Non-pluripotent Cells Research Articles

Embryonic stem cells maintain high origin activity and slow forks to coordinate replication with cell cycle progression

Embryonic stem (ES) cells are pluripotent stem cells that can produce all cell types of an organism. ES cells proliferate rapidly and are thought to experience high levels of intrinsic replication stress. Here, by investigating replication fork dynamics in substages of S phase, we show that mammalian pluripotent stem cells maintain a slow fork speed and high active origin density throughout the S phase, with little sign of fork pausing. In contrast, the fork speed of non-pluripotent cells is slow at the beginning of S phase, accompanied by increased fork pausing, but thereafter fork pausing rates decline and fork speed rates accelerate in an ATR-dependent manner. Thus, replication fork dynamics within the S phase are distinct between ES and non-ES cells. Nucleoside addition can accelerate fork speed and reduce origin density. However, this causes miscoordination between the completion of DNA replication and cell cycle progression, leading to genome instability. Our study indicates that fork slowing in the pluripotent stem cells is an integral aspect of DNA replication.

Accurate Estimation of Single-Cell Differentiation Potency Based on Network Topology and Gene Ontology Information.

One important task in single-cell analysis is to quantify the differentiation potential of single cells. Though various single-cell potency measures have been proposed, they are based on individual biological sources, thus not robust and reliable. It is still a challenge to combine multiple sources to generate a relatively reliable and robust measure to estimate differentiation. In this paper, we propose a New Centrality measure with Gene ontology information (NCG) to estimate single-cell potency. NCG is designed by combining network topology property with edge clustering coefficient, and gene function information using gene ontology function similarity scores. NCG distinguishes pluripotent cells from non-pluripotent cells with high accuracy, correctly ranks different cell types by their differentiation potency, tracks changes during the differentiation process, and constructs the lineage trajectory from human myoblasts into skeletal muscle cells. These indicate that NCG is a reliable and robust measure to estimate single-cell potency. NCG is anticipated to be a useful tool for identifying novel stem or progenitor cell phenotypes from single-cell RNA-Seq data. The source codes and datasets are available at https://github.com/Xinzhe-Ni/NCG.

Differential Expression Pattern of linc-ROR Spliced Variants in Pluripotent and Non-Pluripotent Cell Lines.

The human large intergenic non-coding RNA-regulator of reprogramming program (linc-ROR) is known as a stem cell specific linc-RNA. linc-ROR counteracts differentiation via sequestering microRNA-145 (miR-145) that targets OCT4 transcript. Despite the research on the expression and function, the exact structure of linc-ROR transcripts is not clear. Considering the contribution of alternative splicing in transcripts structures and function, identifying different spliced variants of linc-ROR is necessary for further functional analyses. We aimed to find the alternatively spliced transcripts of linc-ROR and investigate their expression pattern in stem and cancer cell lines and during neural differentiation of NT2 cells as a model for understanding linc-ROR role in stem cell and differentiation. In this experimental study, linc-ROR locus was scanned for identifying novel exons. Different primer sets were used to detect new spliced variants by reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing. Quantitative PCR (qPCR) and RT-PCR were employed to profile expression of linc-ROR transcripts in different cell lines and during neural differentiation of stem cells. We could discover 13 novel spliced variants of linc-ROR harboring unique array of exons. Our work uncovered six novel exons, some of which were the product of exonized transposable elements. Monitoring expression profile of the linc-ROR spliced variants in a panel of pluripotent and non-pluripotent cells exhibited that all transcripts were primarily expressed in pluripotent cells. Moreover, the examined linc-ROR spliced variants showed a similar downregulation during neural differentiation of NT2 cells. Altogether, our data showed despite the difference in the structure and composition of exons, various spliced variants of linc-ROR showed similar expression pattern in stem cells and through differentiation.

Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells.

Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells.

Human ES and iPS cells display less drug resistance than differentiated cells, and naïve-state induction further decreases drug resistance.

Pluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.

