iPS Cell Reprogramming Research Articles

Epigenetic control and inheritance of rDNA arrays.

Ribosomal RNA (rRNA) genes exist in multiple copies arranged in tandem arrays known as ribosomal DNA (rDNA). The total number of gene copies is variable, and the mechanisms buffering this copy number variation remain unresolved. We surveyed the number, distribution, and activity of rDNA arrays at the level of individual chromosomes across multiple human and primate genomes. Each individual possessed a unique fingerprint of copy number distribution and activity of rDNA arrays. In some cases, entire rDNA arrays were transcriptionally silent. Silent rDNA arrays showed reduced association with the nucleolus and decreased interchromosomal interactions, indicating that the nucleolar organizer function of rDNA depends on transcriptional activity. Methyl-sequencing of flow-sorted chromosomes, combined with long read sequencing, showed epigenetic modification of rDNA promoter and coding region by DNA methylation. Silent arrays were in a closed chromatin state, as indicated by the accessibility profiles derived from Fiber-seq. Removing DNA methylation restored the transcriptional activity of silent arrays. Array activity status remained stable through the iPS cell re-programming. Family trio analysis demonstrated that the inactive rDNA haplotype can be traced to one of the parental genomes, suggesting that the epigenetic state of rDNA arrays may be heritable. We propose that the dosage of rRNA genes is epigenetically regulated by DNA methylation, and these methylation patterns specify nucleolar organizer function and can propagate transgenerationally.

Knockdown H19 Accelerated iPSCs Reprogramming through Epigenetic Modifications and Mesenchymal-to-Epithelial Transition.

H19 is an essential imprinted gene that is expressed to govern normal embryonic development. During reprogramming, the parental pronuclei have asymmetric reprogramming capacities and the critical reprogramming factors predominantly reside in the male pronucleus. After inhibiting the expression of H19 and Gtl2, androgenetic haploid ESCs (AG-haESCs) can efficiently and stably support the generation of healthy SC pups at a rate of ~20%, and double-knockout parthenogenetic haESCs can also produce efficiently. Induced pluripotent stem (iPS) cell reprogramming is thought to have a characteristic epigenetic pattern that is the reverse of its developmental potential; however, it is unclear how H19 participates in iPS cell reprogramming. Here, we showed that the expression of H19 was transiently increased during iPSC reprogramming. H19 knockdown resulted in greater reprogramming efficiency. The genes associated with pluripotency showed enhanced expression during the early reprogramming process, and the Oct4 promoter was demethylated by bisulfite genomic sequencing analysis. Moreover, expression analysis revealed that the mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT) were downregulated during reprogramming in H19 knockdown. These findings provide functional insight into the role of H19 as a barrier to the early reprogramming process.

Cbx7 promotes the generation of induced pluripotent stem cells

The iPS cells were discovered in 2006. With their ability to differentiate into cells of all three germ layers, iPS cells have great potential for clinical applications. Oct4, Sox2, c-Myc, and Klf4 were identified as the most effective factors for generating iPS cells. Despite this, iPS cells manufactured with these factors would still be inefficient. As a member of the chromobox family, chromobox protein homolog 7 (Cbx7) binds to PRC1 and PRC2 to inhibit genes involved in differentiation. A decrease in the expression of Cbx7 is observed during embryonic stem cell differentiation. Currently, no report discusses the role of Cbx7 in the production of iPS cells. In this study, we hypothesized that Cbx7 could increase iPS cell generation. We confirmed that Cbx7 is highly expressed in pluripotent stem cells (including ES and iPS cells). In addition, transfecting Cbx7 into fibroblasts increased Oct4, Sox2, c-Myc, and Klf4 expression. Moreover, we describe a novel approach to producing iPS cells using Cbx7 in combination with Oct4, Sox2, c-Myc, and Klf4. In summary, we have demonstrated that Cbx7 enhances the reprogramming of iPS cells and characterized the stemness and pluripotency of iPS cells.

Invivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient invivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression invivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Abstract PO-10: Histone 1 deficiency drives lymphoma through disruption of 3D chromatin architecture

Abstract Linker histone H1 binds to nucleosomes and causes chromatin compaction, yet little is known about their biologic function. Somatic missense mutations in histone 1 genes (HIST1H1B-E) occur in ~30% of follicular lymphomas and DLBCL, with significant mutual co-occurrence among these alleles, most frequently involving H1C and H1E. Examining whole exomes in 547 DLBCL patients, we found significant enrichment for H1 gene SNVs and copy number loss in the MCD class of ABC-DLBCLs as compared to other subtypes. Next we performed a genetic driver analysis in 101 DLBCLs with matched germline control to identify mutations significantly enriched over background somatic variation. We find that lymphoma-associated H1 alleles H1C and H1E are true genetic driver mutations in lymphomas. Lymphoma H1 mutations affect the H1 globular domain or C-terminus. We found that the globular domain mutants fail to bind to chromatin whereas C-ter mutants fail to compact chromatin as shown by atomic force microscopy, in vitro assembled nucleosome arrays, and FRAP assays in live cells. Hence both types of mutation confer loss of function. Constitutive H1C/E knockout mice are healthy and have no overt phenotype. However, immunization with T cell-dependent antigen caused significant GC hyperplasia (p=0.013) and disruption of polarity due to expansion in the number of centrocytes. Notably, H1C/EDKO GC B-cells readily outcompeted WT GC B-cells in mixed chimera experiments, indicating that they have superior fitness (p=0.0086). We performed RNA-seq in H1C/EDKO GC B-cells, which revealed an aberrant gene derepression signature. Strikingly, these same genes are upregulated during induced pluripotency (iPS cell) reprogramming and are normally silenced during early development by the PRC2 complex (p <0.05 to 1e-05). Indeed, histone mass spectrometry showed reduced H3K27me3 (p=0.0003) and increased H3K36me2 (p =0.001) in H1C/EDKO GC B cells. This prompted us to characterize the epigenome of purified H1C/EDKO vs WT GC B cells using Hi-C, ATAC-seq, and ChIP-seq for multiple histone marks. H1 deficiency causes profound architectural remodeling of the genome characterized by large-scale yet focal compartment shifts from compartment B to compartment A. The degree of decompaction results in distinct epigenetic states, primarily due to gain of H3K36me2 and/or loss of repressive H3K27me3. Strikingly, the H1C/EDKO primitive stem cell gene expression signature was highly significantly enriched (NES 1.24, FDR<0.001) in the RNA-seq profiles of independent cohorts of DLBCL patients with H1C and H1E mutations. We crossed H1C+/-H1E+/- mice with VavP-Bcl2 transgenic mice and observed significant acceleration of lymphomagenesis (p=0.0001). Consistent with acquisition of stem cell characteristics, H1C+/-H1E+/-;VavP-Bcl2 but not VavP-Bcl2 primary lymphoma cells manifested lymphoma-initiating functionality after secondary transplantation into recipient animals. Citation Format: Nevin Z. Yusufova, Matt Teater, Andreas Kloetgen, Alexey Soshnev, Adewola Osunade, Christopher Chin, Ashley Doane, Louis Staudt, David Scott, Neil Kelleher, Aristotelis Tsirigos, Marcin Imielinski, Yael David, David Allis, Ethel Cesarman, Ari Melnick. Histone 1 deficiency drives lymphoma through disruption of 3D chromatin architecture [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-10.

Development of a High-Efficacy Reprogramming Method for Generating Human Induced Pluripotent Stem (iPS) Cells from Pathologic and Senescent Somatic Cells.

