Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes.

Transcriptional enhancers are critical for maintaining cell-type-specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.

The role of miR-17-92 cluster in the expression of tumor suppressor genes in unrestricted somatic stem cells.

The miR-17-92 cluster consisted of seven miRNAs (mir-17-5p, -17-3p, -18a, -19a, -20a, -19b-1, and -92a-1). Previous studies have shown this cluster has been over-expressed in several cancers. The aim of this study was to evaluate the over-expression impacts of miR-17-92 on stem cells. In the current work, the effect of miR-17-92 cluster which was cloned in Lentiviral vector has been investigated on unrestricted somatic stem cells (USSCs). Tumor suppressor genes (p53, p15, RBL1, SMAD2, SMAD4, and MAPK-1) expression, especially p53, was considerably reduced. These data show the potential of miR-17-92 for oncogenesis regulation in stem cells. In conclusion, the role of miR-17-92 in USSCs may provide a better understanding of its function in tumorigenesis and for the possible use in cell therapy of the anti-mir-17-92 cluster.

Transcription factor decoy: a pre-transcriptional approach for gene downregulation purpose in cancer.

Gene therapy as a therapeutic approach has been the dream for many scientists around the globe. Many strategies have been proposed and applied for this purpose, yet the void for a functional safe method is still apparent. Since most of the diseases are caused by undesirable upregulation (oncogenes) or downregulation (tumor suppressor genes) of genes, major gene therapy's techniques affect gene expression. Most of the methods are used in post-transcriptional level such as RNA inhibitory (RNAi) and splice-switching oligonucleotides (SSOs). RNAi blocks messenger RNA (mRNA) translation by mRNA degradation or interruption between attachments of mRNA with ribosomes' subunits. However, one of the novel methods is the usage of transcription factor targeted decoys. DNA decoys are the new generation of functional gene downregulatory oligonucleotides which compete with specific binding sites of transcription factors. Considering the exponential growth of this technique in both in vitro and in vivo studies, in this paper, we aim to line out the description, design, and application of decoys in research and therapy.

MiR-371-373 cluster acts as a tumor-suppressor-miR and promotes cell cycle arrest in unrestricted somatic stem cells.

Recent advances in small RNA research have implicated microRNAs (miRNAs) as important regulators of proliferation and development. The miR-371-373 cluster is prominently expressed in human embryonic stem cells (ESCs) and rapidly decreases after cell differentiation. MiR-371-373 cluster was investigated as one of the key factors of stem cell maintenance and pluripotency in unrestricted somatic stem cells (USSCs) using a lentivirus system. Gene expression showed a dual effect on proliferation, which revealed a transient cell cycle progression and consequent repression in pluripotency factors and cell cycle genes. Cell proliferation analysis with CFU, MTT, and DNA content assays further confirmed the dual effect of cluster after prolonged exposure. Analyzing the course of action, it seems that miR-371-373 cluster acts as an onco/tumor suppressor-miR. MiR371-373 cluster acts by modulating the function of these factors and limiting the excessive cell cycle propagation upon oncogenic stimuli to protect cells from replicative stress, but also activate CDK inhibitors and transcriptional repressors of the retinoblastoma family to cause cell cycle arrest. In contrast to the previous studies, we believe that miR-371-373 cluster functions as a self-renewal miRNA to induce and maintain the pluripotent state but also to potentially inhibit dysregulated proliferation through cell cycle arrest. It seems that miR-371-373 cluster presents with a dual effect in this cellular context which may possess different actions in various cells. This not only expands the basic knowledge of the cluster but may offer a great chance for therapeutic interventions.

Mir-302 cluster exhibits tumor suppressor properties on human unrestricted somatic stem cells.

