Testicular somatic cell-like cells derived from embryonic stem cells induce differentiation of epiblasts into germ cells.

Regeneration of the testis from pluripotent stem cells is a real challenge, reflecting the complexity of the interaction of germ cells and somatic cells. Here we report the generation of testicular somatic cell-like cells (TesLCs) including Sertoli cell-like cells (SCLCs) from mouse embryonic stem cells (ESCs) in xeno-free culture. We find that Nr5a1/SF1 is critical for interaction between SCLCs and PGCLCs.

Increased vimentin in human α- and β-cells in type 2 diabetes.

Type 2 diabetes (T2DM) is associated with pancreatic islet dysfunction. Loss of β-cell identity has been implicated via dedifferentiation or conversion to other pancreatic endocrine cell types. How these transitions contribute to the onset and progression of T2DM is unknown.

Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development.

Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling.

Generation of Functional Beta-Like Cells from Human Exocrine Pancreas.

Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells.

Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture.

A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells.

Islet transplantation from a nationally funded UK centre reaches socially deprived groups and improves metabolic outcomes.

Aims/hypothesis: Type 1 diabetes complicated by hypoglycaemia is prevalent in socioeconomically deprived populations. Islet transplantation is of proven efficacy in type 1 diabetes complicated by hypoglycaemia, but it is not known if nationally funded programmes reach the socioeconomically deprived. Our aim was to determine: (1) socioeconomic indices in participants referred to our nationally funded programme; and (2) if metabolic outcomes in our transplant recipients were improved.

A comparison of in vitro nucleosome positioning mapped with chicken, frog and a variety of yeast core histones.

Using high-throughput sequencing, we have mapped sequence-directed nucleosome positioning in vitro on four plasmid DNAs containing DNA fragments derived from the genomes of sheep, drosophila, human and yeast. Chromatins were prepared by reconstitution using chicken, frog and yeast core histones. We also assembled yeast chromatin in which histone H3 was replaced by the centromere-specific histone variant, Cse4.

Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing β-like cells.

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer.

Pancreatic transcription factors containing protein transduction domains drive mouse embryonic stem cells towards endocrine pancreas.

Protein transduction domains (PTDs), such as the HIV1-TAT peptide, have been previously used to promote the uptake of proteins into a range of cell types, including stem cells. Here we generated pancreatic transcription factors containing PTD sequences and administered these to endoderm enriched mouse embryonic stem (ES) cells under conditions that were designed to mimic the pattern of expression of these factors in the developing pancreas. The ES cells were first cultured as embryoid bodies and treated with Activin A and Bone morphogenetic protein 4 (BMP4) to promote formation of definitive endoderm.

Palmitate enhances the differentiation of mouse embryonic stem cells towards white adipocyte lineages.

The number of adipocyte progenitors is determined early in foetal and neonatal development in a process which may be altered by gender and excess nutrient intake, and which in turn determines fat mass in adulthood and the risk of developing obesity. Here we investigate the hypothesis that excess nutrients, in this case the long chain fatty acid palmitate, can program differentiating stem cells towards white fat lineages. The experiments were performed on mouse embryonic stem cells in chemically defined media (CDM) supplemented with bone morphogenetic protein 4 (BMP4) and all trans-retinoic acid (RA).

Aperiodic interferometer for six degrees of freedom position measurement.

We present a new class of interferometer system that is capable of simultaneous measurement of absolute position and rotation in all six degrees of freedom (DOF) with nanometer precision. This novel capability is due to the employment of a system of interference fringes that is not periodic. One of the key strengths offered by this new approach is that the absolute position of the system can be determined with a single measurement, rather than by counting fringes during displacement from a known location.

Efficient differentiation of AR42J cells towards insulin-producing cells using pancreatic transcription factors in combination with growth factors.

The AR42J-B13 rat pancreatic acinar cell line was used to identify pancreatic transcription factors and exogenous growth factors (GFs) that might facilitate the reprogramming of exocrine cells into islets. Adenoviruses were used to induce exogenous expression of the pancreatic transcription factors (TFs) Pdx1, MafA, Ngn3 and Pax4. Individually Pdx1, MafA and Pax4 had no effect on the expression of endocrine markers, whilst adeno-Ngn3 on its own increased the expression of Pax4, Ngn3 and NeuroD.

A factor(s) secreted from MIN-6 beta-cells stimulates differentiation of definitive endoderm enriched embryonic stem cells towards a pancreatic lineage.

In the mouse the developing pancreas is controlled by contact with, and signalling molecules secreted from, surrounding cells. These factors are best studied using explant cultures of embryonic tissue. The present study was undertaken to determine whether embryonic stem (ES) cells could be used as an alternative model in vitro system to investigate the role of cell-cell interactions in the developing pancreas.

An engineered zinc finger protein reveals a role for the insulin VNTR in the regulation of the insulin and adjacent IGF2 genes.

