Keywords

FormalPara What You Will Learn in This Chapter

During regenerative processes, cells are required to restructure parts of a damaged or worn-out organ and tissue. Here, you will become acquainted with the strategies that organisms developed to provide the material for tissue and organ repair. On the one hand, somatic cells can become dedifferentiated to increase their developmental potential and produce the plasticity required to replace the entire cellular complexity of a damaged part. On the other hand, organisms retain organ-specific stem cells with a restricted developmental potency and use these to provide the “spare parts” for replacing damaged cells. In all cases, a substantial reprogramming of the epigenome of these cells accompanies the restructuring process. In vitro strategies have been developed to drive cells back to a pluripotent state, allowing a better understanding of the underlying chromatin adjustments and providing a rich source for cellular therapies.

7.1 Types of Regenerative Phenomena

Epigenetic mechanisms mostly sustain long-term processes, like maintenance of gene expression patterns, keeping either the maternal or the paternal X chromosome repressed within a cell lineage, maintaining cellular memory over developmental time, or tightly silence transposon-riddled genome sections during the life of a cell and of its progenitors. Many of these mechanisms have evolved to be exceptionally stable, approaching sometimes the perseverance of the genetic system. Yet, organisms also adapt to changing environments and physiological requirements and, therefore, have to adjust or reverse some of these long-term epigenetic commitments. A prime example requiring restructuring of the epigenomic landscape is the process of regeneration. Damaged body parts, organs, or tissues can be faithfully repaired contributing to the survival of the adult organism. In adults, development has been completed and cell types and the anatomy of tissues in the organs is maintained with minimal changes. Additionally, the process of ageing leads to a degradation of tissue function. Hence, new strategies to provide the materials necessary for the repair process evolved. The body can carry a reservoir of stem cells from which replacement of parts can form through cell differentiation, by re-running developmental cascades to achieve the necessary structures and functions. Alternatively, differentiated cells in the neighborhood can change their own identity and attain the morphological structures and physiological functions required to replace the damaged parts. In low complexity organs, hyperproliferation of the basic structural and functional constituents can often fill the gaps left by the damage. This chapter deals with the obstacles that regenerative processes encounter when epigenetically encoded, stable states have to be changed and reprogrammed.

While plants have extraordinary regenerative capacities and there is a large body of literature describing the underlying epigenetic processes (Ikeuchi et al. 2019), this chapter will focus on animal regenerative mechanisms (Tanaka and Reddien 2011; Carlson 2005). Different phenomena have been observed in animal phyla, like morphallaxis in Planaria or Hydra, approaching the repair capacities observed in the plant world. Tissue fragments retain the ability to reconstitute an entire functional organism or organ. In general, compensatory growth restores the size of an organ by increasing cell size (compensatory hypertrophy) or by accelerating cell division (compensatory hyperplasia). Limited to the usage of the existing pool of differentiated cells, such regulatory processes can be observed after damage of a number of human organs, like liver or kidney. Physiological regeneration describes the continuing replacement of damaged or ageing cells or body parts. In humans, examples for this form of tissue homeostasis are the process of blood cell replacement, the renewal of the lining of the intestine, or the surface of the skin.

7.1.1 Regenerating from a Blastema

Reparative regeneration represents the most spectacular form of restoration observed in higher animals. The capability to regrow an entire limb structure after amputation, as observed in newts, is certainly such a remarkable process, regarded with envy by us humans. Not surprisingly, these reparative processes were studied for decades by scientists in a number of model organisms, like the amphibian axolotlFootnote 1 or newts. The repair and regenerative process after amputation of the limb can be described in the following series of key events (Carlson 2005):

  1. 1.

    Wound Healing: Epidermal migration to cover the amputation surface and prevent infections by pathogens.

  2. 2.

    Demolition and Phagocytosis: Inflammatory response and enzymatic removal of matrix components just beneath the wound epithelium.

  3. 3.

    Dedifferentiation: The loss of differentiated tissue types beneath the wound epithelium and the appearance of embryonic-looking (dedifferentiated) cells in the same area.

  4. 4.

