Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.

A gain-of-function mutation in Tnni2 impeded bone development through increasing Hif3a expression in DA2B mice.

Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder.

Promoting reprogramming by FGF2 reveals that the extracellular matrix is a barrier for reprogramming fibroblasts to pluripotency.

Leukemia inhibitory factor and bone morphogenetic protein signaling pathways play important roles in maintaining the self-renewal of mouse embryonic stem cells (ESCs). In contrast, the supplementation of fibroblast growth factor 2 (FGF2) in culture promotes mouse ESC differentiation. It has been proposed that factors that are adverse for maintaining the self-renewal of ESCs might play detrimental roles in the transcription factor-mediated reprogramming of somatic cells to pluripotency. However, recent evidence has revealed that reprogramming efficiency could be improved by FGF2, while the underlying molecular mechanism remains unknown. In this study, we dissected the roles of FGF2 in promoting mouse fibroblast reprogramming and disclosed the molecular mechanism behind this process. We used both primary induction and secondary inducible reprogramming systems and demonstrated that supplementation with FGF2 in the early phase of induced pluripotent stem cell induction could significantly increase reprogramming efficiency. Moreover, we discovered that many extracellular matrix candidate genes were significantly downregulated in fibroblasts treated with FGF2, and in particular, the synthesis of collagen could be greatly reduced by FGF2 treatment. Subsequently, we demonstrated that collagen is a barrier for reprogramming fibroblast cells to pluripotency, and the decreasing of collagen either by collagenase treatment or downregulation of collagen gene expression could significantly improve the reprogramming efficiency. Our results reveal a novel role of the extracellular matrix in mediating fibroblasts reprogramming.

Proteolytic Cleavage of the ENaC γ Subunit - Impact Upon Na + and K + Handling.

The ENaC gamma subunit is essential for homeostasis of Na + , K + , and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (P O ), in vitro . Cleavage proximal to the tract occurs at a furin recognition sequence ( 143 RKRR 146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143 RKRR 146 mutation to 143 QQQQ 146 ( Q4 ) in 129/Sv mice would reduce ENaC P O , impair flow-stimulated flux of Na + (J Na ) and K + (J K ) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (I SC ), and impair Na + , K + , and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na + diet did not exhibit altered ENaC P O or flow-induced J Na , though flow-induced J K modestly decreased. Colonic amiloride-sensitive I SC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na + diet. Blood Na + and K + were unchanged on a regular, low Na + , or high K + diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel P O and the influence of ENaC on Na + , K + , and fluid volume homeostasis in 129/Sv mice, in vivo .

Proteome of mouse oocytes at different developmental stages.

The mammalian oocyte possesses powerful reprogramming factors, which can reprogram terminally differentiated germ cells (sperm) or somatic cells within a few cell cycles. Although it has been suggested that use of oocyte-derived transcripts may enhance the generation of induced pluripotent stem cells, the reprogramming factors in oocytes are undetermined, and even the identified proteins composition of oocytes is very limited. In the present study, 7,000 mouse oocytes at different developmental stages, including the germinal vesicle stage, the metaphase II (MII) stage, and the fertilized oocytes (zygotes), were collected. We successfully identified 2,781 proteins present in germinal vesicle oocytes, 2,973 proteins in MII oocytes, and 2,082 proteins in zygotes through semiquantitative MS analysis. Furthermore, the results of the bioinformatics analysis indicated that different protein compositions are correlated with oocyte characteristics at different developmental stages. For example, specific transcription factors and chromatin remodeling factors are more abundant in MII oocytes, which may be crucial for the epigenetic reprogramming of sperm or somatic nuclei. These results provided important knowledge to better understand the molecular mechanisms in early development and may improve the generation of induced pluripotent stem cells.

Reprogramming of trophoblast stem cells into pluripotent stem cells by Oct4.

ESCs and trophoblast stem (TS) cells are both derived from early embryos, yet these cells have distinct differentiation properties. ESCs can differentiate into all three germ layer cell types, whereas TS cells can only differentiate into placental cells. It has not been determined whether TS cells can be converted into ES-like pluripotent stem (PS) cells. Here, we report that overexpression of a single transcription factor, Oct4, in TS cells is sufficient to reprogram TS cells into a pluripotent state. These Oct4-induced PS (OiPS) cells have the epigenetic characteristics of ESCs, including X chromosome reactivation, elevated H3K27 me3 modifications, and hypomethylation of promoter regions in Oct4 and Nanog genes. Meanwhile, methylation of promoter region in the Elf5 gene occurred during reprogramming of TS cells. The gene expression profile of OiPS cells was very similar to ESCs. Moreover, OiPS cells can differentiate into the three germ layer cell types in vitro and in vivo. More importantly, chimeric mice with germline transmission could be efficiently produced from OiPS cells. Our results demonstrate that one single transcription factor, Oct4, could reprogram the nonembryonic TS cells into PS cells.

