Somatic Cell Nuclei Research Articles

Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer.

Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection.

Maternal Factors Involved in Nuclear Reprogramming by Eggs and Oocytes

When a somatic cell nucleus is transplanted into an egg or an oocyte, the transplanted nucleus can be reprogrammed to support early embryonic develop- ment so that the reconstructed embryo gives rise to a cloned animal. Nuclear reprogramming of somatic nuclei is induced by maternal components stored in eggs and oocytes. These endogenous reprogramming factors and mechanisms have been explored for decades in mam- mals and amphibia. There are several ways of investi- gating reprogramming mechanisms, including nuclear transfer to eggs/oocytes and incubation in egg/oocyte extracts. In this review I describe the type of reprogram- ming events induced in each system and what factors in eggs and oocytes are responsible for these. Based on our current knowledge, I propose a model for the early phase of nuclear reprogramming in eggs and oocytes.

Treatment of donor cells with trichostatin A improves in vitro development and reprogramming of buffalo (Bubalus bubalis) nucleus transfer embryos

It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 μM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4–0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos.

Sc65 Is a Novel Endoplasmic Reticulum Protein That Regulates Bone Mass Homeostasis

Members of the Leprecan family of proteins include enzymes, prolyl 3-hydroxylase 1 (P3h1), P3h2, and P3h3, and nonenzymatic proteins, Crtap and Sc65. Mutations in CRTAP and LEPRE1 (encoding P3H1) have been associated with human disease such as recessive osteogenesis imperfecta; however, the function of Sc65, which is closely related and highly homologous to Crtap, is unknown. Sc65 has been described as a synaptonemal complex protein, a nucleolar protein, and a cytoplasmic adapter protein. In light of its high sequence similarity with Crtap, an endoplasmic reticulum (ER)-associated protein, and the importance of post-translational modifications such as collagen prolyl 3-hydroxylation in bone metabolism, we hypothesized that Sc65 was an ER-resident protein that would have an important role in bone homeostasis. In this study, we demonstrate that Sc65 is a previously unrecognized ER protein and that it does not localize in the nucleus of somatic cells. Moreover, Sc65 is expressed and functional during skeletal development because loss of Sc65 results in a progressive osteopenia that affects both trabecular and cortical bone. Bone loss is the result of increased bone resorption mediated by a non-cell-autonomous effect on osteoclasts. Therefore, Sc65, like its related family member Crtap, is an important modulator of bone homeostasis, acting as a negative regulator of osteoclastogenesis.

Establishment of totipotency does not depend on Oct4A

SUMMARYOct4A is a core component of the regulatory network of pluripotent cells, and by itself can reprogram neural stem cells into pluripotent cells in mouse and humans. However, its role in defining totipotency and inducing pluripotency during embryonic development is still unclear. We genetically eliminated maternal Oct4A using a Cre-lox approach in mouse and found that the establishment of totipotency was not affected, as shown by the generation of live pups. After complete inactivation of both maternal and zygotic Oct4A expression, the embryos still formed Oct4-GFP– and Nanog–expressing inner cell masses, albeit non-pluripotent, indicating that Oct4A is not a determinant for the pluripotent cell lineage separation. Interestingly, Oct4A-deficient oocytes were able to reprogram fibroblasts into pluripotent cells. Our results clearly demonstrate that, in contrast to its role in the maintenance of pluripotency, maternal Oct4A is crucial for neither the establishment of totipotency in embryos, nor the induction of pluripotency in somatic cells using oocytes.

The ups and downs of somatic cell nucleus transfer (SCNT) in humans

Achieving successful somatic cell nuclear transfer (SCNT) in the human and subhuman primate relative to other mammals has been questioned for a variety of technical and logistical issues. Here we summarize the gradual evolution of SCNT technology from the perspective of oocyte quality and cell cycle status that has recently led to the demonstration of feasibility in the human for deriving chromosomally normal stem cells lines. With these advances in hand, prospects for therapeutic cloning must be entertained in a conscientious, rigorous, and timely fashion before broad spectrum clinical applications are undertaken.

Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds

Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell resource, with potential for studying disease and use in regenerative medicine. However, genetic manipulation and technically challenging strategies such as nuclear transfer used in reprogramming limit their clinical applications. Here, we show that pluripotent stem cells can be generated from mouse somatic cells at a frequency up to 0.2% using a combination of seven small-molecule compounds. The chemically induced pluripotent stem cells resemble embryonic stem cells in terms of their gene expression profiles, epigenetic status, and potential for differentiation and germline transmission. By using small molecules, exogenous "master genes" are dispensable for cell fate reprogramming. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications.

Sequential addition of reprogramming factors improves efficiency

Addition of a specific set of transcription factors reprograms somatic cell nuclei to a pluripotent state. Sequential addition of these factors, rather than the simultaneous exposure used in standard protocols, improves reprogramming efficiency. This sequential method favours a transition through a state with enhanced mesenchymal characteristics before driving an epithelial transformation on the way to the pluripotent state.

Proteomic Analysis of Early Reprogramming Events in Murine Somatic Cells Incubated withXenopus laevisOocyte Extracts Demonstrates Network Associations with Induced Pluripotency Markers

The reprogramming of somatic cells into a pluripotent/embryonic-like state holds great potential for regenerative medicine, bypassing ethical issues associated with embryonic stem cells (ESCs). Numerous methods, including somatic cell nuclear transfer (SCNT), fusion to pluripotent cells, the use of cell extracts, and expression of transcription factors, have been used to reprogram cells into ES-like cells [termed induced pluripotent stem cells (iPSCs)]. This study investigated early events in the nuclei of permeabilized murine somatic cells incubated in cytoplasmic extract prepared from Xenopus laevis germinal vesicle-stage oocytes by identifying proteins that showed significant quantitative changes using proteomic techniques. A total of 69 protein spots from two-dimensional electrophoresis were identified as being significantly altered in expression after treatment, and 38 proteins were identified by tandem mass spectrometry. Network analysis was used to highlight pathway connections and interactions between these identified proteins, which were found to be involved in many functions--primarily nuclear structure and dynamics, transcription, and translation. The pluripotency markers Klf4, c-Myc, Nanog, and POU5F1 were highlighted by the interaction network analysis, as well as other compounds/proteins known to be repressed in pluripotent cells [e.g., protein kinase C (PRKC)] or enhanced during differentiation of ESCs (e.g., retinoic acid). The network analysis also indicated additional proteins and pathways potentially involved in early reprogramming events.

Improving development of cloned goat embryos by supplementing α-lipoic acid to oocyte in vitro maturation medium

α-Lipoic acid (LA) is a powerful antioxidant for clinical therapy of some metabolic diseases, but there are few reports about the effect of LA on animal occyte in vitro maturation (IVM). The objective of this study is to investigate the effect of supplementing LA to IVM medium on subsequently developmental competence of goat cloning embryos after somatic cell nucleus transfer (SCNT). Twenty-five micromolars LA significantly increased 12% oocyte maturation rate from control 57.8% to treated group 69.8% (P < 0.05). The reconstructed rate of cloning embryos in LA supplement group (67.3%) was significantly higher than control (56.5%, P < 0.05). Although the SCNT embryo cleavage rates did not have significant difference between the two groups (42.0% vs. 47.9%, P > 0.05), LA supplement group had significantly higher blastocyst formation rate and hatched rate than control (24.0% vs. 18.4% and 37.0% vs. 30.9%, respectively, P < 0.05). In addition, supplementing LA significantly reduced the cellular apoptosis rate of nucleus transfer blastocysts by inhibiting the expression of apoptotic activators, such as Bax, Bad, Caspase-3, and CytC genes and promoting cumulus-oocyte complexes to synthesize glutathione (GSH) and express antioxidant enzymes such as GPX4 and SOD genes. In conclusion, supplement of LA to oocyte IVM medium could improve the maturation rate and antioxidant ability of oocytes and increase the developmental competence of oocytes after SCNT.

