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

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Simple SummaryCurrently, not enough is known about the effect of histone modification on the epigenetic reprogramming of somatic cells, and the lack of basic study limits the development of somatic cell nuclear transfer technology. The aim of this study was to explore the influence of lysine crotonylation, a newly discovered histone post-translational modification, on the reprogramming of somatic cells from Cashmere goats. The results showed that the crotonylation level was increased in somatic cells with sodium crotonate treatment. At the same time, the treatment of somatic cells improved the cloned embryo cleavage rate. In conclusion, an increasing crotonylation level could promote the reprogramming of somatic cells and cloned embryo development. This finding provides an important reference for future improvements in the efficiency of in vitro Cashmere goat somatic cell nuclear transfer embryo production.Failure in the epigenetic reprogramming of somatic cells is considered the main reason for lower cloned embryo development efficiency. Lysine crotonylation (Kcr) occupies an important position in epigenetic modification, while its effects on somatic cell reprogramming have not been reported. In this study, we detected the influence of sodium crotonate (NaCr) on the Kcr levels in three types of somatic cells (muscle-derived satellite cells, MDSCs; fetal fibroblast cells, FFCs; and ear tip fibroblast cells, EFCs). The three types of somatic cells were treated with NaCr for cloned embryo construction, and the cleavage rates and Kcr, H3K9cr, and H3K18cr levels in the cloned embryos were analyzed. The results showed that the abnormal levels of Kcr, H3K9cr, and H3K18cr were corrected in the treatment groups. Although there was no significant difference in the cloned embryo cleavage rate in the FFC treatment group, the cleavage rates of the cloned embryos in the MDSCs and EFCs treatment groups were increased. These findings demonstrated that the Kcr level was increased with NaCr treatment in somatic cells from Cashmere goat, which contributed to proper reprogramming. The reprogramming of somatic cells can be promoted and cloned embryo development can be improved through the treatment of somatic cells with NaCr.

PRDM14 promotes the bovine somatic stem cell reprogramming through enhancing oxidative phosphorylation at the initial stage.

BackgroundAlong with the reorganization of epigenetic and transcriptional networks, somatic cell reprogramming brings about numerous changes at the level of RNA processing. These include the expression of specific transcript isoforms and 3’ untranslated regions. A number of studies have uncovered RNA processing factors that modulate the efficiency of the reprogramming process. However, a comprehensive evaluation of the involvement of RNA processing factors in the reprogramming of somatic mammalian cells is lacking.ResultsHere, we used data from a large number of studies carried out in three mammalian species, mouse, chimpanzee and human, to uncover consistent changes in gene expression upon reprogramming of somatic cells. We found that a core set of nine splicing factors have consistent changes across the majority of data sets in all three species. Most striking among these are ESRP1 and ESRP2, which accelerate and enhance the efficiency of somatic cell reprogramming by promoting isoform expression changes associated with mesenchymal-to-epithelial transition. We further identify genes and processes in which splicing changes are observed in both human and mouse.ConclusionsOur results provide a general resource for gene expression and splicing changes that take place during somatic cell reprogramming. Furthermore, they support the concept that splicing factors with evolutionarily conserved, cell type-specific expression can modulate the efficiency of the process by reinforcing intermediate states resembling the cell types in which these factors are normally expressed.

Rapid and Efficient Conversion of Human Fibroblasts into Functional Neurons by Small Molecules.

