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Objectives Patients with urethral stricture due to any type of trauma, hypospadias or gender dysphoria suffer immensely from impaired capacity to urinate and are in need of a new functional urethra. Tissue engineering with decellularization of a donated organ recellularized with cells from the recipient patient has emerged as a promising alternative of advanced therapy medicinal products. The aim of this pilot study was to develop an ovine model of urethral transplantation and to produce an individualized urethra graft to show proof of function in vivo. Methods Donated urethras from ram abattoir waste were decellularized and further recellularized with autologous buccal mucosa epithelial cells excised from the recipient ram and expanded in vitro. The individualized urethral grafts were implanted by reconstructive surgery in rams replacing 2.5 ± 0.5 cm of the native penile urethra. Results After surgery optimization, three ram had the tissue engineered urethra implanted for one month and two out of three showed a partially regenerated epithelium. Conclusions Further adjustments of the model are needed to achieve a satisfactory proof-of-concept; however, we interpret these findings as a proof of principle and a possible path to develop a functional tissue engineered urethral graft with de- and recellularization and regeneration in vivo after transplantation.

Objective Large cartilage defects and osteoarthritis (OA) cause cartilage loss and remain a therapeutic challenge. Three-dimensional (3D) bioprinting with autologous cells using a computer-aided design (CAD) model generated from 3D imaging has the potential to reconstruct patient-specific features that match an articular joint lesion. Design To scan a human OA tibial plateau with a cartilage defect, retrieved after total knee arthroplasty, following clinical imaging techniques were used: (1) computed tomography (CT), (2) magnetic resonance imaging (MRI), and (3) a 3D scanner. From such a scan, a CAD file was obtained to generate G-code to control 3D bioprinting in situ directly into the tibial plateau lesion. Results Highest resolution was obtained using the 3D scanner (2.77 times more points/mm2 than CT), and of the 3 devices tested, only the 3D scanner was able to detect the actual OA defect area. Human chondrocytes included in 3D bioprinted constructs produced extracellular matrix and formed cartilage tissue fragments after 2 weeks of differentiation and high levels of a mature splice version of collagen type II (Col IIA type B), characteristic of native articular cartilage and aggrecan (ACAN). Chondrocytes had a mean viability of 81% in prints after day 5 of differentiation toward cartilage and similar viability was detected in control 3D pellet differentiation of chondrocytes (mean viability 72%). Conclusion Articular cartilage can be formed in 3D bioprints. Thus, this 3D bioprinting system with chondrocytes simulating a patient-specific 3D model provides an attractive strategy for future treatments of cartilage defects or early OA.

Activin A plays a central role in the differentiation of stem cells into definitive endoderm, the first step in embryonic development and function development in many organ systems. The aims of this study were to induce controlled and fine-tuned cell differentiation using a gradient nanotechnology and compare this with a classic protocol and to investigate how induced pluripotent stem cells differentiated depending on the gradual increase of Activin A. The density difference was tested by attaching Activin A to a gold nanoparticle gradient for high-precision density continuity. Cells expressed the definitive endoderm markers SRY-box transcription factor 17 and transcription factor GATA-4 to different extents along the gradient, indicating a density-dependent cell response to Activin A. In both the gradient and the classic differentiation setups, the protein expression increased from days 1 to 5, but a significant increase already on day 3 was found only in the gradient-based setup. By utilizing the gradient technology to present the right amount of active biomolecules to cells in vitro, we were able to find an optimal setting for differentiation into definitive endoderm. The use of gradient surfaces for differentiation allows for improvements, such as efficiency and faster differentiation, compared with a classic protocol.

