The aging signature: a hallmark of induced pluripotent stem cells?

The discovery that somatic cells can be induced into a pluripotent state by the expression of reprogramming factors has enormous potential for therapeutics and human disease modeling. With regard to aging and rejuvenation, the reprogramming process resets an aged, somatic cell to a more youthful state, elongating telomeres, rearranging the mitochondrial network, reducing oxidative stress, restoring pluripotency, and making numerous other alterations. The extent to which induced pluripotent stem cell (iPSC)s mime embryonic stem cells is controversial, however, as iPSCs have been shown to harbor an epigenetic memory characteristic of their tissue of origin which may impact their differentiation potential. Furthermore, there are contentious data regarding the extent to which telomeres are elongated, telomerase activity is reconstituted, and mitochondria are reorganized in iPSCs. Although several groups have reported that reprogramming efficiency declines with age and is inhibited by genes upregulated with age, others have successfully generated iPSCs from senescent and centenarian cells. Mixed findings have also been published regarding whether somatic cells generated from iPSCs are subject to premature senescence. Defects such as these would hinder the clinical application of iPSCs, and as such, more comprehensive testing of iPSCs and their potential aging signature should be conducted.     

In vitro models of multiple system atrophy from primary cells to induced pluripotent stem cells

Multiple system atrophy (MSA) is a rare neurodegenerative disease with a fatal outcome. Nowadays, only symptomatic treatment is available for MSA patients. The hallmarks of the disease are glial cytoplasmic inclusions (GCIs), proteinaceous aggregates mainly composed of alpha‐synuclein, which accumulate in oligodendrocytes. However, despite the extensive research efforts, little is known about the pathogenesis of MSA. Early myelin dysfunction and alpha‐synuclein deposition are thought to play a major role, but the origin of the aggregates and the causes of misfolding are obscure. One of the reasons for this is the lack of a reliable model of the disease. Recently, the development of induced pluripotent stem cell (iPSC) technology opened up the possibility of elucidating disease mechanisms in neurodegenerative diseases including MSA. Patient specific iPSC can be differentiated in glia and neurons, the cells involved in MSA, providing a useful human disease model. Here, we firstly review the progress made in MSA modelling with primary cell cultures. Subsequently, we focus on the first iPSC‐based model of MSA, which showed that alpha‐synuclein is expressed in oligodendrocyte progenitors, whereas its production decreases in mature oligodendrocytes. We then highlight the opportunities offered by iPSC in studying disease mechanisms and providing innovative models for testing therapeutic strategies, and we discuss the challenges connected with this technique.     

iPSC technology-Powerful hand for disease modeling and therapeutic screen

Cardiovascular and neurodegenerative diseases are major health threats in many developed countries. Recently, target tissues derived from human embryonic stem (hES) cells and induced pluripotent stem cells (iPSCs), such as cardiomyocytes (CMs) or neurons, have been actively mobilized for drug screening. Knowledge of drug toxicity and efficacy obtained using stem cell-derived tissues could parallel that obtained from human trials. Furthermore, iPSC disease models could be advantageous in the development of personalized medicine in various parts of disease sectors. To obtain the maximum benefit from iPSCs in disease modeling, researchers are now focusing on aging, maturation, and metabolism to recapitulate the pathological features seen in patients. Compared to pediatric disease modeling, adult-onset disease modeling with iPSCs requires proper maturation for full manifestation of pathological features. Herein, the success of iPSC technology, focusing on patient-specific drug treatment, maturation-based disease modeling, and alternative approaches to compensate for the current limitations of patient iPSC modeling, will be further discussed. [BMB Reports 2015; 48(5): 256-265]     

Establishment of induced pluripotent stem cells from centenarians for neurodegenerative disease research

Induced pluripotent stem cell (iPSC) technology can be used to model human disorders, create cell-based models of human diseases, including neurodegenerative diseases, and in establishing therapeutic strategies. To detect subtle cellular abnormalities associated with common late-onset disease in iPSCs, valid control iPSCs derived from healthy donors free of serious late-onset diseases are necessary. Here, we report the generation of iPSCs from fibroblasts obtained immediately postmortem from centenarian donors (106- and 109-years-old) who were extremely healthy until an advanced age. The iPSCs were generated using a conventional method involving OCT4, SOX2, KLF4, and c-MYC, and then differentiated into neuronal cells using a neurosphere method. The expression of molecules that play critical roles in late-onset neurodegenerative diseases by neurons differentiated from the centenarian-iPSCs was compared to that of neurons differentiated from iPSCs derived from familial Alzheimer's disease and familial Parkinson's disease (PARK4: triplication of the α synuclein gene) patients. The results indicated that our series of iPSCs would be useful in neurodegeneration research. The iPSCs we describe, which were derived from donors with exceptional longevity who were presumed to have no serious disease risk factors, would be useful in longevity research and as valid super-controls for use in studies of various late-onset diseases.     

iPS cells: a game changer for future medicine

The induced pluripotent stem cell (iPSC) technology is instrumental in advancing the fields of disease modeling and cell transplantation. We herein discuss the various issues regarding disease modeling and cell transplantation presented in previous reports, and also describe new iPSC‐based medicine including iPSC clinical trials. In such trials, iPSCs from patients can be used to predict drug responders/non‐responders by analyzing the efficacy of the drug on iPSC‐derived cells. They could also be used to stratify patients after actual clinical trials, including those with sporadic diseases, based on the drug responsiveness of each patient in the clinical trials. iPSC‐derived cells can be used for the identification of response markers, leading to increased success rates in such trials. Since iPSCs can be used in micromedicine for drug discovery, and in macromedicine for actual clinical trials, their use would tightly connect both micro‐ and macromedicine. The use of iPSCs in disease modeling, cell transplantation, and clinical trials could therefore lead to significant changes in the future of medicine.     