    Blastema Formation: The aggregation of the dedifferentiated cells into a structure reminiscent of embryonic limb structure.

  5. 5.

    Morphogenesis and Growth: Growth of the blastema and shaping and differentiation of the blastemal cells into the cells and tissues comprising the normal limb. In larger appendages, after morphogenesis is completed, growth of the miniature regenerate continues until it is of the same size as the original limb structure that was lost.

Central to the process is the formation of a blastema containing the progenitor cells for the regrowth of the full appendage (epimorphic regeneration). Cells forming the blastema derive from tissues surrounding the wound. Though resembling the morphology of embryonic cells, these precursors nevertheless appear not be fully dedifferentiated to a pluripotent state (see below) but gain a multipotent condition required to rebuilt the resident organ or tissue. Lineage tracing experiments in axolotl reveal that the cells of the blastema retain certain developmental restrictions and only have a limited differentiation potential (Tanaka and Reddien 2011). By tracing cells from each major tissue during axolotl limb regeneration, dermal cells turned out to have the potency to form cartilages and tendons, but never switched the fate across embryonic germ layers. Other blastema cells derived from epidermal cells, muscles, Schwann cells, and cartilage cells, retained their original tissue identity during regeneration. Hence, a rather limited dedifferentiation and change in fate appears to occur at the blastema.

7.1.2 Changing Potency by Transdifferentiation

Other processes of regeneration, omitting the formation of a blastema but still utilizing partial dedifferentiation steps, have been observed. For example, zebrafish hearts can regenerate after amputation of up to 20% of the ventricle. During the process, the newly formed heart muscle cells are derived from the dedifferentiation and proliferation of pre-existing cardiomyocytes. Dedifferentiation promotes proliferation and the activation of embryonic cardiogenesis genes. Conversely, a decrease in expression of sarcomeric components leads to the disassembly of sarcomeric structures (Barrero and Izpisua Belmonte 2011).

In other cases, a complete change in cellular fate has been described and termed “transdifferentiation”. Glucagon-producing α-cells can transdifferentiate into pancreatic insulin-producing β-cells after ablation of β-cells with diphtheria toxin. This transition process seems to be direct, not involving undifferentiated intermediatesFootnote 2. Transdifferentiation can also occur through an undifferentiated intermediate, however, as observed during the regeneration of the eye lens in newts. Lens regeneration takes place through transdifferentiation of the pigmented epithelial cells (PECs) of the iris. After removal of the lens, the PECs re-enter the cell cycle, dedifferentiate, and lose their characteristic pigmentation. During this early stage, the expression of developmental regulators, such as the transcription factors Pax6 and Sox2, is activated. Later, the dedifferentiated proliferating PECs start to express crystallins and differentiate into lens fibers.

Taken together, these results suggest that the process of dedifferentiation and transdifferentiation during regeneration involves the silencing of tissue-specific genes, as well as the induction of genes involved in embryonic programs and the control of the cell cycle. These changes in gene expression might facilitate the acquisition of a limited extended plasticity that allows cells to proliferate and rearrange into the new structures by a limited iteration of developmental pathways. Potentially, embryonic genes involved in regeneration might remain poised for activation in regenerating animals, but irreversibly silenced in non-regenerating animals. The questions remaining are: what signaling mechanisms drive this change in potency and what mechanisms repattern the cells to reproduce a full organ?

7.1.3 Signaling in the Blastema

Despite substantial interest in understanding the basic mechanisms regulating regenerative capacity, in many of the classical model organisms it has been difficult to study the underlying molecular mechanisms. Conversely, Drosophila melanogaster offers substantial advantages for regenerative studies since it is less complex than amphibians and humans and a wide palette of research tools is available. Drosophila, unlike some other arthropods, is not able to regenerate damaged legs or wings in adults. However, the larvae harbour regeneration abilities in the imaginal discs (see also book ► Chap. 3 of Paro). Imaginal discs can regenerate to form normal adult appendages even after massive lesion of disc cells are caused by X-rays irradiation at larval stages or by local induction of cell death (Hariharan and Serras 2017; Ahmed-de-Prado and Baonza 2018).

Regeneration of the imaginal discs is also observed under ex vivo conditions (◘ Fig. 7.1). When imaginal discs are manually fragmented, transplanted, and cultured in the abdomen of an adult fly, the disc cells at the wound site regenerate the missing parts. Remember that the imaginal disc cells consist of already specifically determined but undifferentiated cells until metamorphosis, hence they are not uniform cells. Each imaginal disc established during embryogenesis is destined to follow a specific developmental pathway. The regional identities and specific cell fates in each disc are precisely determined in a stepwise manner throughout the larval stage. At the time of damage, the expression of master regulatory genes shows already an intricate developmental pattern. The steps in the regeneration process of fragmented imaginal discs are, in principle, analogous to those of amphibian limb regeneration, consisting of wound healing (closure), localized cell proliferation (regeneration blastema formation), and pattern formation. Collectively, the observations demonstrate that disc regeneration induces limited cellular reprogramming, enabling the reconstitution of the lost tissue while disc identity is maintained independently of the local environment.

Fig. 7.1
figure 1

Signaling in the blastema of the regenerating imaginal disc. a Leg imaginal disc dissected from third instar larva at 100 h after egg deposition, wounded and cultured in adult abdomen (ex vivo). Activation of JNK signaling in cells shown by a continuous labeling of a GFP-reporter (bottom panel) in the blastema at different time points after wounding; (C0P) 0 hours (un-cultured), (C6P) 6 hours, (C12P) 12 hours, (C24P) 24 hours, and (C48P) 48 hours of culturing. Yellow arrowheads indicate the fragmented positions. Scale bar = 100 μm. b JNK signaling at the wound site is required to downregulate PcG silencing and up-regulate ligands for Dpp, JAK-STAT, and Wg signaling cascades. Dpp signaling initiates wound closure. The concerted action of JAK-STAT and Wg signaling induces cell proliferation and tissue repatterning in the blastema

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Studies of imaginal disc transplantation have uncovered the process of transdetermination, i.e., neighbouring groups of cells in regeneration blastema sometimes become more plastic and acquire alternative organ identities from different imaginal discs (comparable to transdifferentiation; see book ► Chap. 3 of Paro). This suggested that the mechanism of cellular memory has to be changed and reprogrammed during the regenerative process. Indeed, disc regeneration and transdetermination are coupled to the regulation of Polycomb (PcG) function. Down-regulation of PcG function, as monitored by the derepression of a silent PcG regulated reporter gene, was observed in proliferating, regenerating cells of the blastema (Lee et al. 2005). Furthermore, only cells with compromised PcG function were found to transdetermine more frequently, suggesting that PcG modulation is a prerequisite for cellular reprogramming.

The healing at wound sites involves the activation of the Jun N-terminal kinase (JNK) signalling pathway in several rows of cells at the edge of the wound (◘ Fig. 7.1). The blastema cells are derived from cells in which JNK has been activated. It was found that the JNK signalling pathway directly controls the down-regulation of PcG silencing (Lee et al. 2005). Clonal activation of the JNK pathway in imaginal discs was sufficient to reduce PcG-mediated silencing function, as can be visualized by ectopic expression of a Hox gene, a target of the PcG system. Hence, the down-regulation of PcG silencing by JNK signaling appears to result in a specific, yet, not global reactivation of PcG target genes. These appear to be driven into a poised state and only upon the activation by tissue-specific transcription factors, the cells are directed towards the required differentiated state (Beira et al. 2018).

An important finding shows that in epimorphic regenerative processes observed in many animals, the formation of a blastema provides the starting source of cells used for the repatterning. However, while changing their differentiation state to a more multipotent character, the cells of the blastema never acquire a fully pluripotent capacity. Unlike the exogenous expression of reprogramming factors to induce pluripotent stem cells (see below), the natural regeneration signals are able to reprogram the chromatin state of the blastema cells to more lineage-restricted progenitor states, circumventing the problem of tumorigenic deregulation often observed in pluripotent cells.

7.2 Stem Cells in the Adult

A different concept for providing cells to regenerating parts are stem cells. By definition, the division of a stem cell will produce another stem cell as well as a precursor for a particular lineage. Hence, on the one side, the constant availability of a stem cell pool is guaranteed. On the other side, the stem cell has to maintain the developmental potential to produce all the cell types of the lineage(s) that is contributing to. In mammals at the early blastocyst stage, cells from epiblast of the inner cell mass can be maintained in culture. They have the capacity to develop and contribute to cell types of all three germ layers when subjected in vitro to a differentiation program or transplanted back into the corresponding developmental stage of the organism. As such, they are pluripotent since they have the potential to develop into any part of the organism, except the extraembryonic tissue giving rise to the placenta. Conversely, adult organisms maintain pools of stem cells with a more restricted developmental capacity (multipotent). Here, the classical example are hematopoietic stem cells. The “fluid” nature of the blood system allowed the study of the various cell types and their pedigrees in great detail. The original source is the multipotential hematopoietic stem cells in the bone marrow. The fact that we can use normal hematopoietic stem cells to repopulate the full blood system in a leukemic patient crippled with cancerous blood cells is a clear indication that our body carries a pool of cells with highly specific regenerative capacities. Is this true for every organ? The search for an answer to this question has occupied generations of scientists. Hampered by their apparently limited cell number and the difficulty to isolate and cultivate them, for long time it has been challenging to identify molecular markers characteristic for adult stem cells. In particular, by analysing organs with a high cellular turnover like skin or gut, caused, for example, by mechanical or chemical strain, the corresponding source of adult stem cells could be localized and the differentiation path their progeny follows could be studied. The intestinal epithelium is composed at the basis of crypts encompassing the cradle of stem cells (expressing, among others, the marker Lgr5) (Tetteh et al. 2015). Progenitors of the stem cells leaving the cradle are subjected to intricate signalling cascades and start to differentiate along their path to the villus (the intestinal surface exposed to the inside of the stomach). Lgr5-expressing cells can be cultivated in vitro. Eventually, they will self-assemble into a mini-gut organoid, manifesting their developmental capability to structure a specific part of an organ. This example demonstrates that also the intestine harbours its own “spare parts” to counter the constant degradation and turnover of cells and thereby maintains tissue homeostasis. Other pools of adult stem cells have been identified in different organs (Clevers and Watt 2018), but their cultivation in vitro and molecular characterization has been exceedingly difficult. This is in part caused by the exclusive neighbourhood required by the stem cells. Stem cells clearly must have a different epigenetic status than most of their differentiated neighbours. They seem to only be able to maintain this status in a special environment, termed “niche”. A niche represents a highly protective microenvironment, located in a specific anatomic location where the stem cells are found. At these sites adult stem cells are maintained in a quiescent state by a combination of cell-cell or cell-matrix interactions, endocrine, paracrine, and autocrine signalling pathways, specific physical interactions, neuronal connections and particular physico-chemical conditions. However, after tissue injury or to maintain tissue homeostasis, the surrounding micro-environment actively signals to stem cells to induce either self-renewal or differentiation to form cells required for regeneration. As soon as stem cells leave the niche, they are subjected to differentiation signals, become first transient amplifying cells, and then start to develop fates similar to their neighbours. Recreating niche conditions in a dish to cultivate the multipotent stem cells has been a major challenge, though.

7.3 Sources of Pluripotent Stem Cells

Why bother with finicky adult stem cells if pluripotent embryonic stem cells can produce all the cell types of a mammalian organism? Indeed, already 40 years ago, mouse embryonic stem cells (mESC ) from the inner cell mass of blastocysts could be established as an in vitro culture. Eventually, these cells could be induced in vitro by specific combinations of developmental factors to produce almost all cell types of a mouse, demonstrating their pluripotent capacity. Experiments with human embryonic stem cells demonstrated a similar capacity. The possibility to cultivate and drive theses pluripotent cells through specific differentiation paths to produce functionally and morphologically elaborate cell types, offered seemingly extraordinary opportunities for regenerative medicine. In parallel, alternative methods to collect or produce pluripotent cells were developed (◘ Fig. 7.2). Besides the described cell mass from blastocysts before implantation, in vivo, two other sources of mouse pluripotent stem cells exist. Embryonic germ cells can be derived from primordial germ cells during mid-gestation (mouse embryonic day 8.5–12.5). Germline-derived pluripotent stem cells can be isolated from spermatogonial stem cells of neonatal and adult testesFootnote 3.

Fig. 7.2
figure 2

Pluripotent stem cells can be derived from several sources. There are three sources of pluripotent stem cells in vivo (see the figure, top half). Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst, before embryo implantation. Embryonic germ (EG) cells are derived from primordial germ cells (PGCs) during mid-gestation (embryonic days 8.5–12.5 in the mouse) and germline-derived pluripotent stem (gPS) cells are derived from spermatogonial stem cells of neonatal and adult testes. In addition, three major routes for somatic cell reprogramming to pluripotency have been described (see the figure, bottom half): fusion between a somatic cell and an ES cell giving rise to reprogrammed hybrid cells; the generation of nuclear transfer embryonic stem (NT-ES) cells, produced by reprogramming of a somatic nucleus by an enucleated oocyte, which is then cultured to the blastocyst stage to allow derivation of ES cells; and the production of induced pluripotent stem (iPS) cells, derived by somatic cell overexpression of reprogramming transcription factors, most commonly OCT4 (also known as POU5F1), Sry-box containing gene 2 (SOX2), myelocytomatosis oncogene (MYC) and Krüppel-like factor 4 (KLF4). (From (Gaspar-Maia et al. 2011)

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The study of murine germ cell development allowed to observe the dramatic reshuffling of epigenetic information not only during germ cell maturation but also after fertilization (◘ Fig. 7.3) (Saitou et al. 2012). The massive changes of the epigenetic landscape detected over time reflect the substantial remodelling germ cells undertake to bring the epigenome back to the ground state (see also book ► Chap. 3 of Paro).

Fig. 7.3
figure 3

A schematic of mouse pre-implantation and germ cell development. (Top) A schematic of pre-implantation and germ-cell development in mice. a Pre-implantation development stages; b post-implantation embryonic development, following blastocyst implantation at around E4.5; and c postnatal germ cell development and maturation. Primordial germ cell (PGC) precursors (E6.25) and PGCs are shown as green circles in embryos from E6.25 to E12.5. (Bottom) Key genetic and epigenetic events are shown that are associated with pre-implantation and germ cell development, together with relative levels of 5-methylcytosine (5mC) at different developmental stages. Al allantois, Epi epiblast, ExE extraembryonic ectoderm, ICM inner cell mass, PB polar body, PGCs primordial germ cells, Sm somite, TE trophectoderm, VE visceral endoderm, ZP zona pelucida. (From (Saitou et al. 2012)

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In primordial germ cells, different waves of DNA de- and re-methylation together with chromatin modifying mechanisms ensure that genomic imprints are reset, inactive X chromosomes reactivated, and somatic epigenetic signatures eliminated (see also book ► Chap. 5 of Grossniklaus). Similar mechanisms will apply to the somatic cell lineages. Hence, organisms have developed very elaborate mechanisms to reprogram, in a substantial and directional manner, the epigenetic landscape of some of their cell types.

In the past decades, three in vitro methods have been developed to generate cells with a pluripotent capacity (Gaspar-Maia et al. 2011). Fusions between a differentiated somatic cell and an embryonic stem cell reprograms the nucleus restricted in developmental potential to a pluripotent state. Here, reprogramming processes could be studied in vitro and the ensuing changes of the epigenetic states could be pursued at the molecular level. The disadvantage, in particular for therapeutic purposes, was that the resulting cell contains both genomes of the fusion partners and, thus, a tetraploid state is obtained. Transferring a nucleus from a somatic cell to an enucleated oocyte also changes the epigenome to a pluripotent ground state. Somatic cell nuclear transfer was most spectacularly demonstrated with the birth of the cloned sheep Dolly, providing evidence that a transferred somatic cell nucleus from the mammary epithelium of an ewe could be fully reprogrammed to restart development and produce an entire mammal. Reprogramming utilizing transcription factors is nowadays the most powerful technique to obtain large quantities of cells with a pluripotent developmental potential, however. Furthermore, this method is ethically also less controversial because it does not require access to mammalian oocytes.

