Cell Development Research Articles

IRF8-driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma.

Glioblastoma (GBM) has a highly immunosuppressive tumor immune microenvironment (TIME), largely mediated by myeloid-derived suppressor cells (MDSCs). Here, we utilized a retroviral replicating vector (RRV) to deliver Interferon Regulatory Factor 8 (IRF8), a master regulator of type 1 conventional dendritic cell (cDC1) development, in a syngeneic murine GBM model. We hypothesized that RRV-mediated delivery of IRF8 could "reprogram" intratumoral MDSCs into antigen-presenting cells (APCs) and thereby restore T-cell responses. Effects of RRV-IRF8 on survival and tumor growth kinetics were examined in the SB28 murine GBM model. Immunophenotype was analyzed by flow cytometry and gene expression assays. We assayed functional immunosuppression and antigen presentation by ex vivo T-cell-myeloid co-culture. Intratumoral injection of RRV-IRF8 in mice bearing intracerebral SB28 glioma significantly suppressed the tumor growth and prolonged survival. RRV-IRF8 treated tumors exhibited significant enrichment of cDC1s and CD8+ T-cells. Additionally, myeloid cells derived from RRV-IRF8 tumors showed decreased expression of the immunosuppressive markers Arg1 and IDO1 and demonstrated reduced suppression of naïve T-cell proliferation in ex vivo co-culture, compared to controls. Furthermore, DCs from RRV-IRF8 tumors showed increased antigen presentation compared to those from control tumors. In vivo treatment with azidothymidine (AZT), a viral replication inhibitor, showed that IRF8 transduction in both tumor and non-tumor cells is necessary for survival benefit, associated with a reprogrammed, cDC1- and CD8 T-cell-enriched TIME. Our results indicate that reprogramming of glioma-infiltrating myeloid cells by in vivo expression of IRF8 may reduce immunosuppression and enhance antigen presentation, achieving improved tumor control.

Structural insights into the cooperative nucleosome recognition and chromatin opening by FOXA1 and GATA4

Mouse FOXA1 and GATA4 are prototypes of pioneer factors, initiating liver cell development by binding to the N1 nucleosome in the enhancer of the ALB1 gene. Using cryoelectron microscopy (cryo-EM), we determined the structures of the free N1 nucleosome and its complexes with FOXA1 and GATA4, both individually and in combination. We found that the DNA-binding domains of FOXA1 and GATA4 mainly recognize the linker DNA and an internal site in the nucleosome, respectively, whereas their intrinsically disordered regions interact with the acidic patch on histone H2A-H2B. FOXA1 efficiently enhances GATA4 binding by repositioning the N1 nucleosome. Invivo DNA editing and bioinformatics analyses suggest that the co-binding mode of FOXA1 and GATA4 plays important roles in regulating genes involved in liver cell functions. Our results reveal the mechanism whereby FOXA1 and GATA4 cooperatively bind to the nucleosome through nucleosome repositioning, opening chromatin by bending linker DNA and obstructing nucleosome packing.

Tumor-secreted extracellular vesicles counteract therapy response by triggering inflammatory mesenchymal stem cell development.

Therapy resistance is a major clinical hurdle in bone cancer treatment and seems to be largely driven by poorly understood microenvironmental factors. Recent evidence suggests a critical role for a unique subpopulation of mesenchymal stem cells with inflammatory features (iMSCs), though their origin and function remained unexplored. We demonstrate that cancer-secreted extracellular vesicles (EVs) trigger the development of iMSCs, which hinder therapy response in vivo, and set out to identify strategies to counteract their function. The role of iMSCs in therapy resistance was evaluated in an orthotopic xenograft mouse model of osteosarcoma. EV-induced alterations of the MSC transcriptome were analyzed and compared with scRNA-seq data of osteosarcoma and multiple myeloma patient biopsies. Functional assays identified EV components driving iMSC development. We assessed the efficacy of clinical drugs in blocking iMSC-induced resistance in vivo. We found that iMSCs are induced by interaction with cancer EVs and completely abrogate the antimetastatic effect of TGFb signaling inhibition. Importantly, EV-induced iMSCs faithfully recapitulate the inflammatory single-cell RNA signature of stromal cells enriched in multiple myeloma and osteosarcoma patient biopsies. Mechanistically, cancer EVs act through two distinct mechanisms. EV-associated TGFb induces IL6 production, while the EV-RNA cargo enhances TLR3-mediated chemokine production. We reveal that simultaneous blockade of downstream EV-activated pathways with ladarixin and tocilizumab disrupts metastasis formation and overcomes iMSC-induced resistance. Our observations establish iMSCs as major contributors to drug resistance, reveal EVs as physiological triggers of iMSC development and highlight a promising combination strategy to improve therapy response in bone cancer patients.

Transcriptome Profiling of Gene Expression in Atlantic Salmon (Salmo salar) at Early Stage of Development.

For Atlantic salmon development, the most critical phase is the early development stage from egg to fry through alevin. However, the studies investigating the early development of Atlantic salmon based on RNA-seq are scarce and focus only on one stage of development. Therefore, using the RNA-seq technology, the assessment of different gene expressions of various early development stages (egg, alevin, and fry) was performed on a global scale. Over 22GB of clean data was generated from 9 libraries with three replicates for each stage with over 90% mapping efficiency. A total of 5534 genes were differentially expressed, among which 19, 606, and 826 genes were specifically expressed in each stage, respectively. The transcriptome analysis showed that the number of differentially expressed genes (DEGs) increased as the Atlantic salmon progressed in development from egg to fry stage. In addition, gene ontology enrichment demonstrated that egg and alevin stages are characterized by upregulation of genes involved in spinal cord development, neuron projection morphogenesis, axonogenesis, and cytoplasmic translation. At the fry stage, upregulated genes were enriched in the muscle development process (muscle cell development, striated muscle cell differentiation, and muscle tissue development), immune system (defense response and canonical NF-kappaB signal transduction), as well as epidermis development. These results suggest that the early development of Atlantic salmon is characterized by a dynamic shift in gene expression and DEGs between different stages, which provided a solid foundation for the investigation of Atlantic salmon development.

The Pivotal Role of the Key Angiogenic Factors in the Development of Endometrioid Pathologies of the Uterus and Ovary.

A characteristic feature of uterine pathologies is a specific change in cell metabolism, which predominantly manifests as a shift in the need for nutrients, thereby directing cells to engage in different angiogenic marker activities. Angiogenesis is one of the main signals supporting the survival and development of cells and tissues not only under physiological conditions. Therefore, it is necessary that we understand pathological hyperactivation in all uterine diseases, from endometriosis through ovarian endometrioid adenocarcinoma to malignant transformed cells of the uterine epithelium and body. This work presents the gene expression results of selected angiogenesis targets (VEGF-A, TGF-β1, ANG1/2, and HIF-1α), cell migration, and cell-cell interaction determined in vitro. Our results suggest that angiogenesis varies in the tested pathological conditions (ectopic endometriosis-12Z; ovarian endometrioid adenocarcinoma-A2780; tumors-SK-UT-1 and RL-95-2) compared to physiological angiogenesis (HME1). The differential expression of angiogenic factors may contribute (or is a contributing factor) to the observed differences to acknowledge an inherent variability in angiogenesis among cell lines. Determining the genomic phenomena responsible for processes associated with inadequate angiogenesis in the pelvic region could help us to develop individual treatment strategies and explain resistance to treatment.

Biologically Relevant Laminin-511 Moderates the Derivation and Proliferation of Human Lens Epithelial Stem/Progenitor-Like Cells.

The role of specific extracellular matrix (ECM) molecules in lens cell development and regeneration is poorly understood, as appropriate cellular models are lacking. Here, a laminin-based lens cell in vitro induction system was developed to study the role of laminin in human lens epithelial stem/progenitor cell (LES/PC) development. The human embryonic stem cell-based lens induction system followed a three-stage protocol. The expression profile of laminins during lens induction was screened, and laminin-511 (LN511) was tested as a candidate substitute. LN511 induction system cellular and molecular features, including induction efficiency, transcription factor expression related to different lens development stages, ECM alterations, and Hippo/YAP signaling, were evaluated. LAMA5, LAMB1, and LAMC1 were highly expressed around the time of LES/PC derivation. We chose LN511 (product of LAMA5, LAMB1, and LAMC1) and found that it considerably enhanced lens cell induction efficiency, compared to that in Matrigel-coated culture, as more and larger lentoid bodies were detected. Notably, LES/PC induction efficiency improved by promoting lens specification-related transcription factor expression and cell proliferation. Transcriptome analysis revealed that compared to those with Matrigel, ECM accumulation and cell adhesion were downregulated in the LN511 system. Hippo/YAP signaling was hypoactive during LES/P-like cell generation, and small molecule inhibitors of YAP/TAZ activity upregulated LES/PC marker expression and promoted the efficiency of LES/P-like cell derivation. The laminin isoform LN511 is a reliable substitute for the LES/P-like cell induction system, and LN511-YAP acted as efficient modulators of LES/PC derivation; this contributes to knowledge of the role of the ECM in human lens development.

