Human pluripotent stem cells (hPSCs) can be used as a renewable source of endothelial cells for treating cardiovascular disease and other ischemic conditions. Here, we present the derivation and characterization of a panel of distinct clonal embryonic endothelial progenitor cells (eEPCs) lines that were differentiated from human embryonic stem cells (hESCs). The hESC line, ESI-017, was first partially differentiated to produce candidate cultures from which eEPCs were cloned. Endothelial cell identity was assessed by transcriptomic analysis, cell surface marker expression, immunocytochemical marker analysis, and functional analysis of cells and exosomes using vascular network forming assays. The transcriptome of the eEPC lines was compared to various adult endothelial lines as well as various non-endothelial cells including both adult and embryonic origins. This resulted in a variety of distinct cell lines with functional properties of endothelial cells and strong transcriptomic similarity to adult endothelial primary cell lines. The eEPC lines, however, were distinguished from adult endothelium by their novel pattern of embryonic gene expression. We demonstrated eEPC line scalability of up to 80 population doublings (pd) and stable long-term expansion of over 50 pd with stable angiogenic properties at late passage. Taken together, these data support the finding that hESC-derived clonal eEPC lines are a potential source of scalable therapeutic cells and cell products for treating cardiovascular disease. These eEPC lines offer a highly promising resource for the development of further preclinical studies aimed at therapeutic interventions. Cardiovascular diseases including atherosclerosis, ischemic stroke, myocardial infarction, and ischemic cardiomyopathy currently represent a large economic and societal burden due to their high mortality and disability rates . ImpairmPCDHB2, and their lack of fetal/adult gene markers such as COX7A1 that are preferentially expressed in cells that have traversed the embryonic-fetal transition [We have previously developed a method of generating purified and embryonic site-specific somatic cell types through the propagation of hPSC-derived clonal human embryonic progenitor cell lines to overcome current issues of purity and scale . We desiansition . These hansition . Due to ansition ,23, seveansition ,25, and ansition  from ouransition ,22,23.In the present study, we derived novel hEP cell lines with endothelial properties and characterized a panel of 14 embryonic endothelial progenitor cell (eEPC) lines to explore their potential as a scalable source of human endothelial cells and cell products for further therapeutic development. We compared the eEPC lines to a diverse panel of adult endothelial cell (AEC) lines using comprehensive transcriptomic analysis. We found that 13 out of 14 eEPC lines expressed a broad array of endothelial specific genes shared across various endothelial subtypes. We further assessed these eEPC lines for endothelial identity using cell surface markers and well-established vascular tube forming assays. Interestingly, the eEPCs were clearly distinguished from AECs based on embryonic specific gene expression and molecular network analysis. In addition, we were able to achieve high scalability typical of previously identified hEP cell lines and stable secretion of angiogenic exosomes, suggesting that eEPCs may be advantageous for developmental research and for treating cardiovascular disease in a large patient population.Research grade hESCs (ESI-017) were differentiated using a two-step progenitor derivation protocol. In the first step, human embryonic stem cells were exposed to a 7-day procedure on Matrigel coated plates involving specific differentiation factors, followed by exposure to two different growth media to generate two cultures of heterogeneous cells designated as Candidate Cultures (CC). Afterwards, each CC subpopulation was placed at clonal densities in their same growth medium for two weeks. Then, individual clonal colonies were selected with cloning cylinders and expanded from a 24-well plate to multiple T225 flasks A.As described above and in 2 and 5% O2.A total of 14 eEPC lines and 15 human adult endothelial cell (AEC) lines were cultured in endothelial growth medium  on gelatin-coated plates. All the information on cell lines including cell origins and sources were described in t-test and p (Corr) cut-off = 0.05 while applying Pearson metric. The dendrogram was created using hierarchical cluster analysis using the average agglomeration method. For each cell culture sample, the differential expression values were calculated against a selection of 14 eEPC clones versus 15 adult endothelial cell lines versus 17 non-endothelial cell lines including embryonic and adult origin. All the information on samples is described in RNA was prepared upon lysis with RLT with 1% β-mercaptoethanol, using Qiagen RNeasy mini kits (Cat# 74104) following manufacturer’s directions. The extracted RNA was then quantitated using a NanoDrop (ND-1000) spectrophotometer. Library Construction was performed using the Illumina Truseq mRNA library prep kit following manufacturer’s directions. Library QC and library pooling were accomplished using an Agilent Technologies 2100 Bioanalyzer to assay the library fragments. qPCR was used to quantify the libraries. Libraries were pooled, which had different barcodes/indexing and sequencing, in one lane. The paired-end sequencing was performed using the Illumina HiSeq4000 sequencing instrument, yielding 100-bp paired-end reads. The sequencing was performed by BGI Americas Corporation. Data analysis of the transcription levels  was performed using Gene Spring GX Suite . Hierarchical and correlation analysis were performed with a moderate Total DNA was extracted from cells using Qiagen DNeasy mini kits according to instructions supplied by the manufacturer. DNA concentrations were measured using a Nanodrop spectrophotometer. Whole-genome expression was obtained using Illumina Human HT-12 v4 BeadArrays. In preparation for Illumina BeadArrays, total DNA was linearly amplified and biotin-labeled using Illumina TotalPrep kits . The sample quality was measured using an Agilent 2100 Bioanalyzer before being hybridized to Illumina BeadChips, processed, and read by an iScan microarray scanner according to the manufacturer’s instructions . Values under 130 relative fluorescence units (RFUs) were considered as nonspecific background signal. Analysis of microarray data was performed using the GeneSpring suite. Raw microarray data were normalized with the R BeadArray library . Mergingt-test. Independent experiments of samples over time were analyzed by repeated measures of ANOVA with the Holm adjustment. Differences were considered significant at probability values of p < 0.05.All data are expressed as mean ± standard deviation. Statistical analysis between groups at each time point was performed by the unpaired student’s 6 cells per 1 mL for 10 min. The cells were stained by incubating them with monoclonal antibodies  for 30 min. Primary antibodies were diluted according to the manufacturer’s instructions. Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), Mouse IgG1 Isotype-FITC were used as controls. The cells were scanned with an LSRII flow cytometer  and analyzed with the FlowJo software version 10.6 .Cells were dissociated into a single-cell suspension by using TrypLE  and fixed in BD Cytofix buffer  for 20 min at room temperature. The cells were permeabilized by washing and incubating them with BD Permeabilization/Wash  buffer at 1 × 10For detection of CXCR4 and CD31, cells were washed once with PBS and fixed in 4% paraformaldehyde for 30–60 min at room temperature (RT). Fixed cells were washed three times with PBS, permeabilized, and blocked by incubation in blocking buffer  for 1 h at RT. The cells were then incubated overnight at 4 °C with CD31 monoclonal antibody with Alexa Fluor 488  and primary rabbit anti-human CXCR4 polyclonal antibody  at a dilution of 1:500 in 5% normal donkey serum, 0.5% BSA, and 0.05% Triton X-100 in PBS. Then, the cells were washed four times with PBS plus 0.05% Triton X-100 (PBS-Triton) and incubated for 1 h at RT with Alexa Fluor 568 donkey anti-rabbit IgG antibody  at a 1:500 dilution in PBS-Triton. Isotype controls were stained under identical conditions except that total rabbit IgG  was used as primary antibody. Cells were counterstained with DAPI at 0.1 ng/mL for 10 min at RT and imaged on a Nikon Eclipse TE2000-U inverted microscope.2). Exosomes for tube formation assay were isolated from eEPC and BM-MSC  culture medium using the precipitation method following the manufacturer’s instructions . Total exosome particles were counted using nanoparticle tracking analysis (NTA).Tube formation angiogenesis assay was performed using the Cell Player Angiogenesis PrimeKit  to assess tube network growth in eEPC lines. eEPCs were labeled using TagRFP control vector, which consists of the lentiviral backbone vector, pLKO.1-puro, containing a gene encoding red fluorescence protein (RFP), driven by the CMV promoter . RFP labeled eEPCs were plated with monolayer, and 10ng/mL of recombinant human VEGF protein  was treated to enhance tube formation. Images were taken every 6 h for 10–14 days using an IncuCyte imager. Tube formation was quantified using the IncuCtye Angiogenesis Analysis Module by dividing the lengths of all tube networks by the image area at each time point  to assess tube network growth effect of eEPCs-secreted exosomes. HUVECs with 104 cells/well were seeded in µ-Slide 15 well 3D  coated with Matrigel and followed by the treatment of eEPC-exosomes in PBS, compared with control conditions . Tube formation networks were analyzed using the ImageJ version 1.44 Angiogenesis Analyzer.Tube formation angiogenesis assay was also performed using human umbilical vein cells (HUVECs) that were seeded onto growth factor reduced MatrigelWe have previously demonstrated that a large variety of distinct, highly scalable, and clonally pure progenitors can be isolated from human pluripotent stem cells using a two-step process of differentiation under diverse conditions followed by clonal selection of scalable cell lines . In our To understand the molecular characteristics of the clonally selected hEP lines, we analyzed whole transcriptomic gene expression. We compared three groups: (1) 14 human embryonic endothelial progenitor cell (eEPC) lines, 30-MV2-6, 30-MV2-3, 30-MV2-4, 30-MV2-10, 30-MV2-17, 30-MV2-19, 30-MV2-2, 30-MV2-7, 30-MV2-9, 30-MV2-14, 30-MV2-24, 30-MV2-8, 30-SM2-1, and 30-SM2-3; (2) 15 primary adult endothelial cell (AEC) lines including HBMEC, HCPEC, HDMEC, HDLEC, HLEC, HIMEC, HRGEC, HHSEC, HCMEC, HAEC, HOMEC, HUVEC, HUAEC, HMVEC, and HAEC; and (3) a panel of 17 non-endothelial cell lines . The description of cell origin, cell sources, and ages of RNAseq samples are described in Hierarchical clustering analysis with a representative heatmap in KDR and vWF) (PDGF-B), inhibitor of differentiation 1 (ID1), and C-X-C Motif Chemokine Receptor 4 (CXCR4), with comparable level to AEC lines  31, VE-Cadherin (CD144), vascular endothelial growth factor receptor (VEGFR-2)/KDR, and Von Willebrand factor (vWF), which are all widely recognized as endothelial-specific markers ,29 and Cand vWF) . InteresEC lines . It has culation , and regculation , respectin vitro. To assess tube network growth in eEPC lines, eEPCs were labeled using TagRFP and co-cultured with normal human dermal fibroblasts (NHDF). This model allows us to demonstrate all phases of the angiogenesis process, including proliferation, migration, and, eventually, differentiation and angiogenic networks [We next investigated the angiogenic functionality of eEPC lines for vascular tube formation networks . ImagingAPLNR), differentiation marker of hematopoietic stem and progenitor cells [IGF2) promoting EPC homing [MAFB, controlling endothelial sprouting [IGFBP5), promoting angiogenesis [H19, a long non-coding RNA increasing EC tube formation [CPT1C) promoting human mesenchymal stem cells survival [ACP5), promoting cell differentiation and proliferation [EGLN3), a regulator associated with resistance to stress) [To better understand the molecular signature of eEPC lines compared to AEC lines, we performed a differential gene expression analysis of the transcriptomic RNAseq data. Although the overall global gene expression patterns of the 13 eEPC lines were similar to 15 AEC lines from various origins, we found that there was a significant subset of genes that were differentially expressed in the eEPC lines . The RNAor cells , insulinC homing , MAFB, cprouting , insulinogenesis , H19, a ormation , Carnitisurvival , Tartratferation , and enh stress)   that is almost exclusively expressed in fetal and adult cells [COX7A1 expression level in eEPC lines was observed to be undetectable (Log2 FC = 0.1), compared to AEC lines (Log2 FC = 4.3), suggesting that eEPC lines retained an embryonic progenitor stage (CCL2), which is associated with diabetes [(BACE2), which is linked to increased risk and earlier disease onset such as Alzheimer’s disease [CAT), which plays an important role in endothelial cell shear stress response [CXCL1), which regulates TNF-stimulated endothelial cells [IFI27), which is a stimulator of VEGF-A mediated angiogenesis [MGP), which regulates differentiation of endothelial cells [MEG3) [We previously identified lt cells . COX7A1 or stage B. In adddiabetes , β-site  disease , an antiresponse , chemokial cells , interfeogenesis , matrix al cells , and a ls [MEG3) , were alWe examined cell growth characteristics by measuring scalability and functional stability of a representative eEPC line, 30-MV2-6. It has been suggested that adult EPCs have therapeutic potential and clinical implications in ischemic diseases ,50,51. T6 exosomes/well  of cells was compared to primary MSCs. We found that MSCs begin to senesce and lose proliferative and differentiation capacity after 12 pd, consistent with previous reports ,53. In cmes/well . The res6 exosomes/well of BM-MSC exosomes showed equivalent angiogenic activity as 30MV2-6 exosomes at 200 × 106 exosomes/well, indicating a 6-fold difference in potency  versus late passages (50 pd). The results show that the early passage eEPC exosome angiogenic activity was stable at later passage C. To invPrevious studies have suggested that mesenchymal stem cells (MSCs) ,5, endotin vitro. Interestingly, our study showed that eEPC lines retained embryonic progenitor characteristics. We found a subset of genes that were upregulated in eEPC lines compared to AEC lines. For example, eEPC overexpressed apelin receptor (APLNR), insulin-like growth factor 2 (IGF2), the MAF BZIP transcription factor B (MAFB), insulin-like growth factor binding protein 2 (IGFBP2), non-coding RNA H19, carnitine palmitoyltransferase 1 C (CPT1C), tartrate-resistant acid phosphatase 5 (ACP5) and enhanced prolyl hydroxylase domain (PHD)-3 (EGLN3) relative to AEC. APLNR signaling is required for the generation of cells that give rise to HSCs [IGF2/IGF2R system in promoting endothelial progenitor cells homing [MAFB gene encodes a transcription factor that controls endothelial sprouting in vitro and in vivo [IGFBP2 is known as a developmentally regulated gene that is highly expressed in embryonic and fetal tissues and markedly decreases after birth [H19 is expressed during EC differentiation and has functional effects on EC tube formation [CPT1C promotes human mesenchymal stem cells survival under glucose deprivation through the modulation of autophagy and is strongly associated with the epithelial-mesenchymal program [ACP5 is upregulated by transforming growth factor-β1 (TGF-β1) and subsequently enhances β-catenin signaling in the nucleus, which promotes the differentiation, proliferation, and migration of fibroblasts [EGLN3, a member of the EGLN family of prolyl hydroxylases, increases during skeletal myoblast differentiation [In the present study, we provided comprehensive transcriptomic profiling analysis demonstrating that eEPCs differentiated from hEPs have endothelial phenotypes expressing endothelial markers such as CD31, CXCR4, KDR, and vWR, and EPC marker, CD34, as well as generating tube formation s homing . The MAF in vivo  and play in vivo ,63. IGFBer birth . It promer birth . H19 is ormation . CPT1C p program . ACP5 isroblasts . EGLN3, ntiation  and is antiation . UpregulBACE2), catalase (CAT), CXCL1, Interferon α-inducible protein 27 (IFI27), matrix Gla protein (MGP), and