The Effects of Nuclear Reprogramming on Mitochondrial DNA Replication

Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers.     

Cyclin a(1) is essential for setting the pluripotent state and reducing tumorigenicity of induced pluripotent stem cells

The proper differentiation and threat of cancer rising from the application of induced pluripotent stem (iPS) cells are major bottlenecks in the field and are thought to be inherently linked to the pluripotent nature of iPS cells. To address this question, we have compared iPS cells to embryonic stem cells (ESCs), the gold standard of ground state pluripotency, in search for proteins that may improve pluripotency of iPS cells. We have found that when reprogramming somatic cells toward pluripotency, 1%-5% of proteins of 5 important cell functions are not set to the correct expression levels compared to ESCs, including mainly cell cycle proteins. We have shown that resetting cyclin A(1) protein expression of early-passage iPS cells closer to the ground state pluripotent state of mouse ESCs improves the pluripotency and reduces the threat of cancer of iPS cells. This work is a proof of principle that reveals that setting expression of certain proteins correctly during reprogramming is essential for achieving ESC-state pluripotency. This finding would be of immediate help to those researchers in different fields of iPS cell work that specializes in cell cycle, apoptosis, cell adhesion, cell signaling, and cytoskeleton.     

De novo reestablishment of gap junctional intercellular communications during reprogramming to pluripotency and differentiation

Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ESCs) and various somatic cells. GJIC has been implicated in the regulation of cell proliferation, self-renewal, and differentiation. Recently, a new type of pluripotent stem cells was generated by direct reprogramming of somatic cells. Here, for the first time, we show that during reprogramming events GJIC is re-established upon reaching complete reprogramming. The opposite process of cell differentiation from the pluripotent state leads to the disruption of GJIC between pluripotent and differentiated cell subsets. However, GJIC is subsequently re-established de novo within each differentiated cell type in vitro, forming communication compartments within a histotype. Our results provide the important evidence that reestablisment of functional gap junctions to the level similar to human ESCs is an additional physiological characteristic of somatic cell reprogramming to the pluripotent state and differentiation to the specific cell type.     

Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles

Spermatogonial stem cells (SSCs) isolated from the adult mouse testis and cultured have been shown to respond to culture conditions and become pluripotent, so called multipotent adult germline stem cells (maGSCs). microRNAs (miRNAs) belonging to the 290 and 302 miRNA clusters have been previously classified as embryonic stem cell (ESC) specific. Here, we show that these miRNAs generally characterize pluripotent cells. They are expressed not only in ESCs but also in maGSCs as well as in the F9 embryonic carcinoma cell (ECC) line. In addition, we tested the time-dependent influence of different factors that promote loss of pluripotency on levels of these miRNAs in all three pluripotent cell types. Despite the differences regarding time and extent of differentiation observed between ESCs and maGSCs, expression profiles of both miRNA families showed similarities between these two cell types, suggesting similar underlying mechanisms in maintenance of pluripotency and differentiation. Our results indicate that the 290-miRNA family is connected with Oct-4 and maintenance of the pluripotent state. In contrast, members of the 302-miRNA family are induced during first stages of in vitro differentiation in all cell types tested. Therefore, detection of miRNAs of miR-302 family in pluripotent cells can be attributed to the proportion of spontaneously differentiating cells in cultures of pluripotent cells. These results are consistent with ESC-like nature of maGSCs and their potential as an alternative source of pluripotent cells from non-embryonic tissues.     

