GFP-positive Cells Research Articles

High-Intensity Pulsed Electromagnetic Field-Mediated Gene Electrotransfection In Vitro.

A high-intensity pulsed electromagnetic field (HI-PEMF) is a non-invasive and non-contact delivery method and may, as such, have an advantage over gene electrotransfer mediated by conventional electroporation using contact electrodes. Due to the limited number of in vitro studies in the field of gene electrotransfection by HI-PEMF, we designed experiments to investigate and demonstrate the feasibility of such a technique for the non-viral delivery of genetic material into cells in vitro. We first showed that HI-PEMF causes DNA adsorption to the membrane, a generally accepted prerequisite step for successful gene electrotransfection. We also showed that HI-PEMF can induce gene electrotransfection as the application of HI-PEMF increased the percentage of GFP-positive cells for two different combinations of pDNA size and concentration. Furthermore, by measuring the uptake of larger molecules, i.e., fluorescently labelled dextrans of three different sizes, we showed endocytosis to be a possible mechanism for introducing large molecules into cells by HI-PEMF.

Macrophage-Specific, Mafb-Deficient Mice Showed Delayed Skin Wound Healing.

Macrophages play essential roles throughout the wound repair process. Nevertheless, mechanisms regulating the process are poorly understood. MAFB is specifically expressed in the macrophages in hematopoietic tissue and is vital to homeostatic function. Comparison of the skin wound repair rates in macrophage-specific, MAFB-deficient mice (Mafbf/f::LysM-Cre) and control mice (Mafbf/f) showed that wound healing was significantly delayed in the former. For wounded GFP knock-in mice with GFP inserts in the Mafb locus, flow cytometry revealed that their GFP-positive cells expressed macrophage markers. Thus, macrophages express Mafb at wound sites. Immunohistochemical (IHC) staining, proteome analysis, and RT-qPCR of the wound tissue showed relative downregulation of Arg1, Ccl12, and Ccl2 in Mafbf/f::LysM-Cre mice. The aforementioned genes were also downregulated in the bone marrow-derived, M2-type macrophages of Mafbf/f::LysM-Cre mice. Published single-cell RNA-Seq analyses showed that Arg1, Ccl2, Ccl12, and Il-10 were expressed in distinct populations of MAFB-expressing cells. Hence, the MAFB-expressing macrophage population is heterogeneous. MAFB plays the vital role of regulating multiple genes implicated in wound healing, which suggests that MAFB is a potential therapeutic target in wound healing.

N-acetylcysteine attenuates atherosclerosis progression in aging LDL receptor deficient mice with preserved M2 macrophages and increased CD146

Background and aimsInflammation and reactive oxygen species (ROS) are important to the pathogenesis of atherosclerosis. The effect of antioxidants on atherosclerosis is inconsistent, and sometimes controversial. We aimed to test the hypothesis that attenuation of atherosclerosis by N-acetylcysteine (NAC) depends on NAC treatment timing and duration. MethodsMale LDL receptor deficient (LDLR−/−) mice were fed a normal diet (ND) and divided into controls (on ND for 24 months), models 1–2 (at age of 9 months, starting NAC treatment for 3 or 6 months), and model 3 (at age of 18 months, starting NAC treatment for 6 months). To determine if hyperlipidemia compromises NAC treatment outcome, mice were fed a high fat diet (HFD) starting at age of 6 weeks and treated with NAC starting at 9 months of age for 6 months. ResultsNAC treatment for 6 months, not for 3 months, significantly attenuated atherosclerosis progression, but did not reverse atherosclerotic lesions, in aging LDLR−/− mice on ND. NAC had no effect on atherosclerotic lesions in mice on HFD. NAC treatment significantly decreased aortic ROS production, and the levels of inflammatory cytokines in serum and aorta of aging LDLR−/− mice with increased CD146 level. Bone marrow transplantation study with GFP-positive bone marrow cells showed that NAC treatment preserved M2 population and M2 polarization in the aorta of LDLR−/− mice. ConclusionsEarly and adequate NAC treatment could effectively attenuate inflammation and atherosclerosis progression with preserved M2 population and increased CD146 level in aging LDLR−/− mice without extreme hyperlipidemia.

Embryo manipulation in neotropical characiform fish: incubation system, anaesthetic, and PGC transplantation in Prochilodus lineatus.