An engineered zinc finger protein (eZFP) was isolated from a library based on its ability to activate expression of the endogenous insulin gene in HEK-293 cells. Using a panel of insulin promoter constructs, the eZFP was shown to act through the variable number of tandem repeat (VNTR) region located 365 base pairs upstream of the transcription start site. The eZFP also activated expression of the IGF2 gene that lies close to INS on chromosome 11p15.

Pancreatic stellate cells can form new beta-like cells.

Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of Type 1 and Type 2 diabetes, both of which are associated with decreased islet cell mass. To date, significant progress has been made in deriving insulin-secreting beta-like cells from human ES (embryonic stem) cells. However, the cells are not fully differentiated, and there is a long way to go before they could be used as a replenishable supply of insulin-secreting beta-cells for transplantation.

Combinatorial transcription factor regulation of the cyclic AMP-response element on the Pgc-1alpha promoter in white 3T3-L1 and brown HIB-1B preadipocytes.

Cold stress in rodents increases the expression of UCP1 and PGC-1alpha in brown and white adipose tissue. We have previously reported that C/EBPbeta specifically binds to the CRE on the proximal Pgc-1alpha promoter and increases forskolin-sensitive Pgc-1alpha and Ucp1 expression in white 3T3-L1 preadipocytes. Here we show that in mice exposed to a cold environment for 24 h, Pgc-1alpha, Ucp1, and C/ebpbeta but not C/ebpalpha or C/ebpdelta expression were increased in BAT.

Identification of adjacent binding sites for the YY1 and E4BP4 transcription factors in the ovine PrP (Prion) gene promoter.

The PrP gene encodes the cellular isoform of the prion protein (PrP(c)) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrP(c), exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs.

Biphasic induction of Pdx1 in mouse and human embryonic stem cells can mimic development of pancreatic beta-cells.

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes. Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development. Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse and human ES cell.

Pancreatic transcription factors and their role in the birth, life and survival of the pancreatic beta cell.

In recent years major progress has been made in understanding the role of transcription factors in the development of the endocrine pancreas in the mouse. Here we describe how a number of these transcription factors play a role in maintaining the differentiated phenotype of the beta cell, and in the mechanisms that allow the beta cell to adapt to changing metabolic demands that occur throughout life. Amongst these factors, Pdx1 plays a critical role in defining the region of the primitive gut that will form the pancreas, Ngn3 expression drives cells towards an endocrine lineage, and a number of additional proteins including Pdx1, in a second wave of expression, Pax4, NeuroD1/beta2, and MafA act as beta cell differentiation factors.

Stem cells and metabolic diseases.

Obesity is a metabolic disorder, which has been recognized as a global epidemic. It contributes to insulin resistance, the major cause of Type 2 diabetes, as well as to the development of other related diseases. Our basic premise is that a better understanding of how adult stem cells of the pancreas contribute to the maintenance of the pancreatic beta-cell pool against the increased metabolic demands associated with obesity may provide new therapeutic targets for treating diabetes.

Embryonic stem cell therapy for diabetes mellitus.

There is a compelling need to develop novel therapies for diabetes mellitus. Recent successes in the transplantation of islets of Langerhans are seen as a major breakthrough. However, there is huge disparity between potential recipients and the availability of donor tissue.

C/EBPbeta reprograms white 3T3-L1 preadipocytes to a Brown adipocyte pattern of gene expression.

cAMP-dependent protein kinase induction of PPARgamma coactivator-1alpha (PGC-1alpha) and uncoupling protein 1 (UCP1) expression is an essential step in the commitment of preadipocytes to the brown adipose tissue (BAT) lineage. We studied the molecular mechanisms responsible for differential expression of PGC-1alpha in HIB1B (BAT) and 3T3-L1 white adipose tissue (WAT) precursor cell lines. In HIB1B cells PGC-1alpha and UCP1 expression is cAMP-inducible, but in 3T3-L1 cells, expression is reduced and is cAMP-insensitive.

ATF-2 stimulates the human insulin promoter through the conserved CRE2 sequence.

The insulin promoter contains a number of dissimilar cis-acting regulatory elements that bind a range of tissue specific and ubiquitous transcription factors. Of the regulatory elements within the insulin promoter, the cyclic AMP responsive element (CRE) binds by far the most diverse array of transcription factors. Rodent insulin promoters have a single CRE site, whereas there are four CREs within the human insulin gene, of which CRE2 is the only one conserved between species.

Comparative analysis of insulin gene promoters: implications for diabetes research.

DNA sequences that regulate expression of the insulin gene are located within a region spanning approximately 400 bp that flank the transcription start site. This region, the insulin promoter, contains a number of cis-acting elements that bind transcription factors, some of which are expressed only in the beta-cell and a few other endocrine or neural cell types, while others have a widespread tissue distribution. The sequencing of the genome of a number of species has allowed us to examine the manner in which the insulin promoter has evolved over a 450 million-year period.

Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin/EGFP mouse.

In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.