Defects in Membrane Frizzled-related Protein (MFRP) cause autosomal recessive retinitis pigmentosa (RP). MFRP codes for a retinal pigment epithelium (RPE)-specific membrane receptor of unknown function. In patient-specific induced pluripotent stem (iPS)-derived RPE cells, precise levels of MFRP, and its dicistronic partner CTRP5, are critical to the regulation of actin organization. Overexpression of CTRP5 in naïve human RPE cells phenocopied behavior of MFRP-deficient patient RPE (iPS-RPE) cells. AAV8 (Y733F) vector expressing human MFRP rescued the actin disorganization phenotype and restored apical microvilli in patient-specific iPS-RPE cell lines. As a result, AAV-treated MFRP mutant iPS-RPE recovered pigmentation and transepithelial resistance. The efficacy of AAV-mediated gene therapy was also evaluated in Mfrp(rd6)/Mfrp(rd6) mice--an established preclinical model of RP--and long-term improvement in visual function was observed in AAV-Mfrp-treated mice. This report is the first to indicate the successful use of human iPS-RPE cells as a recipient for gene therapy. The observed favorable response to gene therapy in both patient-specific cell lines, and the Mfrp(rd6)/Mfrp(rd6) preclinical model suggests that this form of degeneration caused by MFRP mutations is a potential target for interventional trials.
Common polymorphisms in the first intron of FTO are associated with increased body weight in adults. Previous studies have suggested that a CUX1-regulatory element within the implicated FTO region controls expression of FTO and the nearby ciliary gene, RPGRIP1L. Given the role of ciliary genes in energy homeostasis, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. We find that Rpgrip1l?/? mice are hyperphagic and fatter, and display diminished suppression of food intake in response to leptin administration. In the hypothalamus of Rpgrip1l?/? mice, and in human fibroblasts with hypomorphic mutations in RPGRIP1L, the number of AcIII-positive cilia is diminished, accompanied by impaired convening of the leptin receptor to the vicinity of the cilium, and diminished pStat3 in response to leptin. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO locus.
Hyperphagia is a central feature of inherited disorders (e.g., Prader-Willi Syndrome) in which obesity is a primary phenotypic component. Hyperphagia may also contribute to obesity as observed in the general population, thus raising the potential importance of common underlying mechanisms and treatments. Substantial gaps in understanding the molecular basis of inherited hyperphagia syndromes are present as are a lack of mechanistic of mechanistic targets that can serve as a basis for pharmacologic and behavioral treatments.
The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.
While the past decade has seen great progress in mapping loci for common diseases, studying how these risk alleles lead to pathology remains a challenge. Age-related macular degeneration (AMD) affects 9 million older Americans, and is characterized by the loss of the retinal pigment epithelium (RPE). Although the closely linked genome-wide association studies ARMS2/HTRA1 genes, located at the chromosome 10q26 locus, are strongly associated with the risk of AMD, their downstream targets are unknown. Low population frequencies of risk alleles in tissue banks make it impractical to study their function in cells derived from autopsied tissue. Moreover, autopsy eyes from end-stage AMD patients, where age-related RPE atrophy and fibrosis are already present, cannot be used to determine how abnormal ARMS2/HTRA1 expression can initiate RPE pathology. Instead, induced pluripotent stem (iPS) cell-derived RPE from patients provides us with earlier stage AMD patient-specific cells and allows us to analyze the underlying mechanisms at this critical time point. An unbiased proteome screen of A2E-aged patient-specific iPS-derived RPE cell lines identified superoxide dismutase 2 (SOD2)-mediated antioxidative defense in the genetic allele's susceptibility of AMD. The AMD-associated risk haplotype (T-in/del-A) impairs the ability of the RPE to defend against aging-related oxidative stress. SOD2 defense is impaired in RPE homozygous for the risk haplotype (T-in/del-A; T-in/del-A), while the effect was less pronounced in RPE homozygous for the protective haplotype (G-Wt-G; G-Wt-G). ARMS2/HTRA1 risk alleles decrease SOD2 defense, making RPE more susceptible to oxidative damage and thereby contributing to AMD pathogenesis.
Generation of surrogate sources of insulin-producing ?-cells remains a goal of diabetes therapy. While most efforts have been directed at differentiating embryonic or induced pluripotent stem (iPS) cells into ?-like-cells through endodermal progenitors, we have shown that gut endocrine progenitor cells of mice can be differentiated into glucose-responsive, insulin-producing cells by ablation of transcription factor Foxo1. Here we show that FOXO1 is present in human gut endocrine progenitor and serotonin-producing cells. Using gut organoids derived from human iPS cells, we show that FOXO1 inhibition using a dominant-negative mutant or lentivirus-encoded small hairpin RNA promotes generation of insulin-positive cells that express all markers of mature pancreatic ?-cells, release C-peptide in response to secretagogues and survive in vivo following transplantation into mice. The findings raise the possibility of using gut-targeted FOXO1 inhibition or gut organoids as a source of insulin-producing cells to treat human diabetes.