Primordial germ cells transplantation is a unique approach for conservation and reconstitution of endangered fish species. This study aimed to establish techniques to culture dechorionated embryos in different incubation systems and also to determine anaesthetic concentration for fish recipients in the larval stage for subsequent primordial germ cell transplantation. Intact and dechorionated embryos were divided into three incubation systems: (1) a control group with manual replacement of the solution; (2) a closed environment with high oxygen with manual replacement of the solution; and (3) constant solution recirculation. This combination resulted in six treatments. For the evaluation of anaesthetics for larvae, the concentrations evaluated were 19.5 mM, 24.4 mM, 29.3 mM, and 34.2 mM of 2-phenoxyethanol. Anaesthesia concentration and recovery at different stages were evaluated. For transplantation, primordial germ cells of Astyanax altiparanae were transplanted into anaesthetised larvae (1 dph) of Prochilodus lineatus. Better results were obtained in the recirculation system for dechorionated embryos of P. lineatus for hatching (54.18%) and normal morphology (50.06%). The 2-phenoxyethanol anaesthetic with a dose of 29.3 mM resulted in shorter induction times, in addition to the recovery time between 5 and 10 min. By using this anaesthetic concentration at transplantation, GFP-positive cells were seen in two recipients, but the cells did not proliferate. This study established an effective incubation system for the development of the dechorionated embryo and determined an effective anaesthetic concentration for P. lineatus larvae. In addition, micromanipulation and transplantation of primordial germ cells in neotropical species were conducted for the first time.

Smooth muscle protein 22α-Cre recombination in resting cardiac fibroblasts and hematopoietic precursors

The Cre-loxP system has been widely used for cell- or organ-specific gene manipulation, but it is important to precisely understand what kind of cells the recombination takes place in. Smooth muscle 22α (SM22α)-Cre mice have been utilized to alter genes in vascular smooth muscle cells (VSMCs), activated fibroblasts or cardiomyocytes (CMs). Moreover, previous reports indicated that SM22α-Cre is expressed in adipocytes, platelets or myeloid cells. However, there have been no report of whether SM22α-Cre recombination takes place in nonCMs in hearts. Thus, we used the double-fluorescent Cre reporter mouse in which GFP is expressed when recombination occurs. Immunofluorescence analysis demonstrated that recombination occurred in resting cardiac fibroblasts (CFs) or macrophages, as well as VSMCs and CMs. Flow cytometry showed that some CFs, resident macrophages, neutrophils, T cells, and B cells were positive for GFP. These results prompted us to analyze bone marrow cells, and we observed GFP-positive hematopoietic precursor cells (HPCs). Taken together, these results indicated that SM22α-Cre-mediated recombination occurs in resting CFs and hematopoietic cell lineages, including HPCs, which is a cautionary point when using SM22α-Cre mice.

Establishment of the Primary Avian Gonadal Somatic Cell Lines for Cytogenetic Studies.

Simple SummaryWe developed a simple method for primary somatic cell culture establishment from the ovaries of the great tits and testes of ten Passerine species. The ovary-derived cell cultures were cultivated until the tenth passage without any noticeable decrease in their proliferative activity, while testis-derived cell cultures demonstrated a decreased proliferation potential. However, sufficient material was available from both cell cultures originating from the ovary and testis to make excellent mitotic metaphase chromosomal preparations. We demonstrated the high efficiency of electroporation for genetic modification of the ovary-derived cell line. Thus, the established ovary-derived cell line could be efficiently used in cytogenetic and genomic studies.The last decade was marked by a steep rise in avian studies at genomic and cellular levels. Cell lines are important tools for in vitro studies in cell biology and cytogenetics. We developed a simple method of primary somatic cell culture establishment from the ovaries of the great tits (Parus major) and testes of ten Passerine species, characterized the cellular composition of the ovary-derived lines using RT-PCR and immunolocalization of the tissue-specific markers and tested the efficiency of two methods of genetic transformation of the ovary-derived cell line. We found that the ovary-derived cell cultures of the great tit were composed of fibroblasts mainly, but also contained interstitial and granulosa cells. They were cultivated until the 10th passage without any noticeable decrease in their proliferative activity. The testis-derived cell cultures had lower proliferative potential. However, both ovary- and testis-derived cell cultures provided enough material for high quality mitotic metaphase chromosome preparations. The efficiency of its transduction with lentivirus containing a GFP reporter was very low, while electroporation with episomal vectors expressing GFP resulted in a high yield of GFP-positive cells. The proposed method could be used for the generation of high quality material for various cytogenetic and genomic studies.