Organs and tissues, such as cartilage and limbs, are formed during development through an orchestration of growth factors that function as morphogens. Examples of growth factors include growth differentiation factor 5 (GDF5) and transforming growth factors beta 1 and 3 (TGFβ-1 and TGFβ-3) which can specify creation of more than one cell type after forming a concentration gradient in vivo. Here, we studied the impact of morphogen gradients during differentiation of induced pluripotent stem cells (iPSCs) into the chondrocyte lineage. Cell budding zones, consisting of condensed cell aggregates, were observed only in gradients of GDF5. T-box transcription factor 3 (TBX3) was detected specifically in the budding zones (ranging from 500–1,500 particles/μm2) of nuclei and cell vesicles. A homogenous density of GDF5 of 900 particles/μm2 on a surface induced budding and expression of TBX3 after five days in iPSCs. Therefore, we conclude that a gradient of GDF5, as well as the specific homogenous density of GDF5, support the induction of TBX3 in iPCSs. Moreover, differentiation of iPSCs first on GDF5 gradient or homogenous surfaces for five days and then in a three-dimensional structure for five weeks resulted in pellets that expressed TBX3., Cell biology; Developmental Biology; Molecular biology; Biomolecules; Cell Culture; Cell Differentiation; Regenerative Medicine; Spectroscopy; Stem Cells Research; GDF5; SOX9; Ln521; Nano Gradient; iPSC; chondrocyte differentiation; limb budding zone; TGFb3; TBX3; TGFb1

Background Today, several hundreds of million people all over the world are suffering from different joint disorders; like osteoarthritis (OA). Further, traumatic cartilage lesions can develop into OA. Recent studies indicate that human-derived induced pluripotent stem cells (iPSCs) can be 3D bioprinted and directed to form cartilage-like tissue, thus offering new approaches to treat cartilage lessions.Nguyen et al. 2017 The advantages of using an established iPSC line are unlimited cell source with regeneration capacity and chondrogenic differentiation potential. Objectives The aim of this study was to improve the generation of cartilage-like tissue when 3D bioprinting of iPSCs by using molecularly modified nanocelullose/alginate bioink to resemble natural environment found in the tissue. Methods In this study the chondrocyte-derived iPSC line “A2B” was used (Borestrom et al. 2014). These cells were bioprinted in combination with a modified nanocellulose and alginate bioink. Constructs obtained after the 3D bioprinting were cultured in chondrogenic medium in order to stimulate chondrogenic differentiation. Cell number and viability inside the prints was studied and histological analyses of the 3D printed constructs were performed. Furthermore, expression of pluripotency and chondrogenic specific genes was assessed by Taqman qPCR before and after differentiation. Results After 3D bioprinting high cell viability was found inside the constructs. 3D printed constructs were positively stained for alcian blue van gieson (AB-vG) staining, showing proteoglycans presence inside the prints (figure 1B). Molecular analyses showed high relative expression levels of the pluripotency-related gene Oct4 before starting the differentiation protocol. Cells inside the constructs express chondrogenic specific genes, such as collagen type 2 and Sox9 after 6 weeks of differentiation. Moreover, 3D printed constructs showed cartilage-resembles (figure 1A). Conclusions The 3D printing of the iPSc and the in vitro generation of cartilage-like tissue was successfully achieved using the modified nanocellulose/alginate bioink. This approach could be use in the future to model OA disease and to perform screenings of different therapeutic compounds. References [1] Nguyen D, et al. Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink. Sci Rep2017;7(1):658. [2] Borestrom C, et al. Footprint-free human induced pluripotent stem cells from articular cartilage with redifferentiation capacity: a first step toward a clinical-grade cell source. Stem Cells Transl Med2014;3;433–447. Disclosure of Interest None declared

Cells from elderly individuals can by scientists be reprogrammed back to totipotent or pluripotent cells. The first method that achieved reprogramming to totipotency (and thereby pluripotency) was cloning of animals, for example, creation of cloned frogs or Dolly. We and others identified that the protein Oct4 encoded by Pou5f1 gene was a key player in that reprogramming process. Further, epigenetic modifications in the control region of Pou5f1 needed to be reset for the cloning procedure to succeed. The iPS (induced pluripotent stem) cell technology generates pluripotent cells. These cells are promising candidates for regenerative medicine, because of their capability to multiply and differentiate into any cell of interest. Today, a clinical trial is ongoing using cells derived from iPS cells for macular degenerative disease. The iPS cells are also useful for studying human diseases in a dish and drug-screening purposes. The purpose of this article is to introduce the iPS technology and to present the so-called Yamanaka factors; Oct4, c-Myc, Klf4 and Sox2 in the context of development, generation of iPS cells and in cancer.

Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes. ispartof: SCIENTIFIC REPORTS vol:7 issue:1 ispartof: location:England status: published

Hepatocytes derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm, hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins that differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, we identified a module of genes that were expressed at higher levels in the liver tissue samples than in the hPSC-HEP. Considering that hub proteins and modules generally are essential and have important roles in the protein-protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bring this technology to a wider use.

Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes.

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models. CC BY 4.0Correspondence should be addressed to Nidal Ghosheh; nidal.ghosheh@his.se

Somatic cell nuclear transfers and the generation of induced pluripotent stem cells provide potential routes towards non-immunogenic cell replacement therapies. Translationally controlled tumor protein (Tpt1) was recently suggested to regulate cellular pluripotency. Here we explore functions of Tpt1 in mouse embryonic stem (ES) cells. We find that Tpt1 is present in the nucleus and cytoplasm of ES cells, and that specifically nuclear Tpt1 decreases upon cell differentiation. We also find that endogenous Tpt1 forms a complex with endogenous nucleophosmin/nucleoplasmin family member 1 (Npm1) in a cell cycle dependent manner. The Tpt1-Npm1 complex peaks sharply during mitosis and is independent of phosphorylation by Polo-like kinase. Differentiation by retinoic acid decreases Tpt1-Npm1 complex levels. Moreover, Tpt1 knock-down or over-expression reduces proliferation whereas Npm1 over-expression increases proliferation in ES cells. Cells depleted for both Tpt1 and Npm1 exhibit significantly reduced proliferation compared to cells depleted for Tpt1 alone, whereas cells over-expressing both Tpt1 and Npm1 show normal proliferation. Our findings reveal a role for the Tpt1-Npm1 complex in cell proliferation and identify the Tpt1-Npm1 complex as a potential biomarker for mitotic ES cells.

Why are stem cells so fascinating? Besides their capacity for unlimited lifespan, one feature that makes stem cells desirable to medicine is a unique ability to repopulate and potentially restore defective tissues and organs. Following injury, cells in the body migrate to replace and fill up the damaged area. Albeit the human body cannot regrow a lost arm or leg. Given that there are animals whose limbs can regrow, such capabilities have likely been lost during evolution. There are two main types of stem cells: embryonic and adult stem cells.

Human induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine; however, clinical applications of iPSCs are restricted because of undesired genomic modifications associated with most reprogramming protocols. We show, for the first time, that chondrocytes from autologous chondrocyte implantation (ACI) donors can be efficiently reprogrammed into iPSCs using a nonintegrating method based on mRNA delivery, resulting in footprint-free iPSCs (no genome-sequence modifications), devoid of viral factors or remaining reprogramming molecules. The search for universal allogeneic cell sources for the ACI regenerative treatment has been difficult because making chondrocytes with high matrix-forming capacity from pluripotent human embryonic stem cells has proven challenging and human mesenchymal stem cells have a predisposition to form hypertrophic cartilage and bone. We show that chondrocyte-derived iPSCs can be redifferentiated in vitro into cartilage matrix-producing cells better than fibroblast-derived iPSCs and on par with the donor chondrocytes, suggesting the existence of a differentiation bias toward the somatic cell origin and making chondrocyte-derived iPSCs a promising candidate universal cell source for ACI. Whole-genome single nucleotide polymorphism array and karyotyping were used to verify the genomic integrity and stability of the established iPSC lines. Our results suggest that RNA-based technology eliminates the risk of genomic integrations or aberrations, an important step toward a clinical-grade cell source for regenerative medicine such as treatment of cartilage defects and osteoarthritis.

The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replication oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop. Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5′ side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex. We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.

The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (DeltaOBP). We have now performed a quantitative analysis of the interaction between DeltaOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of DeltaOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with DeltaOBP in a sequence-specific manner.

Human embryonic stem cells (hESCs) are a very attractive supply in medicine, in particular as a human model system in drug discovery and developmental biology, but could also be used in cell replacement therapy. We are learning about molecular signaling pathways from several animal models, some of which are directly applicable to human beings. However, several pathways are unique for human cells and therefore culture of human tissue is an important tool to decipher molecular events in the human system. Human embryonic stem cells have several advantages including their capacity to multiply indefinitely in tissue culture and their ability to differentiate into any cell type of the body. Today, technology exists for producing embryonic stem cells (ESCs) containing patient-specific genome. These cells can multiply in tissue culture and upon external signals be induced to differentiate into any cell type of interest. In this way, patient-specific dopamine producing nerve cells can be transplanted to patients suffering from Parkinson's disease or patient-specific skin cells can be grafted in individuals suffering from melanoma cancer. Such cells could also provide a unique model system for patient-specific drug screening. This review describes historical breakthroughs underlying these advances and some of the technical obstacles to overcome.