Developmental biology studies identified many transcription factors that act as master regulators for defining cell fates (Graf and Enver 2009). Forced expression of these master regulators can override determined cell identities and respecify cells to a new cell type. An example is Myf5, whose expression in fibroblasts will induce the formation of contractile muscle cells. In parallel with the respecification, reprogramming of the cell’s epigenome can be observed using biochemical tools. Based on these same principles, Yamanaka and co-workers were able to change a fully differentiated mouse fibroblast back to a pluripotent stem cell (Takahashi and Yamanaka 2006). Mouse embryonic stem cells had been extensively characterized and their transcriptome and underlying regulatory network carefully and comprehensively catalogued. Using this information, they selected a combination of four transcription factors which, when expressed artificially in fibroblasts, reversed the entire differentiation path back to the early pluripotent state. However, this process was very inefficient (less than 1% of the cells became pluripotent, termed induced pluripotent stem cells (iPSC)), already providing a hint that differentiated cellular states are very stable and, only under special circumstances and on rare occasions, revert to another state. The trick was to equip the fibroblast with a fluorescent reporter that was controlled by the Nanog promoter (Nanog + green fluorescent protein (GFP)) which would only become activated when the cell entered the pluripotent state. Subsequently, iPS cells were also derived from human fibroblasts. In the following years, this technique became standard in many laboratories, allowing the production of large quantities of iPS cells from different sources, thereby opening outstanding perspectives for regenerative medicine (see below). Interestingly, over these many years of research, the low efficiency of iPS cells reprogramming could not be substantially improved. This illustrates that the cellular memory encoded in the somatic state of the cell is remarkably stable and highly resistant to induced reprogramming (see book ► Chap. 3 of Paro). Hence, the question is: what are the underlying epigenetic hurdles that need to be removed or remodelled for cell reprogramming?

7.4 Chromatin Dynamics During Reprogramming

Already at the microscopic level, the nuclei of ESC show a different morphology compared to nuclei from differentiated cells. In particular, heterochromatic regions are much more discernible and compact in somatic cells, indicating that one accompanying force of differentiation is the compaction of specific regions of chromatin. This suggests that reprogramming of fates need not only changes local chromatin landscapes to activate or repress different sets of genes, but also requires large scale reshuffling of chromatin domains and chromosomal territories. An example of this is the reactivation of the entire repressed X chromosome in female mammalian cellsFootnote 4 (see book ► Chap. 4 of Wutz). The availability of a technique inducing the reprogramming of somatic chromatin to a pluripotent state, hence, offered a great opportunity to study the underlying processes in substantial detail. The facility of growing cells in culture enabled biochemical studies, using classical transcriptome analysis methods, ChIP, and other chromatin mapping techniques to elucidate changes in the epigenetic landscape during the reprogramming process. The observed complexity of cellular transitions during iPSC induction provide a glimpse of why reprogramming takes a sizeable amount of time and why only few cells manage to reach the pluripotent state. The entire process appears to be stochastic and the individual steps difficult to predict. It is thought that the four transcription factors [Oct4, Sox2, KLF4, and Myc (OSKM)] induce a cascade of changes in the transcription program of the starting cell population. Subsequently, the readjustment of gene expression profiles with the accompanying changes in chromatin and DNA modifications cause: (i) inhibition of somatic regulators, (ii) induction of cellular proliferation, (iii) inhibition of senescence and apoptosis pathways, (iv) activation of pluripotency loci, (v) acquisition of factor independence, (vi) immortalization and finally reprogramming of telomere structures, X chromosome reactivation, and cellular memory erasure (see ◘ Fig. 7.4). The exogenously introduced transcription factors are capable of establishing a self-regulatory loop, eventually activating the corresponding endogenous loci and thereby sustaining a self-propagating stem cell identity. This is a crucial step as exogenously expressed factors become often silenced over time. Additionally, in order to differentiate, iPS cells, as for ESC, need to downregulate pluripotency factors and this can be done only when the endogenous genes are expressed.