Purification of human iPSC-derived cells at large scale using microRNA switch and magnetic-activated cell sorting.

SummaryFor regenerative cell therapies using pluripotent stem cell (PSC)-derived cells, large quantities of purified cells are required. Magnetic-activated cell sorting (MACS) is a powerful approach to collect target antigen-positive cells; however, it remains a challenge to purify various cell types efficiently at large scale without using antibodies specific to the desired cell type. Here we develop a technology that combines microRNA (miRNA)-responsive mRNA switch (miR-switch) with MACS (miR-switch-MACS) to purify large amounts of PSC-derived cells rapidly and effectively. We designed miR-switches that detect specific miRNAs expressed in target cells and controlled the translation of a CD4-coding transgene as a selection marker for MACS. For the large-scale purification of induced PSC-derived cardiomyocytes (iPSC-CMs), we transferred miR-208a-CD4 switch-MACS and obtained purified iPSC-CMs efficiently. Moreover, miR-375-CD4 switch-MACS highly purified pancreatic insulin-producing cells and their progenitors expressing Chromogranin A. Overall, the miR-switch-MACS method can efficiently purify target PSC-derived cells for cell replacement therapy.

Vertical sleeve gastrectomy induces enteroendocrine cell differentiation of intestinal stem cells through bile acid signaling.

Vertical sleeve gastrectomy (VSG) results in an increase in the number of hormone-secreting enteroendocrine cells (EECs) in the intestinal epithelium; however, the mechanism remains unclear. Notably, the beneficial effects of VSG are lost in a mouse model lacking the nuclear bile acid receptor farnesoid X receptor (FXR). FXR is a nuclear transcription factor that has been shown to regulate intestinal stem cell (ISC) function in cancer models. Therefore, we hypothesized that the VSG-induced increase in EECs is due to changes in intestinal differentiation driven by an increase in bile acid signaling through FXR. To test this, we performed VSG in mice that express EGFP in ISC/progenitor cells and performed RNA-Seq on GFP-positive cells sorted from the intestinal epithelia. We also assessed changes in EEC number (marked by glucagon-like peptide-1, GLP-1) in mouse intestinal organoids following treatment with bile acids, an FXR agonist, and an FXR antagonist. RNA-Seq of ISCs revealed that bile acid receptors are expressed in ISCs and that VSG explicitly alters expression of several genes that regulate EEC differentiation. Mouse intestinal organoids treated with bile acids and 2 different FXR agonists increased GLP-1–positive cell numbers, and administration of an FXR antagonist blocked these effects. Taken together, these data indicate that VSG drives ISC fate toward EEC differentiation through bile acid signaling.

Impact of in Utero Rat Exposure to 17Alpha-Ethinylestradiol or Genistein on Testicular Development and Germ Cell Gene Expression.

Although the decline in male fertility is believed to partially result from environmental exposures to xenoestrogens during critical developmental windows, the underlying mechanisms are still poorly understood. Experimental in utero exposures in rodents have demonstrated the negative impact of xenoestrogens on reproductive development, long-term adult reproductive function and offspring health. In addition, transcriptomic studies have demonstrated immediate effects on gene expression in fetal reproductive tissues, However, the immediate molecular effects on the developing germ cells have been poorly investigated. Here, we took advantage of a transgenic rat expressing the green fluorescent protein specifically in germ cells allowing purification of perinatal GFP-positive germ cells. Timed-pregnant rats were exposed to ethinylestradiol (EE2, 2 μg/kg/d), genistein (GE, 10 mg/kg/d) or vehicle by gavage, from gestational days (GD) 13–19; testes were sampled at GD20 or post-natal (PND) 5 for histological analysis and sorting of GFP-positive cells. While EE2-exposed females gained less weight during treatment compared to controls, neither treatment affected the number of pups per litter, sex ratio, anogenital distance, or body and gonadal weights of the offspring. Although GE significantly decreased circulating testosterone at GD20, no change was observed in either testicular histology or germ cell and sertoli cell densities. Gene expression was assessed in GFP-positive cells using Affymetrix Rat Gene 2.0 ST microarrays. Analysis of differentially expressed genes (DEGs) (p < 0.05; fold change 1.5) identified expression changes of 149 and 128 transcripts by EE2 and GE respectively at GD20, and 287 and 207 transcripts at PND5, revealing an increased effect after the end of treatment. Only about 1% of DEGs were common to both stages for each treatment. Functional analysis of coding DEG revealed an overrepresentation of olfactory transduction in all groups. In parallel, many non-coding RNAs were affected by both treatments, the most represented being small nucleolar and small nuclear RNAs. Our data suggest that despite no immediate toxic effects, fetal exposure to xenoestrogens can induce subtle immediate changes in germ cell gene expression. Moreover, the increased number of DEGs between GD20 and PND5 suggests an effect of early exposures with latent impact on later germ cell differentiation.