Females and males contribute in opposite ways to the evolution of gene order in Drosophila.

An intriguing association between the spatial layout of chromosomes within nuclei and the evolution of chromosome gene order was recently uncovered. Chromosome regions with conserved gene order in the Drosophila genus are larger if they interact with the inner side of the nuclear envelope in D. melanogaster somatic cells. This observation opens a new door to understand the evolution of chromosomes in the light of the dynamics of the spatial layout of chromosomes and the way double-strand breaks are repaired in D. melanogaster germ lines. Chromosome regions at the nuclear periphery in somatic cell nuclei relocate to more internal locations of male germ line cell nuclei, which might prefer a gene order-preserving mechanism to repair double-strand breaks. Conversely, chromosome regions at the nuclear periphery in somatic cells keep their location in female germ line cell nuclei, which might be inaccessible for cellular machinery that causes gene order-disrupting chromosome rearrangements. Thus, the gene order stability for genome regions at the periphery of somatic cell nuclei might result from the active repair of double-strand breaks using conservative mechanisms in male germ line cells, and the passive inaccessibility for gene order-disrupting factors at the periphery of nuclei of female germ line cells. In the present article, I find evidence consistent with a DNA break repair-based differential contribution of both D. melanogaster germ lines to the stability/disruption of gene order. The importance of germ line differences for the layout of chromosomes and DNA break repair strategies with regard to other genomic patterns is briefly discussed.

Visualization of actin filaments and monomers in somatic cell nuclei

In addition to its long-studied presence in the cytoplasm, actin is also found in the nuclei of eukaryotic cells. The function and form (monomer, filament, or noncanonical oligomer) of nuclear actin are hotly debated, and its localization and dynamics are largely unknown. To determine the distribution of nuclear actin in live somatic cells and evaluate its potential functions, we constructed and validated fluorescent nuclear actin probes. Monomeric actin probes concentrate in nuclear speckles, suggesting an interaction of monomers with RNA-processing factors. Filamentous actin probes recognize discrete structures with submicron lengths that are excluded from chromatin-rich regions. In time-lapse movies, these actin filament structures exhibit one of two types of mobility: 1) diffusive, with an average diffusion coefficient of 0.06-0.08 μm(2)/s, or (2) subdiffusive, with a mobility coefficient of 0.015 μm(2)/s. Individual filament trajectories exhibit features of particles moving within a viscoelastic mesh. The small size of nuclear actin filaments is inconsistent with a role in micron-scale intranuclear transport, and their localization suggests that they do not participate directly in chromatin-based processes. Our results instead suggest that actin filaments form part of a large, viscoelastic structure in the nucleoplasm and may act as scaffolds that help organize nuclear contents.

Immunobiology of embryonic stem cells: Foreign mtDNA as an immunological barrier in SCNT derived embryonic stem cells transplantation

Objectives: Using somatic cell nucleus transfer (SCNT) for embryonic stem cell (ESC) extraction, the nucleus of a somatic cell is transferred to the cytoplasm of an enucleated egg. ESCs created from the inner cell mass of the blastocyst are not genetically identical with the nucleus donor. The mitochondrial DNA (mtDNA) resides inside the cytoplasm of the egg. This study aimed to investigate the immunological response against mtDNA in ESC transplantation.

Recent progress of animal cloning

I was reported that adult somatic cells could be reprogrammed in enucleated oocytes and developed to a normal sheep after nuclear transfer in 1997. But, it is also clarified that the successful rate of animal cloning is still very limited. One of the possible reasons for the low success is the failure of reprogramming of somatic cells in ooplasm after nuclear transfer. Failure of the reprogramming of somatic cell nuclei in ooplasm causes variedabnormalities to SCNT embryos in every developmental stage, including both preimplantation and postimplantation stages, and resulted in the low successful rate at birth. But the mechanism of reprogramming or reprogramming factors in the enucleated oocytes are still unclear.I will review the recent progress of animal cloning in this talk. Especially, whether goodembryos can be selected before embryo transfer, and how to raise the viability of clonedembryos after implantation will be discussed.

