Induced Cell Line Research Articles

Generation of two isogenic control lines by correcting the BAG3 P209L mutation of human induced pluripotent stem cell (hiPSC) lines from patients with myofibrillar myopathy-6.

BAG3 plays a key role in proteostasis as a central component of the chaperone-assisted selective autophagy (CASA) complex. A point mutation (p.P209L; c.626C>T) in the BAG3 gene causes severe myofibrillar myopathy-6 (MFM6), restrictive cardiomyopathy and polyneuropathy leading to muscle weakness and heart failure. Establishing suitable controls for patient-derived BAG3P209L/WT-induced pluripotent stem cells (iPSCs), two isogenic controls were generated by correcting the point mutation c.626C>T in iPSCs from two MFM6-patients. We performed quality control of these lines by differentiation into the three germ layers and pluripotency tests. These isogenic hiPSC-control lines allow the correct analysis of MFM6 using corresponding patient-specific iPSCs.

Generation of an isogenic series of genome-edited hiPSC lines with the BAG3P209L-mutation for modeling myofibrillar myopathy 6.

BAG3 contributes to the maintenance of proteostasis through chaperone-assisted selective autophagy. This function is impaired by a single amino acid exchange (P209L) in the protein, which causes myofibrillar myopathy-6 (MFM6). This disease manifests as severe skeletal muscle weakness, neuropathy and restrictive cardiomyopathy. We generated an isogenic series of heterozygous and homozygous genome-edited human induced pluripotent stem cell (hiPSC) lines with the BAG3P209L-mutation and its control. For quality control, we tested the pluripotency of these hiPSC lines and their ability to differentiate into the three germ layers. Generation of these cell lines enables the analysis of cellular pathomechanisms of BAG3P209L-related MFM6.

Generation of human induced pluripotent stem cell lines (iPSC) from adipose-derived mesenchymal stromal cells from two patients with systemic sclerosis

Systemic sclerosis (SSc) is a rare and complex connective tissue disease associated with high morbidity and mortality. SSc is characterized by ischemic vasculopathy, cutaneous and visceral fibrosis and a dysimmune state (Denton and Khanna, 2017; Volkmann et al., 2023; Barnes and Mayes, 2012). We have derived induced pluripotent stem cell (iPSC) lines from two SSc patients aged 38 and 67 years with severe vascular phenotype. These iPSC lines expressed pluripotent markers, exhibited normal and stable genome, and differentiated into trilineage embryonic layers in teratoma formation assays. These SSc-specific iPSC lines can be differentiated into endothelial cells, providing a valuable model to elucidate vascular dysfunction and develop personalized therapeutic approaches.

Decreased energy production and Ca2+ homeostasis imbalance induce myocardial hypertrophy in PDHA1-deficient human pluripotent stem cell derived cardiomyocytes.

The PDHA1 gene, responsible for regulating the conversion of the glycolytic product pyruvate to acetyl CoA, is significantly reduced in cardiomyocytes of patients with hypertrophic cardiomyopathy. Cardiac-specific PDHA1-deficient mice demonstrate cardiac hypertrophy and heart failure. However, the mechanisms underlying the pathogenesis of PDHA1 deficiency remain unclear. PDHA1 gene in human induced pluripotent stem cell line (iPSC) was knockout (KO) using CRISPR-Cas9 technology and differentiated it into cardiomyocytes (CMs) in vitro. Contractile force was quantified by video analysis, Ca2+ handling was assessed with Ca2+ transient analysis and mitochondrial function was detected using flow cytometry. The PDHA1 KO iPSC-CMs displayed myocardial hypertrophy phenotypes by day 40 post-differentiation, characterized by enlarged cell size, increased contractility, abnormal calcium handling, and progressed to mimic heart failure phenotypes by day 50, including reduced contractility, lower calcium release and increased ROS generation. RNA-seq analysis revealed dysregulated expression of pathways related to cardiac hypertrophy and the calcium signaling pathway in KO iPSC-CMs. Furthermore, KO iPSC-CMs exhibited decreased energy production before the manifestation of myocardial hypertrophic phenotype at day 30, exacerbating intracellular lactate accumulation, leading to increased sodium‑hydrogen and sodium‑calcium exchange, ultimately resulting in elevated diastolic calcium concentration. Augmenting energy production with l-carnitine restored diastolic Ca2+ and prevented the development of myocardial hypertrophy in KO iPSC-CMs. Elevated diastolic Ca2+ resulting from reduced energy production and lactate accumulation can trigger overactivation of the calcium signaling pathway, diastolic dysfunction, mitochondrial damage, which constitutes the core pathogenic mechanism of myocardial hypertrophy in KO iPSC-CMs.