Application of the RBBP9 Serine Hydrolase Inhibitor, ML114, Decouples Human Pluripotent Stem Cell Proliferation and Differentiation.

Retinoblastoma binding protein 9 (RBBP9) is required for maintaining the expression of both pluripotency and cell cycle genes in human pluripotent stem cells (hPSCs). An siRNA-based study from our group showed it does so by influencing cell cycle progression through the RB/E2F pathway. In non-pluripotent cells, RBBP9 is also known to have serine hydrolase (SH) activity, acting on currently undefined target proteins. The role of RBBP9 SH activity in hPSCs, and during normal development, is currently unknown. To begin assessing whether RBBP9 SH activity might contribute to hPSC maintenance, hPSCs were treated with ML114—a selective chemical inhibitor of RBBP9 SH activity. Stem cells treated with ML114 showed significantly reduced population growth rate, colony size and progression through the cell cycle, with no observable change in cell morphology or decrease in pluripotency antigen expression—suggesting no initiation of hPSC differentiation. Consistent with this, hPSCs treated with ML114 retained the capacity for tri-lineage differentiation, as seen through teratoma formation. Subsequent microarray and Western blot analyses of ML114-treated hPSCs suggest the nuclear transcription factor Y subunit A (NFYA) may be a candidate effector of RBBP9 SH activity in hPSCs. These data support a role for RBBP9 in regulating hPSC proliferation independent of differentiation, whereby inhibition of RBBP9 SH activity de-couples decreased hPSC proliferation from initiation of differentiation.

The Regulatory Effect of lncRNA PSORS1C3 on Different Variants of OCT4 in non-Pluripotent Cells

OCT4 is the major regulator of pluripotency in embryonic stem cells and its association with tumorigenesis, cellular stress response, and homeostatic multifactorial diseases have been recently reported. To serve the versatility in its function, OCT4 generates several transcript variants which their expression levels are tightly regulated through different mechanisms. PSORS1C3 is a long non-coding RNA with overlapping genomic location with OCT4 gene. Here, we investigated the effect of PSORS1C3 overexpression on OCT4 expression in different cell lines. Our data revealed that ectopic expression of PSORS1C3 did not affect OCT4 transcripts abundance in NT2 cells, as a model of pluripotent cells. However, in HEK293T cells, PSORS1C3 overexpression led to an increase in OCT4B as a homeostatic isoform and a decrease in OCT4A transcript level. We also observed that manipulating PSORS1C3 in HeLa cells, as a model of epithelial carcinoma line, caused an upregulation in OCT4A, OCT4C which could regulate stemness and proliferation and OCT4B transcripts at different time points. Our findings indicated that PSORS1C3 could affect the expression level of OCT4 spliced variants, according to their functions and the cells molecular context as well as genetic background. Considering these diverse regulatory effects and co-expression of OCT4 and PSORS1C3 in some cell lines, it is safe to consider PSORS1C3 as a modulator of OCT4 expression in non-pluripotent cells and in association with homeostatic pathways.

Defining Human Pluripotency.

Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.

Glutamine independence is a selectable feature of pluripotent stem cells.

Most rapidly proliferating mammalian cells rely on the oxidation of exogenous glutamine to support cell proliferation. We previously found that culture of mouse embryonic stem cells (ESCs) in the presence of inhibitors against MEK and GSK3β to maintain pluripotency reduces cellular reliance on glutamine for tricarboxylic acid (TCA) cycle anaplerosis, enabling ESCs to proliferate in the absence of exogenous glutamine. Here we show that reduced dependence on exogenous glutamine is a generalizable feature of pluripotent stem cells. Enhancing self-renewal, through either overexpression of pluripotency-associated transcription factors or altered signal transduction, decreases the utilization of glutamine-derived carbons in the TCA cycle. As a result, cells with the highest potential for self-renewal can be enriched by transient culture in glutamine-deficient media. During pluripotent cell culture or reprogramming to pluripotency, transient glutamine withdrawal selectively leads to the elimination of non-pluripotent cells. These data reveal that reduced dependence on glutamine anaplerosis is an inherent feature of self-renewing pluripotent stem cells and reveal a simple, non-invasive mechanism to select for mouse and human pluripotent stem cells within a heterogeneous population during both ESC passage and induced pluripotent cell reprogramming.

