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Cell Reports Jun 2024GGGGCC (GC) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic...
GGGGCC (GC) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology, we deleted EXOC2, which encodes an essential exocyst subunit, in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2, suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to, surprisingly, the decreased levels of dipeptide repeat (DPR) proteins and expanded GC repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded GC repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of GC repeats-containing RNA, making it a potential therapeutic target in C9ORF72-ALS/FTD.
PubMed: 38935506
DOI: 10.1016/j.celrep.2024.114375 -
Journal of the American Heart... Jun 2024Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate characteristics of mature...
BACKGROUND
Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet, a risk of arrhythmias exists when hiPSC-CMs are injected into large animal models. Thus, understanding hiPSC-CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC-CM maturation. Here, we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC-CM maturation in 3-dimensional suspension culture.
METHODS AND RESULTS
Three-dimensional cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator, 10 μM), LOM612 (FOXO relocator, 5 μM), AS1842856 (FOXO inhibitor, 1 μM), or RCM-1 (FOXM1 inhibitor, 1 μM), starting 2 days after onset of beating, with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC-CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation, in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility, oxygen consumption, and voltage peak maximum upstroke velocity, in n=3 to 6 per group for all assays. Further, by single-cell RNA sequencing of n=2 LOM612-treated cells compared with dimethyl sulfoxide, LOM612-mediated FOXO activation promoted expression of cardiac maturational pathways.
CONCLUSIONS
We show that p53 activation promotes FOXO and suppresses FOXM1 during 3-dimensional hiPSC-CM maturation. These results expand our understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3-dimensional culture system.
PubMed: 38934864
DOI: 10.1161/JAHA.123.033155 -
Neural Regeneration Research Jun 2024TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon. TAU is missorted and aggregated in an array of diseases known as...
TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon. TAU is missorted and aggregated in an array of diseases known as tauopathies. Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications (PTMs), changes of which affect microtubule stability and dynamics, microtubule interaction with other proteins and cellular structures, and mediate recruitment of microtubule-severing enzymes. As impairment of microtubule dynamics causes neuronal dysfunction, we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics. We therefore aimed to study the effects of a disease-causing mutation of TAU (P301L) on the levels and localization of microtubule PTMs indicative of microtubule stability and dynamics, to assess whether P301L-TAU causes stability-changing modifications to microtubules. To investigate TAU localization, phosphorylation, and effects on tubulin PTMs, we expressed wild-type or P301L-TAU in human MAPT-KO induced pluripotent stem cell-derived neurons (iNeurons) and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU (pR5 mice). Human neurons expressing the longest TAU isoform (2N4R) with the P301L mutation showed increased TAU phosphorylation at the AT8, but not the p-Ser-262 epitope, and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons. P301L-TAU showed pronounced somatodendritic presence, but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU. P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation, but reduced acetylation, of microtubules compared with non-transgenic littermates. In sum, P301L-TAU results in changes in microtubule PTMs, suggestive of impairment of microtubule stability. This is accompanied by missorting and aggregation of TAU in mice but not in iNeurons. Microtubule PTMs/impairment may be of key importance in tauopathies.
PubMed: 38934386
DOI: 10.4103/NRR.NRR-D-23-01742 -
Materials Today. Bio Jun 2024Human induced pluripotent stem cell (hiPSC)-derived mesenchymal stem cells (iMSCs) are ideal candidates for the production of standardised and scalable bioengineered...
UNLABELLED
Human induced pluripotent stem cell (hiPSC)-derived mesenchymal stem cells (iMSCs) are ideal candidates for the production of standardised and scalable bioengineered bone grafts. However, stable induction and osteogenic differentiation of iMSCs pose challenges in the industry. We developed a precise differentiation method to produce homogeneous and fully differentiated iMSCs. In this study, we established a standardised system to prepare iMSCs with increased osteogenic potential and improved bioactivity by introducing a CHIR99021 (C91)-treated osteogenic microenvironment (COOME). COOME enhances the osteogenic differentiation and mineralisation of iMSCs via canonical Wnt signalling. Global transcriptome analysis and co-culturing experiments indicated that COOME increased the pro-angiogenesis/neurogenesis activity of iMSCs. The superior osteogenic differentiation and mineralisation abilities of COOME-treated iMSCs were also confirmed in a Bio3D module generated using a polycaprolactone (PCL) and cell-integrated 3D printing (PCI3D) system, which is the closest model to research. This COOME-treated iMSCs differentiation system offers a new perspective for generating highly osteogenic, bioactive, and anatomically matched grafts for clinical applications.