The ability to produce genetically superior livestock has established somatic cell nuclear transfer (SCNT) as an invaluable tool in commercial livestock production. Successful reprogramming of somatic cells towards pluripotency requires the epigenetic marks characteristic of the differentiated cell type first be erased in order to inactivate the somatic cell program and activate the embryonic program. Several small molecules have been shown to improve both the kinetics and efficiency of reprogramming. These chemical modifiers aid in overcoming the “roadblocks” encountered during the reprogramming process by inducing the necessary epigenetic modifications needed to silence the somatic cell genome and completely reactivate the embryonic stem cell (ESC) genome. If small molecules are used to “prime” the somatic cells to be used as donor cells in SCNT, the efficiency of nuclear reprogramming during SCNT may be enhanced. We first assessed the effect of pre-treatment with small molecules on the expression of Oct-4, Nanog, and Sox-2 in bovine fetal fibroblast (BFF) cells. Chemical treatment consisted of 3 small molecules: PD0325901, a mitogen activated protein kinase/ERK kinase (MEK) inhibitor; CHIR99021, a glycogen synthase kinase-3 (GSK3) inhibitor; and NuP0178, a G9a histone methyltransferase inhibitor. No significant difference in transcript levels for Oct-4, Nanog, or Sox-2 was detected, indicating that this combination of small molecule inhibitors does not have an effect on the expression of Oct-4, Nanog, and Sox-2 in BFF cells. We next sought to assess the effects this combination of small molecule inhibitors has on the epigenetic state of Oct-4, Nanog, and Sox-2 in BFF cells. Chromatin Immunoprecipitation was used to quantify the enrichment of key histone modifications on the promoter regions of Oct-4, Nanog, and Sox-3 in BFF cells treated with and without PD0325901, CHIR99021, and NuP0178 over time. Time, treatment, and a time*treatment interaction were found to have a significant effect on the histone modifications analyzed. Determining how the expression of these factors alters the epigenetic marks in the promoter regions of key pluripotency-associated genes will allow for the development of defined conditions which best mimic the epigenetic landscape of ESC, ultimately leading to engineering the ideal donor cell for successful SCNT.

Significant breakthroughs have been recently achieved in reprogramming somatic cells to a pluripotent embryonic state by the ectopic expression of specific transcription factors. One of the major drawbacks of reprogramming strategies lays in the low efficiency of the process. It is likely that the required complex epigenetic-remodeling events could be cell-type specific and more rational approaches to cell source selection might help to improve the outcome of the procedure. Because the use of somatic stem cells, and specifically neural stem cells (NSCs), as nuclear donors significantly increased the efficiency of somatic cell nuclear transfer, we aimed to determine whether genetically unmodified somatic NSCs could be more easily reprogrammed to pluripotency than unmodified mouse embryonic fibroblasts. Retroviral transduction of the factors Oct4, Sox2, Klf4, and c-Myc successfully reverted NSCs to a pluripotent embryonic stem cell-like state with a 2-fold efficiency increase, faster kinetic, and with a lower number of viral integrations. Quantification analysis of reprogramming-associated genes revealed that NSCs endogenously expressed high levels of Sox2 and c-Myc. Accordingly, NSCs could be successfully induced to pluripotency through the ectopic viral expression of the other two factors (Oct4 and Klf4). These findings suggest that endogenous expression of reprogramming genes could help the reprogramming process and somatic stem cells might be more prone to reprogramming due to their specific genetic background. Genetic-based somatic cell screening might provide essential information for the selection of alternative cell sources more suitable to direct reprogramming.

Targeting mRNA export complex macromolecules THO subunits (Thoc2 and Thoc5) for somatic cell reprograming.

Cell fusion is one approach that has been used to demonstrate nuclear reprogramming of somatic cells to a pluripotent-like state and is a useful tool for screening factors involved in reprogramming. Recent cell fusion studies reported that the overexpression of Nanog and SalI could improve the efficiency of reprogramming, whereas AID was shown to be essential for DNA demethylation and initiation of reprogramming. The aim of this study was to investigate factors affecting the reprogramming efficiency following cell fusion. We conducted fusions of mouse embryonic stem cells (ESCs) with somatic cells carrying a GFP transgene under control of the Oct4 promoter (Oct4-GFP), which is normally repressed in nonpluripotent cells. The effect of somatic cell type on the reprogramming efficiency was investigated using Oct4-GFP expression as an indicator. Different somatic cell types were tested including mesenchymal stem cells (MSCs), adipose tissue-derived cells (ADCs), neural stem cells (NSCs), and these were compared with the mouse embryonic fibroblast (mEF) standard. The reprogramming efficiencies differed greatly, with mEFs (0.477 ± 0.003%) and MSCs (0.313 ± 0.003%) showing highest efficiencies while NSCs (0.023 ± 0.014%), and ADCs (0.006 ± 0.006%) had significantly lower reprogramming efficiencies (p < 0.05). The differences in the reprogramming efficiencies observed could be in part explained by the in vitro age of the somatic cells used. We demonstrated that the reprogramming efficiency of early passage mEFs was significantly higher compared with late passage mEFs (0.330 ± 0.166% vs. 0.021 ± 0.011%, p < 0.05), suggesting that senescence can affect reprogramming potential. In summary, this study shows that different somatic cell types do not have equivalent potential to be reprogrammed following fusion with ESCs. Furthermore, the in vitro age of somatic cells can also affect the reprogrammability of somatic cells. These findings constitute an important consideration for reprogramming studies.

