The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

Generation of an NCS1 gene knockout human induced pluripotent stem cell line using CRISPR/Cas9.

NCS1 (Neuronal calcium sensor protein 1) encodes a highly conserved calcium binding protein abundantly expressed in neurons. It modulates intracellular calcium homeostasis, calcium-dependent signaling pathways as well as neuronal transmission and plasticity. Here, we generated a NCS1 knockout human induced pluripotent stem cell (hiPSC) line using CRISPR-Cas9 genome editing. It shows regular expression of pluripotent markers, normal iPSC morphology and karyotype as well as no detectable off-target effects on top 6 potentially affected genes. This newly generated cell line constitutes a valuable tool for studying the role of NCS1 in the pathophysiology of various neuropsychiatric disorders and non-neurological disease.

Phosphatidylinositol 3,5-bisphosphate facilitates axonal vesicle transport and presynapse assembly.

Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P2 and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.

ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs.

CRISPR-Cas9 technology coupled with human induced pluripotent stem cells allows precise disease modeling in pluripotent cells and subsequently derived specialized cell types. Here, we present an optimized CRISPR-Cas9 pipeline, ASSURED (affordable, successful, specific, user-friendly, rapid, efficient, and deliverable), to produce gene-modified single-cell-derived knockout or single-nucleotide-polymorphism-modified knockin hiPSCs clones. We describe steps for analyzing targeted genomic sequence and designing guide RNAs and homology repair template. We then detail the CRISPR-Cas9 delivery workflow, evaluation of editing efficiency, and automated cell isolation followed by clone screening.

The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation toward the kidney lineage.

During embryonic development, the mesoderm undergoes patterning into diverse lineages including axial, paraxial, and lateral plate mesoderm (LPM). Within the LPM, the so-called intermediate mesoderm (IM) forms kidney and urogenital tract progenitor cells, while the remaining LPM forms cardiovascular, hematopoietic, mesothelial, and additional progenitor cells. The signals that regulate these early lineage decisions are incompletely understood. Here, we found that the centrosomal protein 83 (CEP83), a centriolar component necessary for primary cilia formation and mutated in pediatric kidney disease, influences the differentiation of human-induced pluripotent stem cells (hiPSCs) toward IM. We induced inactivating deletions of CEP83 in hiPSCs and applied a 7-day in vitro protocol of IM kidney progenitor differentiation, based on timed application of WNT and FGF agonists. We characterized induced mesodermal cell populations using single-cell and bulk transcriptomics and tested their ability to form kidney structures in subsequent organoid culture. While hiPSCs with homozygous CEP83 inactivation were normal regarding morphology and transcriptome, their induced differentiation into IM progenitor cells was perturbed. Mesodermal cells induced after 7 days of monolayer culture of CEP83-deficient hiPCS exhibited absent or elongated primary cilia, displayed decreased expression of critical IM genes (PAX8, EYA1, HOXB7), and an aberrant induction of LPM markers (e.g. FOXF1, FOXF2, FENDRR, HAND1, HAND2). Upon subsequent organoid culture, wildtype cells differentiated to form kidney tubules and glomerular-like structures, whereas CEP83-deficient cells failed to generate kidney cell types, instead upregulating cardiomyocyte, vascular, and more general LPM progenitor markers. Our data suggest that CEP83 regulates the balance of IM and LPM formation from human pluripotent stem cells, identifying a potential link between centriolar or ciliary function and mesodermal lineage induction.

Fluorescence Spectroscopy of Low-Level Endogenous ß-Adrenergic Receptor Expression at the Plasma Membrane of Differentiating Human iPSC-Derived Cardiomyocytes.

The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the ß1- and ß2-adrenergic receptors (ß1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of ß-ARs in adult CMs.

High-content analysis of neuronal morphology in human iPSC-derived neurons.

We present a high-content analysis (HCA) protocol for monitoring the outgrowth capacity of human neurons derived from induced pluripotent stem cells (iPSCs). We describe steps to perform HCA imaging, followed by quantifying the morphology of dendrites and axons within a high-throughput system to evaluate neurons obtained through various differentiation approaches. This protocol can be used to screen for modulators of neuronal morphogenesis or neurotoxicity. The approach can be applied to patient-derived iPSCs to identify patient-specific defects and possible therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Zink et al. (2020) and Inak et al. (2021). The protocol can be used in combination with Zink et al. (2022).

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Generation of 20 human induced pluripotent stem cell lines from patients with focal segmental glomerulosclerosis (FSGS).

