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Items 2053 to 2064 of 13914 total
- Reference(Sep 2024) Heliyon 10 18
Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression
Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 ?g/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin. Highlights•TRIB3 boosts erythroid cell maturation.•Key insights into erythropoiesis from hESCs.•Enhanced ubiquitin-proteasome system and downstream mitophagy in erythroid differentiation.Catalog #: Product Name: 05872 ¸é±ð³¢±ð³§¸éâ„¢ 100-0276 mTeSRâ„¢ Plus Catalog #: 05872 Product Name: ¸é±ð³¢±ð³§¸éâ„¢ Catalog #: 100-0276 Product Name: mTeSRâ„¢ Plus Reference(Nov 2024) PLOS ONE 19 11Generation of isogenic models of Angelman syndrome and Prader-Willi syndrome in CRISPR/Cas9-engineered human embryonic stem cells
Angelman syndrome (AS) and Prader-Willi syndrome (PWS), two distinct neurodevelopmental disorders, result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele, respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless, gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences, reprogramming methods, and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here, we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally, these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS.Catalog #: Product Name: 100-0276 mTeSRâ„¢ Plus Catalog #: 100-0276 Product Name: mTeSRâ„¢ Plus Reference(Jun 2025) Nucleic Acids Research 53 11High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes
AbstractConcentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both messenger RNA (mRNA) and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using hybridization chain reaction Flow-FISH (HCR Flow-FISH) for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to define the transcription start and splice sites of common synthetic promoters and independently vary the defined promoter and 5? UTR sequences in HCR Flow-FISH. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for building more robust transgenic systems. Graphical Abstract Graphical AbstractCatalog #: Product Name: 05872 ¸é±ð³¢±ð³§¸éâ„¢ 100-0276 mTeSRâ„¢ Plus Catalog #: 05872 Product Name: ¸é±ð³¢±ð³§¸éâ„¢ Catalog #: 100-0276 Product Name: mTeSRâ„¢ Plus Reference(Sep 2024) EMBO Reports 25 10An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes
Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. Synopsis This study presents human pluripotent stem cell-derived hepatocyte-like cells (HLCs) as a culture system that expands the repertoire of research tools for studying hepatitis B and D viruses (HBV/HDV) and identifies CD63 as a potential HDV co-entry factor. Co-infection with HBV or virus-mediated delivery of HBV surface proteins enables HDV to complete its life cycle in HLCs.Extracellular HDV spread in HLCs enables the evaluation of anti-HDV therapies.HDV permissiveness along HLC differentiation reveals CD63 as a novel co-factor of HDV cell entry. This study presents human pluripotent stem cell-derived hepatocyte-like cells (HLCs) as a culture system that expands the repertoire of research tools for studying hepatitis B and D viruses (HBV/HDV) and identifies CD63 as a potential HDV co-entry factor.Catalog #: Product Name: 05872 ¸é±ð³¢±ð³§¸éâ„¢ 85850 ³¾°Õ±ð³§¸éâ„¢1 05110 STEMdiffâ„¢ Definitive Endoderm Kit Catalog #: 05872 Product Name: ¸é±ð³¢±ð³§¸éâ„¢ Catalog #: 85850 Product Name: ³¾°Õ±ð³§¸éâ„¢1 Catalog #: 05110 Product Name: STEMdiffâ„¢ Definitive Endoderm Kit Reference(Apr 2025) Scientific Reports 15Topological data analysis of pattern formation of human induced pluripotent stem cell colonies
Understanding the multicellular organization of stem cells is vital for determining the mechanisms that coordinate cell fate decision-making during differentiation; these mechanisms range from neighbor-to-neighbor communication to tissue-level biochemical gradients. Current methods for quantifying multicellular patterning tend to capture the spatial properties of cell colonies at a fixed scale and typically rely on human annotation. We present a computational pipeline that utilizes topological data analysis to generate quantitative, multiscale descriptors which capture the shape of data extracted from 2D multichannel microscopy images. By applying our pipeline to certain stem cell colonies, we detected subtle differences in patterning that reflect distinct spatial organization associated with loss of pluripotency. These results yield insight into putative directed cellular organization and morphogen-mediated, neighbor-to-neighbor signaling. Because of its broad applicability to immunofluorescence microscopy images, our pipeline is well-positioned to serve as a general-purpose tool for the quantitative study of multicellular pattern formation.Catalog #: Product Name: 100-0276 mTeSRâ„¢ Plus Catalog #: 100-0276 Product Name: mTeSRâ„¢ Plus Reference(Aug 2024) Nature Communications 15Loss of symmetric cell division of apical neural progenitors drives
Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families. Developmental and epileptic encephalopathies are devastating neurological disorders. Here, the authors establish a cohort of patients with variants in the gene DENND5A and use human stem cells to discover a disease mechanism involving altered cell division.Catalog #: Product Name: 05872 ¸é±ð³¢±ð³§¸éâ„¢ 85850 ³¾°Õ±ð³§¸éâ„¢1 08581 STEMdiffâ„¢ SMADi Neural Induction Kit 05833 STEMdiffâ„¢ Neural Progenitor Medium 05990 °Õ±ð³§¸éâ„¢-·¡8â„¢ Catalog #: 05872 Product Name: ¸é±ð³¢±ð³§¸éâ„¢ Catalog #: 85850 Product Name: ³¾°Õ±ð³§¸éâ„¢1 Catalog #: 08581 Product Name: STEMdiffâ„¢ SMADi Neural Induction Kit Catalog #: 05833 Product Name: STEMdiffâ„¢ Neural Progenitor Medium Catalog #: 05990 Product Name: °Õ±ð³§¸éâ„¢-·¡8â„¢ Safety Data SheetCatalog #: Product Name: 100-0720 TeSRâ„¢-AOF 3D Catalog #: 100-0720 Product Name: TeSRâ„¢-AOF 3D Reference(Mar 2024) Archives of Toxicology 98 6Effects of the nerve agent VX on hiPSC-derived motor neurons
Poisoning with the organophosphorus nerve agent VX can be life-threatening due to limitations of the standard therapy with atropine and oximes. To date, the underlying pathomechanism of VX affecting the neuromuscular junction has not been fully elucidated structurally. Results of recent studies investigating the effects of VX were obtained from cells of animal origin or immortalized cell lines limiting their translation to humans. To overcome this limitation, motor neurons (MN) of this study were differentiated from in-house feeder- and integration-free-derived human-induced pluripotent stem cells (hiPSC) by application of standardized and antibiotic-free differentiation media with the aim to mimic human embryogenesis as closely as possible. For testing VX sensitivity, MN were initially exposed once to 400 µM, 600 µM, 800 µM, or 1000 µM VX and cultured for 5 days followed by analysis of changes in viability and neurite outgrowth as well as at the gene and protein level using µLC-ESI MS/HR MS, XTT, IncuCyte, qRT-PCR, and Western Blot. For the first time, VX was shown to trigger neuronal cell death and decline in neurite outgrowth in hiPSC-derived MN in a time- and concentration-dependent manner involving the activation of the intrinsic as well as the extrinsic pathway of apoptosis. Consistent with this, MN morphology and neurite network were altered time and concentration-dependently. Thus, MN represent a valuable tool for further investigation of the pathomechanism after VX exposure. These findings might set the course for the development of a promising human neuromuscular test model and patient-specific therapies in the future.Catalog #: Product Name: 85850 ³¾°Õ±ð³§¸éâ„¢1 08581 STEMdiffâ„¢ SMADi Neural Induction Kit 05832 STEMdiffâ„¢ Neural Rosette Selection Reagent 05833 STEMdiffâ„¢ Neural Progenitor Medium Catalog #: 85850 Product Name: ³¾°Õ±ð³§¸éâ„¢1 Catalog #: 08581 Product Name: STEMdiffâ„¢ SMADi Neural Induction Kit Catalog #: 05832 Product Name: STEMdiffâ„¢ Neural Rosette Selection Reagent Catalog #: 05833 Product Name: STEMdiffâ„¢ Neural Progenitor Medium Safety Data SheetCatalog #: Product Name: 100-0720 TeSRâ„¢-AOF 3D Catalog #: 100-0720 Product Name: TeSRâ„¢-AOF 3D Reference(Feb 2024) Genome Biology 25 12HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells
Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03187-w.Catalog #: Product Name: 100-0276 mTeSRâ„¢ Plus Catalog #: 100-0276 Product Name: mTeSRâ„¢ Plus Safety Data SheetCatalog #: Product Name: 100-0720 TeSRâ„¢-AOF 3D Catalog #: 100-0720 Product Name: TeSRâ„¢-AOF 3D Reference(Sep 2024) Scientific Reports 14Establishment of human pluripotent stem cell-derived cortical neurosphere model to study pathomechanisms and chemical toxicity in Kleefstra syndrome
In the present study, we aimed to establish and characterize a mature cortical spheroid model system for Kleefstra syndrome (KS) using patient-derived iPSC. We identified key differences in the growth behavior of KS spheroids determined by reduced proliferation marked by low Ki67 and high E-cadherin expression. Conversely, in the spheroid-based neurite outgrowth assay KS outperformed the control neurite outgrowth due to higher BDNF expression. KS spheroids were highly enriched in VGLUT1/2-expressing glutamatergic and ChAT-expressing cholinergic neurons, while TH-positive catecholamine neurons were significantly underrepresented. Furthermore, high NMDAR1 expression was also detected in the KS spheroid, similarly to other patients-derived neuronal cultures, denoting high NMDAR1 expression as a general, KS-specific marker. Control and KS neuronal progenitors and neurospheres were exposed to different toxicants (paraquat, rotenone, bardoxolone, and doxorubicin), and dose-response curves were assessed after acute exposure. Differentiation stage and compound-specific differences were detected with KS neurospheres being the most sensitive to paraquat. Altogether this study describes a robust 3D model system expressing the disease-specific markers and recapitulating the characteristic pathophysiological traits. This platform is suitable for testing developing brain-adverse environmental effects interactions, drug development, and screening towards individual therapeutic strategies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-72791-4.Catalog #: Product Name: 85850 ³¾°Õ±ð³§¸éâ„¢1 Catalog #: 85850 Product Name: ³¾°Õ±ð³§¸éâ„¢1 Items 2053 to 2064 of 13914 total
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