BackgroundWe have previously noted that there were differences in somatic and male germ cell polyadenylation site choices. First, male germ cells showed a lower incidence of the sequence AAUAAA (an important element for somatic polyadenylation site choice) near the polyadenylation site choice. Second, the polyadenylation sites chosen in male germ cells tended to be nearer the 5' end of the mRNA than those chosen in somatic cells. Finally, a number of mRNAs used a different polyadenylation site in male germ cells than in somatic cells. These differences suggested that male germ cell-specific polyadenylation sites may be poor substrates for polyadenylation in somatic cells. We therefore hypothesized that male germ cell-specific polyadenylation sites would be inefficiently used in somatic cells.ResultsWe tested whether pre-mRNA sequences surrounding male germ cell-specific polyadenylation sites (polyadenylation cassettes) could be used to direct polyadenylation efficiently in somatic cells. To do this, we developed a luciferase reporter system in which luciferase activity correlated with polyadenylation efficiency. We showed that in somatic cells, somatic polyadenylation cassettes were efficiently polyadenylated, while male germ cell-specific polyadenylation cassettes were not. We also developed a sensitive, 3' RACE-based assay to analyze polyadenylation site choice. Using this assay, we demonstrated that male germ cell-specific polyadenylation cassettes were not polyadenylated at the expected site in somatic cells, but rather at aberrant sites upstream of the sites used in male germ cells. Finally, mutation of the male germ cell-specific poly(A) signal to a somatic poly(A) signal resulted in more efficient polyadenylation in somatic cells.ConclusionThese data suggest that regulated polyadenylation site choice of male germ cell-specific polyadenylation sites requires one or more factors that are absent from somatic cells.

Somatic cell reprogramming (SCR) is the conversion of differentiated somatic cells into totipotent or pluripotent cells through a variety of methods. Somatic cell reprogramming also provides a platform to investigate the role of chromatin-based factors in establishing and maintaining totipotency or pluripotency, since high expression of totipotency- or pluripotency-related genes usually require an active chromatin state. Several studies in plants or mammals have recently shed light on the molecular mechanisms by which epigenetic modifications regulate the expression of totipotency or pluripotency genes by altering their chromatin states. In this review, we present a comprehensive overview of the dynamic changes in epigenetic modifications and chromatin states during reprogramming from somatic cells to totipotent or pluripotent cells. In addition, we illustrate the potential role of DNA methylation, histone modifications, histone variants, and chromatin remodeling during somatic cell reprogramming, which will pave the way to developing reliable strategies for efficient cellular reprogramming.

Dolly the sheep (Wilmut et al in Nature 385:810–813, [1]), the world’s first mammal to be cloned in 1995 from a somatic cell was the greatest scientific achievement of the 20 century, which fundamentally changed the basic knowledge of biologists for somatic cells. In 2006, Shinya Yamanaka made a groundbreaking discovery that adult somatic cells can be reprogrammed to become pluripotent cells by the introduction of four pluripotent genes into somatic cells—so-called induced pluripotent stem (iPS) cells (Takanashi and Yamanaka in Cell 385:810–813, [2]). Those discoveries have opened promising in the research and applications of genomic reprogramming for regenerative biomedicine, biopharmaceutical, stem cell therapy, bio-organ, conservation of the rare and endangered animals, development of transgenic animals for breeding new animal varieties, etc. … Currently, there are three basic methods to reprogram somatic cells into totipotent stem cells or pluripotent stem cells: the first method is somatic cell nuclear transfer (SCNT) by injection of a somatic cell into an enucleated oocyte in order to produce totipotent cell (cloned animals) (Wakayama et al in Nature 394:369–374, [3]), the second is fusion of somatic cell with embryonic stem cells, and the third method is introducing 2 to 4 pluripotent genes, Oct4, Sox2, Klf4 and c-MyC into somatic cell (Takanashi and Yamanaka in Cell 385:810–813, [2]). Recently, we found that treatment of somatic cells with germinal vesicle (GV) oocytes extracts could reprogram somatic cells to stem cells, we named these cells “gviPS” Cells (Bui et al in Development 141:2235–2244, [4]). In the four methods listed above, only SCNT method can reprogram somatic cell into full-term development (offspring), also known as cloning animals. In this presentation we will focus on the most advanced technology in the world and the latest research technologies in animal cloning and the applicability of those technologies in medicine, recombinant human protein applications in pharmaceuticals, and in agriculture. Besides, we will discuss the latest methods that our team has achieved in 20 years of combined 4 key biotechnology 21st century animal cloning techniques re-cell differentiation, gene transfer in higher animals, biotechnology and modern breeding applications in medicine and agriculture.