Many studies have reported that miR-302-367 cluster acts in different ways in various cell types. For instance, this cluster is shown to have a potential role in stemness regulation in embryonic stem cells (ESCs). On the other hand, this cluster inhibits the tumorigenicity of human pluripotent stem cells by coordinated suppression of CDK2 and CDK4/6 cell cycle pathways. Indeed, this cluster has a significant posttranscriptional impact on cell cycle progression. Previous reports have shown the participation of miR-302-367 cluster in cell cycle regulation of hESCs, MCF7, HepG2, and Teta-2 embryonal teratocarcinoma cells, but its effect on unrestricted somatic stem cells (USSCs) as a new source of human somatic stem cells from the umbilical cord blood remains to be elucidated. Therefore, in this study, we aimed to investigate the effect of miR-302-367 cluster on cell proliferation by MTT assay, cell cycle analysis, and colony formation assay. In addition, the expression of candidate cell cycle regulatory performance and tumor suppressor genes was determined. In this study, for the first time, we found that miR-302-367 cluster not only did not reprogram human USSCs into a pluripotent ESC-like state, but also inhibited the proliferation of human USSCs. Moreover, analyzing the cell cycle curve revealed a significant apoptotic phase upon viral introduction of miR-302-367. Our gene expression study revealed the overexpression of candidate genes after transduction of USSCs with miR-302-367 cluster. In conclusion, the controversial role of miR-302-367 in different cell types may provide better understanding for its role in stemness level and its antitumorigenicity potential in different contexts.

Coating of electrospun poly(lactic-co-glycolic acid) nanofibers with willemite bioceramic: improvement of bone reconstruction in rat model.

We have investigated the combination effects of bioceramics and poly(lactide-co-glycolide) (PLGA) on bone reconstruction in calvarial critical size defects using a rat model. Willemite (Zn2SiO4) ceramics were prepared and coated on the surface of electrospun fabricated scaffolds. After scaffolds and nanoparticles characterization, osteoconductivity of the construct was analyzed using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. Eight weeks after implantation, no sign of inflammation was observed at the site of the osseous defect. The results showed that the ceramics supported bone regeneration and highest bone reconstruction were observed in willemite-coated PLGA. This suggests that electrospun PLGA nanofibers coated with BG are potential candidate implants for bone tissue engineering applications.

Enhanced reconstruction of rat calvarial defects achieved by plasma-treated electrospun scaffolds and induced pluripotent stem cells.

Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.

Induction of differentiation by down-regulation of Nanog and Rex-1 in cord blood derived unrestricted somatic stem cells.

Stem cells with high self-renewal and tissue regeneration potentials are the core components of regenerative medicine. Adult stem cells with many available sources, high repairing ability, and also possessing no ethical issues are popular candidates in the clinical field. In this study we looked upon the effects of two transcription factors Nanog and Rex-1 in self-renewal and differentiation abilities of a subpopulation of cord blood stem cells known as unrestricted somatic stem cells (USSCs). USSCs were expanded and transfected in vitro with siRNAs targeting either Nanog, Rex-1, and in combination. Gene suppressions were achieved at both transcript and proteome level. Differentiations were evaluated by specific Real time PCR and differentiating staining. Nanog knock down revealed a significant increase in osteogenic markers, Osteocalcin and Osteopontin expression as well as a positive Alizarin Red staining, which proposes Osteogenesis. This treatment also became positive for Oil Red staining, implying adipogenic differentiation as well. In contrast, Rex-1 knock down showed an increase in MAP II and Nestin expression, which is a hall mark of neural differentiation. Surprisingly, treatment with both siRNAs did not express any changes in any of the assessed markers. Therefore, our results indicated a bilateral mesenchymal differentiation for Nanog and a neural lineage fate for Rex-1 suppression. Considering that both transcription factors are core activators of self-renewal and also are orchestrating with other factors, our results imply a positive feedback in response to changes in the regulatory network of self-renewal.

A comparison of pluripotency and differentiation status of four mesenchymal adult stem cells.