FC041: Regeneration of Nephrons and Renal Stroma using the Renal Developmental Environment of Animal Fetuses

Abstract BACKGROUND AND AIMS The kidney is derived from three types of progenitor cells: nephron progenitor cells (NPCs), stromal progenitor cells (SPCs) and ureteric buds (UBs). Extensive research in regenerative medicine has made it possible to induce pluripotent stem cells (PSCs) to develop into NPCs and UBs in vitro and generate the organoids of nephrons and collecting ducts. Renal stroma, including mesangial cells and erythropoietin- and renin-producing cells, is essential for maintaining biological functions. However, to our knowledge, the induction of SPC differentiation from PSCs has not been established yet. Moreover, the renal stroma cannot mature in the in vitro environment, making it difficult to generate the whole kidney, including the renal stroma. In this study, we attempted to regenerate the renal stroma in the in vivo environment by transplanting rat SPCs into the nephrogenic zone of mouse fetuses to provide rat SPCs with the renal developmental mechanism of heterologous mouse fetuses. Furthermore, we developed an interspecies dual replacement of progenitor (i-DROP) system wherein two progenitor cell lines, NPCs and SPCs, were genetically modified and replaced with transplanted progenitor cells and examined the feasibility of the regeneration of kidneys, including nephrons and renal stroma. METHOD We crossed Foxd1GC/+ mice with R26RDTR/DTR mice to generate Foxd1GC/+; R26RDTR/+ mice (Foxd–DTR mice) can eliminate Foxd1-positive SPCs. A group of SPC-rich cells extracted from green fluorescent protein (GFP) rat metanephros targeting platelet-derived growth factor receptor alpha (PDGFRa) was transplanted into the kidneys of Foxd1–DTR mouse fetuses. Subsequently, the organ-cultured metanephros or the transplanted metanephros in immunodeficient NOD/Shi- scid IL2rγnull (NOG) mice or Sprague–Dawley (SD) rats were evaluated using immunostaining. Next, we assessed whether the i-DROP system could regenerate interspecies nephrons and stroma. We crossed Six2TGC/+; Foxd1GC/+ mice obtained from Six2TGC/+ mice and Foxd1GC/+ mice with R26RDTR/DTR mice to generate Six2TGC/+; Foxd1GC/+; and R26RDTR/+ mice (Six2/Foxd1–DTR mice) that can simultaneously eliminate Six2-positive NPCs and Foxd1-positive SPCs. The SPCs targeted to PDGFRa+ and NPCs targeted to PDGFRa−/integrin alpha 8 (Itga8)+ from GFP rat metanephros were simultaneously transferred to Six2/Foxd1–DTR mouse metanephros and evaluated similarly. RESULTS In the organ culture of Foxd1–DTR mouse metanephros, SPCs around the cap mesenchyme replaced from mouse to GFP rat. We achieved extensive rat stromal cell regeneration in mouse kidneys, including endocrine functions such as erythropoietin production in the rat in response to anemia in the host mouse. Next, in the organ culture of the hind kidney of Six2/Foxd1–DTR mice equipped with the i-DROP system, the NPCs and SPCs of GFP rats aggregated around the mouse UB and the cap mesenchyme reconstituted. In vivo, rat nephrons and renal stroma were regenerated simultaneously in the mouse kidneys. The newly generated glomeruli were composed of GFP-positive rat cells, both podocytes and mesangial cells of different lineages (Figure 1). Intravenous injection of fluorescently labeled dextran into host mice revealed that the regenerated nephrons had glomerular filtration and tubular reabsorption capabilities. Furthermore, taking the xenotransplantation of mouse metanephros into SD rats as a control, the transplantation of regenerated rat kidneys into SD rats using our method resulted in reduced xenogenic immune rejection. CONCLUSION The newly developed i-DROP system regenerated multifunctional kidneys, composed of nephrons and renal stroma originating from different species, with urine production and endocrine functions. Functional human kidneys could be regenerated in the future using the renal developmental mechanism of large heterologous animals, such as pigs.