Focal segmental glomerulosclerosis (FSGS) is a major cause of familial nephrotic syndrome. We generated 20 induced pluripotent stem cell lines from patients diagnosed with FSGS. The iPSC lines include 8 female and 12 male lines and cover a donor age range from 31 to 78. The lines were generated from peripheral blood mononuclear cells by integration-free reprogramming using Sendai virus vectors. Cell lines were fully characterized regarding their pluripotency and differentiation potential, and quality controlled for karyotypic integrity, identity and clearance of reprogramming vectors. The generated cell lines represent a valuable tool for disease modelling and drug development for FSGS.

Assessment of Ethanol-Induced Toxicity on iPSC-Derived Human Neurons Using a Novel High-Throughput Mitochondrial Neuronal Health (MNH) Assay.

Excessive ethanol exposure can cause mitochondrial and cellular toxicity. In order to discover potential counteracting interventions, it is essential to develop assays capable of capturing the consequences of ethanol exposure in human neurons, and particularly dopaminergic neurons that are crucial for the development of alcohol use disorders (AUD). Here, we developed a novel high-throughput (HT) assay to quantify mitochondrial and neuronal toxicity in human dopaminergic neuron-containing cultures (DNs) from induced pluripotent stem cells (iPSCs). The assay, dubbed mitochondrial neuronal health (MNH) assay, combines live-cell measurement of mitochondrial membrane potential (MMP) with quantification of neuronal branching complexity post-fixation. Using the MNH assay, we demonstrated that chronic ethanol exposure in human iPSC-derived DNs decreases MMP and neuronal outgrowth in a dose-dependent manner. The toxic effect of ethanol on DNs was already detectable after 1 h of exposure, and occurred similarly in DNs derived from healthy individuals and from patients with AUD. We next used the MNH assay to carry out a proof-of-concept compound screening using FDA-approved drugs. We identified potential candidate compounds modulating acute ethanol toxicity in human DNs. We found that disulfiram and baclofen, which are used for AUD treatment, and lithium caused neurotoxicity also in the absence of ethanol, while the spasmolytic drug flavoxate positively influenced MNH. Altogether, we developed an HT assay to probe human MNH and used it to assess ethanol neurotoxicity and to identify modulating agents. The MNH assay represents an effective new tool for discovering modulators of MNH and toxicity in live human neurons.

Generation of two human induced pluripotent stem cell lines derived from myoblasts (MDCi014-A) and from peripheral blood mononuclear cells (MDCi014-B) from the same donor.

We describe the generation and characterization of two human induced pluripotent stem cell (hiPSCs) lines reprogrammed from myoblasts and from peripheral blood mononuclear cells (PBMCs) from the same donor. The donor was free of neuromuscular disorders, male and 18 years of age. For reprogramming we used Sendai-virus delivery of the four Yamanaka factors. The pluripotent identity of the hiPSC lines was confirmed by the expression of pluripotency markers and their capacity to differentiate into all three germ layers. These hiPSCs constitute a tool to study tissue of origin specific differences in the identity of hiPSCs.

Generation of three age and gender matched pairs of human induced pluripotent stem cells derived from myoblasts (MDCi011-A, MDCi012-A, MDCi013-A) and from peripheral blood mononuclear cells (MDCi011-B, MDCi012-B, MDCi013-B) from the same donor.

We describe the generation and characterization of three pairs of human induced pluripotent stem cell (hiPSC) lines reprogrammed from myoblasts and from peripheral blood mononuclear cells (PBMCs) of the same donor. All donors were free of neuromuscular disorders, female and between 47 and 50 years of age. For reprogramming we used Sendai-virus delivery of the four Yamanaka factors. The pluripotent identity of the hiPSC lines was confirmed by the expression of pluripotency markers and their capacity to differentiate into all three germ layers. These hiPSCs constitute a tool to study tissue of origin specific differences in the identity of hiPSCs.

Simple Workflow and Comparison of Media for hPSC-Cardiomyocyte Cryopreservation and Recovery.

Great progress has been made with protocols for the differentiation and functional application of hPSC-cardiomyocytes (hPSC-CMs) in recent years; however, the cryopreservation and recovery of hPSC-CMs still presents challenges and few reports describe in detail the protocols and general workflow. In order to facilitate cryopreservation and recovery of hPSC-CMs for a wide range of applications, we provide detailed information and step-by-step protocols. The protocols are simple and use common reagents. They are comprised of a fast dissociation, cryopreservation using standard equipment, and gentle recovery following thawing. We discuss various features of the protocols, as well as their utilization in the context of common hPSC-CM differentiation and application workflows. Finally, we compare two proprietary and two common in-house formulations of cryopreservation media used for hPSC-CMs, and despite differences in their price and composition find broadly similar recovery rates and cellular function after thawing. © 2019 The Authors. Basic Protocol 1: Dissociation and cryopreservation of hPSC-CMs Basic Protocol 2: Thawing and recovery of cryogenically frozen hPSC-CMs.