Mitochondrial DNA integrity is essential for mitochondrial maturation during differentiation of neural stem cells

Differentiation of neural stem cells (NSCs) involves the activation of aerobic metabolism, which is dependent on mitochondrial function. Here, we show that the differentiation of NSCs involves robust increases in mitochondrial mass, mitochondrial DNA (mtDNA) copy number, and respiration capacity. The increased respiration activity renders mtDNA vulnerable to oxidative damage, and NSCs defective for the mitochondrial 8-oxoguanine DNA glycosylase (OGG1) function accumulate mtDNA damage during the differentiation. The accumulated mtDNA damages in ogg1(-/-) cells inhibit the normal maturation of mitochondria that is manifested by reduced cellular levels of mitochondrial encoded complex proteins (complex I [cI], cIII, and cIV) with normal levels of the nuclear encoded cII present. The specific cI activity and inner membrane organization of respiratory complexes are similar in wt and ogg1(-/-) cells, inferring that mtDNA damage manifests itself as diminished mitochondrial biogenesis rather than the generation of dysfunctional mitochondria. Aerobic metabolism increases during differentiation in wild-type cells and to a lesser extent in ogg1(-/-) cells, whereas anaerobic rates of metabolism are constant and similar in both cell types. Our results demonstrate that mtDNA integrity is essential for effective mitochondrial maturation during NSC differentiation.     

Identification of Specific Pluripotent Stem Cell Death—Inducing Small Molecules by Chemical Screening

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells, free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death, which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore, we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly, three compounds currently used as clinically approved drugs, nortriptyline, benzethonium chloride and methylbenzethonium chloride, induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs, whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline, a tricyclic antidepressant, has also been described as a potent inhibitor of mitochondrial permeability transition, one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline, although with a different behavior. Our results indicate differential sensitivity of ESCs, hiPSCs and fibroblasts to certain chemical compounds, which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases.     

Gene expression signatures defining fundamental biological processes in pluripotent, early, and late differentiated embryonic stem cells

Investigating the molecular mechanisms controlling the in vivo developmental program postembryogenesis is challenging and time consuming. However, the developmental program can be partly recapitulated in vitro by the use of cultured embryonic stem cells (ESCs). Similar to the totipotent cells of the inner cell mass, gene expression and morphological changes in cultured ESCs occur hierarchically during their differentiation, with epiblast cells developing first, followed by germ layers and finally somatic cells. Combination of high throughput -omics technologies with murine ESCs offers an alternative approach for studying developmental processes toward organ-specific cell phenotypes. We have made an attempt to understand differentiation networks controlling embryogenesis in vivo using a time kinetic, by identifying molecules defining fundamental biological processes in the pluripotent state as well as in early and the late differentiation stages of ESCs. Our microarray data of the differentiation of the ESCs clearly demonstrate that the most critical early differentiation processes occur at days 2 and 3 of differentiation. Besides monitoring well-annotated markers pertinent to both self-renewal and potency (capacity to differentiate to different cell lineage), we have identified candidate molecules for relevant signaling pathways. These molecules can be further investigated in gain and loss-of-function studies to elucidate their role for pluripotency and differentiation. As an example, siRNA knockdown of MageB16, a gene highly expressed in the pluripotent state, has proven its influence in inducing differentiation when its function is repressed.     

5-Aminoimidazole-4-carboxyamide ribonucleoside induces G(1)/S arrest and Nanog downregulation via p53 and enhances erythroid differentiation

Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK), a key regulator for controlling energy metabolism, is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest, without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers, Nanog and stage-specific embryonic antigen-1, in the presence of leukemia inhibitory factor, without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently, an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation, an effect inhibited by MG132, a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs, it increased expression levels of erythroid cell lineage markers (Ter119, GATA1, Klf1, Hbb-b, and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR, endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs.     

Delta40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs

Δ40p53 is a transactivation-deficient isoform of the tumor suppressor p53. We discovered that Δ40p53, in addition to being highly expressed in embryonic stem cells (ESCs), is the major p53 isoform during early stages of embryogenesis in the mouse. By altering the dose of Δ40p53 in ESCs, we identified a critical role for this isoform in maintaining the ESC state. Haploinsufficiency for Δ40p53 causes a loss of pluripotency in ESCs and acquisition of a somatic cell cycle, while increased dosage of Δ40p53 prolongs pluripotency and inhibits progression to a more differentiated state. Δ40p53 controls the switch from pluripotent ESCs to differentiated somatic cells by controlling the activity of full-length p53 at critical targets such as Nanog and the IGF-1 receptor (IGF-1R). The IGF axis plays a central role in the switch between pluripotency and differentiation in ESCs-and Δ40p53, by controlling the level of the IGF-1R, acts as a master regulator of this switch. We propose that this is the primary function of Δ40p53 in cells of the early embryo and stem cells, which are the only normal cells in which this isoform is expressed.     

