Cellular Reprogramming Process Research Articles

Ultrasound-triggered three dimensional hyaluronic acid hydrogel promotes in vitro and in vivo reprogramming into induced pluripotent stem cells

Cellular reprogramming technologies have been developed with different physicochemical factors to improve the reprogramming efficiencies of induced pluripotent stem cells (iPSCs). Ultrasound is a clinically applied noncontact biophysical factor known for regulating various cellular behaviors but remains uninvestigated for cellular reprogramming. Here, we present a new reprogramming strategy using low-intensity ultrasound (LIUS) to improve cellular reprogramming of iPSCs in vitro and in vivo. Under 3D microenvironment conditions, increased LIUS stimulation shows enhanced cellular reprogramming of the iPSCs. The cellular reprogramming process facilitated by LIUS is accompanied by increased mesenchymal to epithelial transition and histone modification. LIUS stimulation transiently modulates the cytoskeletal rearrangement, along with increased membrane fluidity and mobility to increase HA/CD44 interactions. Furthermore, LIUS stimulation with HA hydrogel can be utilized in application of both human cells and in vivo environment, for enhanced reprogrammed cells into iPSCs. Thus, LIUS stimulation with a combinatorial 3D microenvironment system can improve cellular reprogramming in vitro and in vivo environments, which can be applied in various biomedical fields.

A Molecular Perspective on HIF-1α and Angiogenic Stimulator Networks and Their Role in Solid Tumors: An Update.

Hypoxia-inducible factor-1α (HIF-1α) is a major transcriptional factor, which plays an important role in cellular reprogramming processes under hypoxic conditions, which facilitate solid tumors' progression. HIF-1α is directly involved in the regulation of the angiogenesis, metabolic reprogramming, and extracellular matrix remodeling of the tumor microenvironment. Therefore, an in-depth study on the role of HIF-1α in solid tumor malignancies is required to develop novel anti-cancer therapeutics. HIF-1α also plays a critical role in regulating growth factors, such as the vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor, in a network manner. Additionally, it plays a significant role in tumor progression and chemotherapy resistance by regulating a variety of angiogenic factors, including angiopoietin 1 and angiopoietin 2, matrix metalloproteinase, and erythropoietin, along with energy pathways. Therefore, this review attempts to provide comprehensive insight into the role of HIF-1α in the energy and angiogenesis pathways of solid tumors.

A chemical approach facilitates CRISPRa-only human iPSC generation and minimizes the number of targeted loci required.

Aim: We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). Methods: Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). Results: iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by in vitro and invivo. Conclusion: CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine.

Advances in the relationship between ferroptosis and epithelial-mesenchymal transition in cancer.

Epithelial-mesenchymal transition (EMT) is a cellular reprogramming process that converts epithelial cells into mesenchymal-like cells with migratory and invasive capabilities. The initiation and regulation of EMT is closely linked to a range of transcription factors, cell adhesion molecules and signaling pathways, which play a key role in cancer metastasis and drug resistance. The regulation of ferroptosis is intricately linked to various cell death pathways, intracellular iron homeostasis, and the protein network governing iron supply and storage. The ability of ferroptosis to disrupt cancer cells and overcome drug resistance lies in its control of intracellular iron ion levels. EMT process can promote the accumulation of iron ions, providing conditions for ferroptosis. Conversely, ferroptosis may impact the regulatory network of EMT by modulating transcription factors, signaling pathways, and cell adhesion molecules. Thus, ferroptosis related genes and signaling pathways and oxidative homeostasis play important roles in the regulation of EMT. In this paper, we review the role of ferroptosis related genes and their signaling pathways in regulating cancer EMT to better understand the crosstalk mechanism between ferroptosis and EMT, aiming to provide better therapeutic strategies for eradicating cancer cells and overcoming drug resistance.

Role of Epithelial to Mesenchymal Transition in Colorectal Cancer.