Targeting p97-Npl4 interaction inhibits tumor Treg cell development to enhance tumor immunity.

Targeting tumor-infiltrating regulatory T (TI-Treg) cells is a potential strategy for cancer therapy. The ATPase p97 in complex with cofactors (such as Npl4) has been investigated as an antitumor drug target; however, it is unclear whether p97 has a function in immune cells or immunotherapy. Here we show that thonzonium bromide is an inhibitor of the interaction of p97 and Npl4 and that this p97-Npl4 complex has a critical function in TI-Treg cells. Thonzonium bromide boosts antitumor immunity without affecting peripheral Treg cell homeostasis. The p97-Npl4 complex bridges Stat3 with E3 ligases PDLIM2 and PDLIM5, thereby promoting Stat3 degradation and enabling TI-Treg cell development. Collectively, this work shows an important role for the p97-Npl4 complex in controlling Treg-TH17 cell balance in tumors and identifies possible targets for immunotherapy.

Textile-Based Membraneless Microfluidic Double-Inlet Hybrid Microbial-Enzymatic Biofuel Cell.

This study reports the development of a textile-based colaminar flow hybrid microbial-enzymatic biofuel cell. Shewanella MR-1 was used as a biocatalyst on the anode, and bienzymatic system catalysts based on glucose oxidase and horseradish peroxidase were applied on an air-breathing cathode to address the overpotential loss in a body-friendly way. A single-layer Y-shaped channel configuration with a double-inlet was adopted. Microchannels of biofuel cells were patterned by silk screen printing with Ecoflex to maintain the flexibility of textile substrates without harm to the human body. The electrodes were fabricated with poly(3,4-ethylenedioxythiophene):polystyrene sulfonate and a mixture of multiwalled carbon nanotubes and single-walled carbon nanotubes by screen printing. The effects of electrode materials, catalyst type, catalyst concentration, and glucose concentration in the catholyte were investigated to optimize the fuel cell performance. The peak power density (44.9 μW cm-2) and maximum current density (388.9 μA cm-2) of the optimized hybrid biofuel cell were better than those of previously reported textile- or paper-substrate microscale single microbial fuel cells. The developed biofuel cell will be a useful platform as a microscale power source that is harmless to the environment and living organisms.

Abnormal amino acid synthesis and glutathione metabolism may affect PCOS blastocyst development: an examination of in vitro mouse blastocysts model utilizing RNA-sequencing

BackgroundExtensive research has been conducted on embryonic developmental disorders linked to Polycystic Ovary Syndrome (PCOS), a pathological condition that affects 5−10% of women and is characterized by irregularities in the menstrual cycle and infertility. By employing RNA sequencing (RNA-seq), we performed an in-depth investigation of PCOS-related changes in gene expression patterns at the mouse blastocyst stage.MethodsThe zygotes of female B6D2 mice were obtained and then differentiated into blastocysts in K + Simplex Optimised Medium (KSOM) cultures containing exo-NC (negative control for exosomes) or exo-LIPE-AS1 (a novel exosomal marker of PCOS). Subsequently, blastocysts were collected for RNA-seq. The bioinformatics was performed to analyze and compare the differences of gene expression profile between blastocysts of control and PCOS group.ResultsThere were 1150 differentially expressed genes (DEGs) between the two groups of mouse blastocysts; 243 genes were upregulated and 907 downregulated in the blastocysts of the exo-LIPE-AS1 group compared to those of the exo-NC group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the genes involved in amino acid synthesis and glutathione metabolic pathways were down-regulated in exo-LIPE-AS1 group.ConclusionThis study has revealed that blastocyst developmental retardation may be associated with the downregulation of amino acid synthesis and glutathione metabolism, which may affect energy metabolism, biosynthesis, cellular osmotic pressure, antioxidant synthesis, ROS clearance or mitochondrial function, and ultimately cause blastocyst cell development abnormalities. Our research offers encouraging data on the mechanisms underlying aberrant embryonic development in patients with PCOS as well as potential treatment strategies.

