New Insights into X-Chromosome Reactivation during Reprogramming to Pluripotency

Dosage compensation between the sexes results in one X chromosome being inactivated during female mammalian development. Chromosome-wide transcriptional silencing from the inactive X chromosome (Xi) in mammalian cells is erased in a process termed X-chromosome reactivation (XCR), which has emerged as a paradigm for studying the reversal of chromatin silencing. XCR is linked with germline development and induction of naive pluripotency in the epiblast, and also takes place upon reprogramming somatic cells to induced pluripotency. XCR depends on silencing of the long non-coding RNA (lncRNA) X inactive specific transcript (Xist) and is linked with the erasure of chromatin silencing. Over the past years, the advent of transcriptomics and epigenomics has provided new insights into the transcriptional and chromatin dynamics with which XCR takes place. However, multiple questions remain unanswered about how chromatin and transcription related processes enable XCR. Here, we review recent work on establishing the transcriptional and chromatin kinetics of XCR, as well as discuss a model by which transcription factors mediate XCR not only via Xist repression, but also by direct targeting of X-linked genes.

Expression of SSEA4 and TRA1-60 as Marker of Induced Pluripotent Stem Cells by Small Molecule Compound VC6TFZ on Peripheral Blood Mononuclear Cell

IntroductionIt is possible to induce pluripotent stem cells from somatic cells, offering an infinite cell resource with the potential for disease research and use in regenerative medicine. Due to ease of accessibility, minimum invasive treatment, and can be kept frozen, peripheral blood mononuclear cells (PBMC) were an attractive source cell. VC6TFZ, a small molecule compound, has been successfully reprogrammed from mouse fibroblast induced pluripotent stem cells (iPSCs). However, it has not been confirmed in humans.ObjectiveThe aim of this research is to determine whether the small molecule compound VC6TFZ can induced pluripotency of PBMC to generate iPSCs detected with expression of SSEA4 and TRA1-60.MethodsUsing the centrifugation gradient density process, mononuclear cells were separated from peripheral venous blood. Mononuclear cells were cultured for 6 days in the expansion medium. The cells were divided into four groups; group 1 (P1), which was not exposed to small molecules (control group) and groups 2-4 (P2-P4), the experimental groups, subjected to various dosages of the small molecule compound VC6TFZ (VPA, CHIR, Tranylcypromine, FSK, Dznep, and TTNPB). The induction of pluripotency using small molecule compound VC6TFZ was completed within 14 days, then for 7 days the medium shifted to 2i medium. iPSCs identification in based on colony morphology and pluripotent gene expression, SSEA4 and TRA1-60 marker, using immunocytochemistry.ResultsColonies appeared on reprogramming process in day 7th. These colonies had round, large, and cobble stone morphology like ESC. Gene expression of SSEA4 and TRA 1-60 increased statisticaly significant than control group (SSEA4 were P2 p=0.007; P3 p=0.001; P4 p=0.009 and TRA 1-60 were P2 p=0.002; P3 p=0.001; P4 p=0.001).ConclusionSmall molecule compound VC6TFZ could induced pluripotency of human PBMC to generate iPSCs. Pluripotxency marker gene expression, SSEA 4 and TRA 1-60, in the experimental group was statistically significantly higher than in the control group.

Induced Pluripotency: A Powerful Tool for In Vitro Modeling

One of the greatest breakthroughs of regenerative medicine in this century was the discovery of induced pluripotent stem cell (iPSC) technology in 2006 by Shinya Yamanaka. iPSCs originate from terminally differentiated somatic cells that have newly acquired the developmental capacity of self-renewal and differentiation into any cells of three germ layers. Before iPSCs can be used routinely in clinical practice, their efficacy and safety need to be rigorously tested; however, iPSCs have already become effective and fully-fledged tools for application under in vitro conditions. They are currently routinely used for disease modeling, preparation of difficult-to-access cell lines, monitoring of cellular mechanisms in micro- or macroscopic scales, drug testing and screening, genetic engineering, and many other applications. This review is a brief summary of the reprogramming process and subsequent differentiation and culture of reprogrammed cells into neural precursor cells (NPCs) in two-dimensional (2D) and three-dimensional (3D) conditions. NPCs can be used as biomedical models for neurodegenerative diseases (NDs), which are currently considered to be one of the major health problems in the human population.

A mini review on production of pluripotency factors (Oct4, Sox2, Klf4 and c-Myc) through recombinant protein technology

The four transcription factors OCT4, SOX2, KLF4 and c-MYC are highly expressed in embryonic stem cells (ESC) and their overexpression can induce pluripotency, the ability to differentiate into all cell types of an organism. The ectopic expression such transcription factors could reprogram somatic stem cells become induced pluripotency stem cells (iPSC), an embryonic stem cells-like. Production of recombinant pluripotency factors gain interests due to high demand from generation of induced pluripotent stem cells in regenerative medical therapy recently. This review will focus on demonstrate the recent advances in recombinant pluripotency factor production using various host.

Sox2 modulation increases naïve pluripotency plasticity

Induced pluripotency provides a tool to explore the molecular mechanisms underlying the establishment, maintenance and differentiation of naïve pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, Sox2-low cells also differentiate towards the trophoblast lineage both in vitro and in vivo. At the single-cell level self-renewing Sox2-low nPSCs exhibit a homogeneous naïve molecular signature. However, they also display a basal trophoblast molecular signature and decreased ability of Oct4 to bind naïve-associated regulatory sequences compared to control cells. These features underlie observed enhanced cell potency upon the removal of self-renewing cues. In sum, this work defines Sox2 as a restrictor of developmental potential and suggests perturbation of the naïve pluripotent network as an underlying cause of increased cell potency.HighlightsLow Sox2 expression is sufficient for naïve pluripotent stem cell self-renewalLow Sox2 expression does not impair neurectoderm differentiation in vitroLow Sox2 expression impairs Oct4 genomic occupancyLow Sox2 expression increases naïve pluripotent cell plasticity in vitro and in vivo

A versatile bulk electrotransfection protocol for mouse embryonic fibroblast and iPS cells

A square-wave pulsing protocol was developed using OptiMEM-GlutaMAX for high efficient transfection of mouse embryonic fibroblast (MEF) and induced pluripotency stem (iPS) cells. An electrotransfection efficiency of > 95% was repeated for both MEF and iPS cells using reporter-encoding plasmids. The protocol was very efficient for plasmid size ranging from 6.2 to 13.5 kb. A high rate of targeted gene knockout (> 95 %) was produced in Venus transgenic cells using indels formation. Targeted deletions in the Venus transgene were performed by co-electroporation of two gRNA-encoding plasmids. In conclusion, this plasmid electrotransfection protocol is straight-forward, cost-effective, and efficient for CRISPRing mouse primary cells.