Novel importin-alpha family member Kpna7 is required for normal fertility and fecundity in the mouse.

Nuclear importing system and nuclear factors play important roles in mediating nuclear reprogramming and zygotic gene activation. However, the components and mechanisms that mediate nuclearly specific targeting of the nuclear proteins during nuclear reprogramming and zygotic gene activation remain largely unknown. Here, we identified a novel member of the importin-α family, AW146299(KPNA7), which is predominantly expressed in mouse oocytes and zygotes and localizes to the nucleus or spindle. Mutation of Kpna7 gene caused reproductivity reduction and sex imbalance by inducing preferential fetal lethality in females. Parthenogenesis analysis showed that the cell cycle of activated one-cell embryos is loss of control and ahead of schedule but finally failed to develop into blastocyst stage. Further RT-PCR and epigenetic modification analysis showed that knocking out of Kpna7 induced abnormalities of gene expression (dppa2, dppa4, and piwil2) and epigenetic modifications (down-regulation of histone H3K27me3). Biochemical analysis showed that KPNA7 interacts with KPNB1 (importin-ß1). In summary, we identified a novel Kpna7 gene that is required for normal fertility and fecundity.

Generation of histocompatible androgenetic embryonic stem cells using spermatogenic cells.

Androgenetic embryonic stem (aES) cells, produced by pronuclear transplantation, offer an important autologous pluripotent stem cell source. However, the isolation of aES cells, particularly individual-specific aES cells, with the use of fertilized embryos has limited the practical applications of this technology in humans. In this study, we applied a new approach, essentially described as somatic cell nuclear transfer, and generated three aES cell line types with the use of spermatogenic cells including primary spermatocytes, round spermatids, and mature spermatozoa as donor cells, omitting the need to use fertilized embryos. Although abnormality of chimeras and absent germline competency indicated that all three types of aES cells exhibited limited pluripotency, the epigenetic status of the aES cell lines tended to resemble normal ES cells during long-term culture, and some parental-specific imprinted genes were expressed at levels comparable to those of normal ES cells. Furthermore, the histocompatibility of the aES cells was investigated by transplanting the differentiation progenies of the aES cells into major histocompatibility (MHC)-matched and -mismatched recipient mice. The results indicated that these aES cells were histocompatible with MHC-matched mice after transplantation. Our study provides evidence that MHC-competent autologous aES cells could be generated from different spermatogenic cells using nuclear transfer into oocytes, a process that could avoid the use of fertilized embryos.

Defective chromatin structure in somatic cell cloned mouse embryos.

Epigenetic reprogramming plays a central role in the development of cloned embryos generated by somatic cell nuclear transfer, and it is believed that aberrant reprogramming leads to the abnormal development of most cloned embryos. Recent studies show that trimethylation of H3K27 (H3K27me3) contributes to the maintenance of embryonic stem cell pluripotency because the differentiation genes are always occupied by nucleosomes trimethylated at H3K27, which represses gene expression. Here, we provide evidence that differential H3K27me3 modification exists between normal fertilization-produced blastocysts and somatic cell nuclear transfer cloned blastocysts; H3K27me3 was specifically found in cells of the inner cell mass (ICM) of normal blastocysts, whereas there was no modification of H3K27me3 in the ICM of cloned blastocysts. Subsequently, we demonstrated that the differentiation-related genes, which are marked by H3K27me3 in embryonic stem cells, were expressed at significantly higher levels in cloned embryos than in normal embryos. The polycomb repressive complex 2 (PRC2) component genes (Eed, Ezh2, and Suz12), which are responsible for the generation of H3K27me3, were expressed at lower levels in the cloned embryos. Our results suggest that reduced expression of PRC2 component genes in cloned embryos results in defective modification of H3K27me3 to the differentiation-related genes in pluripotent ICM cells. This results in premature expression of developmental genes and death of somatic cloned embryos shortly after implantation. Taken together, these studies suggest that H3K27me3 might be an important epigenetic marker with which to evaluate the developmental potential of cloned embryos.