LncRNA PSORS1C3 is regulated by glucocorticoids and fine-tunes OCT4 expression in non-pluripotent cells

OCT4 is a transcription factor known for its regulatory roles in stemness, tumorigenesis and stress response. Considering its versatile functions, expression of OCT4 is regulated at different levels. PSORS1C3, a long non-coding RNA overlapped with OCT4, has a putative association with immune mediated diseases; however, its exact functions remained to be elucidated. Here, we demonstrated that PSORS1C3 is regulated by glucocorticoids (GC), has two endogenously active promoters, promoter 0 and 1, and two sets of transcripts, short and long variants. According to our findings, PSORS1C3 promoters behaved differently during neural differentiation of NT2 cells and glucocorticoid receptor (GR) activation. In both processes the expression pattern of short variants differed from that of long variants and was similar to OCT4 expression. Furthermore, our data revealed that PSORS1C3’s promoter 0 could act as an enhancer for OCT4 in non-pluripotent cells, where its deletion caused a significant decrease in OCT4 expression. Meanwhile, during GR activation promoter 0 functioned as a negative regulator and alleviated transcription induction of OCT4 after GC treatment. Altogether, our work clarified the structure and regulation of PSORS1C3, explained its relation to immune-related disease through GR signaling and introduced it as a novel fine-tuner of OCT4 expression in non-pluripotent cells.

Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

Introduction Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

Preparation of iPSCs for Targeted Proteomic Analysis.

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, extensive analysis of iPSC are required before their therapeutic applications. With recent developments in mass spectrometry and proteomics, this technique can become a great alternative to traditional genomic approaches for iPSC analysis. Here, we describe preparation of iPSC for targeted proteomic analysis, and measurement of pluripotency markers allowing for classification into either pluripotent or nonpluripotent cells.

ERBB3-Binding Protein 1 (EBP1) Is a Novel Developmental Pluripotency-Associated-4 (DPPA4) Cofactor in Human Pluripotent Cells.

Developmental Pluripotency-Associated-4 (DPPA4) is one of the few core pluripotency genes lacking clearly defined molecular and cellular functions. Here, we used a proteomics screening approach of human embryonic stem cell (hESC) nuclear extract to determine DPPA4 molecular functions through identification of novel cofactors. Unexpectedly, the signaling molecule ERBB3-binding protein 1 (EBP1) was the strongest candidate binding partner for DPPA4 in hESC. EBP1 is a growth factor signaling mediator present in two isoforms, p48 and p42. The two isoforms generally have opposing functions, however their roles in pluripotent cells have not been established. We found that DPPA4 preferentially binds p48 in pluripotent and NTERA-2 cells, but this interaction is largely absent in non-pluripotent cells and is reduced with differentiation. The DPPA4-EBP1 interaction is mediated at least in part in DPPA4 by the highly conserved SAF-A/B, Acinus and PIAS (SAP) domain. Functionally, we found that DPPA4 transcriptional repressive function in reporter assays is significantly increased by specific p48 knockdown, an effect that was abolished with an interaction-deficient DPPA4 ΔSAP mutant. Thus, DPPA4 and EBP1 may cooperate in transcriptional functions through their physical association in a pluripotent cell specific context. Our study identifies EBP1 as a novel pluripotency cofactor and provides insight into potential mechanisms used by DPPA4 in regulating pluripotency through its association with EBP1. Stem Cells 2018;36:671-682.

MiR-290 contributes to the low abundance of cyclin D1 protein in mouse embryonic stem cells.