Induced pluripotent stem (iPS) cells are a type of artificial pluripotent stem cell induced by the epigenetic silencing of somatic cells by the Yamanaka factors. Advances in iPS cell reprogramming technology will allow aging or damaged cells to be replaced by a patient’s own rejuvenated cells. However, tissue that is senescent or pathologic has a relatively low reprogramming efficiency as compared with juvenile or robust tissue, resulting in incomplete reprogramming; iPS cells generated from such tissue types do not have sufficient differentiation ability and are therefore difficult to apply clinically. Here, we develop a new reprogramming method and examine it using myofibroblasts, which are pathologic somatic cells, from patient skin tissue and from each of the four heart chambers of a recipient heart in heart transplant surgery. By adjusting the type and amount of vectors containing transcriptional factors for iPS cell reprogramming, as well as adjusting the transfection load and culture medium, the efficiency of iPS cell induction from aged patient skin-derived fibroblasts was increased, and we successfully induced iPS cells from myocardial fibroblasts isolated from the pathologic heart of a heart transplant recipient.

Prediction for Morphology and States of Stem Cell Colonies using a LSTM Network with Progressive Training Microscopy Images.

We present a new LSTM (P-LSTM: Progressive LSTM) network, aiming to predict morphology and states of cell colonies from time-lapse microscopy images. Apparent short-term changes occur in some types of time-lapse cell images. Therefore, long-term-memory dependent LSTM networks may not predict accurately. The P-LSTM network incorporates the images newly generated from cell imaging progressively into LSTM training to emphasize the LSTM short-term memory and thus improve the prediction accuracy. The new images are input into a buffer to be selected for batch training. For real-time processing, parallel computation is introduced to implement concurrent training and prediction on partitioned images.Two types of stem cell images were used to show effectiveness of the P-LSTM network. One is for tracking of ES cell colonies. The actual and predicted ES cell images possess similar colony areas and the same transitions of colony states (moving, merging or morphology changing), although the predicted colony mergers may delay in several time-steps. The other is for prediction of iPS cell reprogramming from the CD34+ human cord blood cells. The actual and predicted iPS cell images possess high similarity evaluated by the PSNR and SSIM similarity evaluation metrics, indicating the reprogramming iPS cell colony features and morphology can be accurately predicted.

Histone 1 Mutations Drive Lymphomagenesis By Inducing Primitive Stem Cell Functions and Epigenetic Instructions through Profound 3D Re-Organization of the B-Cell Genome

Melnick: Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.

Identification and characterization of iPS clones via automated live-imaging and in-process analysis for quality control using high throughput robotic system

Background & Aim Induced pluripotent stem (iPS) cells are being developed for a broad range of research and therapeutic applications. However, reprogrammed iPS clones are inevitably heterogeneous. Current value judgements are made by visual inspection and manual selection. These methods are prone to large variation and lack the standardization or scalability needed for clinical translation. The goal of this work is to develop and validate Cell XTM, a robust robotic platform that will enable automated, and standardized iPS cell fabrication. Methods, Results & Conclusion We used DF6-9-9T.B hiPSC cell line, reprogrammed skin fibroblasts and peripheral blood mononuclear cells to develop our image processing parameters and picking and weeding protocols. To define and quantify critical quality attributes (CQAs) of “completely” reprogrammed iPS cells in systematic repeatable and reproducible manner, the Cell XTMPlatform is integrated with ColonyzeTM4.0, a very user-friendly WindowsTM-based automated quantitative cell and colony analysis software. The principles and nomenclature enabling this approach are outlined in ASTM Standard F2944-12. To automate manipulation of iPS cells in rapid, precise, repeatable and rigorously documented manner, the Cell XTMdevice integrates imaging, cell-manipulation (biopsy, pick, weed), fluid handling, and motion control systems. Using the Cell XTMgraphical user-interface, imaging, media changes and automated image processing were performed as illustrated in Figure 1. Picking operations were effective, and transplanted cells continued to proliferate effectively with morphology that is unchanged from that of the original colony. Automated image processing will display output metrics (colony area, the x,y,z-centroid for each colony, colony density, roundness, perimeter etc), as well as highlight the periphery of individual colonies or cell boundaries in the montage image. Based on these metrics, one can quantitatively define the cells or colonies of interest and selectively pick and transfer the cells for future experiments. The Cell XTMPlatform provides an integrated system of automated quantitative cell and colony imaging and analysis as well as tools for precision manipulation (biopsy, picking and weeding) driven by quantitative protocols that objectively determine the CQAs for repeatable, reproducible and quality iPS cell manufacturing.