STATEMENT OF SIGNIFICANCE
Although human induced pluripotent stem cell-derived MSCs (iMSCs) are ideal seed cells for synthetic bone implants, the challenges of stable induction and osteogenic differentiation hinder their clinical application. This study established a standardised system for the scalable preparation of iMSCs with improved osteogenic potential by combining our precise iMSC differentiation method with the CHIR99021 (C91)-treated osteocyte osteogenic microenvironment (COOME) through the activation of canonical Wnt signalling. Moreover, COOME upregulated the pro-angiogenic and pro-neurogenic capacities of iMSCs, which are crucial for the integration of implanted bone grafts. The superior osteogenic ability of COOME-treated iMSCs was confirmed in Bio3D modules generated using PCL and cell-integrated 3D printing systems, highlighting their functional potential . This study contributes to tissue engineering by providing insights into the functional differentiation of iMSCs for bone regeneration.
PubMed: 38933413
DOI: 10.1016/j.mtbio.2024.101111 -
Frontiers in Cellular Neuroscience 2024Human cerebral organoids (hCOs) derived from pluripotent stem cells are very promising for the study of neurodevelopment and the investigation of the healthy or diseased...
INTRODUCTION
Human cerebral organoids (hCOs) derived from pluripotent stem cells are very promising for the study of neurodevelopment and the investigation of the healthy or diseased brain. To help establish hCOs as a powerful research model, it is essential to perform the morphological characterization of their cellular components in depth.
METHODS
In this study, we analyzed the cell types consisting of hCOs after culturing for 45 days using immunofluorescence and reverse transcriptase qualitative polymerase chain reaction (RT-qPCR) assays. We also analyzed their subcellular morphological characteristics by transmission electron microscopy (TEM).
RESULTS
Our results show the development of proliferative zones to be remarkably similar to those found in human brain development with cells having a polarized structure surrounding a central cavity with tight junctions and cilia. In addition, we describe the presence of immature and mature migrating neurons, astrocytes, oligodendrocyte precursor cells, and microglia-like cells.
DISCUSSION
The ultrastructural characterization presented in this study provides valuable information on the structural development and morphology of the hCO, and this information is of general interest for future research on the mechanisms that alter the cell structure or function of hCOs.
PubMed: 38933177
DOI: 10.3389/fncel.2024.1406839 -
Life (Basel, Switzerland) Jun 2024Parkinson's disease (PD) caused by gene triplication (3X) leads to early onset, rapid progression, and often dementia. Understanding the impact of 3X and its absence is...
Parkinson's disease (PD) caused by gene triplication (3X) leads to early onset, rapid progression, and often dementia. Understanding the impact of 3X and its absence is crucial. This study investigates the differentiation of human induced pluripotent stem cell (hiPSC)-derived floor-plate progenitors into dopaminergic neurons. Three different genotypes were evaluated in this study: patient-derived hiPSCs with 3X, a gene-edited isogenic line with a frame-shift mutation on all alleles ( 4KO), and a normal wild-type control. Our aim was to assess how the substantia nigra pars compacta (SNpc) microenvironment, damaged by 6-hydroxydopamine (6-OHDA), influences tyrosine hydroxylase-positive (Th+) neuron differentiation in these genetic variations. This study confirms successful in vitro differentiation into neuronal lineage in all cell lines. However, the 4KO line showed unusual LIM homeobox transcription factor 1 alpha (Lmx1a) extranuclear distribution. Crucially, both 3X and 4KO lines had reduced Th+ neuron expression, despite initial successful neuronal differentiation after two months post-transplantation. This indicates that while the SNpc environment supports early neuronal survival, gene alterations-either amplification or knock-out-negatively impact Th+ dopaminergic neuron maturation. These findings highlight 's critical role in PD and underscore the value of hiPSC models in studying neurodegenerative diseases.