Ki67 deficiency impedes chromatin accessibility and BCR gene rearrangement.

The proliferation marker Ki67 has been attributed critical functions in maintaining mitotic chromosome morphology and heterochromatin organization during the cell cycle, indicating a potential role in developmental processes requiring rigid cell-cycle control. Here, we discovered that despite normal fecundity and organogenesis, germline deficiency in Ki67 resulted in substantial defects specifically in peripheral B and T lymphocytes. This was not due to impaired cell proliferation but rather to early lymphopoiesis at specific stages where antigen-receptor gene rearrangements occurred. We identified that Ki67 was required for normal global chromatin accessibility involving regulatory regions of genes critical for checkpoint stages in B cell lymphopoiesis. In line with this, mRNA expression of Rag1 was diminished and gene rearrangement was less efficient in the absence of Ki67. Transgenes encoding productively rearranged immunoglobulin heavy and light chains complemented Ki67 deficiency, completely rescuing early B cell development. Collectively, these results identify a unique contribution from Ki67 to somatic antigen-receptor gene rearrangement during lymphopoiesis.

Supramolecular Cucurbit[5]uril Modulates the Buried SnO2/Perovskite Interface for Efficient and Stable Perovskite Solar Cells

AbstractReducing non‐radiative recombination caused by defects at buried interfaces is crucial to the development of efficient and stable perovskite solar cells (PSCs). Herein, supramolecular cucurbit[5]uril (CB[5]) is introduced into the SnO2 layer, where it engages in host–guest interactions to suppress oxygen vacancies in SnO2, prevent particle aggregation, and enhance the electron mobility of SnO2. By serving as a bridging agent at the buried interface between SnO2 and the perovskite layer, CB[5] reduces the defect density and improves the carrier extraction efficiency. It also reduces the surface energy of the SnO2 substrate, facilitates the formation of large grains in the perovskite film, alleviates residual lattice stresses, and enhances the film quality. Consequently, the PSC with CB[5] shows a champion power conversion efficiency of 24.83%. Moreover, an unencapsulated device incorporating CB[5] retains more than 87% of its initial PCE under continuous illumination at the maximum power point tracking for 1000 h. This study pioneers the utilization of cucurbiturils in PSCs and provides insights into how supramolecular compounds can regulate buried interfaces.

Elucidation of mode of anticancerous activity of metal guanidinobenzimidazoles: A computational and experimental screening

DNA‐binding agents often exhibits dual behavior of simultaneous binding with protein associated with cancerous cell development pathways. This simultaneous protein binding if contributed to malfunctioning of pro‐apoptotic proteins will reduce anticancerous potential of drugs. The elucidation of binding target for anticancerous agent can give an insight into the cancer cell apoptotic pathway. Therefore, anticancerous Zn(II) and Ni(II) complexes of 2‐guanidinobenzimidazole (2GBz) were subjected to DNA‐binding mode assay along with protein‐binding interference assay. The stoichiometry Zn‐2GBz and Ni‐2GBz were determined through density functional theory (DFT) and UV–Vis spectroscopy, followed by structural verification by X‐ray crystallography. The 2GBz and Ni‐2GBz were found to bind with grooves of DNA while groove binding of Zn‐2GBz induced unwinding of DNA through interactive pi‐stacking. Binding constant revealed the M‐2GBz to be a strong binder of DNA molecule with the effect of enhanced cell killing potential of M‐2GBz against MCF‐7 cell lines. Protein‐binding assay revealed that despite of significant interaction of M‐2GBz with chick serum albumin, DNA binding was not altered by simultaneous protein binding. The most potent Zn‐2GBz was found to be sphingosine 1 kinase 2 (SPhK2) which can be a cause of enhanced cancer cell apoptosis with lesser normal cell apoptosis than standard fluorouracil in MCF‐7 cell lines. Co‐administered Zn‐2GBz increased cancer cell sensitivity towards fluorouracil as potent anticancerous agent.