Mouse miR-290 cluster miRNAs are expressed specifically in early embryos and embryonic germ cells. These miRNAs play critical roles in the maintenance of pluripotency and self-renewal. Here, we showed that Cyclin D1 is a direct target gene of miR-290 cluster miRNAs. Negative relationships between the expression of Cyclin D1 protein and miR-290 cluster miRNAs in pluripotent and non-pluripotent cells, as well as in differentiating CGR8 cells were observed. Inhibition of miR-290 cluster miRNAs could arrest cells at the G1 phase and slow down the cell proliferation in CGR8 mouse stem cells. Since miR-290 cluster miRNAs are the most dominant stem-cell-specific miRNAs, our results revealed an important cause for the absence of Cyclin D1 in mouse embryonic stem cells.

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

Back to basics: Refined nuclear reprogramming techniques yield higher-quality stem cells.

Back to basics: Refined nuclear reprogramming techniques yield higher-quality stem cells.

Specific detection of OCT4 isoforms in inflammatory bowel disease.

BackgroundDevelopmentally early cells are mobilized into peripheral blood in Crohn’s disease (CD) patients. OCT4, is considered to be important in sustaining the pluripotency of stem cells. OCT4 splicing variants are differentially expressed in pluripotent and non-pluripotent cells. Our study aims to investigate the expression pattern of OCT4 variants and SOX-2, an essential factor implicated in self-renewal and pluripotency, in tissue and blood samples from patients with IBD.MethodsPeripheral blood and tissue samples were collected from patients with active CD and ulcerative colitis (UC), and from healthy individuals. OCT4 expression was documented by Western blot, immunohistochemistry and by reverse transcription-real-time PCR. OCT4 isoform determination was documented using specific primers. SOX-2 expression levels were also evaluated.ResultsOCT4 protein levels were significantly higher in CD tissue samples than in CD blood samples, and in UC tissue samples. OCT4 protein was localized mainly in the cytosol. In all samples, only the OCT4 pseudogenes and the OCT4B1 variant were detected. OCT4B1 expression levels were elevated in both tissue and blood samples from CD and UC cases compared to healthy controls. In CD patients only SOX-2 mRNA levels were found slightly increased compared to healthy controls.ConclusionOur results suggest that OCT4 is expressed in patients with IBD. Furthermore, we found the presence of the OCT4B1 isoform in IBD in both tissue and blood samples. Our results have shown, that developmentally early cells might be mobilized into peripheral blood as result of tissue damage, indicating a possible role of these cells in repair of injured intestinal tract.

Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells.

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity.

Epigenetic biomarker to support classification into pluripotent and non-pluripotent cells.

Quality control of human induced pluripotent stem cells (iPSCs) can be performed by several methods. These methods are usually relatively labor-intensive, difficult to standardize, or they do not facilitate reliable quantification. Here, we describe a biomarker to distinguish between pluripotent and non-pluripotent cells based on DNA methylation (DNAm) levels at only three specific CpG sites. Two of these CpG sites were selected by their discriminatory power in 258 DNAm profiles – they were either methylated in pluripotent or non-pluripotent cells. The difference between these two β-values provides an Epi-Pluri-Score that was validated on independent DNAm-datasets (264 pluripotent and 1,951 non-pluripotent samples) with 99.9% specificity and 98.9% sensitivity. This score was complemented by a third CpG within the gene POU5F1 (OCT4), which better demarcates early differentiation events. We established pyrosequencing assays for the three relevant CpG sites and thereby correctly classified DNA of 12 pluripotent cell lines and 31 non-pluripotent cell lines. Furthermore, DNAm changes at these three CpGs were tracked in the course of differentiation of iPSCs towards mesenchymal stromal cells. The Epi-Pluri-Score does not give information on lineage-specific differentiation potential, but it provides a simple, reliable, and robust biomarker to support high-throughput classification into either pluripotent or non-pluripotent cells.

Pluripotent stem cells in mice.

Pluripotent stem cells are able to self-renew and to differentiate into all adult cell types. Many studies report data describing these cells and characterize them in molecular terms. Gene expression data of pluripotent and non-pluripotent cells from mouse were assembled. Machine learning was applied to classify samples into pluripotent and non-pluripotent cells. To identify minimal sets of best biomarkers, three methods were used: information gain, random forests, and genetic algorithm.