Fig. 7.4
figure 4

Dynamics of key molecular events during direct reprogramming. A summary of cellular, transcriptional and epigenetic changes (colored bars) that occur during induced pluripotent stem cell (iPSC) formation from fibroblasts and examples of candidate regulators that have been associated with the depicted chromatin marks in the context of direct reprogramming (right). Red arrows indicate the time points of exogenous factor induction (+OKSM) and withdrawal (−OKSM). 5hmC 5-hydroxymethylcytosine, 5mC 5-methylcytosine, MET mesenchymal-to-epithelial- transition, OKSM Oct4, Klf4, Sox2, c-Myc. (From (Apostolou and Hochedlinger 2013))

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OSKM acting as pioneer factors in the reprogramming process require the support from different epigenetic modifiers including histone post-translational modifying enzymes, nucleosome remodeling factors, histone chaperones, and DNA modifying enzymes (◘ Fig. 7.4).

Initially, OSKM bind to active somatic genes, eventually inducing the change of their active histone modifications like H3K4me1 and H3K27ac into repressive marks. Conversely, genes characteristic for embryonic stages lose their negative histone marks and, subsequently, become reactivated. During the entire process, a massive replacement of histone variants can be observed, either by reactivating the quiescent part of the genome (i.e. H2A.X, TH2A/2B) or by removing repressing variants like macroH2A (see book ► Chap. 1 of Wutz and book ► Chap. 2 of Paro). Similarly, histone chaperones play an import role in the genome-wide realignment of histones and nucleosomes. Inhibition of CAF-1 can, for example, increase the reprogramming efficiency. Differentiation is inherently connected to a specific deposition of CpG dinucleotide methylation (see book ► Chap. 1 of Wutz). The entire DNA methylation and demethylation apparatus is heavily engaged over the length of the reprogramming process. DNA dioxygenases like TET1/2, which are involved in the demethylation process, can partly replace OSKM in chromatin remodeling. Not surprisingly, modulating the amount and activity of chromatin factors (writer, readers, and erasers) has a substantial impact on the efficiency of the reprogramming process. For example, adding the DNA methylation inhibitor 5-aza-cytidine during reprogramming substantially increases the production of iPS cells more than 30-fold. As expected, the reprogramming process does not only lead to a readjustment of the somatic epigenetic landscape to an ESC-like state but also to a complete re-organization of the 3D chromatin architecture and topology (Apostolou and Stadtfeld 2018).

7.5 Regenerative Therapies

Stem cell technologies certainly represent a major breakthrough development in regenerative medicine (Cherry and Daley 2012). The availability of large quantities of cells with a pluripotent developmental capacity provide exceptional opportunities to establish novel cell therapies. Additionally, iPS cells can be derived from patients with specific syndromes and used as models for studying disease. In this way, specific drug screens and diagnostic tests for personalized medicine can be developed. A very exciting development based on stem cells in general and iPS cells in particular is the possibility to create organoid structures in vitro (Akkerman and Defize 2017). Originally, small three-dimensional (3D) tissue structures were produced in vitro using tissue culture cells. In most cases these cells are considered to have a tumorigenic state as they can proliferate indefinitely in the dish and therefore the usefulness of these mini-organs was limited. The availability of self-renewing embryonic stem cells and eventually of iPS cells allowed the development of complex culturing and differentiation conditions, leading to organoid structures of remarkable morphological and functional resemblance to the real organ. Here also, the availability of iPS cells from patients as a source for specific organoid construction allows researchers to study the effect of human syndromes on the early development of the corresponding organ.