Interaction between microRNA-195 and HuR regulates Paneth cell function in the intestinal epithelium by altering SOX9 translation.

Paneth cells at the bottom of small intestinal crypts secrete antimicrobial peptides, enzymes, and growth factors and contribute to pathogen clearance and maintenance of the stem cell niche. Loss of Paneth cells and their dysfunction occur commonly in various pathologies, but the mechanism underlying the control of Paneth cell function remains largely unknown. Here we identified microRNA-195 (miR-195) as a repressor of Paneth cell development and activity by altering SOX9 translation via interaction with RNA-binding protein HuR. Tissue-specific transgenic expression of miR-195 (miR195-Tg) in the intestinal epithelium decreased the levels of mucosal SOX9 and reduced the numbers of lysozyme-positive (Paneth) cells in mice. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restored Paneth cell development ex vivo. miR-195 did not bind to Sox9 mRNA but it directly interacted with HuR and prevented HuR binding to Sox9 mRNA, thus inhibiting SOX9 translation. Intestinal mucosa from mice that harbored both Sox9 transgene and ablation of the HuR locus exhibited lower levels of SOX9 protein and Paneth cell numbers than those observed in miR-195-Tg mice. Inhibition of miR-195 activity by its specific antagomir improved Paneth cell function in HuR-deficient intestinal organoids. These results indicate that interaction of miR-195 with HuR regulates Paneth cell function by altering SOX9 translation in the small intestinal epithelium.

Hansenula polymorpha methanol metabolism genes enhance recombinant protein production in Komagataella phaffi

Recombinant protein production in Komagataella phaffi (K. phaffi), a widely utilized host organism, can be optimized by enhancing the metabolic flux in the central carbon metabolism pathways. The methanol utilization pathway (MUT) during methanol-based growth plays a crucial role in providing precursors and energy for cell growth and development. This study investigated the impact of boosting the methanol dissimilation pathway, a branch of MUT that plays a vital role in detoxifying formaldehyde and providing energy in the form of NADH, in K. phaffi. This was achieved by integrating two orthologous genes from Hansenula polymorpha into the K. phaffi genome: formaldehyde dehydrogenase (HpFLD) and formate dehydrogenase (HpFMDH). The HpFLD and HpFMDH genes were isolated from the Hansenula polymorpha genome and inserted under the regulation of the pAOX1 promoter in the genome of recombinant K. phaffi that already contained a single copy of model protein genes (eGFP or EGII). The expression levels of these model proteins were assessed through protein activity assays and gene expression analysis. The findings revealed that while both orthologous genes positively influenced model protein production, HpFMDH exhibited a more pronounced upregulation in expression compared to HpFLD. Co-expression of both orthologous genes demonstrated synergistic effects, resulting in approximately a twofold increase in the levels of the model proteins detected. This study provides valuable insights into enhancing the production capacity of recombinant proteins in K. phaffi.Graphical abstract

Temperature influences immune cell development and body length in purple sea urchin larvae

Anthropogenic climate change has increased the frequency and intensity of marine heatwaves that may broadly impact the health of marine invertebrates. Rising ocean temperatures lead to increases in disease prevalence in marine organisms; it is therefore critical to understand how marine heatwaves impact immune system development. The purple sea urchin (Strongylocentrotus purpuratus) is an ecologically important, broadcast-spawning, omnivore that primarily inhabits kelp forests in the northeastern Pacific Ocean. The S. purpuratus life cycle includes a relatively long-lived (∼2 months) planktotrophic larval stage. Larvae have a well-characterized cellular immune system that is mediated, in part, by a subset of mesenchymal cells known as pigment cells. To assess the role of environmental temperature on the development of larval immune cells, embryos were generated from adult sea urchins conditioned at 14 ˚C. Embryos were then cultured in either ambient (14 ˚C) or elevated (18 ˚C) seawater. Results indicate that larvae raised in an elevated temperature were slightly larger and had more pigment cells than those raised at ambient temperature. Further, the larval phenotypes varied significantly among genetic crosses, which highlights the importance of genotype in structuring how the immune system develops in the context of the environment. Overall, these results indicate that larvae are phenotypically plastic in modulating their immune cells and body length in response to adverse developmental conditions.