The groundbreaking use of transcription factors (Oct4, Sox2, Klf4, c-Myc) in reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides novel ways in regenerative medicine and disease modeling. The reprogramming process is a stepwise process involving global epigenetic remodeling. In recent years, small molecules like DNA methyltransferase inhibitor that alter the epigenetic status of cells were shown to enhance the reprogramming efficiency. It was postulated that chromatin modifying enzymes played an important role during the reprogramming process, and microRNAs (miRNAs) were the upstream regulators. The objectives of this study involve the identification of potential miRNAs regulating the expression of chromatin modifying enzymes and the study of their roles during reprogramming.&#13;\n&#13;\nPrimary mouse embryonic fibroblasts (1o MEFs) were used for the establishment of a reprogramming system, where the delivery of transcription factors Oct4, Sox2, klf4 and cMyc was mediated by lentivirus. Another established secondary MEFs (2o MEFs) reprogramming system was also included in the study. Mouse iPSCs (miPSCs) derived from both systems were shown to express pluripotent markers. In-silico analysis predicted a set of miRNAs (miR-101, miR-135a, miR-148a and miR-148b) commonly targeted the chromatin modifying enzymes in mouse genome. Among them, miR-101 and miR-135a overexpression were found to inhibit the reprogramming efficiency significantly in both 1o and 2o MEFs. Conversely, the inhibition of miR-135a but not miR-101 expression significantly enhanced the reprogramming efficiency in both systems.&#13;\n&#13;\nIn this study, it was postulated that miR-101 regulated enhancer of zeste homolog 2 (Ezh2) during reprogramming. Ezh2 was confirmed to be negatively regulated by miR-101 at protein level. The expression of Ezh2 was high in mouse embryonic stem cells (mESCs) but time dependently depressed during mESC differentiation, while its expression was increased during reprogramming of MEFs. Ezh2 expression was found to negatively correlate with miR-101 expression in these conditions. In addition, the knockdown of Ezh2 mimicked the inhibitory effect of miR-101 overexpression on reprogramming efficiency.&#13;\n&#13;\nThe inhibitory role of miR-135a on reprogramming was linked to its potential target, Sirtuin 1 (Sirt1). Sirt1 was negatively regulated by miR-135a. The expression of miR-135a was upregulated upon mESC differentiation and decreased during reprogramming. Together with the previous finding in this laboratory, miR-135a expression was negatively correlated with Sirt1. Furthermore, miR-135a inhibition increased the proliferation rate of MEFs. More importantly, miPSCs reprogrammed from miR-135a knockdown MEFs maintained the pluripotent state.&#13;\n&#13;\nTo further analyze the pluripotency of the miPSCs, the tetraploid complementation assay was established. Preliminary studies were performed to optimize the conditions for electrofusion. Although single electrofusion with a lower field strength (1000V/cm) resulted in lower fusion rate, the development of the mESC aggregated embryo was the best when compared to higher field strength and those with double electrofusion. Lastly, the mESCs aggregated into tetraploid embryo were mainly localize in the inner cell mass of the embryo.&#13;\n&#13;\nIn conclusion, negative correlations were found between miR-101/Ezh2, and miR-135a/Sirt1 during somatic cell reprogramming. The identification of small molecules in reprogramming helps to understand the molecular mechanisms of reprogramming.