long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) compared to AEC lines. The BACE2 gene is linked to increased risk and earlier disease onset of Alzheimer’s disease. CAT encodes an antioxidant enzyme that plays an important role in endothelial cell pathophysiology, in shear stress response, and ultimately, in arterial aging [CXCL1 was only expressed by old osteoblasts but not young cells [IFI27 is known as an oncogene, a strong stimulator of angiogenesis, by increasing secretion of vascular endothelial growth factor (VEGF-A) [MGP stimulates differentiation of endothelial cells [MEG3 negatively regulates angiogenesis and proliferation in vascular endothelial cells. MEG3 overexpression was significantly suppressed by inhibition of miR-9 resulting in proliferation and in vitro angiogenesis in vascular endothelial cells [We also found a subset of genes that were downregulated in eEPCs. For example, eEPC lines expressed little to no β-site APP cleavage enzyme 2  . MGP stial cells , but alsal cells . MEG3 neal cells . DownregIFITM1, NNAT, H19, PLVAP, and IGF2, which were highly upregulated and CYTL1, KRT7, CXCL1, and CXCL8, which were downregulated in HUVECs and eEPC lines, but not the AEC lines  have been a robust source of ECs with proven capability of forming stable microvessels, which are capable of capillary morphogenesis compared to hPSC-ECs . From ouEC lines . Our finDerivation of eEPC lines that are homogeneous clonal cells with batch-to-batch consistency in production provides a unique competitive advantage for unparalleled industrial scale production that is needed for therapeutic application in various ischemic diseases. The overall advantages of hEP lines are (1) identity and homogeneity, (2) scalability and stability, and (3) diversity  stem cells such as endothelial progenitor cells (EPCs), MSCs, and CDCs for clinical application in the treatment of ischemic disease. However, the source of these primary cell lines results in cellular heterogeneity both within a donor line and between donor lines. Thus, there is a manufacturing bottleneck for the consistent production of therapeutic ECs on an industrial scale needed to treat a large cardiovascular disease patient population. We addressed this issue by developing a bank of embryonic progenitor cell lines derived by early-stage clonal isolation from partially differentiated hPS cells . This meHere, we demonstrate the ability to derive clonally pure endothelial cells (eEPCs) from hPS cells using our two-step process. We derived 13 eEPC lines and characterized them as endothelial by transcriptomic analysis, biochemical markers, and vascular function. Further, we have shown early evidence of the angiogenic potential of exosomes secreted from eEPC lines. We are currently developing eEPC exosomes manufacturing methods and investigating the potential therapeutic uses of eEPC exosomes for treating ischemic conditions like stroke by improving vascular and neurological recovery. Our findings suggest that eEPC lines will be a valuable source of both cells and exosomes for use in therapeutic vascular regeneration.Exosomes from Clonal Progenitor Cells, 2022 (US 11274281); Exosomes from Clonal Progenitor Cells, 2019 ; Methods and compositions for targeting progenitor cell lines, 2019 .


TP53, RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I (Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel therapeutic approach in OS. The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years. Mutations in the genes  Osteosarcoma (OS) is the most frequently occurring primary malignant bone tumour in children and young adults, with the highest peak incidence during the second decade of life . Currentc-Myc and mTOR) and tumour suppressors  promote Pol I transcription activity [Dysregulated cell growth is one of the representative hallmarks of cancer  and accoactivity ,14,15. ICX-5461, the first-in-class small molecule inhibitor of Pol I-mediated transcription, suppresses rRNA transcription by preventing the binding of the Pol I-specific transcription initiation factor, SL-1, to the rDNA promoter region ,17. CX-5TP53 and RB1 are highly mutated [50) values for CX-5461 in two OS cell lines, and a high concentration of CX-5461 was used in cell cycle and autophagy studies [TP53 mutant human OS xenograft, (ii) a TP53 wild-type human OS xenograft and (iii) a TP53 wild-type murine allograft.Recent studies in OS indicate that Pol I transcription activity is very likely to be dysregulated in OS. c-Myc protein, a key upstream regulator of ribosome biogenesis , is freq mutated ,28,29,30 mutated . A previ studies . These r2PO4 (pH4.5) and diluted in growth media immediately prior to use in vitro. For the in vivo studies, 30 mg/kg of CX-5451 was prepared in 50 mM NaH2PO4 (pH4.5).CX-5461 was purchased from SYNkinase , and 10 mM stocks of CX-5461 were prepared in 50 mM NaH®, Manassas, VA, USA), and a further five patient-derived osteosarcoma cell lines, KOS-1, KOS-2, KOS-3, KOS-6 and KOS-7, were kindly provided by HS KIM from Seoul National University Hospital [2.Five commercially available human osteosarcoma cell lines, HOS, 143B, MNNG/HOS, U-2-OS and SJSA-1, were purchased from American Tissue Culture Collection . The assay was conducted following the manufacturer’s instructions (Single-nucleotide variants (SNVs) and copy number variants (CNVs) in ten OS cell lines were analysed using The Ampliseqructions .2; HOS: 6.0 × 102; MNNG/HOS: 6.0 × 102; U-2-OS: 1.0 × 103; SJSA-1: 1.0 × 103; KOS-1: 1.0 × 103; KOS-2: 1.0 × 103; KOS-3: 1.0 × 103; KOS-6: 1.0 × 103; and KOS-7: 1.0 × 103) and murine OS cells (2.0 × 103) following exposure to fresh medium containing vehicle or various concentrations of CX-5461 for 72 h was undertaken as previously described [50) were calculated using Graph-Pad Prism (version 9.1.0).The proliferation of human OS cells (143B: 4.0 × 10escribed . The dos6) and human fibroblast cells (1.0 × 106) as previously described [The protein levels of p53, MDM2, c-Myc and RB1 were assessed in the ten OS cell lines  was estimated by quantitative real-time PCR (qRT-PCR) as previously described [TM Thermocycler , and then cDNA synthesis was performed with SuperScriptTM IV Reverse Transcriptase , Hexameric Random Primers  and dNTPs  in a T100TM Thermocycler. All synthesised cDNA samples were loaded into 96-well PCR plates with SYBRTM Green master mix, primers that targeted the external transcribed spacer-2 (ETS-2) of the pre-47S ribosomal RNA and the housekeeping gene, beta-2-microglobulin (β2M) . All settings for cDNA synthesis and RT-PCR are described in 50) were calculated using Graph-Pad Prism (version 9.1.0).rDNA transcription in 1.0 × 10escribed ,34. Briein (β2M) , and rea50 CX-5461 concentrations (determined from the cell proliferation assay), then labelled with 10 µM BrdU , harvested and stained with PI. Flow cytometry (Becton Dickinson LSR II) was used to assess the level of staining, and FlowJo software (version 10.0) was used for data analysis.All OS cell lines were analysed for cell cycle progression using Bromodeoxyuridine (BrdU) and Propidium iodide (PI) staining as previously described . Briefly6) in 50 µL of Hank’s Balanced Salt Solution (HBSS)  or SJSA-1-Luc cells (5 × 106) in 50 µL of HBSS containing 50% (v/v) Matrigel  were injected subcutaneously into the right flanks of mice. For the allograft mouse model, K7M2 cells (1 × 106) in 50 µL of HBSS were injected subcutaneously into BALB/c wild-type mice on the right flank. Tumour-bearing mice were monitored daily. Once the average tumour volume reached 30~50 mm3, mice were randomised to receive either CX-5461 or vehicle. Thrice (3 times) weekly for 3 weeks, 30 mg/kg of CX-5461 in 50 mM NaH2PO4 (pH4.5) or the corresponding vehicle (50:50 mixture of 50 mM NaH2PO4 and 50 mM Citric Acid) was administered via oral gavage. The experiments were terminated 15 days following the start of drug administration or prior if mice reached the ethical endpoint.All in vivo experiments were conducted following approval from the Australian National University Animal Experimentation Ethics Committee (A2017/16 and A2020/36). Rag 2 knockout (KO) mice were obtained at 6 to 8 weeks of age from the Animal Resource Centre . 