ACAP3 negatively regulated by HDAC2 inhibits the malignant development of papillary thyroid carcinoma cells

ArfGAP with coiled-coil, ankyrin repeat and PH domains 3 (ACAP3) level has been confirmed to be downregulated in papillary thyroid carcinoma (PTC). Histone deacetylase inhibitors (HDACIs) have therapeutic effects on PTC. Accordingly, this study probed into the potential relation of HDACIs and ACAP3 in PTC. Expressions of ACAP3 and histone deacetylase 2 (HDAC2) in PTC were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between HDAC2 and ACAP3 was predicted by Pearson analysis. Cell functional assays (cell counting kit-8, transwell, wound healing and flow cytometry assays) and rescue assay were carried out to determine the effects of HDAC2/ACAP3 axis on biological behaviors of PTC cells. Expressions of apoptosis-, epithelial-mesenchymal transition-, Protein Kinase B (AKT)-, and P53-related proteins were measured by Western blot. ACAP3 level was downregulated in PTC tissues and cells. ACAP3 overexpression (oe-ACAP3) suppressed viability, proliferation, migration and invasion of PTC cells, facilitated apoptosis, downregulated the expressions of Protein Kinase B (Bcl-2) and N-cadherin, upregulated the expressions of Bcl-2 associated protein X (Bax) and E-cadherin, diminished the p-AKT/AKT ratio and elevated the p-p53/p53 ratio; however, ACAP3 silencing or HDAC2 overexpression (oe-HDAC2) did the opposite. HDAC2 negatively correlated with ACAP3. The tumor-suppressing effect of oe-ACAP3 in PTC was reversed by oe-HDAC2. Collectively, ACAP3 negatively regulated by HDAC2 suppresses the proliferation and metastasis while facilitating apoptosis of PTC cells.

Design, synthesis and biological evaluation of novel cyano-cinnamate derivatives as mitochondrial pyruvate carrier inhibitors

Mitochondrial pyruvate carrier (MPC) inhibitors promote the development of hair follicle stem cells without affecting normal cells, which is promising for the treatment of hair loss. Herein, a series of cyano-cinnamate derivatives of UK-5099 were designed and synthesized. All these new compounds have been tested for their ability to promote cellular lactate production in vitro. Compound 4i (LA content:0.322 μmol/106cell) showed better cellular lactate production activity than UK-5099 (LA content:0.185 μmol/106cell). Further compound 4i was also tested on shaved mice by topical treatment and promoted obvious hair growth on mice.

Synthesis and Processing Strategy for High-Bandgap PbS Quantum Dots: A Promising Candidate for Harvesting High-Energy Photons in Solar Cells.

The wide tunability of the energy bandgap of colloidal lead sulfide (PbS) quantum dots (QDs) has uniquely positioned them for the development of single junction and tandem solar cells. While there have been substantial advancements in moderate and narrow bandgap PbS QDs-ideal for single junction solar cells and the bottom cell in tandem solar cells, respectively; progress has been limited in high-bandgap PbS QDs that are ideally suited for the formation of the top cell in tandem solar cells. The development of appropriate high bandgap PbS QDs would be a major advancement toward realizing efficient all-QD tandem solar cells utilizing different sizes of PbS QDs. Here, we report a comprehensive approach encompassing synthetic strategy, ligand engineering, and hole transport layer (HTL) modification to implement high-bandgap PbS QDs into solar cell devices. We achieved a greater degree of size homogeneity in high-bandgap PbS QDs through the use of a growth retarding agent and a partial passivation strategy. By adjusting the ligand polarity, we successfully grow HTL over the QD film to fabricate solar cells. With the aid of an interface modifying layer, we incorporated an organic HTL for the realization of high-performance solar cells. These solar cells exhibited an impressive open-circuit voltage of 0.824 V and a power conversion efficiency of 10.7%, marking a 360% improvement over previous results.

Control of epigenomic landscape and development of fetal male germ cells through L-serine metabolism

Control of epigenomic landscape and development of fetal male germ cells through L-serine metabolism

Human induced pluripotent stem cells for live cell cycle monitoring and endogenous gene activation

The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.