43 PATTERN OF NUCLEOLI FORMATION IN RACCOON-PORCINE INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Recently, great focus has been on the rescue of endangered animals through somatic cell nuclear transfer (SCNT). Because it is difficult to obtain the oocytes of endangered species, interspecies SCNT (iSCNT) methods have been attempted. Numerous iSCNT embryos have shown unsuccessful development due to aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). In particular, aberrant EGA may cause the arrest of nucleoli formation and developmental block in embryos. According to this concept, we performed raccoon iSCNT using porcine oocytes and analyzed iSCNT embryo development pattern and formation of nucleoli. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. Cleavage and blastocyst formation were evaluated under a stereomicroscope at 48 and 168 h post-activation (hpa), respectively. To confirm the formation of nucleoli, which can be detected by C23 antibody labeling in many mammalian species, C23 immunocytochemistry was performed at 48 and 72 hpa. A total of 158 iSCNT embryos were cultured; 68.5% of the raccoon iSCNT embryos were cleaved at 48 hpa (1-cell stage: 9.7%; 2-cell stage: 14.4%; 4-cell stage: 34.1%; 6-cell stage: 12.7%; 8-cell stage: 7.3%; fragmented: 21.8%). But, the embryos seen as 5- to 8-cell stage did not have the same number of nuclei as their blastomere number. When raccoon iSCNT embryos were stained by Hoechst 33342, 5- to 8-blastomere raccoon iSCNT embryos had only 4 nuclei. The raccoon iSCNT embryos did not develop past the 4-cell stage and failed to form blastocysts. In the control group, 65.2% of pig SCNT embryos were cleaved at 48 hpa (1-cell stage: 8.0%; 2-cell stage: 4.2%; 4-cell stage: 23.6%; 6-cell stage: 13.6%; 8-cell stage: 23.8%; fragmented: 26.8%), and 10.0% of pig SCNT embryos developed to blastocysts. In raccoon iSCNT embryos, raccoon nuclei failed to form nucleoli at 48 and 72 hpa. By contrast, pig SCNT embryos showed 18.8 and 87.9% nucleoli formation at 48 and 72 hpa. Our results demonstrate that 4-cell-stage embryos of raccoon-porcine hybrid embryos may be produced by SCNT methods. The pig oocytes partly supported the remodeling and reprogramming of the raccoon somatic cell nuclei, but they were unable to support nucleoli formation. Moreover, aberrant nucleoli formation caused the unsuccessful development of raccoon SCNT embryos to the blastocyst stage. This work was supported by a grant from the Next Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