143B-Luc cells  was assessed using two-way ANOVA. Statistical analyses were performed using GraphPad Prism software, version 9.1.0. The dose–response curve fit and 50% inhibitory values were obtained using non-linear regression. Comparisons of GICTP53 are reported to confer chemo-resistance and a poor prognosis in OS patients [TP53 gene and normal p53 protein expression) (TP53 gene or dysregulated p53 protein expression)   A and abnression) B OS cell 139 nM) C. There 50) range in normal p53  and abnormal p53 OS cell lines   B. In the50 of CX-5461 for 72 h and analysed by flow cytometry. CX-5461 treatment was found to induce G2-phase cell cycle arrest in all OS cell lines analysed, both p53 normal and abnormal cell lines  cell lines, and U-2-OS, SJSA-1 and KOS-3 were identified as normal p53 (p53 wild-type) cell lines . Previoull lines . p < 0.05) after the first week (Day 8) of drug dosing (The anti-tumour activity of CX-5461 was investigated in two human OS xenograft mouse models in Rag 2 knockout (KO) mice using 143B (p53 mutant-type) and SJSA-1 (p53 wild-type) cell lines . Each exg dosing B. Similag dosing C. CX-546g dosing D,E. To investigate whether CX-5461 had the same in vivo therapeutic efficacy in the presence of a functional immune system, the anti-tumour effect of CX-5461 was evaluated in an allograft mouse model using the murine OS cell line K7M2 . As seen50 and tIC50 values for each OS cell line, (ii) demonstrated the on-target effect of CX-5461 on Pol I activity, (iii) analysed the effect of CX-5461 on cell division in a range of cell lines and (iv) demonstrated the therapeutic effect of CX-5461 in vivo using three different mouse models. The advancement of diagnostic and therapeutic strategies for OS has not improved outcomes over the last 30 years, and chemo-resistance is a major problem. There is, therefore, an urgent need for new therapeutic strategies against this disease ,7,8,37. TP53, RB1, TSC1, TSC2 and ATRX and amplifications of c-Myc, MDM2 and CDK4. Additionally, the dysregulated expression of p53, RB1, c-Myc and MDM2 proteins was demonstrated, all of which have the potential to impact Pol I activity. These molecular results are consistent with other genetic studies of OS and highlight the significant diversity in gene and protein alterations that may lead to OS and enhanced Pol I activity, and they emphasise the potential for Pol I inhibitors to be effective against the majority of OS cases.Alterations in tumour suppressors and oncogenes that occur in many cancers influence Pol I transcription activity ,14. The Multiple studies have revealed the mechanism of the therapeutic effect of CX-5461 on human cancers ,36,42,43TP53 gene mutation status [TP53 mutations had a significantly higher risk of death within 2 years than patients with wild-type TP53 tumours [In clinical studies, the response to chemotherapy and the survival rate of OS patients varied according to the n status ,46. A me tumours . Our in  tumours . The dru50 value in the cancer cell lines of 147 nM (range 3–5500 nM), whereas the 5 normal cell lines all had a GIC50 value of approximately 5000 nM [50 values obtained for the 10 OS cell lines in this study (33–188 nM), therefore, closely align with the median values previously reported for other cancer cell lines. More importantly, these values are well below those required to impact the proliferation of normal cell lines; thus, these doses could be considered selective for OS cells. The lack of obvious side effects in our mouse studies, with a maximum weight loss of 10% measured, reflects the results of early phase trials in humans in which CX-5461 was well tolerated [Previous studies of the anti-proliferative effect of CX-5461 in 50 cancer cell lines and 5 normal cell lines demonstrated a median GIC 5000 nM . The GIColerated . InteresAs with most chemotherapy agents, resistance to RNA Pol I inhibition may develop as a result of new mutations or compensatory mechanisms. Combining therapies that target different pathways increases the treatment effect and decreases the likelihood of resistance occurring. CX-5461 was successfully combined with the mTORC1 inhibitor everolimus to demonstrate a synergistic anti-tumour effect in lymphoma-bearing mice . Future In conclusion, this study has identified mutations in genes associated with Pol I activity in 10 OS cell lines and has confirmed the alteration of proteins well recognised to be involved in the regulation of Pol I transcription. Furthermore, this study has demonstrated that targeting elevated Pol I transcription in OS shows significant promise as an effective novel therapeutic strategy. Given the pre-clinical results provided and the safety profile of CX-5461 already obtained in human clinical studies, CX-5461 should be considered a potential therapeutic option for OS.


Peripheral hIAPP plays a major role in the pathogenesis of AD, and high circulating hIAPP level increase AD risk in T2DM patients. However, there is no hard evidence for the role of brain-derived hIAPP in the pathogenesis of AD. Nevertheless, oxidative stress, mitochondrial dysfunction, chaperon-mediated autophagy, heparan sulfate proteoglycan (HSPG), immune response, and zinc homeostasis in T2DM could be the possible mechanisms for the induction of the aggregation of hIAPP which increase AD risk. In conclusion, increasing hIAPP circulating levels in T2DM patients predispose them to the development and progression of AD. Dipeptidyl peptidase 4 (DPP4) inhibitors and glucagon-like peptide-1 (GLP-1) agonists attenuate AD in T2DM by inhibiting expression and deposition of hIAP.Human Islet amyloid polypeptide (hIAPP) from pancreatic β cells in the islet of Langerhans has different physiological functions including inhibiting the release of insulin and glucagon. Type 2 diabetes mellitus (T2DM) is an endocrine disorder due to relative insulin insufficiency and insulin resistance (IR) is associated with increased circulating hIAPP. Remarkably, hIAPP has structural similarity with amyloid beta (Aβ) and can engage in the pathogenesis of T2DM and Alzheimer’s disease (AD). Therefore, the present review aimed to elucidate how hIAPP acts as a link between T2DM and AD. IR, aging and low β cell mass increase expression of hIAPP which binds cell membrane leading to the aberrant release of Ca Human Islet amyloid polypeptide (hIAPP) also called amylin is one of the main secretory products of pancreatic β cells in the islet of Langerhans . hIAPP hhIAPP is co-secreted with insulin from pancreatic β cells, and due to its ability to aggregate into pancreatic islet cells in type 2 diabetes mellitus (T2DM) so-called hIAPP .Notably, hIAPP was initially described in 1901 by weichselbam et al. and Opie et al. as an islet amyloid called islet hyalinization in patients with T2DM . HoweverExpression of hIAPP is not limited to the pancreatic β cells but is also expressed on α cells of the islet . In addiT2DM is an endocrine disorder due to relative insulin insufficiency and IR, characterized by hyperglycemia and cardiometabolic complications . T2DM reDeficiency of IDE which is involved in the degradation of hIAPP induces the development of hyperamylinemia which triggers the development of IR . In vitrNotably, hIAPP unlike rat IAPP is highly subjected to amyloidogenesis by forming amyloid fibril. The amyloidogenic propensity of hIAPP is related to the presence of 20–29 and 8–18 residues which have no proline as in rat IAPP as proline prevents aggregation of hIAPP . Higher In addition, a monoclonal antibody to hIAPP could be an early diagnostic biomarker for T2DM diagnosis . These fTherefore, the oligomerization of hIAPP is controlled by the balance between processing and degradation pathways . GeneticIt has been shown that the incidence and prevalence of T2DM are increased with age due to increase aggregation of hIAPP and the development of pancreatic β cells . Of inteThe underlying causative factors that convert soluble hIAPP oligomers to toxic non-soluble fibrils are largely unknown . Neverth2+ and activation of proteolytic enzyme calpain leading to a series of events causing loss of β cells [.Monomeric and oligomeric hIAPP disrupt cell membrane fluidity by increasing the production of ROS causing membrane leakage and cytotoxicity . Monomer β cells  Fig. 3)2+ and acIn response to oxidative stress and ER stress, chaperon-mediated autophagy is