TLK1 Phosphorylates RAD54 To Promote Homology Driven DSB Repair

BackgroundEnvironmental agents like Ionizing radiation (I.R) can cause severe damage to DNA often causing double‐strand break (DSB). If unrepaired, DSB elicits chromosomal rearrangements, tumorigenesis, or apoptosis. The main mechanism to maintain the fidelity of the genetic information after DSBs is homologous recombination repair (HRR). The regulation of this process is unclear. Tousled‐like kinase 1 (TLK1), a Ser/Thr kinase that regulates DNA damage checkpoint and DSB repair has been found to interact with RAD54, a central DNA translocase enzyme in HRR. Our work aims to study how TLK1 regulates RAD54 activities during HRR? We hypothesize that phosphorylation of RAD54 at both NTD and CTD by TLK1 promotes HRR through RAD51‐nucleofilament interaction or during Branch Migration through RAD54 translocase activity.Material and methodsImmunoprecipitation and pull‐down assays show that TLK1 interacts with RAD54 in cells. We performed in‐vitro kinase assays with recombinant TLK1 to phosphorylate purified human RAD54 followed by mass spectrometry (MS). To understand the effect of TLK1 and mutation on RAD54 at the novel sites in HR efficiency, we use DR‐GFP assay in HeLa cells.ResultsWe find with pull‐downs and IP that TLK1 and RAD54 interact in vitro and ex vivo. Invitro TLK1 kinase assay with RAD54 followed by LC‐MS/MS reveals three novel sites of phosphorylation on RAD54 at T41, T59, and T700. The T41 and T59 lie in the unstructured domain (USD) region of N‐terminus (NTD). T700 lies in the Zn2+ finger motif in the C‐terminus (CTD) which contacts the DNA backbone for the translocase (t) function. HRR activity is monitored through the DR‐GFP assay which results in GFP positive cells after an efficient HR event. Our results with TLK1 specific inhibitor (J54‐10uM) or knockdown using TLK1 shRNA confirm that inhibition or depletion of TLK1 suppresses HRR activity in cells. To mimic the de‐phosphorylated or phosphorylated state of RAD54, we did a T to A/D site‐directed mutagenesis and generated DR‐GFP integrated HeLa‐RAD54 mutant cell lines in which RAD54 was genetically deleted. RAD54‐T700A/D, T2A/D (NTD double mutant‐T41, 59A/D), T3A/D (triple point) mutants expressed in HeLa‐RAD54K.O cell line were used to test the DNA repair proficiency in MMC sensitivity assay. We find T3A/D survival is lower than WT at the lowest concentration of MMC (0.1uM). All the D‐mutants are sensitive at 0.9uM MMC. Our data shows that compared to WT, T700D protein has 3‐fold higher basal ATPase activity in presence of dsDNA (‐ RAD51) than T2D and T3D. With RAD51, the activity of T700D was 2‐fold lower than T2D and T3D and 4‐fold lower than WT. RAD51 binding affinity was determined for all the mutant proteins and T700D had 20‐30% higher binding affinity than WT, T2D &amp; T3D. This implies that 1) interaction of phosphorylated RAD54‐T700 with RAD51 can downregulate the ATPase activity during HR completion or, 2) pRAD54‐T700 may have higher translocase activity and greater RAD51 affinity that needs to be turned off in later HRR steps.ConclusionWe expect that pRAD54 by TLK1 will impact gene conversion events and shed light on HRR regulation at the post‐translational level of RAD54

Application of transposon systems in the transgenesis of bovine somatic and germ cells