PubMed: 38929711
DOI: 10.3390/life14060728 -
Life (Basel, Switzerland) May 2024The escalating prevalence of retinal diseases-notably, age-related macular degeneration and hereditary retinal disorders-poses an intimidating challenge to ophthalmic... (Review)
Review
The escalating prevalence of retinal diseases-notably, age-related macular degeneration and hereditary retinal disorders-poses an intimidating challenge to ophthalmic medicine, often culminating in irreversible vision loss. Current treatments are limited and often fail to address the underlying loss of retinal cells. This paper explores the potential of stem-cell-based therapies as a promising avenue for retinal regeneration. We review the latest advancements in stem cell technology, focusing on embryonic stem cells (ESCs), pluripotent stem cells (PSCs), and mesenchymal stem cells (MSCs), and their ability to differentiate into retinal cell types. We discuss the challenges in stem cell transplantation, such as immune rejection, integration into the host retina, and functional recovery. Previous and ongoing clinical trials are examined to highlight the therapeutic efficacy and safety of these novel treatments. Additionally, we address the ethical considerations and regulatory frameworks governing stem cell research. Our analysis suggests that while stem-cell-based therapies offer a groundbreaking approach to treating retinal diseases, further research is needed to ensure long-term safety and to optimize therapeutic outcomes. This review summarizes the clinical evidence of stem cell therapy and current limitations in utilizing stem cells for retinal degeneration, such as age-related macular degeneration, retinitis pigmentosa, and Stargardt's disease.
PubMed: 38929652
DOI: 10.3390/life14060668 -
Animals : An Open Access Journal From... Jun 2024Embryonic stem cells (ESCs) are remarkably undifferentiated cells that originate from the inner cell mass of the blastocyst. They possess the ability to self-renew and... (Review)
Review
Embryonic stem cells (ESCs) are remarkably undifferentiated cells that originate from the inner cell mass of the blastocyst. They possess the ability to self-renew and differentiate into multiple cell types, making them invaluable in diverse applications such as disease modeling and the creation of transgenic animals. In recent years, as agricultural practices have evolved from traditional to biological breeding, it has become clear that pluripotent stem cells (PSCs), either ESCs or induced pluripotent stem cells (iPSCs), are optimal for continually screening suitable cellular materials. However, the technologies for long-term in vitro culture or establishment of cell lines for PSCs in livestock are still immature, and research progress is uneven, which poses challenges for the application of PSCs in various fields. The establishment of a robust in vitro system for these cells is critically dependent on understanding their pluripotency maintenance mechanisms. It is believed that the combined effects of pluripotent transcription factors, pivotal signaling pathways, and epigenetic regulation contribute to maintaining their pluripotent state, forming a comprehensive regulatory network. This article will delve into the primary mechanisms underlying the maintenance of pluripotency in PSCs and elaborate on the applications of PSCs in the field of livestock.
PubMed: 38929361
DOI: 10.3390/ani14121742 -
International Journal of Molecular... Jun 2024CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the...
Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody.
CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147 THP-1 cells and evaluated their differentiation properties compared to the wild type. A humanized anti-CD147 antibody, HuM6-1B9, was also constructed for investing the phagocytic function of CD147 THP-1 cells mediated by HuM6-1B9 in the phagocytosis of Jurkat T cells. The CD147 THP-1 was generated by CRISPR/Cas9 and maintained polarization profiles. HuM6-1B9 was produced in CHO-K1 cells and effectively bound to CD147 with high binding affinity (K: 2.05 ± 0.30 × 10 M). Additionally, HuM6-1B9 enhanced the phagocytosis of Jurkat T cells by CD147 THP-1-derived LPS-activated macrophages (M-LPS), without self-ADCP. The formation of THP-1-derived mMDSC was limited in CD147 THP-1 cells, highlighting the significant impact of CD147 deletion. Maintaining expression markers and phagocytic function in CD147 THP-1 macrophages supports future engineering and the application of induced pluripotent stem cell-derived macrophages. The combination of HuM6-1B9 and CD147 monocyte-derived macrophages holds promise as an alternative strategy for T-ALL.
Topics: Humans; Phagocytosis; Jurkat Cells; Basigin; Cell Differentiation; THP-1 Cells; Myeloid-Derived Suppressor Cells; Antibodies, Monoclonal, Humanized; Animals; CHO Cells; Cricetulus; Monocytes; Macrophages; CRISPR-Cas Systems
PubMed: 38928332
DOI: 10.3390/ijms25126626 -
International Journal of Molecular... Jun 2024An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na currents by removing the inactivation of voltage-gated Na...
An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na currents by removing the inactivation of voltage-gated Na channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na currents significantly stronger as compared to peak Na currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na channels expressed in cardiomyocytes may be relevant for the generation of late Na currents following oxidative stress.
Topics: Myocytes, Cardiac; Humans; Animals; Tetrodotoxin; Mice; Induced Pluripotent Stem Cells; HEK293 Cells; Oxidation-Reduction; Chloramines; Reactive Oxygen Species; NAV1.5 Voltage-Gated Sodium Channel; Sodium; Action Potentials; Tosyl Compounds
PubMed: 38928302
DOI: 10.3390/ijms25126596