, Volume 14, Issue 6, pp 603–608

Stress cycles in stem cells/iPSCs development: implications for tissue repair


    • French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert ResearchBen-Gurion University of the Negev
Review article

DOI: 10.1007/s10522-013-9445-4

Cite this article as:
Grafi, G. Biogerontology (2013) 14: 603. doi:10.1007/s10522-013-9445-4


Stem cells have become a major topic, both publicly and scientifically, owing to their potential to cure diseases and repair damaged tissues. Particular attention has been given to the so-called “induced pluripotent stem cells” (iPSCs) in which somatic cells are induced by the expression of transcription factor encoding transgenes—a methodology first established by Takahashi and Yamanaka (Cell 126:663–676, 2006)—to acquire pluripotent state. This methodology has captured researchers’ imagination as a potential procedure to obtain patient-specific therapies while also solving both the problem of transplant rejection and the ethical concerns often raised regarding the use of embryonic stem cells in regenerative medicine. The study of the biology of stem cells/iPSCs, in recent years, has uncovered some fundamental weaknesses that undermine their potential use in transplantation therapies.


Induced pluripotent stem cellsDedifferentiationStress-induced dedifferentiationStress cyclesAgingChromatinTransposable elements



Induced pluripotent stem cells


Embryonic stem cells


Hematopoietic stem cells


Cyclin dependent kinase


Transposable elements


Hematopoietic stem cells


Long-interspersed nuclear element-1


Ultra violet

What are stem cells?

A major problem in stem cell biology is the definition of stem cells by developmental means, namely, self-renewal and multitype differentiation, rather than by their inherent features (Potten and Loeffler 1990; McKay 2000). This has often led to the erroneous assumption that the reentry of stem cells into the cell cycle for the purpose of “self-renewal” represents an inherent feature of stem cells. Consequently, the idea of a “stem cell culture” has been established, leading biologists to incorrectly assume that stem cell features can be fully maintained under culture conditions (Grafi and Avivi 2004). As a result, the attempts to uncover the “stem cell signature” or the “stemness genes” via transcriptome analysis of different stem cell culture lines have failed as these experiments yielded different “signatures” and non-overlapping “stemness genes” (Ramalho-Santos et al. 2002; Ivanova et al. 2002; Fortunel et al. 2003). Thus, when stem cells enter the cell cycle, either for the purpose of “self renewal” or for establishing the so-called “stem cell culture”, they lose their identity and they are no longer stem cells.

Contrary to the idea that stem cells represent a unique cell type characterized by the expression of a specific set of “stemness” genes, it has been suggested that stem cells represent a unique transient state characterized by the promiscuous expression of marker/transcription factor genes (Zipori 2004). It appears that many differentiation/lineage-specific genes, which are not expressed or expressed at a very low level, assume a transcriptionally competent chromatin state (Zipori 2004; Meshorer and Misteli 2006; Efroni et al. 2008). This possibility is supported by the fact that stem cells, like dedifferentiating cells, acquire an open, decondensed chromatin architecture, which is essential, though not sufficient, for initiating gene transcription (Zhao et al. 2001; Williams et al. 2003; Grafi 2004; Gaspar-Maia et al. 2011). It also gains support from the finding that some non-expressed genes or genes expressed at low levels in ESCs are primed, but their transcription is attenuated epigenetically by a unique combination of permissive and restrictive chromatin marks (Azuara et al. 2006; Bernstein et al. 2006). An apparently open chromatin configuration appears to be a fundamental feature of stem cells that provides a chromatin environment, which is susceptible to genomic restructuring.

