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.     

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.     

Nuclei of embryonic stem cells reprogram somatic cells

The restricted potential of a differentiated cell can be reverted back to a pluripotent state by cell fusion; totipotency can even be regained after somatic cell nuclear transfer. To identify factors involved in resetting the genetic program of a differentiated cell, we fused embryonic stem cells (ESCs) with neurosphere cells (NSCs). The fusion activated Oct4, a gene essential for pluripotency, in NSCs. To further identify whether cytoplasmic or nuclear factors are responsible for its reactivation, we fused either karyoplasts or cytoplasts of ESCs with NSCs. Our results show that ESC karyoplasts could induce Oct4 expression in the somatic genome, but cytoplasts lacked this ability. In addition, mitomycin C-treated ESCs, although incapable of DNA replication and cell division, could reprogram 5-azacytidine-treated NSCs. We therefore conclude that the Oct4 reprogramming capacity resides in the ESC karyoplast and that gene reactivation is independent of DNA replication and cell division.     

Cell extract-derived differentiation of embryonic stem cells

Various means have been used to encourage the differentiation of embryonic stem cells (ESCs) toward specific lineages, including growth factor administration, genetic modification, and coculture with relevant cells/tissues. Cell extract-based reprogramming has recently been used to derive mature cells from nonrelated phenotypes. In this communication, we tested whether this in vitro reprogramming approach can be used to direct ESC differentiation. Permeabilized murine ESCs exposed to extracts of murine type II pneumocytes showed increased expression of surfactant protein C and its corresponding mRNA, reflecting enhanced differentiation of pneumocytes. Subsequent differentiation to a type I phenotype was demonstrated by expression of aquaporin 5. Pneumocyte formation occurred quicker than with growth factor-induced differentiation. Our findings establish that ESCs can be differentiated in vitro using cellular extracts. This model provides a tool for analysis of the key factors involved in the differentiation of ESCs to type II pneumocytes.     

Role of Gap Junctions in Embryonic and Somatic Stem Cells

Stem cells provide an invaluable tool to develop cell replacement therapies for a range of serious disorders caused by cell damage or degeneration. Much research in the field is focused on the identification of signals that either maintain stem cell pluripotency or direct their differentiation. Understanding how stem cells communicate within their microenvironment is essential to achieve their therapeutic potentials. Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ES cells) and various somatic stem cells. GJIC has been implicated in regulating different biological events in many stem cells, including cell proliferation, differentiation and apoptosis. This review summarizes the current understanding of gap junctions in both embryonic and somatic stem cells, as well as their potential role in growth control and cellular differentiation.     

Effects of Akt signaling on nuclear reprogramming

Reprogramming of the epigenetic state from differentiated to pluripotent cells can be attained by cell fusion of differentiated somatic cells with embryonic stem (ES) cells or transfer of the nucleus of a differentiated cell into an enucleated oocyte. Activation of Akt signaling is sufficient to maintain pluripotency of ES cells and promotes derivation of embryonic germ (EG) cells from primordial germ cells (PGCs). Here we analyzed the effects of Akt signaling on somatic cell nuclear reprogramming after cell fusion and nuclear transfer. We found that forced activation of Akt signaling stimulated reprogramming after cell fusion of ES cells with thymocytes or mouse embryonic fibroblasts. These hybrid cells showed ES cell characteristics, including in vitro and in vivo differentiation capacity. In contrast, Akt signaling significantly reduced the efficiency of reprogramming with nuclear transfer. Our results demonstrate that Akt signaling plays important roles on the nuclear reprogramming of somatic cells.     