The histone demethylase JMJD2C is stage-specifically expressed in preimplantation mouse embryos and is required for embryonic development.

Epigenetic modifications play a pivotal role in embryonic development by dynamically regulating DNA methylation and chromatin modifications. Although recent studies have shown that core histone methylation is reversible, very few studies have investigated the functions of the newly discovered histone demethylases during embryonic development. In the present study, we investigated the expression characteristics and function of JMJD2C, a histone demethylase that belongs to the JmjC-domain-containing histone demethylases, during preimplantation embryonic development of the mouse. We found that JMJD2C is stage-specifically expressed during preimplantation development, with the highest activity being observed from the two-cell to the eight-cell stage. Depletion of JMJD2C in metaphase II oocytes followed by parthenogenetic activation causes a developmental arrest before the blastocyst stage. Moreover, consistent with a previous finding in embryonic stem (ES) cells, depletion of JMJD2C causes a significant down-regulation of the pluripotency gene Nanog in embryos. However, contrary to a previous report in ES cells, we observed that other pluripotency genes, Pou5f1 and Sox2, are also significantly down-regulated in JMJD2C-depleted embryos. Furthermore, the depletion of JMJD2C in early embryos also caused significant down-regulation of the Myc and Klf4 genes, which are associated with cell proliferation. Our data suggest that the deregulation of these critical genes synergistically causes the developmental defects observed in JMJD2C-depleted embryos.

Mice cloned from induced pluripotent stem cells (iPSCs).

Differentiated somatic cells of various species can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopically expressing a combination of several transcription factors that are highly enriched in embryonic stem cells (ESCs). The generation of iPSCs in large animals has raised the possibility of producing genetically modified large animals through the nuclear transplantation approach. However, it remains unknown whether iPSCs could be used for generating cloned animals through the nuclear transfer method. Here, we show the successful production of viable cloned mice from inducible iPSCs through the nuclear transfer approach, and the efficiency is similar to that of using ESCs derived via normal fertilization. Furthermore, the cloned mice are fertile and can produce second-generation offspring. These efforts strengthen the possibility of utilizing iPSCs to generate gene-modified large animals for pharmaceutical purposes in the future.

Differentiation of reprogrammed somatic cells into functional hematopoietic cells.

Recent advances have demonstrated that the differentiated somatic cells could be reprogrammed into pluripotent state. Consequently, the reprogrammed somatic cells recapitulate the capacity to differentiate into specific cell lineages under appropriate culture conditions, which provides unlimited cell sources for cell transplantation-based therapy. In the present study, testicular Sertoli cells were successfully reprogrammed into pluripotent stem cells through somatic cell nuclear transfer (SCNT). Hematopoietic differentiation potential of the reprogrammed somatic cells was investigated in parallel to fertilization-derived ES (F-ES) cells. Our results demonstrated that the reprogrammed Sertoli cells (NT-ES) could efficiently differentiate into hematopoietic embryoid bodies (EBs). The hematopoietic-related genes including FLK-1, Bmp4, Runx1, etc. were dynamically expressed during the differentiation of the reprogrammed somatic cells in vitro. Transplantation of these differentiated reprogrammed cells into the bone marrow of irradiated mice could allow differentiation into different functional hematopoietic lineages in vivo. Moreover, blast-colony-forming cells (BL-CFCs) could be generated from both NT-ES and F-ES cells with similar efficiency in vitro. Our study indicates that the reprogrammed somatic cells possess the equivalent potency as F-ES cells in differentiating into functional hematopoietic cells.

Differential methylation status of imprinted genes in nuclear transfer derived ES (NT-ES) cells.

Compared with fertilized embryo derived ES (F-ES) cells, somatic cell nuclear transfer (SCNT) produced ES (NT-ES) cells were proposed appropriate for cell transplantation based therapies. Although previous studies indicated that NT-ES cells and F-ES cells were transcriptionally and functionally indistinguishable, characterization of DNA methylation patterns of imprinted genes in NT-ES cells is lacking. Here, we show that DNA methylation patterns in the differentially methylated region (DMR) of paternally imprinted gene, H19, displayed distinct abnormalities in certain NT-ES and F-ES cell lines after long-term culture in vitro. DNA methylation profiles of H19 appeared very dynamic in most ES cell lines examined, either hypermethylation or hypomethylation could be observed in specific ES cell lines. In contrast to H19, maternally imprinted genes, Mest and Peg3, showed relatively stable methylation patterns in ES cells, especially Peg3, which displayed better capability in enduring long-term culture in vitro. Our results indicate that abnormal methylation profiles of certain imprinted genes could be observed in both NT-ES and F-ES cell lines after long-term culture in vitro although these cell lines were proved to be pluripotent with germline transmission competent. Stringent screening of epigenetically normal NT-ES cells might be potentially necessary for further therapeutic application of these cells.