BackgroundSeveral DNA transposons including PiggyBac (PB), Sleeping Beauty (SB), and Tol2 have been applied as effective means for of transgenesis in many species. Cattle are not typically experimental animals, and relatively little verification has been presented on this species. Thus, the goal here was to determine the applicability of three transposon systems in somatic and embryo cells in cattle, while also investigating which of the three systems is appropriate for each cell type. Green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, the GFP-positive cells or blastocysts were observed through fluorescence, while the transfection efficiency was calculated by FACS.ResultsIn bovine somatic cells, the PB (63.97 ± 11.56) showed the highest efficiency of the three systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Conversely, Tol2 (75.00%) and SB (70.00%) presented a higher tendency in the embryonic cells compared to PB (42.86%).ConclusionsThese results demonstrate that these three transposon systems can be used in bovine somatic cells and embryos as gene engineering experimental methods. Moreover, they demonstrate which type of transposon system to apply depending on the cell type.

Residues 140-142, 199-200, 222-223, and 262 in the Surface Glycoprotein of Subgroup A Avian Leukosis Virus Are the Key Sites Determining Tva Receptor Binding Affinity and Infectivity.

Subgroup A avian leukosis virus (ALV-A) invades cells through gp85-encoded surface glycoprotein (SU) via specifically recognizing the cellular receptor Tva. To identify the key residues of ALV-A SU that determine the Tva binding affinity and infectivity in DF-1 cells, a strategy of substituting corresponding residues of SU between ALV-A RSA and ALV-E ev-1 (using Tvb as the receptor) was adopted. A series of chimeric soluble gp85 proteins were expressed for co-immunoprecipitation (co-IP) analysis and blocking analysis of viral entry, and various recombinant viruses based on replication-competent avian retrovirus vectors containing Bryan polymerase (RCASBP) were constructed for transfection into DF-1 cells and measurement of the percentage of GFP-positive cells. The results revealed that the substitution of residues V138, W140, Y141, L142, S145, and L154 of host range region 1 (hr1), residues V199, G200, Q202, R222, and R223 of host range region 2 (hr2), and residue G262 of variable region 3 (vr3) reduced the viral infectivity and Tva binding affinity, which was similar to the effects of the −139S, −151N, −155PWVNPF, −201NFD, Δ214–215, and −266S mutations. Our study indicated that hr1 and hr2 contain the principal receptor interaction determinants, with new identified-vr3 also playing a key role in the receptor binding affinity of ALV-A.

Regenerative Effects of Locally or Intra-Arterially Administered BMSCs on the Thin Endometrium.

Stem cell–based therapy plays a pivotal role in the regeneration of damaged endometrium. Previous studies have demonstrated the therapeutic potential of bone marrow mesenchymal stem cells (BMSCs) through diverse administration ways. However, the homing, survival, and differentiation potential of these differently administered BMSCs are poorly defined, and the best route of administration is not well-defined. Herein, we aim to compare the engraftment, retaining time, and therapeutic efficiency of differently administered BMSCs. To achieve this, GFP/Luc-labeled BMSCs administered in two modes were assessed in a thin endometrium rat model: either into the damaged horns directly or through the ipsilateral iliac artery. The retaining time and hemi-quantitative distribution were evaluated by in vivo bioluminescence imaging and immunohistological analysis. Locally administered BMSCs were strongly detected in the abdomen at the first 4 days post treatment but underwent a rapid decrease in luminescent signal afterward and were rarely found 28 days after treatment. In contrast, the retaining time of BMSCs injected through the iliac artery was longer, reflected by more GFP-positive cells detected in the uterine section 28 days post treatment. Differentiation toward endometrial stromal cells was observed. Both routes of administration contributed to the restoration of the damaged endometrium, showing a comparable increase in the endometrial thickness and a decrease in fibrosis. However, more importantly, higher expression of LIF and VEGF, better recruitment, and longer retainment were found in the intra-arterial administration, contributing to the establishment of the optimal administration mode in clinical practice.

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

SummaryFragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Beneficial Impact of Interspecies Chimeric Renal Organoids Against a Xenogeneic Immune Response.

