were generated cues that could be removed to boost the endogenous from mouse somatic cells using a chemical repro- regenerative potential. gramming strategy, based on a combination of Assessing functional maturation also implies ensur- seven small-molecule compounds without any trans- ing that the newly generated cells will hold no memory gene overexpression [144]. A small molecule was also of their previous phenotype, so that they do not revert found to induce an immortalised pancreatic a-cell line to their original identity. In fact, reprogramming could to express insulin and convert to a b-cell-like state lead to aberrations in which a cell would have a [145]. Similarly, in vitro, it was possible to achieve hybrid epigenetic landscape, with residual elements of chemical-mediated conversion of fibroblasts into both the origin cell and functional regions of the adopted multipotent neural stem cell-like cells and prolifera- identity [142]. We now need to better understand tive early cardiac progenitors [146,147]. When trans- DNA methylation, histone tail marks, three-dimen- planted into the infarcted heart, these cardiac sional chromatin structure and other mechanisms of progenitors could survive and develop into cardiomy- epigenetic regulation in transdifferentiating and repro- ocytes, smooth muscle cells, and endothelial cells gramming cells. [146,147]. Furthermore, it has been shown that speci- fic small molecules cocktails can enhance regeneration Conclusions and future perspectives following acute injury of the liver in vivo [148]. In particular, improved liver function was associated Regenerative capabilities vary extensively across the with intraperitoneal administration of a cocktail con- animal kingdom. Some organisms, such as amphibians taining inhibitors of: (a) histone deacetylases (e.g. val- and cold-blooded vertebrates, can repair tissues even proic acid, sodium butyrate); (b) glycogen synthase after massive damage. In humans, there is physiologi- kinase 3 beta (GSK-3b, e.g. chiron, lithium chloride); cal turnover of cells of the epidermis and intestinal (c) transforming growth factor beta (TGF-b, e.g. mucosa; apart from this, however, regenerative capa- Repsox, SD-208). Remarkably, cocktails with the bility is inherently low and limited to a few tissues, same composition are also able to promote most notably the liver.

12 The FEBS Journal (2018) ª 2018 Federation of European Biochemical Societies M. Pesaresi et al.

Decades of painstaking studies have revealed that and Alberto Oliveri (Botliveri) for his help with the successful tissue regeneration involves processes such as graphical design of the figures. We apologise to the col- dedifferentiation, transdifferentiation, reprogramming leagues whose work could not be cited due to space limi- and cell fusion. Importantly, these regenerative mecha- tations. Work in the MPC lab is supported by an ERC nisms are not mutually exclusive; on the contrary, they grant (242630-RERE to MPC), the Ministerio de could occur independently from each other under differ- Economia y Competitividad and FEDER funds ent conditions or, alternatively, they could coexist and (SAF2011-28580, BFU2014-54717-P and BFU2015- work synergistically to enhance regeneration. 71984-ERC to MPC), an AGAUR grant from Secre- Various approaches aimed at the enhancement of taria d’Universitats i Investigacio0 del Departament mammalian regenerative abilities are currently being d’Economia I Coneixement de la Generalitat de Catalu- explored, each one with its own advantages and disad- nya (2014SGR1137 to MPC), Velux Stiftung (MPC), vantages. Such approaches are collectively referred to Secretaria d’Universitats i Recerca del Departament as regenerative medicine. Regenerative medicine can- d’Empresa i Coneixement de la Generalitat de Catalu- not be seen as an isolated, stand-alone science; on the nya ref. 2018FI_B_00637 (to RS-P), Subprograma esta- contrary, it relies on contributions from multiple and tal de Formacion del Ministerio de Economıa y various fields, ranging from surgery and nanotechnol- Competitividad ref. BES-2015-075802 (to M.P.), and ogy to tissue engineering, gene therapy and clinical the co-finance of Fondo Social Europeo (FSE to RS-P procedures. Among them, induction of in vivo dediffer- and MP). We acknowledge the support of the Spanish entiation and reprogramming stands out for its Ministry of Economy, Industry and Competitiveness tremendous potential in the clinic. (MEIC) to the EMBL partnership. We also acknowl- Nonetheless, numerous questions in the field remain edge support of the CERCA Programme (Generalitat open. For instance, most of the published work has de Catalunya), and of the Spanish Ministry of Economy focused on overexpression of pioneering transcription and Competitiveness, “Centro de Excelencia Severo factors. Even though new potential routes for repro- Ochoa”. gramming are emerging, delivery methods for such fac- tors are, at least for the time being, predominantly Competing financial interests viral and, therefore, unsuitable for clinical applica- tions. Additionally, cell identity changes into a specific The authors declare no competing financial interests. lineage have been fully accomplished only in vitro. In our opinion, in vitro studies can (and will) pro- Author contributions vide valuable insights into the molecular mechanisms and the pathways associated with cell fate and regener- MP, RS-P and MPC wrote the review. ation; however, in order to truly understand the mech- anisms controlling pluripotency and reprogramming, References more extensive in vivo investigations are required. To conclude, even though several technical chal- 1 Newmark PA & Sanchez Alvarado A (2000) lenges need to be overcome and the feasibility of Bromodeoxyuridine specifically labels the regenerative 220 – approaches promoting endogenous repair carefully stem cells of planarians. Dev Biol , 142 153. evaluated, the outcomes of several studies from differ- 2 Kumar A, Velloso CP, Imokawa Y & Brockes JP (2000) Plasticity of retrovirus-labelled myotubes in the ent fields is slowly, but encouragingly opening up the newt limb regeneration blastema. Dev Biol 218, 125– possibility of using in vivo reprogramming for the 136. treatment of pathologies such as diabetes, muscle 3 Potten CS & Loeffler M (1990) Stem cells: attributes, degeneration and neurodegenerative diseases. Indeed, cycles, spirals, pitfalls and uncertainties. Lessons for in our view, integrating this approach with other and from the crypt. Development 110, 1001–1020. aspects of medicine, cell therapy, tissue engineering, 4 Blanpain C, Lowry WE, Geoghegan A, Polak L & molecular medicine and nanotechnology would max- Fuchs E (2004) Self-renewal, multipotency, and the imise our chances of achieving satisfactory results in a existence of two cell populations within an epithelial not-so-distant future. stem cell niche. Cell 118, 635–648. 5 Nishimura EK, Jordan SA, Oshima H, Yoshida H, Acknowledgements Osawa M, Moriyama M, Jackson IJ, Barrandon Y, Miyachi Y & Nishikawa S (2002) Dominant role of We thank the members of the MPC lab, in particular the niche in melanocyte stem-cell fate determination. Sergi A. Bonilla-Pons for critical reading of the review; Nature 416, 854–860.

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