Reprogramming fibroblasts and peripheral blood cells from a C9ORF72 patient: A proof-of-principle study

As for the majority of neurodegenerative diseases, pathological mechanisms of amyotrophic lateral sclerosis (ALS) have been challenging to study due to the difficult access to alive patients' cells. Induced pluripotent stem cells (iPSCs) offer a useful in vitro system for modelling human diseases. iPSCs can be theoretically obtained by reprogramming any somatic tissue although fibroblasts (FB) remain the most used cells. However, reprogramming peripheral blood cells (PB) may offer significant advantages. In order to investigate whether the choice of starting cells may affect reprogramming and motor neuron (MNs) differentiation potential, we used both FB and PB from a same C9ORF72-mutated ALS patient to obtain iPSCs and compared several hallmarks of the pathology. We found that both iPSCs and MNs derived from the two tissues showed identical properties and features and can therefore be used interchangeably, giving the opportunity to easily obtain iPSCs from a more manageable source of cells, such as PB.     

The tumourigenicity of iPS cells and their differentiated derivates

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC‐derivates), iPSC‐derivated cardiomyocyte (iPSC‐CMs) were subcutaneously injected into the back of nude mice. Non‐invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC‐derivates and iPSC‐CMs survived in receipts. Both iPSCs and iPSC‐derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC‐CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC‐derivates transplanted mice, but not in iPSC‐CM transplanted ones. In vitro study showed the long‐term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC‐like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage‐specific differentiation is necessary prior to therapeutic use of iPSCs.     

Mechanism and methods to induce pluripotency

Pluripotent stem cells are able to self-renew indefinitely and differentiate into all types of cells in the body. They can thus be an inexhaustible source for future cell transplantation therapy to treat degenerative diseases which currently have no cure. However, non-autologous cells will cause immune rejection. Induced pluripotent stem cell (iPSC) technology can convert somatic cells to the pluripotent state, and therefore offers a solution to this problem. Since the first generation of iPSCs, there has been an explosion of relevant research, from which we have learned much about the genetic networks and epigenetic landscape of pluripotency, as well as how to manipulate genes, epigenetics, and microRNAs to obtain iPSCs. In this review, we focus on the mechanism of cellular reprogramming and current methods to induce pluripotency. We also highlight new problems emerging from iPSCs. Better understanding of the fundamental mechanisms underlying pluripotenty and refining the methodology of iPSC generation will have a significant impact on future development of regenerative medicine.     

Induced pluripotent stem cells’ self-renewal and pluripotency is maintained by a bovine granulosa cell line-conditioned medium

Induced pluripotent stem cells (iPSCs) are a promising type of stem cells, comparable to embryonic stem cells (ESCs) in terms of self-renew and pluripotency, generated by reprogramming somatic cells. These cells are an attractive approach to supply patient-specific pluripotent cells, for producing in vitro models of disease, drug discovery, toxicology and potentially treating degenerative disease circumventing immune rejection. In spite of the great advance since iPSCs’ establishment, their obtention and propagation is an increasing area of great interest.In a recent work, we have shown that the conditioned medium from a bovine granulosa cell line (BGC-CM) is able to preserve the basic properties of mESCs. Therefore, based on our previous results and the reported resemblance between iPSCs and ESCs, we hypothesized that BGC-CM could provide a favorable context to culturing iPSCs. In this work, we have reprogrammed mouse embryonic fibroblasts obtaining iPSC lines, and showed that they can be propagated in BGC-CM while maintaining self-renewal and pluripotency, evidenced by expression of specific gene markers and capability of in vitro and in vivo differentiation to cell types from the three germ layers. We believe that these findings may provide a novel context to propagate iPSCs to study the molecular mechanisms involved in self-renewal and pluripotency.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.     

New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood

Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.     

Defining pluripotent stem cells through quantitative proteomic analysis

Embryonic stem cells (ESCs) are at the center stage of intense research, inspired by their potential to give rise to all cell types of the adult individual. This property makes ESCs suitable candidates for generating specialized cells to replace damaged tissue lost after injury or disease. However, such clinical applications require a detailed insight of the molecular mechanisms underlying the self-renewal, expansion and differentiation of stem cells. This has gained further relevance since the introduction of induced pluripotent stem cells (iPSCs), which are functionally very similar to ESCs. The key property that iPSCs can be derived from somatic cells lifts some of the major ethical issues related to the need for embryos to generate ESCs. Yet, this has only increased the need to define the similarity of iPSCs and ESCs at the molecular level, both before and after they are induced to differentiate. In this article, we describe the proteomic approaches that have been used to characterize ESCs with regard to self-renewal and differentiation, with an emphasis on signaling cascades and histone modifications. We take this as a lead to discuss how quantitative proteomics can be deployed to study reprogramming and iPSC identity. In addition, we discuss how emerging proteomic technologies can become a useful tool to monitor the (de)differentiation status of ESCs and iPSCs.     

The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.     

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.