The epithelial-mesenchymal transition (EMT) is a cellular reprogramming process that occurs during embryonic development and adult tissue homeostasis. This process involves epithelial cells acquiring a mesenchymal phenotype. Through EMT, cancer cells acquire properties associated with a more aggressive phenotype. EMT and its opposite, mesenchymal-epithelial transition (MET), have been described in more tumors over the past ten years, including colorectal cancer (CRC). When EMT is activated, the expression of the epithelial marker E-cadherin is decreased and the expression of the mesenchymal marker vimentin is raised. As a result, cells temporarily take on a mesenchymal phenotype, becoming motile and promoting the spread of tumor cells. Epithelial-mesenchymal plasticity (EMP) has become a hot issue in CRC because strong inducers of EMT (such as transforming growth factor β, TGF-β) can initiate EMT and regulate metastasis, microenvironment, and immune system resistance in CRC. In this review, we take into account the significance of EMT-MET in CRC and the impact of the epithelial cells' plasticity on the prognosis of CRC. The analysis of connection between EMT and colorectal cancer stem cells (CCSCs) will help to further clarify the current meager understandings of EMT. Recent advances affecting important EMT transcription factors and EMT and CCSCs are highlighted. We come to the conclusion that the regulatory network for EMT in CRC is complicated, with a great deal of crosstalk and alternate paths. More thorough research is required to more effectively connect the clinical management of CRC with biomarkers and targeted treatments associated with EMT.

Cyclic Stretch Promotes Cellular Reprogramming Process through Cytoskeletal-Nuclear Mechano-Coupling and Epigenetic Modification.

Advancing the technologies for cellular reprogramming with high efficiency has significant impact on regenerative therapy, disease modeling, and drug discovery. Biophysical cues can tune the cell fate, yet the precise role of external physical forces during reprogramming remains elusive. Here the authors show that temporal cyclic-stretching of fibroblasts significantly enhances the efficiency of induced pluripotent stem cell (iPSC) production. Generated iPSCs are proven to express pluripotency markers and exhibit in vivo functionality. Bulk RNA-sequencing reveales that cyclic-stretching enhances biological characteristics required for pluripotency acquisition, including increased cell division and mesenchymal-epithelial transition. Of note, cyclic-stretching activates key mechanosensitive molecules (integrins, perinuclear actins, nesprin-2, and YAP), across the cytoskeletal-to-nuclear space. Furthermore, stretch-mediated cytoskeletal-nuclear mechano-coupling leads to altered epigenetic modifications, mainly downregulation in H3K9 methylation, and its global gene occupancy change, as revealed by genome-wide ChIP-sequencing and pharmacological inhibition tests. Single cell RNA-sequencing further identifies subcluster of mechano-responsive iPSCs and key epigenetic modifier in stretched cells. Collectively, cyclic-stretching activates iPSC reprogramming through mechanotransduction process and epigenetic changes accompanied by altered occupancy of mechanosensitive genes. This study highlights the strong link between external physical forces with subsequent mechanotransduction process and the epigenetic changes with expression of related genes in cellular reprogramming, holding substantial implications in the field of cell biology, tissue engineering, and regenerative medicine.

Long-chain noncoding RNA sequencing analysis reveals the molecular profiles of chemically induced mammary epithelial cells.

Long noncoding RNAs (lncRNAs) were important regulators affecting the cellular reprogramming process. Previous studies from our group have demonstrated that small molecule compounds can induce goat ear fibroblasts to reprogram into mammary epithelial cells with lactation function. In this study, we used lncRNA-Sequencing (lncRNA-seq) to analyze the lncRNA expression profile of cells before and after reprogramming (CK vs. 5i8 d). The results showed that a total of 3,970 candidate differential lncRNAs were detected, 1,170 annotated and 2,800 new lncRNAs. Compared to 0 d cells, 738 lncRNAs were significantly upregulated and 550 were significantly downregulated in 8 d cells. Heat maps of lncrnas and target genes with significant differences showed that the fate of cell lineages changed. Functional enrichment analysis revealed that these differently expressed (DE) lncRNAs target genes were mainly involved in signaling pathways related to reprogramming and mammary gland development, such as the Wnt signaling pathway, PI3K-Akt signaling pathway, arginine and proline metabolism, ECM-receptor interaction, and MAPK signaling pathway. The accuracy of sequencing was verified by real-time fluorescence quantification (RT-qPCR) of lncRNAs and key candidate genes, and it was also demonstrated that the phenotype and genes of the cells were changed. Therefore, this study offers a foundation for explaining the molecular mechanisms of lncRNAs in chemically induced mammary epithelial cells.