HUMAN INDUCED PLURIPOTENT STEM CELL REGION DETECTION IN BRIGHT-FIELD MICROSCOPY IMAGES USING CONVOLUTIONAL NEURAL NETWORKS

Human induced pluripotent stem (iPS) cells represent an ideal source for patient specific cell-based regenerative medicine. For practical uses of iPS cells, large-scale, cost- and time-effective production of fully reprogrammed iPS cells from a number of patients should be achieved. To achieve this goal, culture protocols for inducing iPS cells as well as methods for selecting fully reprogrammed iPS cells in a mixture of cells which are still in reprogramming and non-iPS differentiated cells, should be improved. This paper proposes a convolutional neural network (CNN) structure to classify a bright-field microscopy image as respective probability images. Each probability image represents regions of differentiated cells, fully reprogrammed iPS cells or cells still in reprogramming, respectively. The CNN classifier was trained by multiple types of image patches which represent differentiated, reprogramming and reprogrammed iPS cells, etc. Classification of an image containing the confirmed iPS cells by the trained CNN classifier shows that high classification accuracy can be achieved. Classifications of sets of time-lapse microscopy images show that growth and transition from CD34[Formula: see text] human cord blood cells through reprogramming to reprogrammed iPS cells can be visualized and quantitatively analyzed by the output time-series probability images. These experiment results show our CNN structure yields a potential tool to detect the differentiated cells that possibly undergo reprogramming to iPS cells for screening reagents or culture conditions in human iPS induction, and ultimately further understand the ideal culturing conditions for practical use in regenerative medicine.

SU32 - PATIENT-SPECIFIC AD IN VITRO MODELS FOR THE ANALYSIS OF LATE-ONSET ALZHEIMER'S DISEASE

Background Alzheimer´s Disease (AD) is the most common form of dementia. Environmental and genetic factors contribute to the risk for AD, but the underlying disease mechanisms are poorly understood. AD is genetically complex and shows heritability of up to 80%. Glia cells including astrocytes and microglia are affected in AD patients contributing to the tau pathology and the accumulation of neurotoxic amyloid as well. In recent years, Genome-Wide Association Studies (GWAS) allowed the identification of DNA variations associated with an elevated risk for AD. Together with other groups, we identified a number of AD susceptibility genes including CD33, SORL1, ABCA7 and TREM2, which highly recommends that the immune system plays a major role in onset, progression and treatment of AD. Reprogramming of EBV-transformed patient specific cell lines for the generation of induced pluripotent stem cells (iPS) enables the patient-specific differentiation into neurons, astrocytes, and microglia. Patient-specific cells can be used as a model to functionally characterize disease associated variants. Methods Blood was collected from AD patients carrying risk variants in AD susceptibility genes for the generation of iPS cells. SNP variants in those genes were genotyped based on their potential function according to literature and genomic localization (exonic non-synonymous, binding domain, or promoter affecting SNPs). Reprogrammed iPS cells carried the genetic background of a certain AD patient. Pluripotency was characterized by alkaline phosphatase staining, the expression of pluripotency markers, and the differentiation into the three germ layers. Crucial pluripotency markers are OCT4, SOX2, NANOG, KLF4, MYC, and LIN28. AD iPS cells were differentiated into astrocytes and microglia and the differentiation status was characterized by the expression of crucial glia cell markers. Further, AD susceptibility genes recently published in GWAS were analysed. Results The protein expression was successfully induced shown by immunofluorescence and western-blot analysis. Cells were also screened for the most efficient induction of the three germ layers and the induction of neural cell fates including glia cell fates. The established AD-specific iPS cell lines from AD patients represent a powerful tool for the analysis of molecular and cellular disease mechanisms. We established 4-step protocol for the generation of AD-specific microglia enabling the focused analysis of AD-associated risk variants. The protocol was verified by morphology, FACS analysis, immunofluorescence and RNA expression of crucial microglia marker, including hematopoetic lineage markers and the induction of well-described microglia markers including IBA1. The disease-specific analysis of the genetic background of AD patients represents a completely new approach for the understanding of AD genetics and AD-associated risk variants. Discussion Together, combining molecular genetics of AD for the investigation of risk variants and iPS cell technology for the generation of patient- and disease-specific stem cells provides a promising approach to characterize known disease mechanisms, to deepen the understanding of known disease mechanisms, and to discover unknown disease aspects.