upregulated to prevent cell injury . ChaperoFurthermore, heparan sulfate proteoglycan (HSPG) can increase hIAPP aggregation . PerlecaMoreover, the immune response to the formed hIAPP fibrils triggers the release of pro-inflammatory cytokines mainly IL-1β which increases hIAPP fibrils-induced β cytotoxicity and development of IR and T2DM . In vitrIndeed, zinc prevents protein misfolding and aggregation of hIAPP . Zinc deIn sum, hIAPP-induced dysfunction of pancreatic β cells through induction of oxidative stress and ER stress. Zinc deficiency and exaggerated immune response against hIAPP aggregation trigger more injury of pancreatic β cells.AD is the most common type of dementia characterized by progressive neurodegeneration leading to memory and cognitive deficits . The patT2DM patients have been reported to hold a higher incidence of cognitive decline and AD; T2DM has been intensely associated with an increased risk of developing all types of dementia, including AD . A systeDisturbances in brain insulin signaling mechanisms represent early and progressive abnormalities and could account for the majority of molecular, biochemical, and histopathological lesions in AD . Increasvia a combination of four β-sheet-to-β-sheet interfaces and two packing orientations, focusing on a comparison of different matches of β-sheet layers [Amyloid formation is a hallmark in both AD and T2DM due to the deposition of hIAPP in both pancreatic β islets and the brain . Pancreat layers . Two crot layers . Both Aβt layers , 85. Inft layers . The inft layers . In contt layers .However, there was no correlation between peripheral hIAPP and AD risk in animal and human studies . In the Peripheral hIAPP serum level is not different in AD and controls . TherefoDeposition of hIAPP in both T2DM and AD is due to metabolic dysfunction, microvascular injury, and failure of Aβ clearance . Besides42 level [42 level even in non-diabetic status, hIAPP disrupts neurovascular injury, interacts with Aβ42 and acts as a seed to increase deposition of Aβ plaques [42 by increasing the production of ROS. Likewise, Aβ42 interacts, cross seed and increases the toxic effects of hIAPP [42 increases tau protein hyperphosphorylation through induction of oxidative stress and inflammatory changes causing progressive synaptic loss and synaptic dysfunction [In the normal state, unmodified (soluble) hIAPP crosses BBB and enhances clearance of Aβ4 to prevent the development and progression of AD neuropathology . When un42 level . Both hI42 level . The cor42 level . Interes42 level . In cont42 level , 91. In  plaques . In addiof hIAPP . Co-aggrfunction . IAPP cofunction , 95. Synfunction . Given tfunction , 96. How42 is prevented by hIAPP and hIAPP analogue pramlintide which attenuate the development of AD and other neurodegenerative diseases [A recent study involved postmortem brain tissues of AD patients together with in vitro and experimental studies illustrated that hIAPP promotes tau protein deposition . In addidiseases . Taken tInterestingly, hIAPP undergoes tertiary structural changes and can form aggregates that deposit in the pancreatic islet causing β cells dysfunction even in the prediabetic period . Both ADThe mode of toxicity by hIAPP and Aβ despite is mediated in a receptor-dependent manner. Receptor activity modifying protein 3 (RAMP3) and calcitonin receptors which are highly expressed in the brain act as a receptor for hIAPP to increase Aβ toxicity . In addiInterestingly, HSPG and perlecan which increase hIAPP aggregation  are dysrFurthermore, abnormal immune response to the aggregated hIAPP triggers releases of pro-inflammatory cytokines and expression of inflammatory signaling pathways that augment hIAPP-induced cytotoxicity in both T2DM  and AD . Of noteIn addition, zinc has a protective effect against protein misfolding and aggregation of hIAPP . Zinc deTaken together, oxidative stress, mitochondrial dysfunction, chaperon-mediated autophagy, HSPG, immune response, and zinc homeostasis in T2DM could be the possible mechanisms for induction of the aggregation of hIAPP which increase AD risk.It has been detected that IR is linked with mild cognitive impairment in AD patients compared to controls due to dysfunction of brain insulin signaling . AdminisFurthermore, the thiazolidinediones (TZDs), a family of peroxisome proliferator–activated receptor γ (PPARγ) activators like rosiglitazone and pioglitazone are antidiabetic agents that primarily act by improving peripheral insulin sensitivity . TZDs imAPOE-Ε4-negative or other analysis populations [It has been demonstrated that hIAP formation in vivo is markedly reduced by the effects of rosiglitazone and metformin in hIAPP transgenic male mice fed a moderate-fat diet for 1 year . Rosigliulations . These fDipeptidyl peptidase 4 (DPP4) inhibitors like sitagliptin are widely used and tolerated in the management of T2DM with minimal or no hypoglycemia as an adverse effect , 149. Pr2+ and activation of the proteolytic enzyme leading to series of events causing loss of β cells. hIAPP-induced dysfunction of pancreatic β cells through induction of oxidative stress and ER stress. Zinc deficiency and exaggerated immune response against hIAPP aggregation trigger more injury of pancreatic β cells. Peripheral hIAPP plays a major role in the pathogenesis of AD, and high circulating hIAPP level increase AD risk in T2DM patients. However, there is no hard evidence for the role of brain-derived hIAPP in the pathogenesis of AD. In sum, oxidative stress, mitochondrial dysfunction, chaperon-mediated autophagy, HSPG, immune response and zinc homeostasis in T2DM could be the possible mechanisms for induction the aggregation of hIAPP which increase AD risk. In addition, DPP4 inhibitors and GLP-1 agonists attenuate AD in T2DM by inhibiting the expression and deposition of hIAP. Despite these evidences the underlying mechanism linking pancreatic islet hIAPP in the development of AD need to be elucidated. Therefore, retrospective and prospective studies are recommended in this state.hIAPP is one of the main secretory products of pancreatic β cells in the islet of Langerhans. hIAPP has different physiological functions including inhibiting gastric empty, regulation of satiety and inhibiting the release of insulin and glucagon. T2DM is an endocrine disorder due to relative insulin insufficiency and IR, characterized by hyperglycemia and cardiometabolic complications. Interestingly, hIAPP has structural similarity with Aβ and can involve in the pathogenesis of T2DM and AD. The underlying causative factors that convert soluble hIAPP oligomers to toxic non-soluble fibrils are largely unknown. IR, aging and low β cell mass increases the expression of hIAPP which binds the cell membrane leading to the aberrant release of Ca


V12, provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center.Expression of activated Ras, Ras Fruit flies are widely used in the life and biomedical sciences as models of animal biology. They are small in size and easy to care for in a laboratory, making them ideal for studying how the body works. There are, however, some experiments that are difficult to perform on whole flies and it would be advantageous to use populations of fruit fly cells grown in the laboratory – known as cell cultures – instead.Ras in fruit fly cells helps the cells to survive and grow in cultures, making it simpler to generate new fruit fly cell lines compared with traditional methods. However, the methods used to express activated Ras result in cell lines that can be a mixture of many different types of cell, which limits how useful they are for research.Unlike studies in humans and other mammals, which – for ethical and practical reasons –heavily rely on cell cultures, few studies have used fruit fly cell cultures. Recent work has shown that having an always active version of a gene called Ras to generate a collection of cell lines from specific types of fruit fly cells in the muscle, nervous system, blood and other parts of the body. The experiments show that selectively expressing activated Ras in an individual type of cell enables them to outcompete other cells in culture to generate a cell line consisting only of the cell type of interest.Here, Coleman-Gosser, Hu, Raghuvanshi, Stitzinger et al. aimed to