Application of Metabolic Reprogramming to Cancer Imaging and Diagnosis.

Cellular metabolism governs the signaling that supports physiological mechanisms and homeostasis in an individual, including neuronal transmission, wound healing, and circadian clock manipulation. Various factors have been linked to abnormal metabolic reprogramming, including gene mutations, epigenetic modifications, altered protein epitopes, and their involvement in the development of disease, including cancer. The presence of multiple distinct hallmarks and the resulting cellular reprogramming process have gradually revealed that these metabolism-related molecules may be able to be used to track or prevent the progression of cancer. Consequently, translational medicines have been developed using metabolic substrates, precursors, and other products depending on their biochemical mechanism of action. It is important to note that these metabolic analogs can also be used for imaging and therapeutic purposes in addition to competing for metabolic functions. In particular, due to their isotopic labeling, these compounds may also be used to localize and visualize tumor cells after uptake. In this review, the current development status, applicability, and limitations of compounds targeting metabolic reprogramming are described, as well as the imaging platforms that are most suitable for each compound and the types of cancer to which they are most appropriate.

Intracellular metabolic reprogramming mediated by micro-RNAs in differentiating and proliferating cells under non-diseased conditions.

Intracellular metabolic reprogramming is a critical process the cells carry out to increase biomass, energy fulfillment and genome replication. Cells reprogram their demands from internal catabolic or anabolic activities in coordination with multiple genes and microRNAs which further control the critical processes of differentiation and proliferation. The microRNAs reprogram the metabolism involving mitochondria, the nucleus and the biochemical processes utilizing glucose, amino acids, lipids, and nucleic acids resulting in ATP production. The processes of glycolysis, tricarboxylic acid cycle, or oxidative phosphorylation are also mediated by micro-RNAs maintaining cells and organs in a non-diseased state. Several reports have shown practical applications of metabolic reprogramming for clinical utility to assess various diseases, mostly studying cancer and immune-related disorders. Cells under diseased conditions utilize glycolysis for abnormal growth or proliferation, respectively, affecting mitochondrial paucity and biogenesis. Similar metabolic processes also affect gene expressions and transcriptional regulation for carrying out biochemical reactions. Metabolic reprogramming is equally vital for regulating cell environment to maintain organs and tissues in non-diseased states. This review offers in depth insights and analysis of how miRNAs regulate metabolic reprogramming in four major types of cells undergoing differentiation and proliferation, i.e., immune cells, neuronal cells, skeletal satellite cells, and cardiomyocytes under a non-diseased state. Further, the work systematically summarizes and elaborates regulation of genetic switches by microRNAs through predominantly through cellular reprogramming and metabolic processes for the first time. The observations will lead to a better understanding of disease initiation during the differentiation and proliferation stages of cells, as well as fresh approaches to studying clinical onset of linked metabolic diseases targeting metabolic processes.

Multi-Functional Genetic Engineering of Pluripotent Cell Lines for Universal Off-the-Shelf Natural Killer Cell Cancer Immunotherapy

Since the initial identification of natural killer (NK) cells, considerable effort has been made to harness their inherent anti-tumor capacities for the treatment of cancer. Despite recent clinical advancements utilizing the adoptive transfer of allogeneic NK cells, particularly with haplo-identical NK cells for acute myeloid leukemia, significant opportunities remain to further augment the anti-tumor activity of NK cells. Clinical trials have shown the persistence of adoptively transferred NK cells to be generally correlated with improved patient outcome. Natural killer cell persistence can be enhanced through several strategies, including by the administration of long-lived sub-populations such as adaptive-memory NK cells or of NK cells that are genetically modified to prolong survival or attenuate rejection by the host immune system. However, conventional sources of primary allogeneic NK cells have proven to be highly variable and difficult to genetically modify.Human induced pluripotent stem cells (hiPSCs) are unique in their capacity to self-renew and differentiate into any specialized cell type of the body. In addition, hiPSCs can be readily genetically-edited, and single hiPSCs can be clonally selected for the creation of engineered master pluripotent cell lines. As such, hiPSCs represent a renewable and reliable source for generating off-the-shelf engineered cell therapy products that are homogenous and display augmented anti-tumor properties.To enhance the persistence of hiPSC-derived NK cells, we employed a multi-dimensional strategy for hiPSC modification. First, hiPSCs were derived from fibroblast cells through a proprietary cellular reprogramming process that promotes telomere elongation. Second, hiPSCs were genetically-engineered to express cell surface-bound IL-15 to promote NK cell expansion during hiPSC differentiation and survival upon adoptive transfer. Third, additional hiPSC modifications were introduced to disrupt cell surface HLA Class I presentation and to drive cell surface expression of the non-classical HLA molecule HLA-G, to protect against host-mediated rejection by CD8+ T cells and NK cells, respectively. Lastly, we explored the influence of NKG2C to promote the adaptive-memory NK cell phenotype.We observed that hiPSC-derived NK cells displayed telomere length that was on average ~2-fold longer as compared to peripheral blood NK cells. The introduction of surface-bound IL-15 supported enhanced proliferation during differentiation with minimal requirements for cytokine support. Disruption of HLA-Class I expression decreased the proliferation of allogeneic CD8+ T cells upon challenge with modified hiPSCs as compared to wild-type hiPSCs (64% for wild-type vs. 29% for modified). This modification increased the cytotoxic activity of NK cells against modified hiPSCs (50% killing at 66 hours for modified as compared to 94 hours for wild-type). To this end, we further modified the hiPSCs to express the non-classical HLA molecule HLA-G. HLA-G protected against NK cell cytotoxicity and extended median survival of modified hiPSCs from 35 hours to 58 hours in an in vitro co-culture system. Finally, engineered hiPSC-derived NK cells can be effectively expanded (>1000x in 14 days) to support an efficient central manufacturing and banking method that enables multi-dose strategies to further support product persistence. Combined, the data suggest that our multi-engineered strategy can support the generation of a universal hiPSC backbone for the derivation of NK cells with improved persistence and anti-tumor efficacy. We are currently adding targeting modalities such as enhanced CD16 or chimeric antigen receptor expression to the discussed universal hiPSC backbone to generate master pluripotent cell lines to enable the manufacture of off-the-shelf NK cell cancer immunotherapy products. DisclosuresBjordahl:Fate Therapeutics: Employment, Equity Ownership. Clarke:Fate Therapeutics Inc.: Employment, Equity Ownership. Gaidarova:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics Inc.: Employment, Equity Ownership. Groff:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment, Equity Ownership. Lan:Fate Therapeutics Inc.: Employment, Equity Ownership. Abujarour:Fate Therapeutics Inc.: Employment. Bonello:Fate Therapeutics Inc.: Employment. Robinson:Fate Therapeutics Inc.: Employment. Hardy:Fate Therapeutics: Employment. Valamehr:Fate Therapeutics: Employment, Equity Ownership.

Nanomaterial Shape Influence on Cell Behavior.

Nanomaterials are proven to affect the biological activity of mammalian and microbial cells profoundly. Despite this fact, only surface chemistry, charge, and area are often linked to these phenomena. Moreover, most attention in this field is directed exclusively at nanomaterial cytotoxicity. At the same time, there is a large body of studies showing the influence of nanomaterials on cellular metabolism, proliferation, differentiation, reprogramming, gene transfer, and many other processes. Furthermore, it has been revealed that in all these cases, the shape of the nanomaterial plays a crucial role. In this paper, the mechanisms of nanomaterials shape control, approaches toward its synthesis, and the influence of nanomaterial shape on various biological activities of mammalian and microbial cells, such as proliferation, differentiation, and metabolism, as well as the prospects of this emerging field, are reviewed.

Deeper Insight in Metastatic Cancer Progression; Epithelial-to- Mesenchymal Transition and Genomic Instability: Implications on Treatment Resistance.

Therapy resistance remains the major obstacle to successful cancer treatment. Epithelial-to- mesenchymal transition [EMT], a cellular reprogramming process involved in embryogenesis and organ development and regulated by a number of transcriptional factors [EMT-TFs] such as ZEB1/2, is recognized for its role in tumor progression and metastasis. Recently, a growing body of evidence has implicated EMT in cancer therapy resistance, but the actual mechanism that underlies this finding has remained elusive. For example, whether it is, the EMT states in itself or the EMT-TFs that modulate chemo or radio-resistance in cancer is still contentious. Here, we summarise the molecular mechanisms of the EMT program and chemotherapeutic resistance in cancer with specific reference to DNA damage response [DDR]. We provide an insight into the molecular interactions that exist between EMT program and DNA repair machinery in cancer and how this interaction influences therapeutic response. We review conflicting studies linking EMT and drug resistance via the DNA damage repair axis. We draw scientific evidence demonstrating how several molecular signalling, including EMT-TFs, work in operational harmony to induce EMT and confer stemness properties on the EMT-susceptible cells. We highlight the role of enhanced DNA damage repair system associated with EMT-derived stem cell-like states in promoting therapy resistance and suggest a multi-targeting modality in combating cancer treatment resistance.

Differential expression of piRNAs in reprogrammed pluripotent stem cells from mouse embryonic fibroblasts.

piRNAs are a large class of small noncoding RNA that interact with an animal-specific class of Argonaute proteins, P-element induced wimpy proteins. piRNAs were initially discovered in mouse testes to be a fundamental component of spermatogenesis. Outside of the germline, piRNAs were found to function in embryogenesis, development, regeneration and cancer cells. However, despite a decade of scrutiny, functional understanding of this class of small RNAs remains very limited. To determine whether there are piRNAs present and involved in the cellular reprogramming process, we extracted piwi-interacting RNA (piRNA) signatures from a small RNA deep sequencing data set of mouse embryonic fibroblasts (MEFs), mouse embryonic stem cells (mESCs) and reprogrammed stem cells by three different technologies. We successfully identified three piRNA families specifically expressed in these reprogrammed stem cells. Meanwhile, there were almost no piRNAs observed in MEFs and mESCs. Further analysis indicated that these piRNAs may associate with the reprogramming process but not cellular pluripotency. Target gene prediction suggested that at least one of piRNAs, piR-mmu-64162, may take part in the reprogramming process by regulating cell senescence. Overall, we firstly identified the potential reprogramming associated piRNAs, shedding new light on piRNA functions.

Endogenous mechanism of right ventricular repair in pulmonary hypertension

IntroductionRight‐sided heart failure is the major cause of death among patients with pulmonary hypertension (PH) as well as children who suffer from various forms of congenital heart disease. Thus, agents that repair the damaged right ventricle (RV) have therapeutic potential to treat right‐sided heart failure. The biology of RV, however, is not well understood. The present study describes an endogenous mechanism for RV repair that was discovered in an experimental animal model of PH.Methods & ResultsThe treatment of Sprague‐Dawley (SD) rats with the injection of SU5416 (VEGF inhibitor) and 3 weeks of chronic hypoxia, followed by the maintenance in normoxia, resulted in progressive PH and RV remodeling. At 17 weeks after the initiation of the treatment, RV cardiomyocytes were damaged and the fibrotic region was found to be 12% of the total RV. However, at 35 weeks, RV fibrosis was significantly reduced and new cardiomyocytes were regenerated, despite RV pressure remained elevated. Before the RV repair occurs, α‐smooth muscle actin (αSMA) was found to be expressed in myofibroblasts. In addition, we noted the expression of αSMA in some of RV cardiomyocytes. Normal terminally differentiated cardiomyocytes do not express αSMA, but induced cardiomyocytes (iCMs) derived from induced pluripotent stem cells (iPSCs) do. Thus, we hypothesized that the damaged RV is repaired by activating the cellular reprogramming process that converts resident cardiac fibroblasts into iCMs perhaps through an iPSC‐like mechanism. This hypothesis was substantiated by our observations that Sox2, stem cell‐specific transcription factor, was also expressed in RV cardiomyocytes being repaired. Further, transmission electron microscopy observations revealed that extracellular matrix contains fibroblast‐line cells with myofilaments, which are organized into myofibrils and formed into the sarcomere‐like structure similar to iCMs.ConclusionsThe RV damage promoted by PAH can be repaired through a naturally occurring process that may involve cardiomyocyte regeneration through cell reprogramming. Understanding such an endogenous repair mechanism should contribute to the development of therapeutic strategies to treat right‐sided heart failure.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of cellular reprogramming and epigenetic dysregulation in acquired chemoresistance in breast cancer.

Acquired resistance to chemotherapy is a major limitation in clinical treatment for breast cancer. Accumulating evidence from in vitro, in vivo and clinical studies suggest that acquired chemoresistance is progressive, multifactorial and involve genetic and epigenetic aberrations. Among various mechanisms that contribute to chemoresistance, cellular reprogramming has extensively been implicated in breast cancer resistance lately. Cellular reprogramming events such as acquisition of epithelial to mesenchymal transition (EMT) and cancer stemness (CSCs) not only provide cancer cells with reversible phenotypic plasticity and survival advantage against cytotoxicity but also leads to aggressiveness, metastasis, clinical resistance, tumor recurrence and poor survival. The transient and reversible nature of cellular reprogramming processes and their controlled interaction with epigenetic regulatory complexes strongly support the involvement of dynamic epigenetic regulatory network in governing the cellular reprogramming and associated acquired chemoresistance. Further, epigenetic modulations are also gaining interest as promising interventions addressing the cancer cell reprogramming machinery to overcome acquired chemoresistance. This review discusses the previous reports and our recent findings that lead to current understanding of epigenetic dysregulation dictating the cellular reprogramming processes such as acquisition of EMT and CSCs phenotype and how they co-ordinate to establish acquired drug resistance in breast cancer.

Modeling Cellular Differentiation and Reprogramming with Gene Regulatory Networks.

Gene expression regulation is a fundamental cellular process that enables robust functioning of cells. How different genes interact among themselves to coordinate and maintain the overall gene expression profile observed in a cell is a key question in cellular biology. However, the immense complexity arising due to the scale and the nature of gene-gene interactions often hinders obtaining a global understanding of gene regulation. In this regard, network models of gene regulation based on gene-gene interactions, commonly referred to as gene regulatory networks (GRNs), serve important purpose of describing the overall interactions within a cell and provide a systematic approach to study their global behavior. In particular, in the context of cellular differentiation and reprogramming, where regulated changes in gene expression play a crucial role, precise knowledge of a cell type-specific GRN can enable control of the eventual cell fates with potential clinical applications. In this chapter, we describe our computational methodologies that we have tailor-made with purpose of applications to cell fate control. Briefly, we introduce the process of cellular differentiation and reprogramming, describe GRNs and common strategies to model them, and, finally, introduce the concept of determinants of cellular reprogramming and differentiation. In the Methods section, we elaborate on the different steps involved in the computational pipeline, including initial gene expression data processing, characterization of prior knowledge network, algorithm to remove non-cell type-specific edges, topological characterization of the inferred network, and Boolean network simulations to mimic cellular transitions. Finally, we provide a strategy to identify determinants of cellular reprogramming and differentiation based on the proposed computational methods.

Withaferin A inhibits Epithelial to Mesenchymal Transition in Non-Small Cell Lung Cancer Cells

Lung cancer is the leading cause of cancer-related deaths worldwide and in the United States. Despite recent advancements in treatment approaches, metastasis remains a major therapeutic challenge in lung cancer and explains the extremely poor prognosis. Epithelial to mesenchymal transition (EMT), a complex process of cellular reprogramming has become an attractive drug target because it plays a crucial role in the metastasis of non-small cell lung cancer (NSCLC). In the present study, we examined the effects of withaferin A (WFA), a plant-derived steroidal lactone on EMT in human NSCLC cell lines. First, we demonstrated that WFA displayed time- and concentration-dependent cytotoxicity on A549 and H1299 NSCLC cells. Then, cells were exposed to ≤ 0.5 µM WFA for ≤ 4 h to minimize cytotoxicity and determined its effects on EMT, cell adhesion, motility, migration, and invasion. EMT induction was performed by culturing cells in serum-free media containing TGFβ1 (5 ng/mL) and TNFα (25 ng/mL) for 48 h. We observed that pretreatment of cells with WFA inhibited cell adhesion, migration, and invasion of A549 and H1299 cells. Using western blot, immunofluorescence, and qRT-PCR analysis, we demonstrated that WFA suppressed TGFβ1 and TNFα-induced EMT in both cell lines. Mechanistically, WFA suppressed the phosphorylation and nuclear translocation of Smad2/3 and NF-κB in A549 and H1299 cells. Together, our study provides additional evidence demonstrating the inhibitory effects of WFA on EMT induction in NSCLC cells and further demonstrates the therapeutic potential of WFA against the metastasis in NSCLC.