Stress cycles in stem cell/iPSC development

The study of stem cells/iPSCs uncovers their weaknesses and susceptibility to hazardous genomic modifications, which could challenge their potential use in regenerative medicine. Animal embryonic stem cells, as well as iPSCs, were shown to acquire genomic and epigenomic abnormalities in culture, which often leads to malignant transformations (Lefort et al. 2009; Mayshar et al. 2010; Ben-David and Benvenisty 2011; Lund et al. 2012; Nguyen et al. 2013). These genetic and epigenetic aberrations may be related to the source from which the somatic cells for reprogramming were derived or may originate from transgene-induced insertional mutagenesis or during cell culturing processes. A fundamental aspect in stem cell/iPSCs biology that is often overlooked is the way these cells are handled, manipulated and processed and the implications for cell viability. The procedure commonly employed for the generation of iPSCs or for the establishment of a “stem cell culture” is extremely stressful and prone to hazardous genomic modifications. At almost every step in the course of their development, these cells undergo cycles of exposure to stress conditions that might have hazardous consequences (Fig. 1). “Stress” refers to the sum of cell responses elicited by any kind of external/environmental signal/agent or methodology that forces the cell to change behavior or fate. Barbra McClintock (1984) recognized the potential for hazardous genetic variation that can be induced following exposure of cells to stress, such as cell culturing and viral infection. McClintock predicted that abnormal genome responses to stress are likely to be driven by the activation of transposable elements, followed by their transposition into other chromosomal sites. This might explain, at least partly, why various dedifferentiation-driven processes in animals, namely, somatic cell nuclear transfer, as well as iPSCs, have very low rates of success (Tamada and Kikyo 2004; William and Plath 2008). Indeed, it has been shown that reprogramming to pluripotent stem cells is impaired following expression of the four reprogramming factors due to induction of senescence demonstrated by upregulation of cyclin dependent kinase (CDK) inhibitors p16INK4a and p21CIP1 and p53—a tumor suppressor that ensures genome integrity via the induction of cell cycle arrest or apoptosis in damaged cells (Banito et al. 2009); an increased rate of success was observed in cells deficient in p53 (Hong et al. 2009; Utikal et al. 2009; Marión et al. 2009; Li et al. 2009; Kawamura et al. 2009; Banito et al. 2009). This further demonstrated that reprogrammed cells have accumulated hazardous genetic variations, leading to a p53-mediated DNA damage response and p53-dependent apoptosis (Marión et al. 2009; Krizhanovsky and Lowe 2009). Although failure in cell reprogramming is believed to be selective for cells with preexisting DNA damage, it is likely that DNA damage or deleterious mutations accumulate in cells (e.g., primary mouse embryonic fibroblasts) due to their exposure to cycles of stress conditions in the course of their development into iPSCs and further processing for cell transplantation (Fig. 1).
Fig. 1

The stress cycle in stem cell and iPSC development. The removal of somatic cells from the body is stressful and might induce dedifferentiation (reprogramming) accompanied by genetic variation induced by DNA transposition/recombination prior to reentry of cells into the cell cycle. All further manipulation imposed on the cells, including the induction of reprogramming via transgene integration and non-transgene methods, the cell culturing of stem cells/iPSCs, in vitro differentiation and finally transplantation of cells, are stressful, further affecting genome integrity and stability. Conceivably, the genotype of transplanted cells has been modified extensively and is different from the genotype of the original stem cells/somatic cells. Modified from Grafi et al. (2011a)

With regard to the above, it is worth mentioning that almost half of the human genome is composed of TEs, which have a profound effect on genome structure and function. The mobility of TE and inter-TE recombinations are thought to contribute to a large spectrum of mutations and genome reorganization leading to diseases (Deragon and Capy 2000; Schulz et al. 2006). Accordingly, whole-genome profiles of DNA methylation of several human iPSC lines showed the aberrant reprogramming of DNA methylation, particularly in regions proximal to centromeres and telomeres, which are often rich in TEs (Lister et al. 2011).

Stress response meets chromatin

A fundamental theme in adaptation to an ever-changing environment is plasticity, that is, the inherent ability of an organism to change. At the cellular level, much of the response to stress is mediated via changes in chromatin structure and function (Smith and Workman 2012). The structure of chromatin is flexible due to multiple types of reversible chemical modifications that occur on the DNA (cytosine methylation) or on the DNA-interacting core histone proteins. Most modifications occur on multiple amino acid residues at the N-terminal tails of histone proteins and include acetylation, methylation and phosphorylation.

The reversing of DNA and histone modifications, which is associated with chromatin remodeling, both in plants and animals, often occurs following exposure to various biotic and abiotic stress conditions—a topic covered by multiple recent reviews (Kim et al. 2010; Luo et al. 2012; Smith and Workman 2012; Tsai and Wu 2013). Accumulated data support the hypothesis that plant cells may respond to various environmental cues by undergoing dedifferentiation, characterized by chromatin decondensation (reviewed in Grafi et al. 2011b). A special case is the demonstration that plant somatic cells induced to senesce by long exposure to dark acquired stem cell features characterized by open chromatin conformation and overrepresentation of transcription factors (Damri et al. 2009; Florentin et al. 2013). This suggests that senescence represents a unique, transient developmental stage resembling dedifferentiation that occurs prior to a switch in cell fate (e.g., cell cycle or death). Stress-induced dedifferentiation and chromatin relaxation is not unique to plant cells and has also been reported in human cells exposed to oxidative stress (paraquat), UV light and hydrogen peroxide (Abrahan et al. 2009; Halicka et al. 2009). Indeed, it has recently been proposed that mammalian somatic cells may undergo cell dedifferentiation as an adaptation for extreme stress conditions (Shoshani and Zipori 2011).