In 2006, Shinya Yamanaka and colleagues reported that only four transcription factors were needed to reprogram mouse fibroblasts back in development into cells similar to embryonic stem cells (ESCs). These reprogrammed cells were called induced pluripotent stem cells (iPSCs). The year after, iPSCs were successfully produced from human fibroblasts and in 2008 reprogramming cells were chosen as the breakthrough of the year by Science magazine. In particular, this was due to the establishment of patient-specific cell lines from patients with various diseases using the induced pluripotent stem cell (iPSC) technique. IPSCs can be patient specific and therefore may prove useful in several applications, such as; screens for potential drugs, regenerative medicine, models for specific human diseases and in models for patient specific diseases. When using iPSCs in academics, drug development, and industry, it is important to determine whether the derived cells faithfully capture biological processes and relevant disease phenotypes. This chapter provides a summary of cell types of human origin that have been transformed into iPSCs and of different iPSC procedures that exist. Furthermore we discuss advantages and disadvantages of procedures, potential medical applications and implications that may arise in the iPSC field.

Methodologies to reprogram somatic cells into patient-specific pluripotent cells, which could potentially be used in personalized drug discovery and cell replacement therapies, are currently under development. Oct4 activation is essential for successful reprogramming and pluripotency of embryonic stem (ES) cells, albeit molecular details of Oct4 activation are not completely understood. Here we report that endogenous SAF-A is involved in regulation of Oct4 expression, binds the Oct4 proximal promoter in ES cells, and dissociates from the promoter upon early differentiation induced by LIF withdrawal. Depletion of SAF-A decreases Oct4 expression even in the presence of LIF, and results in an increase of the mesodermal marker Brachyury. The overexpression of wild-type human SAF-A rescues the mouse knock-down phenotype and results in increased Oct4 level. We also demonstrate that endogenous SAF-A interacts with the C-terminal domain (CTD) of endogenous RNA polymerase II and that the interaction is independent of CTD phosphorylation and mRNA. Moreover, we show that SAF-A exist in complexes with transcription factors Sox2 and Oct4 as well as STAT3 in ES cells. The number of endogenous SAF-A:Oct4 and SAF-A:Sox2 complexes decreases upon LIF depletion. These discoveries allow us to propose a model for activation of Oct4 transcription.

BACKGROUND: Scaffold attachment factor A (SAF-A) participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II. METHODOLOGY: Here we use co-localization, co-immunoprecipitation (co-IP) and in situ proximity ligation assay (PLA) to identify Brahma Related Gene 1 (BRG1), the ATP-driven motor of the human SWI-SNF chromatin remodeling complex, as another SAF-A interaction partner in mouse embryonic stem (mES) cells. We also employ RNA interference to investigate functional aspects of the SAF-A/BRG1 interaction. PRINCIPAL FINDINGS: We find that endogenous SAF-A protein interacts with endogenous BRG1 protein in mES cells, and that the interaction does not solely depend on the presence of mRNA. Moreover the interaction remains intact when cells are induced to differentiate. Functional analyses reveal that dual depletion of SAF-A and BRG1 abolishes global transcription by RNA polymerase II, while the nucleolar RNA polymerase I transcription machinery remains unaffected. CONCLUSIONS: We demonstrate that SAF-A interacts with BRG1 and that both components are required for RNA Polymerase II Mediated Transcription.

The 6-protein complex shelterin protects the telomeres of human chromosomes. The recent discovery that telomeres are important for epigenetic gene regulation and vertebrate embryonic development calls for the establishment of model organisms to study shelterin and telomere function under normal developmental conditions. Here, we report the sequences of the shelterin-encoding genes in Xenopus laevis and its close relation Xenopus tropicalis. In vitro expression and biochemical characterization of the Xenopus shelterin proteins TRF1, TRF2, POT1, TIN2, RAP1, TPP1, and the shelterin accessory factor PINX1 indicate that all main functions of their human orthologs are conserved in Xenopus. The XlTRF1 and XtTRF1 proteins bind double-stranded telomeric DNA sequence specifically and interact with XlTIN2 and XtTIN2, respectively. Similarly, the XlTRF2 and XtTRF2 proteins bind double-stranded telomeric DNA and interact with XlRAP1 and XtRAP1, respectively, whereas the XlPOT1 and XtPOT1 proteins bind single-stranded telomeric DNA. Real-time PCR further reveals the gene expression profiles for telomerase and the shelterin genes during embryogenesis. Notably, the composition of shelterin and the formation of its subcomplexes appear to be temporally regulated during embryonic development. Moreover, unexpectedly high telomerase and shelterin gene expression during early embryogenesis may reflect a telomere length-resetting mechanism, similar to that reported for induced pluripotent stem cells and for animals cloned through somatic nuclear transfer.