The self-renewal and differentiation status of a stem cell is very important in the applications concerning regenerative medicine. Proliferation capacity, differentiation potentials and epigenetic properties of stem cells differ between sources. Studies have shown the high potentials of stem cells in iPS reprogramming. To examine this; we have compared the stem-ness and differential potential of four adult stem cells from common sources. We show a correlation between pluripotency and differentiation status of each stem cell with available data on the reprogramming efficiency. Four human adult stem cells including, adipose tissue-mesenchymal stem cells (AT-MSC), bone marrow mesenchymal stem cells (BM-MSCs), nasal septum derived multipotent progenitors (NSP) and umbilical cord blood stem cells (USSCs) were isolated and characterized. The self- renewal and differentiation potentials of each stem cell were assessed. Stem-ness transcription factors and the propagation potentials of all cells were analyzed. Furthermore the differentiation potentials were evaluated using treatment with induction factors and specific MicroRNA profile. Real-time PCR results showed that our stem cells express innate differentiation factors, miR145 and Let7g, which regulate the stem-ness and also the reprogramming potentials of each stem cell. To complete our view, we compared the propagation and differentiation potentials by correlating the stem-ness gene expression with differentiation MicroRNAs, also the direct effect of these factors on reprogramming. Our results suggest that the potentials of adipose tissue stem cells for GMP (Good Manufacturing Practice) compliant starting material are adequate for clinical applications. Our results indicate a low risk potential for AT-MSCs as starting material for iPS production. Although let7g and mir145 are well known for their differentiation promoting effects, but function more of a fine tuning system between self-renewal and differentiation status.

miRNAs expressed differently in cancer stem cells and cancer cells of human gastric cancer cell line MKN-45.

Recent studies show that cancers may originate from special cells named cancer stem cells (CSCs). As miRNAs have a prominent role in regulating cell activities, a question arise, that is, if there is any difference in miRNA expression level between CSC and other cancer cells of human gastric cancer cell line MKN-45. In this study, CSCs were isolated by fluorescence-activated cell sorter based on the expression level of cell surface marker CD44. CSC characteristics were checked using spheroid formation assay and soft agar assay. Using reverse transcriptase polymerase chain reaction (RT-PCR), the expression level of some stemness genes was studied. Real-time q-PCR was used for analysis of the expression level of miRNAs. CSCs were able to make spheroids and colonies, whereas other cancer cells failed to show aforementioned features. In addition, RT-PCR resulted in a difference in the expression levels of Nanog, Sox2, Lin28 and Oct-4 between these two kinds of cells. Real-time RT-PCR analysis demonstrated an increase in mir-21 and mir-302 expression level in CSCs, relative to cancer cells, whereas let-7a expression level was decreased in CSC in comparison with cancer cells, which may be due to their different differentiation level. On the other hand, mir-372, mir-373 and mir-520c-5p were markedly increased in cancer cells in comparison with CSCs. This study shows that there is a difference in miRNA expression level between CSCs and other cancer cells, which reflects dissimilar molecular pathways in these cells. These miRNAs may be promising objects for targeting CSCs specifically and efficiently.

Pluripotency Crossroads: Junction of Transcription Factors, Epigenetic Mechanisms, MicroRNAs, and Long Non-coding RNAs.

Embryonic stem cells (ESCs) are derived from inner cell mass (ICM) and have the potency to differentiate into three germ layers (ectoderm, endoderm, and mesoderm). This potency of ESCs, called pluripotency, is critical for maintaining stemness. Transcriptional regulatory circuitry preserving stemness consists of transcription factors (TFs), epigenetic mechanisms, microRNAs (miRNAs or miRs), and long non-coding RNAs (lncRNAs). In this circuitry, components assist each other to activate essential genes for maintaining pluripotency and suppressing lineage-specific genes. TFs act directly by binding to their binding sites in the genome or indirectly by activating another gene (such as a miR), epigenetic mechanisms play their role by providing an activatory or inhibitory context for transcription, miRNAs regulate gene expression at the post-transcriptional level, and lncRNAs act as a scaffold function for epigenetic elements, regulating gene expression in ESCs. All these factors create a crossroad and collaborate to sustain stemness in the ESCs. Herein, we explain the role of each member in this circuitry and demonstrate the significance of the crossroad for keeping stemness.