use The new cell lines offer models for experiments that more closely reflect their counterparts in flies. For example, the team were able to recapitulate how fly muscles develop by treating one of the cell lines with a hormone called ecdysone, which triggered the cells to mature into active muscle cells that spontaneously contract and relax.In the future, the new cell lines could be used for various experiments including high throughput genetic screening or testing the effects of new drugs and other compounds. The method used in this work may also be used by other researchers to generate more fruit fly cell lines. The use of cell cultures has been important for studying biological processes that are not easily accessible in whole organisms . A numbeDrosophila melanogaster) cell cultures are among the most widely used invertebrate cell cultures   . The majr (DGRC) . Furtherr (DGRC) . All linr (DGRC) . Thus, tpathways , exploripathways , establipathways , charactpathways , nucleospathways  among mupathways .D. melanogaster cell lines listed in the DGRC catalog were derived from whole embryos and the remainder are from various larval imaginal discs, the larval central nervous system, larval hemocytes, or adult ovaries. The potential of cells from these different sources to differentiate into adult cell types is not known. However, temporal transcriptional profiling of the Ecdysone response of 41 cell lines  we identified lines with neuronal and hemocyte characteristics. Collectively, these cell lines provide in vitro models for five different cell types and are expected to be a valuable resource for high-throughput and biochemical approaches, which rely on large numbers of homogeneous cells.Here, we describe a second-generation version of the Ras method in which Raspression . As we sV12UAS-Ras expression was restricted to glial, tracheal epithelial, and mesodermal cells using lineage-specific Gal4 drivers (V12UAS-Ras (Drosophila Gene Expression Tool (DGET) database (https://www.flyrnai.org/tools/dget/web/), which is the bulk RNAseq data portal at Drosophila RNAi Screening Center (DRSC)  . The TM4r (DRSC) . As exper (DRSC)  and new r (DRSC) . Moreover (DRSC) ; Table 2r (DRSC)  reveals repo-Gal4 driver that recapitulates Repo expression was used to express V12UAS-Ras  line has been passaged more than 50 times. The parental Rbr6 line and three clonal derivatives  have an elongated morphology and stained positive for Repo , which encodes a potassium-dependent sodium/calcium exchanger, is upregulated in all three clones, consistent with the literature  a master regulator of tracheal identity  and is expressed in mesoderm and muscle cells  and control larvae  showed that RasV12 expression disrupted muscle development, suggesting that the prevalent amorphous GFP-positive cells observed in primary cultures were abnormal muscle cells  and twist (twi) (myoblast city (mbo), which encodes an unconventional bipartite GEF with a role in myoblast fusion  ; Table 2t fusion . The capV12UAS-Ras with the pan-neural drivers scratch-Gal4 and elav-Gal4, however none of the primary cultures resulted in continuous cell lines , slowpoke (slo), 5-hydroxytryptamine (serotonin) receptor 1A (5-HT1A), Protein C kinase 53E (Pkc53E), Diuretic hormone 31 Receptor (Dh31-R), and straightjacket (stj). In addition, comparison of the top upregulated genes in these cells to marker genes from scRNAseq data identifies a cell type of neuronal origin as the best match , Hemese (He), eater, u-shaped (ush), Cecropin A2 (CecA2), and Cecropin C (CecC). Comparison of top upregulated genes with scRNAseq data showed that the cells have a strong match to the top marker genes of hemocytes  were diploid, or near diploid , co-expression of the p35 baculovirus survival factor, or growth stimulation by downregulation of tumor suppressors (dsRNA for warts or brat). One line, also harboring a Gal80ts transgene, reached passage 25; however, the line was unstable and in early passages the cells variably lost Repo expression and changed morphologically. The one continuous glial line generated expresses a transgene that targets the tumor suppressor, V12; UAS-bratdsRNAbrat . Given a single success, it is not clear if downregulation of brat contributed to derivation of the line. Moreover, there is no evidence that these genotypic variations enhanced cell line generation with other drivers, as primary cultures expressing RasV12 without modulation or a survival factor produced lines with similar success rates for the btl-Gal4 or 24B/how-Gal4 drivers  will provide good models for investigating assay conditions that promote polarization and 3D cell interactions that could allow the cells to manifest a more complex tissue architecture. In keeping with this possibility, treating these cells with ecdysone to induce differentiation showed cell clumping suggestive of a multicellular structure .RNAseq analysis of cells from the ActGSI-3 cell clone showed a striking similarity to hemocytes, and the cells may be a good model for studying immunity . The celglial cell missing (gcm) is not differentially expressed in the three glial-lineage cell clones and gcm2 is differentially expressed in only two of the three clones. Further, trachealess (trh) is only differentially expressed in one of the three tracheal-lineage cell lines. Similarly, the muscle-specific transcription factors twist (twi), nautilus (nau), snail (sna), and Mef2 show variable expression in the muscle-lineage cell clones. It should also be noted that the expression patterns were determined for undifferentiated cells and expression levels could change after hormone exposure.The most significantly upregulated marker genes in each cell line are significantly enriched for top marker genes from expected cell types based on the single-cell RNAseq data from Fly Cell Atlas in most cases. This indicates the potential value of these cell lines as corresponding in vitro models for studying these cell types. While the cells will prove to be valuable models, it should be noted that even those showing a clear differentiated phenotype exhibit unexpected patterns of gene expression. For example, some cells in the mesodermal clone, 24B5-B8, are positive for HRP  and the Drosophila have only been performed in S2 cells, which have hemocyte-like features. The availability of new cell lines with muscle, glial, and epithelial characteristics will enable screens designed to interrogate biological processes specific to these cell types.The cells will have value for both low- and high-throughput approaches, including genetic or compound screens for which screening in the relevant cell type will result in identifying targets that are more likely to be of physiological relevance. Most of the cells have an attP-flanked cassette , which mDrosophila cell lines; however, the number corresponding to known cell types is low. This is due in part to the lack of a method for generating cell lines from specific tissues. We expect that the method described here, using restricted expression of RasV12, will be a tractable approach for investigators to generate lines of cell types of interest. Single-cell cloning followed by cell characterization (immunohistochemistry and RNAseq) also proved to be a useful method to identify cell-type-specific lines and this approach could identify additional valuable lines in the existing collection at the DGRC. In summary, we show that lineage-restricted Ras expression and cell cloning has produced a set of new cell lines that will be of immediate value for analyses in the five cell types they represent.There are hundreds of Gal4 drivers: 24B/how-Gal4, w[*]; P(w[+mW.hs]=GawB)how[24B] (BL 1767); repo-Gal4, P(GAL4)repo (BL 7415); btl-Gal4, P(GAL4-btl.S)3-2 (BL 78328); Act5C-GeneSwitch-Gal4, P(UAS-GFP.S65T)Myo31DF[T2]; P(Act5C(-FRT)GAL4.Switch.PR)3 (BL 9431). Transgenes: UAS-RasV12 (3), P(w[+mC]=UAS-Ras85D.V12)TL1 (BL 64195); UAS-RasV12 (2), P(w[+mC]=UAS-Ras85D.V12)2 (BL 64196); UAS-RasV12 with RMCE site (3), P(w[+mC]=UAS-Ras85D.V12)TL1, P(w[+mC]=attP.w[+].attP)JB89B (BL 64197); UAS-GFP nuclear, P(UAS-GFP.nls)14 (BL 4775); bratdsRNA, P(y[+t7.7] v[+t1.8]=TRiP.HMS01121)attP2 (BL 34646); UAS-p35 baculovirus death inhibitor, P(w[+mC]=UAS-p35.H)BH1 (BL 5072) and Gal80ts, w[*]; P(w[+mC]=tubP-GAL80[ts])20 (BL 7019).The following fly stocks were used to create primary cell lines: V12 lines. Some RasV12 stocks had additional alleles as noted in g. The supernatant was discarded, and the pellet was resuspended in 3-ml Schneider’s S2 medium and centrifugation step and washing with Schneider’s S2 medium was repeated twice more. The final pellet size was estimated and plated in 1 or more 12.5 cm2 T-flasks with 2–3 ml Schneider’s S2 medium. The number of flasks needed for a given pellet size can also be estimated from the volume of packed embryos with approximately 30 µl of packed embryos being sufficient for one flask.This follows a detailed method, which has additional information , except 2 T-flasks at 25°C in Schneider’s S2 medium and were passaged at between 90% and full confluence  using Effectene Transfection Reagent (QIAGEN). After 24 hr, cells were selected with puromycin at 0.5–2.5 µg/ml for 5 days. After 2–4 weeks, colonies were isolated and expanded. For dilution cloning, cells were seeded into a 96-well plate at a concentration of 0.5–1 cell/well in 100 µl conditioned media  at 1 µg/ml separated by 24 hr in non-supplemented medium.Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min or 3.5% formaldehyde (Sigma) for 30 min at room temperature, and then rinsed twice with 0.1% Tween-20 in phosphate-buffered saline (PBS-T). Cells were permeabilized (0.2% Triton X-100 in PBS) for 10 min at room temperature. Cells were blocked  for 30 min at room temperature and incubated with diluted primary antibodies overnight at 4°C. Cells were washed three times with PBS-T and incubated with diluted secondary antibodies in blocking buffer for 1 hr at room temperature or overnight at 4°C. Cells were washed three times with PBS-T and mounted in VectaShield with DAPI (Vector Laboratories). For the Dcad2 antibody, cells were fixed and processed as described in 5 cells were plated in a 12-well plate. Cells were counted from triplicate wells every 3 days over a 9-day period. Doubling time was calculated using log2 cell numbers (1–2 × 10 numbers .Cells were grown to 50–90% confluence and incubated with 0.05 µg/ml KaryoMAX (Gibco-Thermo Fisher 15212012) for 3–18 hr. Cells were processed for analysis using the method in Cells in a 6-well plate (approximately 70% confluent) were transfected with 0.4 µg of an Actin5C-EGFP plasmid  using Effectene Transfection Reagent (QIAGEN). The fraction of GFP-positive cells was scored after 48 hr.Drosophila Medium (Gibco Cat # 21720001), supplemented with 10% fetal bovine serum (Cytiva Hyclone Cat SH30070.03). For Act5C-GS>Ras attP-GFP-LI-Clone 2, Act5C-GS>Ras attP-GFP-LI-Clone 3, and Act5C-GS>Ras attP-GFP-LB-Clone 6 cultures were grown in the same basal media supplemented with 10 nM of Mifepristone (Thermo Fisher Cat# H11001). Cultures were allowed to grow in T-25 flasks to become confluent before treatment with trypsin (Gibco Cat# 12604013) for 4 min to dislodge the cell monolayer from the growth surface. The cells were resuspended in 4 ml of their respective media and 1 ml of the cell suspension was collected for pelleting, followed by washing in 1× PBS, and then flash-freezing in liquid nitrogen. All cell samples were processed in triplicates.Cell cultures were grown and expanded in their respective media. All cell lines were cultured in Schneiders A260/A280 ratio >1.8 and RIN >7.Total RNA was isolated from the pellets using the TRIzol reagent  as per the manufacturer’s instructions. The isolated total RNA was subjected to further purification using the RNeasy Mini Kit  and the RNA post-cleanup was eluted in RNase-free water. The eluted total RNA was confirmed to have a Upon passing the quality control parameters, Illumina TruSeq libraries were constructed using TruSeq stranded mRNA HT kit . Paired end sequencing was performed on an Illumina NextSeq 500 with a 150-cycle high output kits .https://github.com/alexdobin/STAR; Drosophila genome and featureCounts were used to get gene counts from all samples into a count matrix for downstream analysis. A principal component analysis plot was produced using heatmaply. FPKM values were calculated using fpkm(DEseq2) using gene length output by featureCounts. The reference genome used was FB2022_05, dmel_r6.48 (FlyBase) (https://www.flyrnai.org/tools/dget/web/).Raw data processing was performed using the STAR sequence aligner (FlyBase) . Both raEach sample was compared against all other samples by using DESeq2   to deterDrosophila cell lines  at Indiana University. The lines are available for distribution to the research community.All cell lines described here have been deposited to the  Drosophila. The evidence supporting the claims of the authors is convincing, with rigorous characterization of the cell lines and incorporation of their transcriptomes into Drosophila Gene Expression Tool website for user-friendly access. These lines will be a valuable resource that complements in vivo Drosophila genetics, improving biochemistry and facilitating high-throughput screening.This valuable work describes the establishment and characterization of new cell lines derived from specific tissues of the fruit fly  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Drosophila research" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Erika A Bach as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Claude Desplan as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: David Bilder (Reviewer #2).Thank you for submitting your article "Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. Increase the 'identity' of the cell lines– Provide information and possibly new data to increase the utility of Table 2– Provide information on what expected marker genes are NOT expressed in the new cell lines– Compare the new lines to existing lines, especially the workhorse S2 cells.– Provide information/commentary on growing in new cells line in large numbers or employing them in high-throughput screening.2. Improve user access to genomics data. Downloading and accessing GEO files are not user-friendly. Genomic data should be accessible to the potential users of these cell lines.– Provide tabular genome-wide data for each cell line.– Consider generating a website with these data3. Improve the images and figure legends– Provide higher magnification views to support the conclusions.– Figure 2: show Ecad staining on a non-epithelial cell line with comparable density.– Figure 3D: show more clearly the multinucleate cells.– Provide information on magnification for images.4. Methods– Mention how specific the GAL4 drivers are in embryos .– Provide a detailed description of how the new cell lines were generated .– Provide a detailed description of the culture conditions for the new cell lines.– Provide reagent tables and RRIDs for all reagents5. Improve karyotyping.Reviewer #1 (Recommendations for the authors):1. The authors should review their reference manager because not all of the references are cited in the same way. Most are last name comma first initial but A. Simcox is frequently cited.2. Under the Methods section, the authors should provide a description of how they generated the cell lines .3. Is it possible to put scale bars on the micrographs? If not, could the authors please state the magnification?Reviewer #2 (Recommendations for the authors):1) The critical assignment of the 'identity' of the cell lines would benefit from more detail and explanation. The process is a bit confusing. For instance, in Table 2, why is the dataset Whole body for some and a specific organ for others? In the latter case, why does Btl8 map to the oenocyte sample? On line 182, the authors say that the glial lines did not result in as clear a similarity as other lines, but that is not obvious from the values in Table 2. Is it possible/useful to compare the enrichment scores of Table 2 to those of well-characterized tissue-specific mammalian cell lines and their in vivo equivalents  for perspective?2). Related, the paper would benefit from an explicit critical discussion of the cell lines. For instance, what expected marker genes are NOT expressed? E.g. the tracheal-derived cell lines L8 do not express trh, L7 downregulates trh. In what ways are these not simple immortalized cells of in vivo equivalents -that may be important for people thinking about using