The aim of this contribution is to relate amphibian nuclear transplantation work to prospects for stem cell creation and hence to the long-term aim of cell replacement in humans. The methods used include the transplantation of single somatic cell nuclei to enudeated unfertilized eggs of Xenopus, and also the transfer of multiple somatic cell nuclei to the nucleus (germinal vesicle) of a growing ovarian oocyte. A key difference between these types of recipient cell is that eggs immediately induce DNA replication in transplanted nuclei, whereas an oocyte induces no DNA replication, but directly reprograms an injected nucleus to a new pattern of transcriptional activity. We summarize the extent and success of past and current nuclear reprogramming in experiments with enucleated frog eggs, and also those carried out with growing oocytes. We discuss possible mechanisms of nuclear reprogramming, and the possible contribution of such knowledge for stem cell creation and cell replacement in humans.

Insulin resistance and type 2 diabetes are associated with decreased expression of genes that regulate oxidative phosphorylation in skeletal muscle. To determine whether this defect might be inherited or acquired, we investigated the association of genetic, epigenetic, and nongenetic factors with expression of NDUFB6, a component of the respiratory chain that is decreased in muscle from diabetic patients. Expression of NDUFB6 was influenced by age, with lower gene expression in muscle of elderly subjects. Heritability of NDUFB6 expression in muscle was estimated to be approximately 60% in twins. A polymorphism in the NDUFB6 promoter region that creates a possible DNA methylation site (rs629566, A/G) was associated with a decline in muscle NDUFB6 expression with age. Although young subjects with the rs629566 G/G genotype exhibited higher muscle NDUFB6 expression, this genotype was associated with reduced expression in elderly subjects. This was subsequently explained by the finding of increased DNA methylation in the promoter of elderly, but not young, subjects carrying the rs629566 G/G genotype. Furthermore, the degree of DNA methylation correlated negatively with muscle NDUFB6 expression, which in turn was associated with insulin sensitivity. Our results demonstrate that genetic, epigenetic, and nongenetic factors associate with NDUFB6 expression in human muscle and suggest that genetic and epigenetic factors may interact to increase age-dependent susceptibility to insulin resistance.

The differentiated state of somatic cells is remarkably stable, but nuclear transfer can efficiently override the stability of cell differentiation by nuclear reprogramming. Genes that were silent in the differentiated state are activated, whereas genes specific for the differentiated state are switched off during the reprogramming process. The epigenetic changes that occur after nuclear transfer to Xenopus oocytes involve chromatin remodelling and DNA demethylation. In particular, we have reported that reactivation of the mouse stem cell specific gene oct-4 depends on demethylation of CpG:s in the proximal oct-4 promoter. Here we discuss molecular mechanisms of nuclear reprogramming, with special emphasis on DNA demethylation.

Nuclear transplantation experiments in amphibia and mammals have shown that oocyte and egg cytoplasm can extensively reprogram somatic cell nuclei with new patterns of gene expression and new pathways of cell differentiation1,2,3; however, very little is known about the molecular mechanism of nuclear reprogramming. Here we have used nuclear and DNA transfer from mammalian somatic cells to analyse the mechanism of activation of the stem cell marker gene oct4 by Xenopus oocytes. We find that the removal of nuclear protein accelerates the rate of reprogramming, but even more important is the demethylation of somatic cell DNA. DNA demethylation seems to precede gene reprogramming, and is absolutely necessary for oct4 transcription. Reprogramming by oocytes occurs in the absence of DNA replication and RNA/protein synthesis. It is also selective, operating only on the promoter, but not enhancers, of oct4; both a putative Sp1/Sp3 and a GGGAGGG binding site are required for demethylation and transcription. We conclude that the demethylation of promoter DNA may be a necessary step in the epigenetic reprogramming of somatic cell nuclei.