49 EFFECTS OF CULTURE CONDITIONS AND GENE TRANSFECTION ON THE DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Somatic cell nucleus transfer (SCNT) and in vitro culture of reconstructed embryos are the pivotal steps for successful cloning and generation of transgenic cattle. The aim of the study was to determine the influence of different cell fusion parameters, maturation, and culture conditions and the type of a cell line (bovine fetal fibroblast cell lines with or without gene transfection) on SCNT blastocyst development. Slaughterhouse-derived oocytes were matured for 17 h in TCM-199 (Sigma, St. Louis, MO, USA) supplemented with 0.05 µg mL–1 of epidermal growth factor (EGF) and 15 IU mL–1 of hCG/eCG (Intervet, PG600) or 10 µg mL–1 of FSH and 12.5 mU mL–1 of LH (Sioux Biochemical Inc., Sioux Center, IA, USA). Four fetal fibroblast cell lines (4 to 5 passages) and identical cell lines transfected with plasmid containing either human erythropoietin, FSH, growth hormone, or insulin-coding cDNA under β-casein promoter (7 to 9 passages) were used for SCNT. Cell fusion was induced by 2 direct-current pulses in 0.5 or 0.2 micro fusion chambers (Eppendorf Multiporator) using one of the following treatments: 100V for 15 µs (F1), 65V for 25 µs (F2), 65V for 20 µs (F3; all in a 0.5-mm chamber), or 36V for 25 µs (F4; 0.2-mm chamber). Fused complexes were activated with 4 µg mL–1 of Ca-ionophore for 4 min and then incubated for 5 h in 2 mM DMAP. The embryos were cultured in SOFaaci medium (Holm et al. 1999) or in commercial SOF medium (Minitüb GmbH, Tiefenbach, Germany) for 7 days. Data were analysed by ANOVA and the chi-square test. The results of the study showed that the cleavage rate of the reconstructed embryos was influenced by the fusion regimen (P &lt; 0.05) but not by the donor cell type (P &lt; 0.05). Treatments F2 and F3 resulted in cleavage rates higher (P &lt; 0.05) than F1 and F4 (77.2, 82.0, 62.8, and 63.1%, respectively). Blastocyst yield was not significantly influenced by the different in vitro maturation (IVM) media – altogether, addition of FSH/LH resulted in 14.6% (158/1079) and EGF + hCG/eCG in 13.2% (73/554) of blastocysts (P &lt; 0.05). The combination of TCM-199 + FSH/LH and SOFaaci resulted in 19.6% (79/403) blastocysts compared with 12.4% (74/596) when the same IVM medium and commercial SOF were used (P &lt; 0.05). The use of transgenic cell lines for cloning led to a lower overall blastocyst rate (10.9%, 38/348) than use of non-transfected cell lines (17.7%, 115/651; P &lt; 0.05), whereas the differences were 5.6 and 4.1 percentage points for SOF and SOFaaci, respectively. There were no significant differences between the individual cell lines within a cell line type. In conclusion, the optimization of the fusion parameters and in vitro culture (IVC) conditions led to improved blastocyst yields. In vivo development potential of the generated embryos still has to be evaluated in further studies. This study was supported by Project EU29023 of Enterprise Estonia, CCRMB, targeted grant SF1080045s07, and grant P8001 from the Estonian University of Life Sciences.

Reprogramming of the MHC‐I and Its Regulation by NFκB in Human‐Induced Pluripotent Stem Cells

The immunogenicity of human pluripotent stem cells plays a major role in their potential use in the clinic. We show that, during their reprogramming, human-induced pluripotent stem (iPS) cells downregulate expression of human leukocyte antigen (HLA)-A/B/C and β2 microglobulin (β2M), the two components of major histocompatibility complex-I (MHC-I). MHC-I expression in iPS cells can be restored by differentiation or treatment with interferon-gamma (IFNγ). To analyze the molecular mechanisms that regulate the expression of the MHC-I molecules in human iPS cells, we searched for correlation between the expression of HLA-A/B/C and β2M and the expression of transcription factors that bind to the promoter of these genes. Our results show a significant positive correlation between MHC-I expression and expression of the nuclear factors, nuclear factor kappa B 1 (NFκB1) and RelA, at the levels of RNA, protein and was confirmed by chromatin binding. Concordantly, we detected robust levels of NFκB1 and RelA proteins in the nucleus of somatic cells but not in the iPS cell derived from them. Overexpression of NFκB1 and RelA in undifferentiated pluripotent stem cells led to induction in expression of MHC-I, whereas silencing NFκB1 and RelA by small hairpin RNA decreased the expression of β2M after IFNγ treatment. Our data point to the critical role of NFκB proteins in regulating the MHC-I expression in human pluripotent stem cells.

Epigenetic reprogramming in somatic cells induced by extract from germinal vesicle stage pig oocytes

Genomic reprogramming factors in the cytoplasm of germinal vesicle (GV) stage oocytes have been shown to improve the efficiency of producing cloned mouse offspring through the exposure of nuclei to a GV cytoplasmic extract prior to somatic cell nuclear transfer (SCNT) to enucleated oocytes. Here, we developed an extract of GV stage pig oocytes (GVcyto-extract) to investigate epigenetic reprogramming events in treated somatic cell nuclei. This extract induced differentiation-associated changes in fibroblasts, resulting in cells that exhibit pluripotent stem cell-like characteristics and that redifferentiate into three primary germ cell layers both in vivo and in vitro. The GVcyto-extract treatment induced large numbers of high-quality SCNT-generated blastocysts, with methylation and acetylation of H3-K9 and expression of Oct4 and Nanog at levels similar to in vitro fertilized embryos. Thus, GVcyto-extract could elicit differentiation plasticity in treated fibroblasts, and SCNT-mediated reprogramming reset the epigenetic state in treated cells more efficiently than in untreated cells. In summary, we provide evidence for the generation of stem-like cells from differentiated somatic cells by treatment with porcine GVcyto-extract.