Empowering self-renewal and differentiation: the role of mitochondria in stem cells

Stem cells are characterized by their multi-lineage differentiation potential (pluripotency) and their ability for self-renewal, which permits them to proliferate while avoiding lineage commitment and senescence. Recent studies demonstrate that undifferentiated, pluripotent stem cells display lower levels of mitochondrial mass and oxidative phosphorylation, and instead preferentially use non-oxidative glycolysis as a major source of energy. Hypoxia is a potent suppressor of mitochondrial oxidation and appears to promote “stemness” in adult and embryonic stem cells. This has lead to an emerging paradigm, that mitochondrial oxidative metabolism is not just an indicator of the undifferentiated state of stem cells, but may also regulate the pluripotency and self-renewal of stem cells. The identification of specific mitochondrial pathways that regulate stem cell fate may therefore enable metabolic programming and reprogramming of stem cells.     

Sirt1, p53, and p38(MAPK) are crucial regulators of detrimental phenotypes of embryonic stem cells with Max expression ablation

c-Myc participates in diverse cellular processes including cell cycle control, tumorigenic transformation, and reprogramming of somatic cells to induced pluripotent cells. c-Myc is also an important regulator of self-renewal and pluripotency of embryonic stem cells (ESCs). We recently demonstrated that loss of the Max gene, encoding the best characterized partner for all Myc family proteins, causes loss of the pluripotent state and extensive cell death in ESCs strictly in this order. However, the mechanisms and molecules that are responsible for these phenotypes remain largely obscure. Here, we show that Sirt1, p53, and p38(MAPK) are crucially involved in the detrimental phenotype of Max-null ESCs. Moreover, our analyses revealed that these proteins are involved at varying levels to one another in the hierarchy of the pathway leading to cell death in Max-null ESCs.     

Knockdown of IKK1/2 Promotes Differentiation of Mouse Embryonic Stem Cells into Neuroectoderm at the Expense of Mesoderm

Activation of nuclear factor kappa B (NF-??B) is accomplished by a specific kinase complex (IKK-complex), phosphorylating inhibitors of NF-??B (I??B). In embryonic stem cells (ESCs), NF-??B signaling causes loss of pluripotency and promotes differentiation towards a mesodermal phenotype. Here we show that NF-??B signaling is involved in cell fate determination during retinoic acid (RA) mediated differentiation of ESCs. Knockdown of IKK1 and IKK2 promotes differentiation of ESCs into neuroectoderm at the expense of neural crest derived myofibroblasts. Our data indicate that RA is not only able to induce neuronal differentiation in vitro but also drives ESCs into a neural crest cell lineage represented by differentiation towards peripheral neurons and myofibroblasts. The NC is a transiently existing, highly multipotent embryonic cell population generating a wide range of different cell types. During embryonic development the NC gives rise to distinct precursor lineages along the anterior-posterior axis determining differentiation towards specific derivates. Retinoic acid (RA) signaling provides essential instructive cues for patterning the neuroectoderm along the anterior-posterior axis. The demonstration of RA as a sufficient instructive signal for the differentiation of pluripotent cells towards NC and the involvement of NF-??B during this process provides useful information for the generation of specific NC-lineages, which are valuable for studying NC development or disease modeling.     