Evaluation and comparison of the in vitro characteristics and chondrogenic capacity of four adult stem/progenitor cells for cartilage cell-based repair.

Cell-based therapy is being considered as a promising approach to regenerate damaged cartilage. Though, autologous chondrocyte implantation is the most effective strategy currently in use, but is hampered by some drawbacks seeking comprehensive research to surmount existing limitations or introducing alternative cell sources. In this study, we aimed to evaluate and compare the in vitro characteristics and chondrogenic capacity of some easily available adult cell sources for use in cartilage repair which includes: bone marrow-derived mesenchymal stem cells (MSC), adipose tissue-derived MSC, articular chondrocyte progenitors, and nasal septum-derived progenitors. Human stem/progenitor cells were isolated and expanded. Cell's immunophenotype, biosafety, and cell cycle status were evaluated. Also, cells were seeded onto aligned electrospun poly (l-lactic acid)/poly (ε-caprolactone) nanofibrous scaffolds and their proliferation rate as well as chondrogenic potential were assessed. Cells were almost phenotypically alike as they showed similar cell surface marker expression pattern. The aligned nanofibrous hybrid scaffolds could support the proliferation and chondrogenic differentiation of all cell types. However, nasal cartilage progenitors showed a higher proliferation potential and a higher chondrogenic capacity. Though, mostly similar in the majority of the studied features, nasal septum progenitors demonstrated a higher chondrogenic potential that in combination with their higher proliferation rate and easier access to the source tissue, introduces it as a promising cell source for cartilage tissue engineering and regenerative medicine. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 600-610, 2016.

Chromatin Dynamics in Lineage Commitment and Cellular Reprogramming.

Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells.

Enhancement of Immune Responses by Co-delivery of CCL19/MIP-3beta Chemokine Plasmid With HCV Core DNA/Protein Immunization.

BACKGROUND: Using molecular adjuvants offers an attractive strategy to augment DNA vaccine-mediated immune responses. Several studies have revealed that an efficient HCV vaccine model should be able to induce both humoral and cell mediated immune responses targeting the conserved regions of the virus to circumvent the immune escape mutants. The beta chemokine Macrophage Inflammatory Protein 3-beta (MIP-3beta) is a key modulator of dendritic cells (DCs) and T-cells interaction, functions during immune response induction and is secreted specifically by cells in the lymphoid tissues. OBJECTIVES: In the present study, we questioned whether co-administration of MIP-3beta gene could enhance the immune responses to HCV core in DNA vaccination. MATERIALS AND METHODS: Expression and biological activity of MIP-3beta expressing plasmid were evaluated by ELISA and transwell migration assays, respectively. HCV core DNA vaccine ± plasmid expressing MIP-3beta were electroporated subcutaneously to the front foot pads of BALB/c mice on days 0 and 14, and HCV core protein booster was applied to all core-DNA-vaccine received mice on the day 28. Both cell mediated immunity (proliferation, IFN-γ and IL-4 cytokine release, IFN-γ ELISpot and cytotoxic Granzyme B release assays) and humoral immune responses (total IgG and IgG2a/IgG1 subtyping) were evaluated ten days after final immunization. RESULTS: Mice covaccinated with MIP-3beta elicited an enhanced Th1 biased systemic immune response as evidenced by higher IFN-γ/IL-4 and anti-core IgG2a/IgG1 ratio, lymphoproliferation, strong cytolytic GrzB release and enhanced population of IFN-γ producing immunocytes. Likewise, the humoral immune response assumed as the total anti-core IgG level was augmented by MIP-3beta co-delivery. CONCLUSIONS: These results exhibited the immuno potentiator effects of MIP-3beta plasmid when coadministrated with the HCV core DNA vaccine. Complimentary studies integrating MIP-3beta as a genetic adjuvant in HCV-core-DNA vaccination models are warranted.