Circular and Circulating DNA in Inflammatory Bowel Disease: From Pathogenesis to Potential Molecular Therapies.

Inflammatory bowel diseases (IBD), including Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic multifactorial disorders which affect the gastrointestinal tract with variable extent. Despite extensive research, their etiology and exact pathogenesis are still unknown. Cell-free DNAs (cfDNAs) are defined as any DNA fragments which are free from the origin cell and able to circulate into the bloodstream with or without microvescicles. CfDNAs are now being increasingly studied in different human diseases, like cancer or inflammatory diseases. However, to date it is unclear how IBD etiology is linked to cfDNAs in plasma. Extrachromosomal circular DNA (eccDNA) are non-plasmidic, nuclear, circular and closed DNA molecules found in all eukaryotes tested. CfDNAs appear to play an important role in autoimmune diseases, inflammatory processes, and cancer; recently, interest has also grown in IBD, and their role in the pathogenesis of IBD has been suggested. We now suggest that eccDNAs also play a role in IBD. In this review, we have comprehensively collected available knowledge in literature regarding cfDNA, eccDNA, and structures involving them such as neutrophil extracellular traps and exosomes, and their role in IBD. Finally, we focused on old and novel potential molecular therapies and drug delivery systems, such as nanoparticles, for IBD treatment.

Direct conversion of mouse fibroblasts into induced neural stem cells.

Terminally differentiated cells can be directly converted into different types of somatic cells by using defined factors, thus circumventing the pluripotent state. However, low reprogramming efficiency, along with the absence of proliferation of some somatic cell types, makes it difficult to generate large numbers of cells with this method. Here we describe a protocol to directly convert mouse fibroblasts into self-renewing induced neural stem cells (iNSCs) that can be expanded in vitro, thereby overcoming the limitations associated with low reprogramming efficiency. The four transcription factors required for direct conversion into iNSCs (Sox2, Klf4, Myc (also known as c-Myc) and Pou3f4 (also known as Brn4)) do not generate a pluripotent cell state, and thus the risk for tumor formation after transplantation is reduced. By following the current protocol, iNSCs are observed 4-5 weeks after transduction. Two additional months are required to establish clonal iNSC cell lines that exhibit retroviral transgene silencing and that differentiate into neurons, astrocytes and oligodendrocytes.

Counteracting activities of OCT4 and KLF4 during reprogramming to pluripotency.

Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) after overexpressing four transcription factors, of which Oct4 is essential. To elucidate the role of Oct4 during reprogramming, we investigated the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types using microarray analysis. By downregulating somatic-specific genes, Oct4 induction influenced each transcriptional program in a unique manner. A significant upregulation of pluripotent markers could not be detected. Therefore, OCT4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Finally, Oct4 overexpression upregulated the gene Mgarp in all the analyzed cell types. Strikingly, Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, OCT4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs.

Unrestricted somatic stem cells (USSC) from human umbilical cord blood display uncommitted epigenetic signatures of the major stem cell pluripotency genes.

Unrestricted somatic stem cells (USSC) from human cord blood display a broad differentiation potential for ectodermal, mesodermal, and endodermal cell types. The molecular basis for these stem cell properties is unclear and unlike embryonic stem cells (ESC) none of the major stem cell factors OCT4, SOX2, and NANOG exhibits significant expression in USSC. Here, we report that these key stem cell genes hold an epigenetic state in between that of an ESC and a terminally differentiated cell type. DNA methylation analysis exhibits partial demethylation of the regulatory region of OCT4 and a demethylated state of the NANOG and SOX2 promoter/enhancer regions. Further genome-wide DNA methylation profiling identified a partially demethylated state of the telomerase gene hTERT. Moreover, none of the pluripotency factors exhibited a repressive histone signature. Notably, SOX2 exhibits a bivalent histone signature consisting of the opposing histone marks dimeH3K4 and trimeH3K27, which is typically found on genes that are "poised" for transcription. Consequently, ectopic expression of OCT4 in USSC led to rapid induction of expression of its known target gene SOX2. Our data suggest that incomplete epigenetic repression and a "poised" epigenetic status of pluripotency genes preserves the USSC potential to be able to react adequately to distinct differentiation and reprogramming cues.

Somatic cell nuclear reprogramming of mouse oocytes endures beyond reproductive decline.

The mammalian oocyte has the unique feature of supporting fertilization and normal development, while capable of reprogramming nuclei of somatic cells toward pluripotency, and occasionally even totipotency. While oocyte quality is known to decay with somatic aging, it is not a given that different biological functions decay concurrently. In this study, we tested whether oocyte's reprogramming ability decreases with aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT), giving rise to higher blastocysts rates compared to young donors ooplasts. Despite the differences in transcriptome between climacteric and young ooplasts, gene expression profiles of SCNT blastocysts were very similar. Importantly, embryonic stem cell lines with capacity to differentiate into tissues from all germ layers were derived from SCNT blastocysts obtained from climacteric ooplasts. Although apoptosis-related genes were down-regulated in climacteric ooplasts, and reprogramming by transcription factors (direct-induced pluripotency) benefits from the inhibition of p53-mediated apoptosis, reprogramming capacity of young ooplasts was not improved by blocking p53. However, more outgrowths were derived from SCNT blastocysts developed in the presence of a p53 inhibitor, indicating a beneficial effect on trophectoderm function. Results strongly suggest that oocyte-induced reprogramming outcome is determined by the availability and balance of intrinsic pro- and anti-reprogramming factors tightly regulated and even improved throughout aging, leading to the proposal that oocytes can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction.