Repressing miR-23a promotes the transdifferentiation of pancreatic α cells to ß cells via negatively regulating the expression of SDF-1α.

Pancreatic ß-cell failure is a pathological feature in type 1 diabetes. One promising approach involves inducing transdifferentiation of related pancreatic cell types, specifically α cells that produce glucagon. The chemokine stromal cell-derived factor-1 alpha (SDF-1α) is implicated in pancreatic α-to-ß like cell transition. Here, the serum level of SDF-1α was lower in T1D with C-peptide loss, the miR-23a was negatively correlated with SDF-1α. We discovered that exosomal miR-23a, secreted from ß cells, functionally downregulates the expression of SDF-1α, leading to increased Pax4 expression and decreased Arx expression in vivo. Adenovirus-vectored miR-23a sponge and mimic were constructed to further explored the miR-23a on pancreatic α-to-ß like cell transition in vitro, which yielded results consistent with our cell-based assays. Suppression of miR-23a upregulated insulin level and downregulated glucagon level in STZ-induced diabetes mice models, effectively promoting α-to-ß like cell transition. Our findings highlight miR-23a as a new therapeutic target for regenerating pancreatic ß cells from α cells.

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation.

Trans-differentiation from an adenocarcinoma to a small cell neuroendocrine state is associated with therapy resistance in multiple cancer types. To gain insight into the underlying molecular events of the trans-differentiation, we perform a multi-omics time course analysis of a pan-small cell neuroendocrine cancer model (termed PARCB), a forward genetic transformation using human prostate basal cells and identify a shared developmental, arc-like, and entropy-high trajectory among all transformation model replicates. Further mapping with single cell resolution reveals two distinct lineages defined by mutually exclusive expression of ASCL1 or ASCL2. Temporal regulation by groups of transcription factors across developmental stages reveals that cellular reprogramming precedes the induction of neuronal programs. TFAP4 and ASCL1/2 feedback are identified as potential regulators of ASCL1 and ASCL2 expression. Our study provides temporal transcriptional patterns and uncovers pan-tissue parallels between prostate and lung cancers, as well as connections to normal neuroendocrine cell states.

Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss.

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.

Potential involvement of miR-183/96/182 cluster-gene target interactions in transdifferentiation of human retinal pigment epithelial cells into retinal neurons.

miR-183/96/182 cluster plays a critical role in the developing retina by regulating many target genes involved in signaling pathways. This study aimed to survey the miR-183/96/182 cluster-target interactions that, potentially contribute to human retinal pigmented epithelial (hRPE) cell differentiation into photoreceptors. Target genes of the miR-183/96/182 cluster were obtained from miRNA-target databases and applied to construct miRNA-target networks. Gene ontology and KEGG pathway analysis was performed. miR-183/96/182 cluster sequence was cloned into an eGFP-intron splicing cassette in an AAV2 vector and overexpressed in hRPE cells. The expression level of target genes including HES1, PAX6, SOX2, CCNJ, and RORΒ was evaluated using qPCR. Our results showed that miR-183, miR-96, and miR-182 share 136 target genes that are involved in cell proliferation pathways such as PI3K/AKT and MAPK pathway. qPCR data indicated a 22-, 7-, and 4-fold overexpression of miR-183, miR-96, and miR-182, respectively, in infected hRPE cells. Consequently, the downregulation of several key targets such as PAX6, CCND2, CDK5R1, and CCNJ and upregulation of a few retina-specific neural markers such as Rhodopsin, red opsin, and CRX was detected. Our findings suggest that the miR-183/96/182 cluster may induce hRPE transdifferentiation by targeting key genes that involve in the cell cycle and proliferation pathways.

Phenotypic and genotypic infidelity in B-lineage neoplasms, including transdifferentiation following targeted therapy: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: Session 2 of the 2021 Society for Hematopathology and European Association for Haematopathology Workshop collected examples of lineage infidelity and transdifferentiation in B-lineage neoplasms, including after targeted therapy. METHODS: Twenty cases were submitted. Whole-exome sequencing and genome-wide RNA expression analysis were available on a limited subsample. RESULTS: A diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) was rendered on at least 1 biopsy from 13 patients. There was 1 case of acute myeloid leukemia (AML); the remaining 6 cases were mature B-cell neoplasms. Targeted therapy was administered in 7 cases of B-ALL and 4 cases of mature B-cell neoplasms. Six cases of B-ALL underwent lineage switch to AML or mixed-phenotype acute leukemia at relapse, 5 of which had rearranged KMT2A. Changes in maturational state without lineage switch were observed in 2 cases. Examples of de novo aberrant T-cell antigen expression (n = 2) were seen among the mature B-cell lymphoma cohort, and their presence correlated with alterations in tumor cell gene expression patterns. CONCLUSIONS: This cohort of cases enabled us to illustrate, discuss, and review current concepts of lineage switch and aberrant antigen expression in a variety of B-cell neoplasms and draw attention to the role targeted therapies may have in predisposing neoplasms to transdifferentiation as well as other, less expected changes in maturational status.

Oxidative stress-triggered Wnt signaling perturbation characterizes the tipping point of lung adeno-to-squamous transdifferentiation.

Lkb1 deficiency confers the Kras-mutant lung cancer with strong plasticity and the potential for adeno-to-squamous transdifferentiation (AST). However, it remains largely unknown how Lkb1 deficiency dynamically regulates AST. Using the classical AST mouse model (Kras LSL-G12D/+;Lkb1flox/flox, KL), we here comprehensively analyze the temporal transcriptomic dynamics of lung tumors at different stages by dynamic network biomarker (DNB) and identify the tipping point at which the Wnt signaling is abruptly suppressed by the excessive accumulation of reactive oxygen species (ROS) through its downstream effector FOXO3A. Bidirectional genetic perturbation of the Wnt pathway using two different Ctnnb1 conditional knockout mouse strains confirms its essential role in the negative regulation of AST. Importantly, pharmacological activation of the Wnt pathway before but not after the tipping point inhibits squamous transdifferentiation, highlighting the irreversibility of AST after crossing the tipping point. Through comparative transcriptomic analyses of mouse and human tumors, we find that the lineage-specific transcription factors (TFs) of adenocarcinoma and squamous cell carcinoma form a "Yin-Yang" counteracting network. Interestingly, inactivation of the Wnt pathway preferentially suppresses the adenomatous lineage TF network and thus disrupts the "Yin-Yang" homeostasis to lean towards the squamous lineage, whereas ectopic expression of NKX2-1, an adenomatous lineage TF, significantly dampens such phenotypic transition accelerated by the Wnt pathway inactivation. The negative correlation between the Wnt pathway and AST is further observed in a large cohort of human lung adenosquamous carcinoma. Collectively, our study identifies the tipping point of AST and highlights an essential role of the ROS-Wnt axis in dynamically orchestrating the homeostasis between adeno- and squamous-specific TF networks at the AST tipping point.

Temporal Analysis Reveals the Transient Differential Expression of Transcription Factors That Underlie the Trans-Differentiation of Human Monocytes to Macrophages.

The activation of monocytes and their trans-differentiation into macrophages are critical processes of the immune response. Prior work has characterized the differences in the expression between monocytes and macrophages, but the transitional process between these cells is poorly detailed. Here, we analyzed the temporal changes of the transcriptome during trans-differentiation of primary human monocytes into M0 macrophages. We find changes with many transcription factors throughout the process, the vast majority of which exhibit a maximally different expression at the intermediate stages. A few factors, including AP-1, were previously known to play a role in immunological transitions, but most were not. Thus, these findings indicate that this trans-differentiation requires the dynamic expression of many transcription factors not previously discussed in immunology, and provide a foundation for the delineation of the molecular mechanisms associated with healthy or pathological responses that involve this transition.

Loss of SUMO-specific protease 2 causes isolated glucocorticoid deficiency by blocking adrenal cortex zonal transdifferentiation in mice.

SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by Adrenocorticotropic Hormone (ACTH)/cAMP-dependent Protein Kinase (PKA) signalling within the zona fasciculata, we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zona fasciculata, a blunted reponse to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/ß-catenin signalling leading to repression of PKA activity and ectopic activation of ß-catenin. At the cellular level, this blocks transdifferentiation of ß-catenin-positive zona glomerulosa cells into fasciculata cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.

From IL-17 to IFN-γ in inflammatory skin disorders: Is transdifferentiation a potential treatment target?

The targeted inhibition of effector cytokines such as interleukin 17 (IL-17) in psoriasis and IL-13 in atopic dermatitis offers impressive efficacy with a favorable side effect profile. In contrast, the downregulation of interferon gamma (IFN-γ) in T helper (Th) 1-dominant skin disorders may lead to more adverse events, given the crucial role of IFN-γ in antiviral and antitumoral immunity. Modulating Th17 and Th2 cell differentiation is performed by blocking IL-23 and IL-4, respectively, whereas anti-IL-12 antibodies are only moderately effective in downregulating Th1 lymphocyte differentiation. Therefore, a targeted approach of IFN-γ-driven disorders remains challenging. Recent literature suggests that certain pathogenic Th17 cell subsets with Th1 characteristics, such as CD4+CD161+CCR6+CXCR3+IL-17+IFN-y+ (Th17.1) and CD4+CD161+CCR6+CXCR3+IL-17-IFN-y+ (exTh17), are important contributors in Th1-mediated autoimmunity. Differentiation to a Th17.1 or exTh17 profile results in the upregulation of IFN-y. Remarkably, these pathogenic Th17 cell subsets are resistant to glucocorticoid therapy and the dampening effect of regulatory T cells (Treg). The identification of Th17.1/exTh17 cells in auto-immune disorders may explain the frequent treatment failure of conventional immunosuppressants. In this review, we summarize the current evidence regarding the cellular plasticity of Th17 cells in inflammatory skin disorders. A deeper understanding of this phenomenon may lead to better insights into the pathogenesis of various skin diseases and the discovery of a potential new treatment target.

Kruppel Like Factor 5 Enhances High Glucose-Induced Renal Tubular Epithelial Cell Transdifferentiation in Diabetic Nephropathy.

Background - Diabetic nephropathy (DN) is a principal reason for kidney disease worldwide. High glucose (HG) is a major factor for DN. Kruppel like factor 5 (KLF5) participates in DN development. In the present study, we aim to elaborate the role of KLF5 in HG-induced renal tubular epithelial cell (RTEC) transdifferentiation in DN. Methods - RTECs (HK-2 cells) were treated with HG and were transfected with si-KLF5 or oe-HMGB1. Afterwards, expression of KLF5 and HMGB1 was detected, HK cell viability was determined, and levels of alpha-smooth muscle actin (α-SMA), E-cadherin, vimentin, and transforming growth factor beta 1 (TGF-ß1) were assessed. Additionally, the binding relation between KLF5 and HMGB1 was analyzed. Results - In HK-2 cells with HG treatment, expression of KLF5 and HMGB1 was upregulated; levels of α-SMA, vimentin, and TGF-ß1 were increased; and E-cadherin level was decreased. Moreover, KLF5 silencing resulted in down-regulated levels of α-SMA, vimentin, and TGF-ß1 but upregulated level of E-cadherin. On the other hand, KLF5 could bind to the HMGB1 promoter and activate HMGB1 transcription. HMGB1 overexpression partially counteracted the inhibitive effect of KLF5 silencing on HG-induced HK-2 transdifferentiation. Conclusion - HG induced overexpressed KLF5 in RTECs, and as a transcription factor, KLF5 could bind to the HMGB1 promoter, thereby promoting HMGB1 transcription and RTEC transdifferentiation.

Human umbilical cord mesenchymal stem cell-derived exosomal miR-335-5p attenuates the inflammation and tubular epithelial-myofibroblast transdifferentiation of renal tubular epithelial cells by reducing ADAM19 protein levels.

BACKGROUND: Renal tubular epithelial-myofibroblast transdifferentiation (EMT) plays a key role in the regulation of renal fibrosis. Exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) play a crucial role in alleviating renal fibrosis and injury. Additionally, hucMSC-derived exosomes contain numerous microRNAs (miRNAs). However, it is unclear whether mesenchymal stem cells can regulate the transforming growth factor (TGF)-ß1-induced EMT of human renal tubular epithelial cells (RTECs) through exosomal miRNAs. METHOD: HK-2, a human RTEC line, was co-treated with TGF-ß1 and hucMSC-derived exosomes. Additionally, TGF-ß1-treated HK-2 cells were transfected with a miR-335-5p mimic and disintegrin and metalloproteinase domain-containing protein 19 (ADAM19)-overexpression plasmid. miR-335-5p expression and ADAM19 protein and inflammation levels were measured via quantitative reverse transcription polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assays, respectively. RESULTS: TGF-ß1 treatment changed the shape of HK-2 cells from a cobblestone morphology to a long spindle shape, accompanied by an increase in interleukin (IL)-6, tumor necrosis factor-α, IL-1ß, collagen I, collagen III, α-smooth muscle actin, vimentin, and N-cadherin protein levels, whereas E-cadherin protein levels were reduced in these HK-2 cells, suggesting that TGF-ß1 treatment induced the inflammation and EMT of HK-2 cells. HucMSC-exosomes improved the inflammation and EMT phenotype of TGF-ß1-induced HK-2 cells by transferring miR-335-5p. miR-335-5p was found to bind the ADAM19 3'-untranslated region to reduce ADAM19 protein levels. Additionally, miR-335-5p improved the inflammation and EMT phenotype of HK-2 cells by reducing ADAM19 protein levels with TGF-ß1 induction. CONCLUSIONS: HucMSC-derived exosomal miR-335-5p attenuates the inflammation and EMT of HK-2 cells by reducing ADAM19 protein levels upon TGF-ß1 induction. This study provides a potential therapeutic strategy and identifies targets for clinically treating renal fibrosis.

Direct Conversion of Cell Fate and Induced Endothelial Cells.

Advances in nuclear reprogramming technology have enabled the dedifferentiation and transdifferentiation of mammalian cells. Forced induction of the key transcription factors constituting a transcriptional network can convert cells back to their pluripotent status or directly to another cell fate without inducing pluripotency. To date, direct conversion to several cell types, including cardiomyocytes, various types of neurons, and pancreatic ß-cells, has been reported. We previously demonstrated direct lineage reprogramming of adult fibroblasts into induced endothelial cells (iECs) in mice and humans. In contrast to induced pluripotent stem cells, for which there is consensus on the criteria defining pluripotency, such criteria have not yet been established in the field of direct conversion. We thus suggest that careful assessment of the status of converted cells using genetic and epigenetic profiling, various functional assays, and the use of multiple readouts is essential to determine successful conversion. As direct conversion does not go through pluripotent status, this technique can be utilized for therapeutic purposes without the risk of tumorigenesis. Further, direct conversion can be induced in vivo by gene delivery to the target tissue or organ in situ. Thus, direct conversion technology can be developed into cell therapy or gene therapy for regenerative purposes. Here, we review the potential and future directions of direct cell fate conversion and iECs.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air-liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

PGE2 and Thrombin Induce Myofibroblast Transdifferentiation via Activin A and CTGF in Endometrial Stromal Cells.

Endometriosis is characterized by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal hemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, menstruation-related factors, PGE2 and thrombin (P/T), a PAR1 agonist induced epithelial-mesenchymal transition (EMT) of endometrial cells under hypoxia. However, the molecular mechanisms by which P/T induce development of endometriosis have not been fully characterized. To investigate the effects of P/T, RNA extracted from endometrial stromal cells (ESCs) treated with P/T were subjected to RNA sequence analysis, and identified activin A, FOS, and GATA2 as upregulated genes. Activin A increased the expression of connective tissue growth factor (CTGF) and mesenchymal marker genes in ESCs. CTGF induced the expression of fibrosis marker type I collagen, fibronectin, and α-smooth muscle actin (αSMA), indicating fibroblast to myofibroblast transdifferentiation (FMT) of ESCs. In addition, activin A, FOS, GATA2, CTGF, and αSMA were localized in endometriosis lesions. Taken together, our data show that P/T induces changes resembling EMT and FMT in ectopic ESCs derived from retrograde menstruation, and that these are associated with fibrotic changes in the lesions. Pharmacological means that block P/T-induced activin A and CTGF signaling may be strategies to inhibit fibrosis in endometriotic lesions.

Sertoli Cells Improve Myogenic Differentiation, Reduce Fibrogenic Markers, and Induce Utrophin Expression in Human DMD Myoblasts.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene translating in lack of functional dystrophin and resulting in susceptibility of myofibers to rupture during contraction. Inflammation and fibrosis are critical hallmarks of DMD muscles, which undergo progressive degeneration leading to loss of independent ambulation in childhood and death by early adulthood. We reported that intraperitoneal injection of microencapsulated Sertoli cells (SeC) in dystrophic mice translates into recovery of muscle morphology and performance thanks to anti-inflammatory effects and induction of the dystrophin paralogue, utrophin at the muscle level, opening new avenues in the treatment of DMD. The aim of this study is to obtain information about the direct effects of SeC on myoblasts/myotubes, as a necessary step in view of a translational application of SeC-based approaches to DMD. We show that (i) SeC-derived factors stimulate cell proliferation in the early phase of differentiation in C2C12, and human healthy and DMD myoblasts; (ii) SeC delay the expression of differentiation markers in the early phase nevertheless stimulating terminal differentiation in DMD myoblasts; (iii) SeC restrain the fibrogenic potential of fibroblasts, and inhibit myoblast-myofibroblast transdifferentiation; and, (iv) SeC provide functional replacement of dystrophin in preformed DMD myotubes regardless of the mutation by inducing heregulin ß1/ErbB2/ERK1/2-dependent utrophin expression. Altogether, these results show that SeC are endowed with promyogenic and antifibrotic effects on dystrophic myoblasts, further supporting their potential use in the treatment of DMD patients. Our data also suggest that SeC-based approaches might be useful in improving the early phase of muscle regeneration, during which myoblasts have to adequately proliferate to replace the damaged muscle mass.

Expression of Piwi, MMP, TIMP, and Sox during Gut Regeneration in Holothurian Eupentacta fraudatrix (Holothuroidea, Dendrochirotida).

Mesodermal cells of holothurian Eupentacta fraudatrix can transdifferentiate into enterocytes during the regeneration of the digestive system. In this study, we investigated the expression of several genes involved in gut regeneration in E. fraudatrix. Moreover, the localization of progenitor cells of coelomocytes, juvenile cells, and their participation in the formation of the luminal epithelium of the digestive tube were studied. It was shown that Piwi-positive cells were not involved in the formation of the luminal epithelium of the digestive tube. Ef-72 kDa type IV collagenase and Ef-MMP16 had an individual expression profile and possibly different functions. The Ef-tensilin3 gene exhibited the highest expression and indicates its potential role in regeneration. Ef-Sox9/10 and Ef-Sox17 in E. fraudatrix may participate in the mechanism of transdifferentiation of coelomic epithelial cells. Their transcripts mark the cells that plunge into the connective tissue of the gut anlage and give rise to enterocytes. Ef-Sox9/10 probably controls the switching of mesodermal cells to the enterocyte phenotype, while Ef-Sox17 may be involved in the regulation of the initial stages of transdifferentiation.

Application of Modified mRNA in Somatic Reprogramming to Pluripotency and Directed Conversion of Cell Fate.

Modified mRNA (modRNA)-based somatic reprogramming is an effective and safe approach that overcomes the genomic mutation risk caused by viral integrative methods. It has improved the disadvantages of conventional mRNA and has better stability and immunogenicity. The modRNA molecules encoding multiple pluripotent factors have been applied successfully in reprogramming somatic cells such as fibroblasts, mesenchymal stem cells, and amniotic fluid stem cells to generate pluripotent stem cells (iPSCs). Moreover, it also can be directly used in the terminal differentiation of stem cells and fibroblasts into functional therapeutic cells, which exhibit great promise in disease modeling, drug screening, cell transplantation therapy, and regenerative medicine. In this review, we summarized the reprogramming applications of modified mRNA in iPSC generation and therapeutic applications of functionally differentiated cells.

MiR-126-3p Is Dynamically Regulated in Endothelial-to-Mesenchymal Transition during Fibrosis.

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFß2 and IL1ß. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.

Transcription factor stoichiometry in cell fate determination.

Transcription factors play very important role in cell fate determination. There are many cell specific transcription factors which when expressed ectopically may lead to cell fate conversion or transdifferentiation. Many of these transcription factors function differently based on their levels and stoichiometry. Many different types of differentiated cells have been generated from other differentiated cell types by expressing different levels and stoichiometry of reprogramming factors. Many methodologies have been developed for efficient cell fate conversion by regulating the levels and stoichiometry of transcription factors in a particular cocktail that have therapeutic values. An approach called phenotypic activation which involves overexpression of putative transcription factors has been developed as a tool to discover new transcription factors and their targets. Transcription factor overexpression may also have toxic effects where nonspecific electrostatic interactions and 'squelching' may lead to inhibition of many genes. Altered levels of transcription factors may have disastrous consequences like cancer. Recent developments like designing of artificial transcription factors, nanotechnology-based transcriptional tools and CRISPR-based transcription modules with capabilities of precise regulation of gene expression patterns hold huge potentials in the field of transcriptional therapeutics.

Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells.

The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor's characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.