Investigating the metabolic load of monoclonal antibody production conveyed to an inducible CHO cell line using a transfer-rate online monitoring system.

Shake flasks are a foundational tool in early process development by allowing high throughput exploration of the design space. However, lack of online data at this scale can hamper rapid decision making. Oxygen transfer rate (OTR) monitoring has been readily applied as an online process characterization tool at the benchtop bioreactor scale. Recent advances in modern sensing technology have allowed OTR monitoring to be available at the shake flask level. It is now possible to multiplex time-of-action (e.g., Induction, temperature shift, pH shift, feeding initiation, point of harvest) characterization studies by relying on careful analysis of OTR profile kinetics. As a result, there is potential to save time and capital expenditures while exploring process intensification studies though accurate and physiologically relevant online data. In this article, we detail the application of OTR monitoring to characterize the impact that recombinant protein production has on an inducible CHO cell line expressing Palivizumab. We then test out time-of-action studies to intensify protein production outcomes. We observe that recombinant protein expression causes a metabolic load that diminishes potential biomass growth. As a result, when compared to a control standard process, delaying induction and temperature shift has the potential to improve viable cell densities (VCD) by 2-fold thus increasing recombinant protein yield by over 30 %. The study also demonstrates that OTR can serve as a useful tool to detect cessation of exponential growth. Consequently, time-of-action points that are characteristic of inducible systems can be formulated accurately and reliably to maximize production performance.

Establishing of human induced pluripotent stem cell line DMSCi002-A from the hematopoietic stem cells of a healthy male donor.

Using the integration-free episomal vector containing the reprogramming components OCT3/4/shp53, Sox2/KLF4, L-MYC/LIN28, and EBNA-1, hematopoietic stem cells obtained from a healthy 33-year-old man were effectively reprogrammed and turned into induced pluripotent stem cells (iPSCs). The reprogrammed iPSCs were grown without the use of feeders. They exhibited a normal karyotype, displayed pluripotency markers, and differentiated into cells from the three germ layers. This DMSCi002-A line may serve as a control for investigating disease mechanisms.

Methods for Detecting Phosphorylated Proteins and Observing Intracellular Localization During Mitosis.

This protocol describes the detection of phosphorylated proteins within cells and the identification of their intracellular localization, with a particular focus on mitotic cells. While the detection of phosphorylated proteins can be achieved using radioactive labeling with 32Pi, this method presents experimental challenges due to the requirement for radioisotopes. Alternatively, detection using phosphorylation-specific antibodies is a potent method; however, it necessitates the identification of phosphorylation sites and further requires the generation of antibodies targeting these sites, making it effective only for thoroughly analyzed phosphorylations. Here, we outline a convenient method for detecting intracellular phosphorylation using the Phos-tag technique. Additionally, we discuss the establishment of inducible stable cell lines using lentivirus, synchronization methods for cells in the mitotic phase using thymidine and nocodazole, as well as some tips for immunoprecipitation, Western blotting, and immunostaining from mitotic cells.

Generation, characterization, and validation of two human induced pluripotent stem cell lines from the peripheral blood of young and older adults.

Aging is a leading risk factor for the development of age-related diseases. However, how aging impacts human induced pluripotent stem cell (hiPSC) reprogramming, age-related epigenetic memory post-reprogramming, differentiation, and its potential applicability to cardiovascular regenerative medicine remains underexplored. We generated, characterized, and validated two hiPSC lines from human peripheral blood mononuclear cells (PBMCs) obtained from whole blood of young and older human donors. The two hiPSC lines expressed four pluripotency markers, have normal karyotypes and trilineage differentiation potential, and genetically match parental PBMCs. These lines are invaluable for regenerative medicine and exploring epigenetic-related molecular mechanisms that underlie aging and aging-related diseases.

Generation of human induced pluripotent stem cell lines HUJIi004 and HUJIi005 from two individuals carrying autosomal-dominant mutations in the CTNBB1 gene resulting in CTNNB1 neurodevelopmental disorder

Generation of human induced pluripotent stem cell lines HUJIi004 and HUJIi005 from two individuals carrying autosomal-dominant mutations in the CTNBB1 gene resulting in CTNNB1 neurodevelopmental disorder

Generation of a human iPSC line (UCLi025-A) from a patient with PHARC syndrome harbouring biallelic variants in ABHD12.