The Histone Variant MacroH2A Blocks Cellular Reprogramming by Inhibiting Mesenchymal-to-Epithelial Transition.

Transcription factor-induced reprogramming of somatic cells to pluripotency is mediated via profound alterations in the epigenetic landscape. The histone variant macroH2A1 (mH2A1) is a barrier to the cellular reprogramming process. We demonstrate here that mH2A1 blocks reprogramming and contributes to the preservation of cell identity by trapping cells at the very early stages of the process, namely, at the mesenchymal-to-epithelial transition (MET). We provide a comprehensive analysis of the genomic sites occupied by the mH2A1 nucleosomes in human fibroblasts and embryonic stem (ES) cells and how they affect the reprogramming of fibroblasts to pluripotency. We have integrated chromatin immunoprecipitation sequencing (ChIP-seq) data with transcriptome sequencing (RNA-seq) data using cells containing reduced levels of mH2A1 and have inferred mH2A1-centered gene-regulatory networks that support the fibroblast and ES cell fates. We found that the exact positions of mH2A1 nucleosomes in regulatory regions of specific network genes with key regulatory roles guarantee the functional robustness of the regulatory networks. Using the reconstructed networks, we can predict and validate several components and their interactions in the establishment of stable cell types by limiting progression to alternative cell fates.

Implications of Cellular Aging in Cardiac Reprogramming.

Aging is characterized by a chronic functional decline of organ systems which leads to tissue dysfunction over time, representing a risk factor for diseases development, including cardiovascular. The aging process occurring in the cardiovascular system involves heart and vessels at molecular and cellular level, with subsequent structural modifications and functional impairment. Several modifications involved in the aging process can be ascribed to cellular senescence, a biological response that limits the proliferation of damaged cells. In physiological conditions, the mechanism of cellular senescence is involved in regulation of tissue homeostasis, remodeling, and repair. However, in some conditions senescence-driven tissue repair may fail, leading to the tissue accumulation of senescent cells which in turn may contribute to tumor promotion, aging, and age-related diseases. Cellular reprogramming processes can reverse several age-associated cell features, such as telomere length, DNA methylation, histone modifications and cell-cycle arrest. As such, induced Pluripotent Stem Cells (iPSCs) can provide models of progeroid and physiologically aged cells to gain insight into the pathogenesis of such conditions, to drive the development of new therapies for premature aging and to further explore the possibility of rejuvenating aged cells. An emerging picture is that the tissue remodeling role of cellular senescence could also be crucial for the outcomes of in vivo reprogramming processes. Experimental evidence has demonstrated that, on one hand, senescence represents a cell-autonomous barrier for a cell candidate to reprogramming, but, on the other hand, it may positively sustain the reprogramming capability of surrounding cells to generate fully proficient tissues. This review fits into this conceptual framework by highlighting the most prominent concepts that characterize aging and reprogramming and discusses how the aging tissue might provide a favorable microenvironment for in vivo cardiac reprogramming.

C-terminal domain small phosphatase-like 2 promotes epithelial-to-mesenchymal transition via Snail dephosphorylation and stabilization.

The epithelial-to-mesenchymal transition (EMT) is a cellular reprogramming process converting epithelial cells into mesenchymal cell morphology. Snail is a critical regulator of EMT by both suppressing epithelial gene expression and promoting mesenchymal gene expression. Expression and activity of Snail are tightly controlled at transcriptional and post-translational levels. It has previously been reported that Snail undergoes phosphorylation and ubiquitin-dependent proteasome degradation. Here, we report nuclear phosphatase SCP4/CTDSPL2 acts as a novel Snail phosphatase. SCP4 physically interacts with and directly dephosphorylates Snail. SCP4-mediated dephosphorylation of Snail suppresses the ubiquitin-dependent proteasome degradation of Snail and consequently enhances TGFβ-induced EMT. The knockdown of SCP4 in MCF10A mammary epithelial cells leads to attenuated cell migration. Collectively, our finding demonstrates that SCP4 plays a critical role in EMT through Snail dephosphorylation and stabilization.