Wolfram syndrome is an autosomal recessive disorder caused by mutations in WFS1 and characterized by insulin-dependent diabetes mellitus, optic atrophy and deafness. To investigate the cause of beta cell failure, we used induced pluripotent stem (iPS) cells to create insulin-producing cells from individuals with Wolfram syndrome. WFS1-deficient beta cells showed increased levels of endoplasmic reticulum (ER) stress molecules, and decreased insulin content. Upon exposure to experimental ER stress, Wolfram beta cells showed impaired insulin processing and failed to increase insulin secretion in response to glucose and other secretagogues. Importantly, 4-phenyl butyric acid, a chemical protein folding and trafficking chaperone, restored normal insulin synthesis and the ability to upregulate insulin secretion. These studies show that ER stress plays a central role in beta cell failure in Wolfram syndrome and indicate that chemical chaperones might have therapeutic relevance under conditions of ER stress in Wolfram syndrome and other forms of diabetes.
ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations.
The remarkable ability of oocytes to reinstate the totipotent state from a unipotent somatic cell, allowing the cloning of animals and the generation of human stem cells, has fascinated scientists for decades. Due to the complexity of oocytes, it has remained challenging to understand the rapid reprogramming following nuclear transfer at a molecular level. Conversely, the detailed characterization of molecular mechanisms is also often insufficient to comprehend the functional relevance of a complex molecular process, such as the dissociation of transcription factors from chromatin during cell division, the role of chromatin modifications in cellular memory, or of cell type-specific DNA replication. This review attempts to bridge the gap between nuclear transfer and molecular biology by focusing on the role of the cell cycle in reprogramming.
Diabetes is a disorder characterized by loss of ? cell mass and/or ? cell function, leading to deficiency of insulin relative to metabolic need. To determine whether stem cell-derived ? cells recapitulate molecular-physiological phenotypes of a diabetic subject, we generated induced pluripotent stem cells (iPSCs) from subjects with maturity-onset diabetes of the young type 2 (MODY2), which is characterized by heterozygous loss of function of the gene encoding glucokinase (GCK). These stem cells differentiated into ? cells with efficiency comparable to that of controls and expressed markers of mature ? cells, including urocortin-3 and zinc transporter 8, upon transplantation into mice. While insulin secretion in response to arginine or other secretagogues was identical to that in cells from healthy controls, GCK mutant ? cells required higher glucose levels to stimulate insulin secretion. Importantly, this glucose-specific phenotype was fully reverted upon gene sequence correction by homologous recombination. Our results demonstrate that iPSC-derived ? cells reflect ? cell-autonomous phenotypes of MODY2 subjects, providing a platform for mechanistic analysis of specific genotypes on ? cell function.
Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13(NEG)SSEA4(POS)Tra-1-60(POS) on day 7-10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and "contaminating" partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.
Germline mutations of the breast cancer 1 (BRCA1) gene are a major cause of familial breast and ovarian cancer. The BRCA1 protein displays E3 ubiquitin ligase activity, and this enzymatic function is thought to be required for tumor suppression. To test this hypothesis, we generated mice that express an enzymatically defective Brca1. We found that this mutant Brca1 prevents tumor formation to the same degree as does wild-type Brca1 in three different genetically engineered mouse (GEM) models of cancer. In contrast, a mutation that ablates phosphoprotein recognition by the BRCA C terminus (BRCT) domains of BRCA1 elicits tumors in each of the three GEM models. Thus, BRCT phosphoprotein recognition, but not the E3 ligase activity, is required for BRCA1 tumor suppression.
Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation.
The New York Stem Cell Foundations "Fifth Annual Translational Stem Cell Research Conference" convened on October 12-13, 2010 at the Rockefeller University in New York City. The conference attracted over 400 scientists, patient advocates, and stem cell research supporters from 16 countries. In addition to poster and platform presentations, the conference featured panels entitled "Road to the Clinic" and "Regulatory Roadblocks."
The exchange of the oocytes genome with the genome of a somatic cell, followed by the derivation of pluripotent stem cells, could enable the generation of specific cells affected in degenerative human diseases. Such cells, carrying the patients genome, might be useful for cell replacement. Here we report that the development of human oocytes after genome exchange arrests at late cleavage stages in association with transcriptional abnormalities. In contrast, if the oocyte genome is not removed and the somatic cell genome is merely added, the resultant triploid cells develop to the blastocyst stage. Stem cell lines derived from these blastocysts differentiate into cell types of all three germ layers, and a pluripotent gene expression program is established on the genome derived from the somatic cell. This result demonstrates the feasibility of reprogramming human cells using oocytes and identifies removal of the oocyte genome as the primary cause of developmental failure after genome exchange.
Nuclear transfer allows the reprogramming of somatic cells to totipotency. The cell cycle state of the donor and recipient cells, as well as their extent of differentiation, have each been cited as important determinants of reprogramming success. Here, we have used donor and recipient cells at various cell cycle and developmental stages to investigate the importance of these parameters. We found that many stages of the cell cycle were compatible with reprogramming as long as a sufficient supply of essential nuclear factors, such as Brg1, were retained in the recipient cell following enucleation. Consistent with this conclusion, the increased efficiency of reprogramming when using donor nuclei from embryonic cells could be explained, at least in part, by reintroduction of embryonic nuclear factors along with the donor nucleus. By contrast, cell cycle synchrony between the donor nucleus and the recipient cell was not required at the time of transfer, as long as synchrony was reached by the first mitosis. Our findings demonstrate the remarkable flexibility of the reprogramming process and support the importance of nuclear transcriptional regulators in mediating reprogramming.
Sponsored by the New York Stem Cell Foundation (NYSCF), the "Fourth Annual Translational Stem Cell Research Conference: Breaking Ground" convened October 13-14, 2009 at The Rockefeller University in New York City to discuss translational stem cell research. Attracting over 400 scientists, patient advocates, and stem cell research supporters from fifteen countries, the two-day conference featured an afternoon of panel discussions, intended for a broad audience, followed by a second day of scientific talks and poster presentations. This report summarizes both days of this exciting conference.
The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.
We developed a digital RNA allelotyping method for quantitatively interrogating allele-specific gene expression. This method involves ultra-deep sequencing of padlock-captured single-nucleotide polymorphisms (SNPs) from the transcriptome. We characterized four cell lines established from two human subjects in the Personal Genome Project. Approximately 11-22% of the heterozygous mRNA-associated SNPs showed allele-specific expression in each cell line and 4.3-8.5% were tissue-specific, suggesting the presence of tissue-specific cis regulation. When we applied allelotyping to two pairs of sibling human embryonic stem cell lines, the sibling lines were more similar in allele-specific expression than were the genetically unrelated lines. We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions. Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.
Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.
It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.
Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle-chromosome complexes. Mitochondrial DNA transferred with the nuclear genome was initially detected at levels below 1%, decreasing in blastocysts and stem-cell lines to undetectable levels, and remained undetectable after passaging for more than one year, clonal expansion, differentiation into neurons, cardiomyocytes or ?-cells, and after cellular reprogramming. Stem cells and differentiated cells had mitochondrial respiratory chain enzyme activities and oxygen consumption rates indistinguishable from controls. These results demonstrate the potential of nuclear genome transfer to prevent the transmission of mitochondrial disorders in humans.
Enucleated oocytes have the distinctive ability to reprogram somatic nuclei back to totipotency. Here, we investigate genome-scale DNA methylation patterns after nuclear transfer and compare them to the dynamics at fertilization. We identify specific targets for DNA demethylation after nuclear transfer, such as germline-associated promoters, as well as unique limitations that include certain repetitive element classes.
The U.S. Food and Drug Administration recently approved phase I/II clinical trials for embryonic stem (ES) cell-based retinal pigmented epithelium (RPE) transplantation, but this allograft transplantation requires lifelong immunosuppressive therapy. Autografts from patient-specific induced pluripotent stem (iPS) cells offer an alternative solution to this problem. However, more data are required to establish the safety and efficacy of iPS transplantation in animal models before moving iPS therapy into clinical trials. This study examines the efficacy of iPS transplantation in restoring functional vision in Rpe65(rd12)/Rpe65(rd12) mice, a clinically relevant model of retinitis pigmentosa (RP). Human iPS cells were differentiated into morphologically and functionally RPE-like tissue. Quantitative real-time polymerase chain reaction (RT-PCR) and immunoblots confirmed RPE fate. The iPS-derived RPE cells were injected into the subretinal space of Rpe65(rd12)/Rpe65(rd12) mice at 2 d postnatally. After transplantation, the long-term surviving iPS-derived RPE graft colocalized with the host native RPE cells and assimilated into the host retina without disruption. None of the mice receiving transplants developed tumors over their lifetimes. Furthermore, electroretinogram, a standard method for measuring efficacy in human trials, demonstrated improved visual function in recipients over the lifetime of this RP mouse model. Our study provides the first direct evidence of functional recovery in a clinically relevant model of retinal degeneration using iPS transplantation and supports the feasibility of autologous iPS cell transplantation for retinal and macular degenerations featuring significant RPE loss.
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