A human induced pluripotent stem cell (hiPSC) line (UCLi025-A) was generated from dermal fibroblast cells from a 42-year-old female donor with polyneuropathy, hearing loss, retinitis pigmentosa and early-onset cataract (PHARC) syndrome carrying a homozygous nonsense variant in ABHD12 c.193C>T, p.(Arg65*). Fibroblasts were confirmed to carry the variant by Sanger sequencing and subsequently reprogrammed using non-integrating episomal plasmids generating a hiPSC line (UCLi025-A). This established cell line was validated for pluripotency markers expression, in vitro differentiation potential and normal karyotype. The utilization of this cell line will serve as a valuable resource for modelling PHARC syndrome and identification of therapeutic targets.

Inducible cell lines producing replication-defective human immunodeficiency virus particles containing envelope glycoproteins stabilized in a pretriggered conformation.

During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, "closed" (State-1) conformation to more "open" (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41)3] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25-50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system.IMPORTANCEA major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies.

STIL Overexpression Is Associated with Chromosomal Numerical Abnormalities in Non-Small-Cell Lung Carcinoma Through Centrosome Amplification.

STIL is a regulatory protein essential for centriole biogenesis, and its dysregulation has been implicated in various diseases, including malignancies. However, its role in non-small-cell lung carcinoma (NSCLC) remains unclear. In this study, we examined STIL expression and its potential association with chromosomal numerical abnormalities (CNAs) in NSCLC using The Cancer Genome Atlas (TCGA) dataset, immunohistochemical analysis, and in vitro experiments with NSCLC cell lines designed to overexpress STIL. TCGA data revealed upregulated STIL mRNA expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), the two major subtypes of NSCLC. Immunohistochemical analysis of cases from our hospital (LUAD, n = 268; LUSC, n = 98) revealed STIL protein overexpression. To elucidate the functional role of STIL, an inducible STIL-overexpressing H1299 NSCLC cell line was generated. Overexpression of STIL in these cells promoted centrosome amplification, leading to chromosomal instability. Finally, analysis of arm-level chromosomal copy number alterations from the TCGA dataset revealed that elevated STIL mRNA expression was associated with CNAs in both LUAD and LUSC. These findings suggest that STIL overexpression is associated with CNAs in NSCLC, likely through centrosome amplification, which is linked to chromosomal instability and might represent a potential therapeutic target for NSCLC treatment.

Generation of hiPSCs lines from three sporadic Parkinson's disease patients.

Here, we described the generation of human induced pluripotent stem cell lines (hiPSCs) from three sporadic Parkinson's disease (sPD) patients by reprogramming of their peripheral blood mononuclear cells (PBMC).

Molecular characterization and sub-retinal transplantation of hypoimmunogenic human retinal sheets in a minipig model of severe photoreceptor degeneration.

Retinal degenerative diseases affect millions of people worldwide, and legal blindness is generally associated with the loss of cone photoreceptors located in the central region of the retina called the macula. Currently, there is no treatment to replace the macula. Addressing this unmet need, we employed control isogenic and hypoimmunogenic induced pluripotent stem cell lines to generate spontaneously polarized retinal sheets (RSs). RSs were enriched in retinal progenitor and cone precursor cells, which could differentiate into mature S- and M/L-cones in long-term cultures. Single-cell RNA-seq analysis showed that RSs recapitulate the ontogeny of the developing human retina. Isolation of neural rosettes for sub-retinal transplantation effectively eliminated unwanted cells such as RPE cells. In a porcine model of chemically induced retinal degeneration, grafts integrated the host retina and formed a new, yet immature, photoreceptor layer. In one transplanted animal, functional and immunohistochemical assays suggest that grafts exhibited responsiveness to light stimuli and established putative synaptic connections with host bipolar neurons. This study underscores the potential and challenges of RSs for clinical applications.