BackgroundAnimal fetal kidneys have the potential to be used as scaffolds for organ regeneration. We generated interspecies chimeric renal organoids by adding heterologous rat renal progenitor cells to single cells from mouse fetal kidneys and applying the renal development mechanism of mouse fetuses to rat renal progenitor cells to examine whether rat renal progenitor cells can differentiate into renal tissues of the three progenitor cell lineages of kidneys between different species. Furthermore, we investigated whether chimeric renal organoids with an increased proportion of recipient cells reduce xenogeneic rejection.MethodsC57BL/6JJmsSlc mice (B6 mice) and Sprague-Dawley-Tg (CAG-EGFP) rat (GFP rats) fetuses were used as donors, and mature male NOD/Shi-scid, IL-2RγKO Jic mice (NOG mice) and Sprague-Dawley rats (SD rats) were used as recipients. First, fetal kidneys were removed from E13.5 B6 mice or E15.5 GFP rats and enzymatically dissociated into single cells. These cells were then mixed in equal proportions to produce chimeric renal organoids in vitro. The chimeric organoids were transplanted under the renal capsule of NOG mice, and maturation of the renal tissues in the organoids was observed histologically. Furthermore, chimeric organoids were prepared by changing the ratio of cells derived from mouse and rat fetal kidneys and transplanted under the renal capsule of SD rats subjected to mild immunosuppression to pathologically analyze the strength of the xenogeneic immune response.ResultsThe cap mesenchyme was reconstructed in vitro, and nephron progenitor cells and ureteric buds were mosaically comprised GFP-negative mouse and GFP-positive rat cells. In the in vivo environment of immunodeficient mice, chimeric renal organoids mosaically differentiated and matured into renal tissues of three lineages. Chimeric renal organoids with high rates of recipient rat cells showed milder rejection than complete xenograft organoids. The vessels of recipient rats entered from the periphery of the transplanted chimeric renal organoids, which might reduce their immunogenicity.ConclusionInterspecies chimeric renal organoids may differentiate into mature renal tissues of each renal progenitor cell lineage. Furthermore, they may reduce transplant rejection compared with xenograft organoids.

Cancer-Associated Stromal Cells Promote the Contribution of MMP2-Positive Bone Marrow-Derived Cells to Oral Squamous Cell Carcinoma Invasion

Simple SummaryBased on its invasiveness, oral squamous cell carcinoma (OSCC) shows two different subtypes: less-invasive verrucous squamous carcinoma (VSCC) or highly invasive squamous cell carcinoma (SCC). The stromal component influences OSCC progression and invasion. On the other hand, bone marrow-derived cells (BMDCs) are recruited into tumors and involved in tumor development. We hypothesized that stromal factors might also affect the relation of BMDCs and tumor invasion. We established the OSCC models transplanted with stromal cells from VSCC and SCC, and we compared the potential stromal factors of VSCC and SCC for the involvement of BMDCs in tumor invasion. Our study showed that stromal factors IL6 and IL1B might promote the contribution of MMP-2 positive BMDCs to OSCC invasion.Tumor stromal components contribute to tumor development and invasion. However, the role of stromal cells in the contribution of bone marrow-derived cells (BMDCs) in oral squamous cell carcinoma (OSCC) invasion is unclear. In the present study, we created two different invasive OSCC patient-derived stroma xenografts (PDSXs) and analyzed and compared the effects of stromal cells on the relation of BMDCs and tumor invasion. We isolated stromal cells from two OSCC patients: less invasive verrucous OSCC (VSCC) and highly invasive conventional OSCC (SCC) and co-xenografted with the OSCC cell line (HSC-2) on green fluorescent protein (GFP)-positive bone marrow (BM) cells transplanted mice. We traced the GFP-positive BM cells by immunohistochemistry (IHC) and detected matrix metalloproteinase 2 (MMP2) expression on BM cells by double fluorescent IHC. The results indicated that the SCC-PDSX promotes MMP2-positive BMDCs recruitment to the invasive front line of the tumor. Furthermore, microarray analysis revealed that the expressions of interleukin 6; IL-6 mRNA and interleukin 1 beta; IL1B mRNA were higher in SCC stromal cells than in VSCC stromal cells. Thus, our study first reports that IL-6 and IL1B might be the potential stromal factors promoting the contribution of MMP2-positive BMDCs to OSCC invasion.