Generation of the induced pluripotent stem cell line CSSi006-A (3681) from a patient affected by advanced-stage Juvenile Onset Huntington's Disease

Juvenile Onset Huntington's Disease (JOHD) is a rare variant of HD withage of onset ≤20 years, accounting for 3–10% of all HD patients. The rarity occurrence of JOHD cases, who severely progress towards mental and physical disability with atypical clinical manifestations compared to classical HD, are responsible of general lack of knowledge about this variant. We obtained a fully reprogrammed iPS cell line from fibroblasts of a JOHD patient carrying 65 CAG repeats and age at onset at age 15. At the biopsy time, the patient showed an advanced stage after 10 years of disease.

Understanding Parkinson's Disease through the Use of Cell Reprogramming.

Recent progress in the field of somatic cell reprogramming offers exciting new possibilities for the study and treatment of Parkinson's disease (PD). Reprogramming technology offers the ability to untangle the diverse contributing risk factors for PD, such as ageing, genetics and environmental toxins. In order to gain novel insights into such a complex disease, cell-based models of PD should represent, as closely as possible, aged human dopaminergic neurons of the substantia nigra. However, the generation of high yields of functionally mature, authentic ventral midbrain dopamine (vmDA) neurons has not been easy to achieve. Furthermore, ensuring cells represent aged rather than embryonic neurons has presented a significant challenge. To date, induced pluripotent stem (iPS) cells have received much attention for modelling PD. Nonetheless, direct reprogramming strategies (either to a neuronal or neural stem/progenitor fate) represent a valid alternative that are yet to be extensively explored. Direct reprogramming is faster and more efficient than iPS cell reprogramming, and appears to conserve age-related markers. At present, however, protocols aiming to derive authentic, mature vmDA neurons by direct reprogramming of adult human somatic cells are sorely lacking. This review will discuss the strategies that have been employed to generate vmDA neurons and their potential for the study and treatment of PD.

Conversion of Prostate Adenocarcinoma to Small Cell Carcinoma-Like by Reprogramming.

The lineage relationship between prostate adenocarcinoma and small cell carcinoma was studied by using the LuCaP family of xenografts established from primary neoplasm to metastasis. Expression of four stem cell transcription factor (TF) genes, LIN28A, NANOG, POU5F1, SOX2, were analyzed in the LuCaP lines. These genes, when force expressed in differentiated cells, can reprogram the recipients into stem-like induced pluripotent stem (iPS) cells. Most LuCaP lines expressed POU5F1, while LuCaP 145.1, representative of small cell carcinoma, expressed all four. Through transcriptome database query, many small cell carcinoma genes were also found in stem cells. To test the hypothesis that prostate cancer progression from "differentiated" adenocarcinoma to "undifferentiated" small cell carcinoma could involve re-expression of stem cell genes, the four TF genes were transduced via lentiviral vectors into five adenocarcinoma LuCaP lines-70CR, 73CR, 86.2, 92, 105CR-as done in iPS cell reprogramming. The resultant cells from these five transductions displayed a morphology of small size and dark appearing unlike the parentals. Transcriptome analysis of LuCaP 70CR* ("*" to denote transfected progeny) revealed a unique gene expression close to that of LuCaP 145.1. In a prostate principal components analysis space based on cell-type transcriptomes, the different LuCaP transcriptome datapoints were aligned to suggest a possible ordered sequence of expression changes from the differentiated luminal-like adenocarcinoma cell types to the less differentiated, more stem-like small cell carcinoma types, and LuCaP 70CR*. Prostate cancer progression can thus be molecularly characterized by loss of differentiation with re-expression of stem cell genes. J. Cell. Physiol. 231: 2040-2047, 2016. © 2016 Wiley Periodicals, Inc.