Stress-induced open chromatin conformation, the chromatin environment necessary for the establishment of the stem cell state, is vulnerable to mutations that can be induced by environmental factors, such as UV radiation, as well as by the activation of potentially hazardous nuclear reactions, such as TE transposition and recombination. Indeed, LINE-1, which was activated during salamander limb regeneration was suggested to serve as a marker for cell dedifferentiation during early stages of limb regeneration (Zhu et al. 2012). Thus, following exposure to acute stress (e.g., genotoxic stress, cell culturing, limb amputation), the genome reacts through extensive epigenetic modifications and chromatin reorganization that may release epigenetic constraints over TEs, resulting in their activation and transposition into other chromosomal sites (Farkash and Luning Prak 2006; Oliver and Greene 2009; Baccarelli et al. 2009), leading to genetic instability and human diseases, including genetic disorders and various forms of cancers (reviewed in Belancio et al. 2009; Moskalev et al. 2013). Stress-induced TE mobilization might have implications for aging as first hypothesized by Murray (1990). Accordingly, transposition of mobile elements into essential genes was hypothesized to lead to death of cells or organism or to malfunction of cells and aging. More recently, several reviews have highlighted the potential deleterious effects of LINE-1 on genome integrity and stability and consequently on aging (St. Laurent et al. 2010; Moskalev et al. 2013). Notably, the expression and transposition of Alu and LINE-1 retroelements were reported in cultured human ESCs and in human neural progenitor cells (Garcia-Perez et al. 2007; Coufal et al. 2009; Macia et al. 2011). This might lead to reduction in cell function and to stem cell aging, which might challenge their use in regenerative medicine.

Concluding remarks

Obviously, differentiated cells are not necessarily terminally differentiated but retain their developmental potentialities and have the inherent capacity for switching fate, as long as their genome is intact. Thus, why to manipulate (and stress) somatic cells through exogenous genes instead of activating endogenous ones to bring about reprogramming? Accumulating data suggest that the reprogramming of primary cells does not require the “four factors” (OCT4, SOX2, KLF4, c-Myc), first demonstrated by Takahashi and Yamanaka (2006), and can be achieved with only one factor OCT4 (Kim et al. 2009) or without any of those factors via incubation of primary cells with an extract of undifferentiated cells (Taranger et al. 2005) or alternatively by somatic cell nuclear transfer (reviewed in Gurdon and Byrne 2003). It has been shown that pretreatment of somatic cells with chromatin modifier inhibitors to DNA methyltransferase and histone deacetylase can facilitate reprogramming and the formation of hESC-like colonies by embryonic stem cell extracts (Han et al. 2010). However, as demonstrated in Fig. 1, the existing methodologies for generating iPSCs and their descending cells for transplantation therapies are highly risky as manipulated cells have reduced ability to cope with repeated cycles of stress conditions and thus accumulate hazardous genetic variations that leads to cell aging (reviewed in Moskalev et al. 2012). This casts doubt on the suitability of these cells for use in regenerative medicine. Thus, for the safe use of stem cells, it is crucial to avoid the implementation of repeated cycles of stress over these cells (e.g., cell culturing, transgene integration etc.) and to select the most suitable method for basic or clinical applications (Gonzalez et al. 2011). Accordingly, a relatively successful procedure of stem cell application for curing diseases, namely, the transplantation of HSCs, does not involve tissue culturing but rather careful freezing (cryopreservation) of the cells for the purpose of storage for prolonged periods. Considering that hematopoietic stem cells are pluripotent, capable of giving rise to almost all cell types of the body (Kanji et al. 2011; Ogawa et al. 2013), a possible approach for safe tissue repair is a direct transplantation of autologous/heterologous HSCs into the damaged tissue (“live” cell therapy, Mezey 2011) without further manipulation by cell culturing or even freezing. An alternative method is to imitate natural rejuvenation processes, that is, the possibility of inducing dedifferentiation and the pluripotent state in somatic cells in vivo (Abramovich et al. 2008).

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© Springer Science+Business Media Dordrecht 2013