SRSF2-P95Hdelays Myelofibrosis Development through Altered JAK/STAT Signaling in JAK2-V617F Megakaryocytes

Background: The gain-of-function JAK2 V617F mutant is the most common driver mutation identified in myeloproliferative neoplasms (MPNs). Additional somatic variants, also found in other malignant hemopathies, are detected in primary myelofibrosis (MF) and supposed to contribute to fibrosis or leukemia development. One of these mutations affects SRSF2, a gene encoding a component of the splicing machinery. SRSF2 heterozygous mutation mainly affects the proline 95 residue of the protein. Its association with JAK2 V617F correlates with a reduced leukemia free survival. Whether and how SRSF2 P95 variants could favor fibrosis development in JAK2 V617F cells remained unknown.Methods & Results: To investigate how homozygous Jak2 V617F and heterozygous Srsf2 P95H could interact in the hematopoietic tissue, we generated conditional knock-in mice in which the CreERt recombinase expression was driven by the HSC-Scl promoter leading to Jak2 V617F and/or Srsf2 P95H hematopoietic-specific expression upon tamoxifen induction.Srsf2 P95H mutation initially exhibited limited effect on Jak2 V617F-induced polycythemia vera (PV) only slightly reducing erythrocytosis and leukocytosis (through a previously described decrease in B220 + B cell number). The expansion of hematopoietic stem cells (SLAM), multipotent progenitors (MPP) and megakaryocyte progenitors (MKP) observed in Jak2 V617F mice was not affected by Srsf2 status. However, while platelet count was decreasing in Jak2 V617F alone mice at later time point due to fibrosis development, Srsf2 P95H/Jak2 V617F combination further increased platelet counts correlating with a significant delay in the development of myelofibrosis. Bone marrow cells (BM) were transplanted into lethally irradiated recipient mice together with GFP-positive wild-type competitor cells and tamoxifen was administrated after transplantation. Double mutant cells initially demonstrated a limited competitive advantage over wild-type cells as compared to Jak2 V617F-only cells. However, serial transplantation revealed a rapid exhaustion of Jak2 V617F single mutant cells leading to lethal pancytopenia, which was not observed in animals transplanted with Jak2 V617F/Srsf2 P95H double mutant cells.As both monocytes and megakaryocytes (MK) were involved in fibrosis development, we further explored the role of these two cell populations. Spectral flow analysis of monocyte subsets in peripheral blood and BM failed to detect any significant change in double compared to single mutant animals. In contrast, double-mutant mice presented a significant delay in MK maturation with normalized expression of c-Mpl and ploidy. Using mass cytometry, we found ex vivo a higher proportion of MKP and MK expressing high levels of P-Stat5 in Jak2 V617F mice, which the addition of Srsf2 P95H tend to reduce, suggesting an altered Mpl/Jak2 signaling pathway.To validate the hypothesis that Srsf2 P95H negatively interfere with Jak2-mediated signaling in MK, we injected high dose of the thrombopoietin-mimetic romiplostim in mice transplanted with wild-type or Srsf2 P95H BM. Both thrombocytosis and myelofibrosis were significantly reduced in Srsf2 P95H transplanted animals.To further decipher the mechanism by which Srsf2 P95H could alter cell signaling, we performed bulk RNA sequencing on sorted MK. Pathway analysis using gene set enrichment analysis identified mostly a down-regulation of signaling pathways, including JAK/STAT signaling, in Jak2 V617F/Srsf2 P95H compared to Jak2 V617F single mutant cells. Further analysis of splicing events in Srsf2 P95H mutant cells identified an increased exon 14 skipping in Jak2, which was validated by RT-qPCR.Summary: Contrary to EZH2 mutation that promotes JAK2 V617F-induced myelofibrosis in mouse models, heterozygous Srsf2 P95H delays myelofibrosis development in Jak2 V617F-transgenic mice. Srsf2 P95H co-mutation prevents the clonal exhaustion induced by serial transplantation of JAK2 V617F BM cells. This effect is associated with a reduced signaling in MK, which may involve abnormal splicing of signaling components including Jak2 exon 14 skipping. DisclosuresAbdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

A Transgenic Murine Model Expressing Hyperactive STAT3 Recapitulates the Features of MDS/AML

Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders.Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs.To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies.Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. [Display omitted] DisclosuresFrank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.