Immunobiology of SCNT Derived Embryonic Stem Cells: Pivotal Role of mtDNA in Rejection of Transplanted Stem Cells

Background: For regenerative therapies, somatic cell nucleus transfer (SCNT) can be used to transfer the nucleus of a somatic cell into the cytoplasm of an enucleated egg. The resulting SCNT-embryo has the capability to develop to the blastocyst stage. Subsequently, embryonic stem cells (ESCs) can be derived from the inner cell mass of these blastocysts. These ESCs are genetically identical with the donor nucleus, with the exception of mitochondrial DNA (mtDNA) from the receiving egg. This study aimed to investigate the role of mtDNA in ESC transplantation and its immunological recognition. Methods: SCNT-ESCs were generated by transferring donor cells from Balb/C background into enucleated oocytes from BDF1 background. 1×106 of these SCNT-ESCs or SCNT-ESC-derived cardiomyocytes (CMs) were injected into Balb/C (mitochondria-incompatible) or BDF1 (nucleus-incompatible) mice (n=10). After 5 days, cellular and humoral responses were investigated by ELISPOT, donorspecific antibodies, histopathology, and confocal immunofluorescence microscopy. Immunodeficient SCIDbeige mice were used as controls (n=6). In vivo bioluminescence imaging was used to visualize cell survival longitudinal. The amount of mitochondria in undifferentiated ESCs was compared to ESC-derived CMs using the TMRM assay (Figure 1).[Figure 1]Results: Using ELISPOT assays for Th1, Th2, and Th17-cells, we found that foreign mitochondria caused a similar immune response compared to ESCs with foreign nuclear DNA, both being significant higher than in SCIDbeige (p< 0.001). Similar results were observed for the humoral response. Re-injection of ESCs resulted in an adaptive immune response, highlighting the role of T-cell mediated rejection as shown by ELISPOT assays and in vivo bioluminescence imaging. Antigenicity of ESCs increased with their differentiation into CMs, correlating with increased MHCI-expression and amount of mitochondrial antigens (Figure 1). Conclusion: mtDNA is coding for 13 proteins, which surprisingly resulted in an adaptive immune response and donorspecific antibody production after transplantation. Upon ESC differentiation into CMs we observed an increase of mitochondrial antigens resulting in a greater immunological recognition of transplanted cells. In summary, SCNT-derivates are not sufficient immune privileged and undergo immune rejection after transplantation.

HIRA dependent H3.3 deposition is required for transcriptional reprogramming following nuclear transfer to Xenopus oocytes

BackgroundNuclear reprogramming is potentially important as a route to cell replacement and drug discovery, but little is known about its mechanism. Nuclear transfer to eggs and oocytes attempts to identify the mechanism of this direct route towards reprogramming by natural components. Here we analyze how the reprogramming of nuclei transplanted to Xenopus oocytes exploits the incorporation of the histone variant H3.3.ResultsAfter nuclear transplantation, oocyte-derived H3.3 but not H3.2, is deposited on several regions of the genome including rDNA, major satellite repeats, and the regulatory regions of Oct4. This major H3.3 deposition occurs in absence of DNA replication, and is HIRA-and transcription-dependent. It is necessary for the shift from a somatic- to an oocyte-type of transcription after nuclear transfer.ConclusionsThis study demonstrates that the incorporation of histone H3.3 is an early and necessary step in the direct reprogramming of somatic cell nuclei by oocyte. It suggests that the incorporation of histone H3.3 is necessary during global changes in transcription that accompany changes in cell fate.