Generation of three human induced pluripotent stem cell lines from retinitis pigmentosa 25 patient and two carriers but asymptomatic daughters

Generation of three human induced pluripotent stem cell lines from retinitis pigmentosa 25 patient and two carriers but asymptomatic daughters

Derivation of two induced pluripotent stem cell lines from a healthy control subject

Two human induced pluripotent stem cell lines, LEIi021-A and LEIi021-B, were derived from dermal fibroblasts from a healthy control subject from an Australian Aboriginal family with retinitis pigmentosa-11. Reprogramming was performed using episomal vectors expressing OCT4, SOX2, LIN28, L-MYC, KLF4 and mp53DD. Pluripotency markers were expressed in both LEIi021-A and LEIi021-B lines. The two cell lines displayed normal karyotypes and demonstrated the ability to differentiate into embryoid bodies with the three primary germ layers and retinal pigment epithelial cells

Generation of human induced pluripotent stem cell lines UKJi001-A and UKJi006-A from patients with heterozygous mutation in the PKP2 gene

One of the main signs we do not know enough about arrhythmogenic right ventricular dysplasia 9/cardiomyopathy (ARVCD9, OMIM #609040 autosomal dominant) is the lack of early markers and therapeutic alternatives. To better study disease pathways in vitro, we generated human induced pluripotent stem cell (hiPSC) lines from the father (UKJi006-A) and son (UKJi001-A), who both shared the same heterozygous mutation in the PKP2 gene (OMIM *602861). While the father had a clinical diagnosis of ARVC, the son lacked the ARVC phenotype. To generate hiPSC lines, non-integrating Sendai virus (SeV) vectors expressing the reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) were used for reprogramming patient peripheral blood mononuclear cells (PBMCs).

SNMF: Integrated Learning of Mutational Signatures and Prediction of DNA Repair Deficiencies.

Many tumours show deficiencies in DNA damage response (DDR), which influence tumorigenesis and progression, but also expose vulnerabilities with therapeutic potential. Assessing which patients might benefit from DDR-targeting therapy requires knowledge of tumour DDR deficiency status, with mutational signatures reportedly better predictors than loss of function mutations in select genes. However, signatures are identified independently using unsupervised learning, and therefore not optimised to distinguish between different pathway or gene deficiencies. We propose SNMF, a supervised non-negative matrix factorisation that jointly optimises the learning of signatures: (1) shared across samples, and (2) predictive of DDR deficiency. We applied SNMF to mutation profiles of human induced pluripotent cell lines carrying gene knockouts linked to three DDR pathways. The SNMF model achieved high accuracy (0.971) and learned more complete signatures of the DDR status of a sample, further discerning distinct mechanisms within a pathway. Cell line SNMF signatures recapitulated tumour-derived COSMIC signatures and predicted DDR pathway deficiency of TCGA tumours with high recall, suggesting that SNMF-like models can leverage libraries of induced DDR deficiencies to decipher intricate DDR signatures underlying patient tumours. https://github.com/joanagoncalveslab/SNMF .

Single-cell RNA sequencing reveals key regulators and differentiation trajectory of iPSC-derived cardiomyocytes

Cardiac differentiation of human pluripotent stem cells is not only a new strategy of regenerative therapy for cardiovascular disease treatment but also provides unique opportunities for the study of in vitro disease models and human heart development. To elucidate the dynamic gene regulatory networks and pivotal regulators involved in the cardiomyocyte differentiation process, we conducted an analysis of single-cell RNA sequencing data obtained from the reprogramming of two human induced pluripotent stem cell (iPSC) lines into cardiomyocytes. The data were collected from 32,365 cells at 4 stages of this process. We successfully identified cardiomyocyte clusters and several other cell clusters with different molecular characteristics derived from iPSC and described the differentiation trajectory of cardiomyocytes during differentiation in vitro. Through differential gene analysis and SCENIC analysis, we identified several candidate genes including CREG and NR2F2 that play an important regulatory role in cardiomyocyte lineage commitment. This study provides the key differentiation trajectory of heart differentiation in vitro at single-cell resolution and reveals the molecular basis of heart development and differentiation of iPSC-derived cardiomyocytes.

An inducible iFUCCI reporter cell line for tracking cell cycle dynamics in human pluripotent stem cells and their differentiated progeny

An ability to monitor cell cycle progression in real time has numerous applications in pluripotent stem cell-based models and therapeutics development. Fluorescence Ubiquitination-based Cell Cycle Indicator (FUCCI) systems enable live monitoring of the cell cycle in stem cells and their differentiated progenies in a non-invasive manner. We describe the generation and characterisation of a cell line with a doxycycline-inducible reporter system, iFUCCI, in the human embryonic stem cell (hESC) line MEL-1. MEL-1 iFUCCI hESCs exhibited a normal karyotype, expressed markers of the undifferentiated state, and demonstrated expected differentiation potential by giving rise to cell populations from all three germ layers.