Reduced oxygen concentration enhances conversion of embryonic stem cells to epiblast stem cells

Recently, an additional type of pluripotent stem cell-line derived from mouse embryos has been established and termed epiblast stem cell (EpiSC), and is expected to be an important tool for studying the mechanisms of maintenance of pluripotency since they depend on basic fibroblast growth factor-MAPK and Activin A-Smad2/3 signaling to maintain pluripotency, unlike mouse embryonic stem cells (ESCs). Further, because of the similarities between mouse EpiSCs and human ESCs, EpiSCs are expected to be effective experimental models for human stem cell therapy. Recently, study for conversion from ESC state to EpiSC state or reversion from EpiSC state to ESC state has attracted interest since these techniques may lead to increasing the potential of pluripotent stem cells and our knowledge about their developmental status. In the present study, we find that a low oxygen concentration in culture environment accelerated, improved, and stabilized the EpiSC state of the converted cells from the ESC state using Oct4ΔPE-GFP transgenic ESCs. Induced EpiSCs (iEpiSCs) in hypoxia possess closer gene expression patterns to native EpiSCs, and bisulfite sequences for the promoter regions of Stella and Oct4 genes have elucidated that the iEpiSC gain EpiSC-specific methylation patterns in hypoxia. Our data provide evidence that oxygen concentration is an important factor for establishment of the EpiSC-specific state.     

DNA hypermethylation in somatic cells correlates with higher reprogramming efficiency

The efficiency of somatic cell reprogramming to pluripotency using defined factors is dramatically affected by the cell type of origin. Here, we show that human keratinocytes, which can be reprogrammed at a higher efficiency than fibroblast [Nat Biotechnol 2008;26:1276-1284], share more genes hypermethylated at CpGs with human embryonic stem cells (ESCs) than other somatic cells frequently used for reprogramming. Moreover, pluripotent cells reprogrammed from keratinocytes (KiPS) are more similar to ESCs than those reprogrammed from fibroblasts (FiPS) in regard to DNA methylation levels, mostly due to the presence of genes that fail to acquire high levels of DNA methylation in FiPS cells. We propose that higher reprogramming efficiency correlates with the hypermethylation of tissue-specific genes rather than with a more permissive pluripotency gene network.     

Identification of developmental pluripotency associated 5 expression in human pluripotent stem cells

Pluripotent embryonic germ cells (EGCs) can be derived from the culture of primordial germ cells (PGCs). However, there are no reports of gonocytes, following the stage of PGC development, becoming stem cell lines. To analyze the gene expression differences between PGCs and gonocytes, we performed cDNA subtractive hybridization with mouse gonads containing either of the two cell populations. We confirmed that developmental pluripotency associated 5 (Dppa5), originally found in mouse embryonic stem cells (ESCs) and mouse embryonic carcinoma cells (ECCs), was strongly expressed in mouse PGCs and the expression was rapidly downregulated during germ cell development. A human sequence homologous to Dppa5 was identified by bioinformatics approaches. Interestingly, human Dppa5 was expressed only in human PGCs, human EGCs, and human ESCs and was not detected in human ECCs. Its expression was downregulated during induced differentiation of human ESCs. These findings confirmed that Dppa5 is specifically and differentially expressed in human cells that have pluripotency. The results strongly suggest that Dppa5 may have an important role in stemness in human ESCs and EGCs and also can be used as a marker of pluripotent stem cells. Human pluripotent stem cells may have their own ways to be pluripotent, as opposed to the much uniform mouse stem cells.     

Recent advancements towards the derivation of immune-compatible patient-specific human embryonic stem cell lines

The derivation of human embryonic stem cell lines from blastocyst stage embryos, first achieved almost a decade ago, demonstrated the potential to prepare virtually unlimited numbers of therapeutically beneficial cells in vitro. Assuming that large-scale production of differentiated cells is attainable, it is imperative to develop strategies to prevent immune responses towards the grafted cells following transplantation. In this review, I will discuss recent advances in the production of pluripotent cell lines using three emerging techniques: somatic cell nuclear transfer into enucleated oocytes and zygotes, parthenogenetic activation of unfertilized oocytes and induction of pluripotency in somatic cells. Importantly, if these techniques can be harnessed for generating patient-specific pluripotent cell lines, then immunological processes are expected to be low or completely absent.