Methods for Automated Single Cell Isolation and Sub-Cloning of Human Pluripotent Stem Cells.

Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical sub-cloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell-derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications. © 2020 The Authors. Basic Protocol: Efficient automated hPSC single cell seeding and clonal expansion using the iotaSciences IsoCell platform Alternate Protocol 1: hPSC single cell seeding and clonal expansion using the Cellenion CellenONE single-cell dispenser Alternate Protocol 2: hPSC single cell seeding and clonal expansion using the Cytena single-cell dispenser Support Protocol 1: Coating cell culture plates with Geltrex Support Protocol 2: hPSC maintenance in defined feeder-free conditions Support Protocol 3: hPSC passaging in clumps Support Protocol 4: Laminin 521 coating of IsoCell plates and 96-well/384-well plates Support Protocol 5: Preparation of medium containing anti-apoptotic small molecules Support Protocol 6: 96- and 384-well target plate preparation prior to single cell seeding Support Protocol 7: Single cell dissociation of hPSCs Support Protocol 8: IsoCell-, CellenONE-, and Cytena-derived hPSC clone subculture and expansion.

CPAP promotes timely cilium disassembly to maintain neural progenitor pool.

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.

Optimized Generation of Functional Neutrophils and Macrophages from Patient-Specific Induced Pluripotent Stem Cells: Ex Vivo Models of X(0)-Linked, AR22(0)- and AR47(0)- Chronic Granulomatous Diseases.

Chronic granulomatous disease (CGD) is an inherited orphan disorder caused by mutations in one of the five genes encoding reduced nicotinamide-adenine-dinucleotide-phosphate oxidase subunits, which subsequently lead to impairment in the production of microbicidal reactive oxygen species (ROS). In order to offer several cell line models of CGD and therefore support research on pathophysiology and new therapeutic approaches, we optimized protocols to differentiate induced pluripotent stem cells (iPSCs) from wild-type, X(0)-, AR22(0)- and AR47(0)-CGD patient's fibroblasts into neutrophils and into macrophages. Aberrant genetic clones were discarded after chromosome karyotyping and array-comparative genomic hybridization analysis. All remaining iPSC lines showed human embryonic stem cell-like morphology, expressed all tested pluripotency markers and formed embryoid bodies that contained cells originating from all three primary germ layers. Furthermore, each CGD patient-specific iPSC line retained the gp91 (phox) , p47 (phox) , and p22 (phox) mutations found in the corresponding patient's neutrophils. The average production of CD34(+) progenitors was of 1.5×10(6) cells after 10 days of differentiation of 10×10(6) iPSCs. They were terminally differentiated into about 3×10(5) neutrophils or into 3×10(7) macrophages. Based on morphological, phenotypical, and functional criteria both phagocyte types were mature and indistinguishable from the native human neutrophils and macrophages. However, neutrophils and macrophages derived from X(0)-, AR22(0)-, and AR47(0)-CGD patient-specific iPSC lines lacked ROS production and the corresponding mutated proteins. To simplify the phagocytes' production upon request, progenitors can be cryopreserved. In conclusion, we describe a reproducible, simple, and efficient way to generate neutrophils and macrophages from iPSCs and provide a new cellular model for the AR22(0)-CGD genetic form that has not been described before.

Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation.

Pluripotent stem cells present an extraordinary powerful tool to investigate embryonic development in humans. Essentially, they provide a unique platform for dissecting the distinct mechanisms underlying pluripotency and subsequent lineage commitment. Modest information currently exists about the expression and the role of ion channels during human embryogenesis, organ development, and cell fate determination. Of note, small and intermediate conductance, calcium-activated potassium channels have been reported to modify stem cell behaviour and differentiation. These channels are broadly expressed throughout human tissues and are involved in various cellular processes, such as the after-hyperpolarization in excitable cells, and also in differentiation processes. To this end, human induced pluripotent stem cells (hiPSCs) generated from plucked human hair keratinocytes have been exploited in vitro to recapitulate endoderm formation and, concomitantly, used to map the expression of the SK channel (SKCa) subtypes over time. Thus, we report the successful generation of definitive endoderm from hiPSCs of ectodermal origin using a highly reproducible and robust differentiation system. Furthermore, we provide the first evidence that SKCas subtypes are dynamically regulated in the transition from a pluripotent stem cell to a more lineage restricted, endodermal progeny.