Embryonic stem cells derived from somatic cloned and fertilized blastocysts are post-transcriptionally indistinguishable: a MicroRNA and protein profile comparison.

Therapeutic cloning, whereby somatic cell nuclear transfer is used to generate customized embryonic stem cells (NT-ES) from differentiated somatic cells of specific individuals, has been successfully performed in mice and non-human primates. Safety concerns have prevented this technology from being potentially applied to humans, as severely abnormal phenotypes have been observed in cloned animals. Although it has been demonstrated that the transcriptional profiles and developmental potentials of ES cells derived from cloned blastocysts are identical to those of ES cells derived from fertilized blastocysts (F-ES), a systematic analysis of the post-transcriptional profiles of NT-ES cell lines has not yet been performed. To investigate whether NT-ES cells are comparable to F-ES cells post-transcriptionally, we compared the microRNA and protein profiles of five NT- and matching F-ES cell lines by microRNA microarray, 2-D DIGE and bioinformatic analyses. Stem-loop real-time PCR and MS/MS assays were further performed to verify the expression of specific microRNAs and characterize differentially expressed proteins. Our results demonstrate that the ES cell lines derived from cloned and fertilized mouse blastocysts have highly similar microRNA and protein expression profiles, consistent with their similar developmental potentials and transcriptional profiles.

Protein Expression Landscape of Mouse Embryos during Pre-implantation Development.

Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst). We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.

High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells.

Somatic cell nuclear transfer and transcription factor mediated reprogramming are two widely used techniques for somatic cell reprogramming. Both fully reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells hold potential for regenerative medicine, and evaluation of the stem cell pluripotency state is crucial for these applications. Previous reports have shown that the Dlk1-Dio3 region is associated with pluripotency in induced pluripotent stem cells and the incomplete somatic cell reprogramming causes abnormally elevated levels of genomic 5-methylcytosine in induced pluripotent stem cells compared to nuclear transfer embryonic stem cells and embryonic stem cells. In this study, we compared pluripotency associated genes Rian and Gtl2 in the Dlk1-Dio3 region in exactly syngeneic nuclear transfer embryonic stem cells and induced pluripotent stem cells with same genomic insertion. We also assessed 5-methylcytosine and 5-hydroxymethylcytosine levels and performed high-throughput sequencing in these cells. Our results showed that Rian and Gtl2 in the Dlk1-Dio3 region related to pluripotency in induced pluripotent stem cells did not correlate with the genes in nuclear transfer embryonic stem cells, and no significant difference in 5-methylcytosine and 5-hydroxymethylcytosine levels were observed between fully and partially reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells. Through syngeneic comparison, our study identifies for the first time that Grb10 is associated with the pluripotency state in nuclear transfer embryonic stem cells.

The effects of different donor cells and passages on development of reconstructed embryos.

In order to study the effects of different donor cells and passages on development of nuclear transfer embryos, we constructed embryos by electrofusing several kinds of donor cells into enucleated M II oocytes from Kun Ming (KM) mouse. These cells include 2-cell embryonic blastomeres, KMW embryonic stem (ES) cells, fetal fibroblast, ear fibroblast, tail tip fibroblast, sertoil cells and spermatogonia. Meanwhile, we compared the effects of passage numbers of fetal fibroblast cells on developmental competency after nuclear transfer. We found that 7.4% of reconstructed embryos from 2-cell embryonic blastomeres and 0.7% from ES cell could develop to blastocyst in vitro; embryos from fetal fibroblast could only develop to morula stage with the rate of 0.2%; embryos from spermatogonia could only develop to 8-cell stage and the rate was 0.3%; embryos respectively from ear fibroblast, sertoli cell and tail tip fibroblast could only develop to 4-cell stage. Although 2-cell development rate of embryos reconstructed from fetal fibroblast in first passage was significantly lower than those from the 2nd, the 3rd and the 4th passage, embryos from different passages could develop to 8-cell stage except the 3rd passage. The result indicated that it is more difficult for terminally differentiated cell nuclei to be reprogrammed in enucleated M II oocytes than for low differentiated cell nuclei. The reason of low development rate from ES cells maybe that most of ES cells was at S stage of the cell cycle, which out of coordination with M II oocytes. We could conclude that culture and passage of donor cells might be benefit to nucleus reprogramming.

Xist deficiency and disorders of X-inactivation in rabbit embryonic stem cells can be rescued by transcription-factor-mediated conversion.

The deficiency of X-inactive specific transcript (XIST) on the inactive X chromosome affects the behavior of female human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), and further chromosomal erosion can occur with continued passaging of these cells. However, X chromosome instability has not been identified in other species. In the present study, we investigated three female rabbit ESC (rbESC) lines and found that two of them expressed Xist normally and obtained both Xist RNA coating and H3K27me3 foci, thus defined as Xi(Xist)Xa. Interestingly, the third female rbESC line lacked Xist expression during ESC maintenance and differentiation. This line showed H3K27me3 foci but no Xist RNA coating in the early passages and was thus defined as Xi(w/oXist)Xa. Similar to Xi(w/oXist)Xa hESCs or hiPSCs, Xi(w/oXist)Xa rbESCs lose H3K27me3 and undergo Xi erosion (Xe) with passaging. Moreover, Xist-deficient rbESCs also exhibit impaired differentiation ability and upregulation of cancer-related genes. By overexpressing OCT4, SOX2, KLF4, and c-MYC in Xist-deficient rbESCs under optimized culture conditions, we successfully obtained mouse ESC-like (mESC-like) cells. The mESC-like rbESCs displayed dome-shaped colony morphology, activation of the LIF/STAT3-dependent pathway, and conversion of disordered X chromosome. Importantly, the defective differentiation potential was also greatly improved. Our data demonstrate that variations in X chromosome inactivation occur in early passage of rbESCs; thus, Xi disorders are conserved across species and are reversible using the proper epigenetic reprogramming and culture conditions. These findings may be very useful for future efforts toward deriving fully pluripotent rbESCs or rabbit iPSCs (rbiPSCs).

Enhanced telomere rejuvenation in pluripotent cells reprogrammed via nuclear transfer relative to induced pluripotent stem cells.

Although somatic cell nuclear transfer (SCNT) and induction of pluripotency (to form iPSCs) are both recognized reprogramming methods, there has been relatively little comparative analysis of the resulting pluripotent cells. Here, we examine the capacity of these two reprogramming approaches to rejuvenate telomeres using late-generation telomerase-deficient (Terc(-/-)) mice that exhibit telomere dysfunction and premature aging. We found that embryonic stem cells established from Terc(-/-) SCNT embryos (Terc(-/-) ntESCs) have greater differentiation potential and self-renewal capacity than Terc(-/-) iPSCs. Remarkably, SCNT results in extensive telomere lengthening in cloned embryos and improved telomere capping function in the established Terc(-/-) ntESCs. In addition, mitochondrial function is severely impaired in Terc(-/-) iPSCs and their differentiated derivatives but significantly improved in Terc(-/-) ntESCs. Thus, our results suggest that SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming. Understanding the basis of this differential could help optimize reprogramming strategies.

An elaborate regulation of Mammalian target of rapamycin activity is required for somatic cell reprogramming induced by defined transcription factors.

The mammalian target of the rapamycin (mTOR) signaling pathway functions in many cellular processes, including cell growth, proliferation, differentiation, and survival. Recent advances have demonstrated that differentiated somatic cells can be directly reprogrammed into the pluripotent state by overexpression of several pluripotency transcription factors. However, whether the mTOR signaling pathway is involved in this somatic cell-reprogramming process remains unknown. Here, we provide evidence that an elaborate regulation of the mTOR activity is required for the successful reprogramming of somatic cells to pluripotency. The reprogramming of somatic cells collected from the Tsc2(-/-) embryo, in which the mTOR activity is hyperactivated, is entirely inhibited. By taking advantage of the secondary inducible pluripotent stem (iPS) system, we demonstrate that either elevating the mTOR activity by Tsc2 shRNA knockdown or using high concentrations of rapamycin to completely block the mTOR activity in cells derived from iPS mice greatly impairs somatic cell reprogramming. Secondary iPS induction efficiency can only be elevated by elaborately regulating the mTOR activity. Taken together, our data demonstrate that the precise regulation of the mTOR activity plays a critical role in the successful reprogramming of somatic cells to form iPS cells.