Despite the extraordinary enthusiasm accompanying iPSC technology, only few clinical trials have so far tested the medical usefulness of stem cell transplants. As mentioned, normal regenerative processes in almost all complex animals do not involve cells with a pluripotent developmental potential but organ- and tissue-specific multipotent stem and progenitor cells. For good reasons, organisms restrain and keep under strict control cells in a pluripotent state (see germ cell development). Cancer cells often attain and maintain their highly proliferative and destructive capability by dedifferentiation, thereby increasing their epigenetic resemblance to embryonic stem cells (see book ► Chap. 3 of Paro and ► Chap. 8 of Santoro). Incidentally, the first pluripotent cells to be cultivated in vitro were from teratocarcinomas (cancers of the germ line). Hence, there is only a fine line between normal development from a pluripotent state and degenerated development. Indeed, when transplanted into the skin of mice, iPS cells develop into carcinomas, but injection into blastocysts allows them to contribute to normal development. Additionally, it was found that iPS cells do not always erase all epigenetic marks from the somatic state from which they were derived, leaving, for example, distinctive somatic DNA methylation patterns. This imposes on the starting cells an unwanted epigenetic history and may, in certain conditions, lead to aberrant differentiation behaviour, aging onset, or susceptibility to degeneration. Using iPSC derived from the same person to install regenerative processes potentially circumvents the well-known problems of organ rejection. However, the delivery and integration of iPS cells to damaged parts in the organism is still a major challenge.

Second generation iPSC technology is attempting to reduce some of these problems by inducing the reprogramming process in vivo (Martinez-Redondo and Izpisua Belmonte 2020). CRISPR/Cas9 technology produced forms of the Cas9 enzyme which allows the alteration of histone modifications and other epigenetic marks in a site-specific manner. Potentially, this approach could aid the entire reprogramming process to the pluripotent state but also partial reprogramming to intermediate or alternative differentiation states. After all, most known regenerative processes in complex organisms use the latter for repopulating damaged organs or tissues, either by using highly specialized stem cells with a restricted potential or by reassigning necessary new fates to cells at the injury site. Thus, regenerative biology and medicine will need to understand these processes in much better detail in order to be able to use cell therapies in a controllable and predictive fashion.

It is very clear that the epigenetic landscape of cells used in therapies is of crucial importance. Hence, an epigenetic profiling of stem cells used in clinical context appears as an obligatory requirement as is the sequencing of the genome to eliminate cell lineages with potential deleterious mutations. Indeed, technically we are approaching capabilities to comprehensively screen cells for genetic and epigenetic integrity. What we are still lacking is a better knowledge of how natural regenerative processes are able to reprogram the epigenome of somatic cells to the desired organ-specific multipotent state. This would permit to apply cellular therapies in a much more controlled fashion.

Take-Home Message

  • Amphibians and many other animals can regenerate complex body structures through a blastema (epimorphic regeneration). In the blastema, ordered signaling events reprogram cells for patterning and regrowth processes.

  • In regenerating Drosophila imaginal discs, JNK is activated at the wound site and in the blastema, resulting in downregulation of PcG silencing and, as a consequence, re-activation of many silenced PcG target genes, like the homeotic master regulators.

  • Dedifferentiation and transdifferentiation of cells/tissues is also utilized in vertebrates to produce cells with structures and functions necessary to regenerate a wound or an organ damage in their neighborhood.

  • Organ-specific adult stem cells contribute to physiological regeneration required for tissue and organs, like gut or skin, subjected to constant mechanical and chemical stress. They undergo specific differentiation programs and extended epigenetic reprogramming to maintain tissue homeostasis.

  • Hematopoietic stem cells are used in the clinic to treat leukemia patients. Stem cells from other organs are difficult to isolate and cultivate, requiring specific conditions found in stem cell niches.

  • Pluripotent stem cells can be induced to differentiate into any other cell type. iPSC technology reprograms differentiated cells to induced pluripotent stem cells. Transcription factor-driven reprogramming encompasses numerous pathways requiring reversal of many epigenetic processes.

  • Some of the inefficiency of iPSC reprogramming can be explained by the need to change the cellular memory system of differentiated cells to the embryonic state. A highly coordinated and sequential erasure of chromatin marks and DNA methylation patterns must occur during reprogramming.

  • iPS cells can be used for cell therapies. Patient-derived iPS cells can be used to study disease or for the development of new drugs. A promising new path is the engineering of self-organizing organoid structures using iPS cell cultures.