Embryonic stem cell extracts: use in differentiation and reprogramming

Stem cells have been studied extensively for decades and they have the inherent capacity to self-renew as well as to generate one or more types of specialized cells. The current focus of research on stem cells, particularly on embryonic stem cells, is on directed differentiation of these cells into specific cell types for future regenerative medicine. For the past few years, the process of reprogramming, which mediates conversion of somatic cells to their pluripotent state, has been given much attention, as it provides a possible source of autologous stem cells. In addition, understanding the molecular mechanism of differentiation and reprogramming has long been a subject of interest. In this article, we have briefly introduced stem cells and discussed the use of embryonic stem cells in reprogramming of somatic cells and differentiation to different lineages. The application of embryonic stem cells extracts in inducing reprogramming and transdifferentiation has also been described and discussed. Should this approach be successful, patient-specific cells will be produced safely and the likelihood of rejection will be decreased when used in cell therapy for many debilitating human diseases for which there is no cure such as Parkinson's disease, Alzheimer's disease, diabetes and others.     

Human induced pluripotent stem cells—from mechanisms to clinical applications

Human pluripotent stem cells hold great promise for basic research and regenerative medicine due to their inherent property to propagate infinitely, while maintaining the potential to differentiate into any given cell type of the human body. Since the first derivation in 1998, pluripotent human embryonic stem cells (ESCs) have been studied intensively, and although these cells provoke ethical and immune rejection concerns, translation of human ESC research into the clinics has been initiated. The generation of embryonic stem cell-like human induced pluripotent stem cells (iPSCs) from somatic cells by virus-mediated overexpression of distinct sets of reprogramming factors (OCT4, SOX2, KLF4, and c-MYC, or OCT4, SOX2, NANOG, and LIN28) in 2007 has opened up further opportunities in the field. While circumventing the major disputes associated with human ESCs, iPSCs offer the same advantages and, in addition, new perspectives for personalized medicine. This review summarizes technical advances toward the generation of potentially clinically relevant human iPSCs. We also highlight key molecular events underlying the process of cellular reprogramming and discuss inherent features of iPSCs, including genome instability and epigenetic memory. Furthermore, we will give an overview of particular envisaged human iPSC applications and point out which improvements are yet to come and what has been achieved so far.     

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.     

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.     

Pluripotential reprogramming of the somatic genome in hybrid cells occurs with the first cell cycle

The fusion of pluripotent embryonic cells with somatic cells results in reprogramming of the somatic cell genome. Oct4-green fluorescent protein (GFP) transgenes that do not contain the proximal enhancer (PE) region are widely used to visualize reprogramming of the somatic to the pluripotent cell state. The temporal onset of Oct4-GFP activation has been found to occur 40-48 hours postfusion. We asked whether activation of the transgene actually reflects activation of the endogenous Oct4 gene. In the current study, we show that activation of an Oct4-GFP transgene that contains the PE region occurs within 22 hours of fusion. In addition, demethylation of the Oct4-GFP transgene and that of the endogenous Oct4 and Nanog genes was found to occur within 24 hours of fusion. As this timing corresponds with the timing of cell cycle completion in embryonic stem cells and fusion hybrids (approximately 22 hours), we postulate that pluripotential reprogramming of the somatic cell genome begins during the first cell cycle after the fusion of a somatic cell with a pluripotent cell and has been completed by day 2 postfusion.     

Reprogramming away from the exhausted T cell state

Induced pluripotent stem cells (iPSCs) describe somatic cells that have been reprogrammed to the pluripotent state from which they can then be differentiated into any cell type of the body. This ability has tremendous implications on a wide number of medical sciences and applications, including cancer treatments. In many cancer patients, tumor infiltrating lymphocytes (TILs) have reached an exhausted state and are unable to exert effector function despite detecting and localizing at the tumor. Although the isolation, ex vivo expansion and transplantation of TILs is effective in a significant group of patients, too many patients do not respond positively to this treatment, in part because the expanded TIL population does not include a sufficient number of cells with the naïve or memory phenotype. Cell reprogramming using iPSC technologies aims to overcome this problem by returning TILs to the pluripotent state from which they can be differentiated into a heterogeneous population of T cells that are best suited to combat the tumor.     

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.