Exploring standards for industrializing human induced pluripotent stem cells

Popular belief assumes that human pluripotent cells can now be obtained in any lab or company by induced pluripotent stem (iPS) cell reprogramming. However, the difficulties in robustly producing human iPS-derived cells that are fit for drug discovery are becoming increasingly apparent. This is because we still have not come up with a strict definition of pluripotency. Our attempts at prospectively identifying differentiation-defective human iPS cells using teratoma assays or marker expression have clearly failed to date. Here, we will revisit how conventional pluripotency tests have failed in evaluating iPS cells adequately for drug discovery and emphasize two aspects of developmental transitions (what we call here a cell's chronological value and the segregation of factors as it differentiates) to elucidate inherent problems with our current understanding of human iPS cells. Finally, we challenge the field by presenting our perspective on distinguishing good human iPS cells from bad ones.

Epigenetic modification in congenital heart diseases by using stem cell technologies

Congenital heart diseases are the most common birth defects, occurring in more than 1% of newborns. The gene regulatory network of cardiac development has been revealed and some cases of congenital heart diseases have been shown to be associated with cardiac-specific gene mutations or related chromosomal abnormalities; however, almost all cases of congenital heart diseases are sporadic and the pathogenesis of heart malformations still remains unknown. Recently, studies of epigenetic regulation have been making progress in the field of cardiac development and the accumulated knowledge helps us understand more about cardiogenesis and congenital heart diseases. Interestingly, pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have attracted attention as tools to dissect epigenetic regulation during differentiation. Reprogramming and differentiation of ES cells and iPS cells can be induced by changes of gene expression patterns and epigenetic regulation, not by changing DNA sequences. We can also modify histone patterns and chromatin structures using chemical reagents. Hence, pluripotent stem cells enable us to dissect and modify epigenetic regulation during cardiogenesis in vitro. Moreover, in terms of iPS cells, they have become disease models because they can be generated from the somatic cells of patients. In this review, we summarize the recent findings of epigenetic regulation in cardiac development and congenital heart diseases. We also review the epigenetic modification during the reprogramming and cardiac differentiation of ES cells and iPS cells, and introduce our study showing that the disease-specific iPS cells of hypoplastic left heart syndrome, one of the severest congenital heart diseases with single ventricular circulation, exhibit unique epigenetic regulation during differentiation. Furthermore, we briefly focus on modifications of epigenetic regulation in cardiac development using chemical reagents, which suggest the possibility of treating congenital heart diseases using drugs. Lastly, we discuss the epigenetic memory of iPS cells, a specific feature that often complicates or prevents study of the differentiation of iPS cells.

Early reprogramming regulators identified by prospective isolation and mass cytometry.

In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1–11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.

Dppa3 expression is critical for generation of fully reprogrammed iPS cells and maintenance of Dlk1-Dio3 imprinting.

Reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) often generates partially reprogrammed iPSCs (pre-iPSCs), low-grade chimera forming iPSCs (lg-iPSCs) and fully reprogrammed, high-grade chimera production competent iPSCs (hg-iPSCs). Lg-iPSC transcriptome analysis revealed misregulated Dlk1-Dio3 cluster gene expression and subsequently the imprinting defect at the Dlk1-Dio3 locus. Here, we show that germ-cell marker Dppa3 is present only in lg-iPSCs and hg-iPSCs, and that induction with exogenous Dppa3 enhances reprogramming kinetics, generating all hg-iPSCs, similar to vitamin C (Vc). Conversely, Dppa3-null fibroblasts show reprogramming block at pre-iPSCs state and Dlk1-Dio3 imprinting defect. At the molecular level, we show that Dppa3 is associated with Dlk1-Dio3 locus and identify that Dppa3 maintains imprinting by antagonizing Dnmt3a binding. Our results further show molecular parallels between Dppa3 and Vc in Dlk1-Dio3 imprinting maintenance and suggest that early activation of Dppa3 is one of the cascades through which Vc facilitates the generation of fully reprogrammed iPSCs.

Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species.

The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells.

331 ABNORMAL DNA METHYLATION PATTERNS AND ALLELE-SPECIFIC EXPRESSION OF IMPRINTED GENES IN BOVINE-INDUCED PLURIPOTENT STEM CELLS

Pluripotency reacquisition of somatic cells has been achieved through nuclear transfer (NT) to oocytes and, more recently, through induction with pluripotency-related factors (iPS cells). However, the epigenetic reprogramming process that enables the derivation of both NT-derived cloned animals and iPS cells is usually incomplete, leading to unhealthy offspring and poorly reprogrammed iPS cell lines. These unfavourable outcomes result in part from abnormal genome DNA methylation that leads to aberrant gene expression patterns. For instance, differentially methylated regions (DMR) and monoalleleic expression of imprinted genes, essential for normal cellular commitment and early development, are thought to be severely disturbed by reprogramming techniques. Indeed, H19 and SNRPN, imprinted genes, were disturbed in bovine NT-derived embryos and fetuses. Herein we investigated whether the DMR and parent-of-origin expression of the imprinted genes H19 and SNRPN are also perturbed in iPS lines. To analyse the DMR methylation patterns and allelic expression of H19 and SNRPN using parental-specific polymorphisms, we derived multiple clones of bovine iPS (biPS) cells from an interspecies (Bos indicus × Bos taurus) fetal fibroblast (bFF) using transduction with a policystronic lentivirus containing mouse Oct4, Sox2 c-Myc, and Klf-4 transcription factors. The DNA methylation patterns were evaluated by bisulfite sequencing and allelic expression by designing allele-specific PCR probes. We also quantified transcript expression by RT-PCR of H19, IGF2, SNRPN, OCT4, and NANOG by normalization with 3 housekeeping genes (GAPDH, NAT1, and ACTB). The biPS lines were characterised by a high nuclear : cytoplasmic ratio, dome-shaped colonies, positive AP activity, embryoid body formation, in vitro and in vivo (teratoma) formation, and expression of pluripotency-related genes. Compared to the bFF cells, methylation analyses of H19 showed partial hypomethylation of the paternal DMR on 1 iPS cell line and partial demethylation of the CTCF-binding region in the DMR of 2 other biPS lines, indicating abnormal demethylation of 3 out of the 4 biPS lines analysed. Methylation analyses of SNRPN revealed a partial hypomethylation in the maternal DMR and partial hypermethylation of the paternal DMR in 2 iPS lines. Gene expression analyses revealed the biallelic expression of H19 and decreased global expression of both H19 and IGF2, as well as the exclusively monoallelic paternal expression and significant increase in global expression of SNRPN. Interestingly, although OCT4 was substantially overexpressed in biPS lines, we identified a hypermethylation of the CG-rich region of the OCT4 exon 1. Endogenous NANOG expression was observed in 2 biPS clones. We conclude that imprinting errors are observed in biPS clones, suggesting that these epigenetic anomalies are related to the reprogramming process and could be directly responsible for the variable phenotypes and low success rates of both cloning and iPS derivation procedures.Financial support was from NSERC, FAPESP (13/13686-8, 11/08376-4, 57877-3/2008, 08.135-2/2013), CNPq (573754/2008-0, 482163/2013-5).