The transplantation of a somatic cell nucleus to an enucleated egg results in a major reprogramming of gene expression and switch in cell fate. We review the efficiency of nuclear reprogramming by nuclear transfer. The serial transplantation of nuclei from defective first-transfer embryos and the grafting of cells from such embryos to normal host embryos greatly increases the proportion of nuclei that can be seen to have been reprogrammed. We discuss possible reasons for the early failure of most nuclear transfers from differentiated cells and describe the potential value of growing oocytes, rather than unfertilized eggs, as a source of nuclear reprogramming molecules and for the eventual identification of these molecules. Nuclear transfer provides a possible route for the creation of stem cells from adult somatic cells.

Nuclear reprogramming by the transplantation of somatic cell nuclei to eggs (in second meiotic metaphase) is always followed by a phase of chromosome replication and cell division before new gene expression is seen. To help understand the mechanism of nuclear reprogramming, we have asked whether the nuclei of normal, nontransformed, nondividing, and terminally differentiated mammalian cells can be directly reprogrammed, without DNA replication, by Xenopus oocytes. We find that nuclei of adult mouse thymocytes and of adult human blood lymphocytes, injected into Xenopus oocytes, are induced to extinguish a differentiation marker and to strongly express oct-4, the most diagnostic mammalian stem cell/pluripotency marker. In the course of 2 days at 18°C, the mammalian oct-4 transcripts are spliced to mature mRNA. We conclude that normal mammalian nuclei can be directly reprogrammed by the nucleus (germinal vesicle) of amphibian oocytes to express oct-4 at a rate comparable to that of oct-4 in mouse ES cells. To our knowledge, this is the first demonstration of a stem cell marker being induced in a differentiated adult human cell nucleus. This is an early step toward the long-term aim of developing a procedure for reprogramming readily accessible human adult cells for cell replacement therapy.

Nuclear transplantation is one of the very few ways by which the genetic content and capacity for nuclear reprogramming can be assessed in individual cells of differentiated somatic tissues. No more than 6% of the cells of differentiated tissues have thus far been shown to have nuclei that can be reprogrammed to elicit the formation of unrelated cell types. In Amphibia, about 25% of such nuclear transfers form morphologically abnormal partial blastulae that die within 24 h. We have investigated the genetic content and capacity for reprogramming of those nuclei that generate partial blastulae, using as donors the intestinal epithelium cells of feeding Xenopus larvae. We have analyzed single nuclear transplant embryos obtained directly from intestinal tissue, thereby avoiding any genetic or epigenetic changes that might accumulate during cell culture. The expression of the intestine-specific gene intestinal fatty acid binding protein is extinguished by at least 10 4 times, within a few hours of nuclear transplantation. At the same time several genes that are normally expressed only in early embryos are very strongly activated in nuclear transplant embryos, but to an unregulated extent. Remarkably, cells from intestine-derived partial blastulae, when grafted to normal host embryos, contribute to several host tissues and participate in the normal 100-fold increase in axial muscle over several months. Thus, cells of defective cloned embryos unable to survive for more than 1 day can be reprogrammed to participate in new directions of differentiation and to maintain indefinite growth, despite the abnormal expression of early genes.

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III–box I hairpin with a 3′ single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III–box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III–box I hairpin in partially unwound DNA.

Positioned nucleosomes are believed to play important roles in transcriptional regulation and for the organization of chromatin in cell nuclei. Here, we have isolated the DNA segments in the mouse genome that form the most stable nucleosomes yet characterized. In separate molecules we find phased runs of three to four adenine nucleotides, extensive CA repeats, and in a few cases phased TATA tetranucleotides. The latter forms the most stable nucleosome yet characterized. One sequence with CAG repeats was also found. By fluorescence in situ hydridization the selected sequences are shown to be localized at the centromeric regions of mouse metaphase chromosomes.

The herpes simplex virus type 1 (HSV-1) origin binding protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding protein, ICP-8. The stimulation is dependent on direct protein-protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a single-stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding protein with high precision onto single-stranded DNA at a replication fork or at an origin of DNA replication.