them? The authors' simple statements e.g. line 212 do a disservice to the nuances of identity.3) The authors could make clear how these new lines compare to existing lines. For instance, S2 was generally thought to be hemocyte-like, how does it compare to Act GSI-3? What reason would researchers interested in hemocyte biology want to use one over the other? An example of this value is the comparison of transfection efficiency to S2 (line 237).4) Any experience with growing in large numbers, or in high-throughput screening format? Since these are promoted as uses, it would be very helpful to have a perspective from the authors, who include those most experienced with the variety of existing cell lines and their use in these contexts.Other points – not absolutely needed for accceptanceIn general, the figures are rather sparse, there is room for more data such as those suggested above.Any speculation as to why so many of the lines correlate with an adult, rather than embryonic, profiles when the tissue source was embryos?Figure 2: would be nice to show Ecad staining on a non-epithelial cell line with comparable density.Would be nice to mention how specific the GAL4 drivers are in embryosThe culture conditions for the cell lines, which is critical information, are buried in a section about 'RNA extraction'.Figure 3D: would be nice to show more clearly the multinucleate cells.Mef2; elav) -is it possible that the levels of active Ras were too high to be compatible with life and/or immortalization?For the lines where there was a failure to derive :The paper is clear and well-written, but I believe that the work needs to be extended to make it useful.1) The RNAseq characterization is perhaps the most important and global aspect of the utility, because someone interested in a particular gene, could check to see if it was well expressed and in which of the novel (or existing) cell lines. I did not see tabular genome-wide data that would fit this bill. The expression data is basically not presented in the manuscript. The authors are used to presenting data to the community through the DGRC, DRSC, and FlyBase. They need to make these data easy to access. It is hard to judge the GEO submission as it is still listed as private.2) The phase images are not sufficient to understand the cell biology nature of these cell lines. High magnification views that show higher content information on the cells are needed. It is nice to show the hormonally induced differentiation onset using one or a few markers, but for a resource, I would hope for more granularity. Again it is difficult to make choices of cell lines for experiments with this resolution.3) Although I do not recall reading it explicitly, I assume that the chromosome spreads were scored based on chromosome size. This is not very satisfying in the era of chromosome paints and especially DNAseq (e.g. PMID: 25262759).4) The figure legends should indicate what is being detected. "Expressed" is too vague.5) Reagent tables are much easier to read than in-text methods with names and undescribed identifiers. Essential revisions:1. Increase the 'identity' of the cell lines– Provide information and possibly new data to increase the utility of Table 2– Provide information on what expected marker genes are NOT expressed in the new cell lines– Compare the new lines to existing lines, especially the workhorse S2 cells.– Provide information/commentary on growing in new cells line in large numbers or employing them in high-throughput screening.In Table 2, we have added the cell-type based on in situ data. This complements the single cell RNAseq and confirms the cell type identity.We have added a section on transcription factors, not expressed in the new cell lines, which might be expected for that cell type. Also, we note that the RNAseq data is for undifferentiated cells and genes not expressed in the proliferative state may be expressed in differentiated cells. This is one reason why information on what is not expressed may not be definitive.We have included figures that show the morphology of the cells, including S2 cells . These show that none of the new lines are similar to S2, including the hemocyte-like cell line ActGSI-3. While both S2 and ActGSI-3 cells are round and grow in suspension, S2 cells grow individually, whereas ActGSI-3 form large cell rafts.6 in a single well of a 12 well plate. In this experiment S2 cells grew to a confluent density of 6.2 X 106. As all cells, except ActGSI-3, are adherent, the cells are also potentially well suited for multi-well plate assays.In Table 3, we have added data about the confluent density of the cells. These numbers show that the cells can be grown to densities of 1.4-8.1 x 102. Improve user access to genomics data. Downloading and accessing GEO files are not user-friendly. Genomic data should be accessible to the potential users of these cell lines.– Provide tabular genome-wide data for each cell line.– Consider generating a website with these dataWe added a supplementary table (Table S3) with genome-wide expression levels for each gene in each cell line .Drosophila Gene Expression Tool (DGET) database (https://www.flyrnai.org/tools/dget/web/), which is the bulk RNAseq data portal at Drosophila RNAi Screening Center (DRSC). DGET can be used to query the expression levels for the genes of interest as well as search the genes with similar expression pattern for any input gene. The expression dataset is called “Ras cell lines”.The dataset has been imported into the 3. Improve the images and figure legends– Provide higher magnification views to support the conclusions.– Figure 2: show Ecad staining on a non-epithelial cell line with comparable density.– Figure 3D: show more clearly the multinucleate cells.– Provide information on magnification for images.The new Figure 1 shows the cells at high magnification. This was a very useful suggestion because it allows comparison of cell morphologies within and between lineages. For example, the glial cells have a long spindle-like shape, and the epithelial cells are cuboidal.Figure 6 shows Btl3 and S2 cells stained for the epithelial marker E-Cadherin/Shotgun. Btl3 cells expressed Ecad at the cell periphery and S2 cells had low level cytoplasmic expression.New panels in Figure 5 show the multinucleate muscle cells.Scale bars are included in Figures 1 and 8, which show phase images of the cells.4. Methods– Mention how specific the GAL4 drivers are in embryos .– Provide a detailed description of how the new cell lines were generated .– Provide a detailed description of the culture conditions for the new cell lines.– Provide reagent tables and RRIDs for all reagentsMef2-Gal4 and 24B/how-Gal4. Only 24B/how-Gal4 produced continuous cell lines. In a new figure we show that RasV12 expression with Mef2-Gal4 in muscle cells disrupted muscle development and that in primary cultures the RasV12-expressing cells (marked with GFP) failed to attach and grow . We. Do not the know the basis for this; however, expression level is a possibility. Even after successful cell line generation it is important to use a battery of assays to determine lineage characteristics because cells change as they adapt for survival in vitro. This is exemplified by some of the 24B-Gal4 derived lines that failed to differentiate after extended passages.We find that additional factors other than specificity of expression of Gal4 are necessary for successful cell line development. This is illustrated for the muscle lineage where we tested two well characterized drivers—A detailed primary culture procedure is provided in the Methods section.Culture conditions and passaging regimes for the new cell lines are provided in the Methods section. This method also refers to Figure 8, which shows the cells at confluence and will help users determine when cells are ready for sub-culturing (passaging).All reagents and RRIDs (when available) are provided.5. Improve karyotyping.We redid the karyotypes for all the cell lines and include karyograms for each. The quality is greatly improved and equivalent to the standard for the field. The figures showing the karyotypes and the chromosomes ordered in a karyogram appear in supplemental figures for each cell line and the refined karyotypes are summarized in the revised Table 3.