Ventral Telencephalic Patterning Protocols for Induced Pluripotent Stem Cells.

The differentiation of human induced pluripotent stem cells (hiPSCs) into specific cell types for disease modeling and restorative therapies is a key research agenda and offers the possibility to obtain patient-specific cells of interest for a wide range of diseases. Basal forebrain cholinergic neurons (BFCNs) play a particular role in the pathophysiology of Alzheimer's dementia and isolated dystonias. In this work, various directed differentiation protocols based on monolayer neural induction were tested for their effectiveness in promoting a ventral telencephalic phenotype and generating BFCN. Ventralizing factors [i.e., purmorphamine and Sonic hedgehog (SHH)] were applied at different time points, time intervals, and concentrations. In addition, caudal identity was prevented by the use of a small molecule XAV-939 that inhibits the Wnt-pathway. After patterning, gene expression profiles were analyzed by quantitative PCR (qPCR). Rostro-ventral patterning is most effective when initiated simultaneously with neural induction. The most promising combination of patterning factors was 0.5 µM of purmorphamine and 1 µM of XAV-939, which induces the highest expression of transcription factors specific for the medial ganglionic eminence, the source of GABAergic inter- and cholinergic neurons in the telencephalon. Upon maturation of cells, the immune phenotype, as well as electrophysiological properties were investigated showing the presence of marker proteins specific for BFCN (choline acetyltransferase, ISL1, p75, and NKX2.1) and GABAergic neurons. Moreover, a considerable fraction of measured cells displayed mature electrophysiological properties. Synaptic boutons containing the vesicular acetylcholine transporter (VACHT) could be observed in the vicinity of the cells. This work will help to generate basal forebrain interneurons from hiPSCs, providing a promising platform for modeling neurological diseases, such as Alzheimer's disease or Dystonia.

Neuronal Differentiation of Induced Pluripotent Stem Cells from Schizophrenia Patients in Two-Dimensional and in Three-Dimensional Cultures Reveals Increased Expression of the Kv4.2 Subunit DPP6 That Contributes to Decreased Neuronal Activity.

Although the molecular underpinnings of schizophrenia (SZ) are still incompletely understood, deficits in synaptic activity and neuronal connectivity have been identified as core pathomechanisms of SZ and other neuropsychiatric disorders. In this study, we generated induced pluripotent stem cell (iPSC) lines from skin fibroblasts from healthy donors and patients diagnosed with idiopathic SZ. We differentiated the human iPSC into cortical neurons both as adherent monolayers and as three-dimensional spheroids. RNA sequencing revealed little overlap in differentially expressed genes between 2D and 3D neuron cultures from SZ iPSC compared with controls. Notably, mRNA transcripts encoding dipeptidyl peptidase-like protein 6 (DPP6), an accessory subunit of Kv4.2 voltage-gated potassium channels, were massively increased in cortical neurons from SZ iPSC in the 2D and 3D model. Consistently, multielectrode array recordings and calcium imaging showed significantly decreased neuronal activity both in 2D and in 3D cultures from SZ neurons. To show a causal relationship, we treated iPSC-derived neurons in 2D cultures with lentiviral DPP6 shRNA vectors and the Kv4.2 channel blocker AmmTx3, respectively. Both treatments successfully reversed neuronal hypoexcitability and hypoactivity in cortical neurons from SZ iPSC. Our data highlight a contribution of DPP6 and Kv4.2 to the deficit in neurotransmission in an iPSC model for SZ, which may be of therapeutic relevance for a subset of SZ patients.

CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 Causes Developmental Deficits in Human Cortical Neurons Derived from Induced Pluripotent Stem Cells.

The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.

Altered calcium dynamics and glutamate receptor properties in iPSC-derived motor neurons from ALS patients with C9orf72, FUS, SOD1 or TDP43 mutations.

The fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characterized by a profound loss of motor neurons (MNs). Until now only riluzole minimally extends life expectancy in ALS, presumably by inhibiting glutamatergic neurotransmission and calcium overload of MNs. Therefore, the aim of this study was to investigate the glutamate receptor properties and key aspects of intracellular calcium dynamics in induced pluripotent stem cell (iPSC)-derived MNs from ALS patients with C9orf72 (n = 4 cell lines), fused in sarcoma (FUS) (n = 9), superoxide dismutase 1 (SOD1) (n = 3) or transactive response DNA-binding protein 43 (TDP43) (n = 3) mutations as well as healthy (n = 7 cell lines) and isogenic controls (n = 3). Using calcium imaging, we most frequently observed spontaneous transients in mutant C9orf72 MNs. Basal intracellular calcium levels and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced signal amplitudes were elevated in mutant TDP43 MNs. Besides, a majority of mutant TDP43 MNs responded to 3.5-dihydroxyphenylglycine as metabotropic glutamate receptor agonist. Quantitative real-time PCR demonstrated significantly increased expression levels of AMPA and kainate receptors in mutant FUS cells compared to healthy and isogenic controls. Furthermore, the expression of kainate receptors and voltage gated calcium channels in mutant C9orf72 MNs as well as metabotropic glutamate receptors in mutant SOD1 cells was markedly elevated compared to controls. Our data of iPSC-derived MNs from familial ALS patients revealed several mutation-specific alterations in glutamate receptor properties and calcium dynamics that could play a role in ALS pathogenesis and may lead to future translational strategies with individual stratification of neuroprotective ALS treatments.

Multielectrode Array (MEA)-Based Detection of Spontaneous Network Activity in Human iPSC-Derived Cortical Neurons.

Multielectrode arrays enable the detection of spontaneous cellular network activity, which can be utilized for the characterization of a neuronal culture. Here, we describe the detection of spontaneous neuronal activity in iPSC-derived cortical neurons using a 24-well plate for a multiwall-MEA system.

Assessing Neuronal Excitability on a Fluorometric Imaging Plate Reader (FLIPR) Following a Defined Electrostimulation Paradigm.

FLIPR-based calcium assay enables the detection and characterization of neuronal excitability by using electrical field stimulation to evoke and record action potential-driven calcium transients in induced pluripotent stem cell (iPSC)-derived cortical forebrain neurons. Here we describe high throughput measurement of neuronal excitability with a defined electrostimulation paradigm in a 384-well plate format using FLIPR.

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro.

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 G93A -dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.

Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation.

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.

HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)-mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.

Functional effects of cannabinoids during dopaminergic specification of human neural precursors derived from induced pluripotent stem cells.

Among adolescents cannabis is one of the most widely used illicit drugs. In adolescence brain development continues, characterized by neuronal maturation and synaptic plasticity. The endocannabinoid system plays an important role during brain development by modulating neuronal function and neurogenesis. Changes in endocannabinoid signaling by Δ9 -tetrahydrocannabinol (THC), the psychoactive component of cannabis, might therefore lead to neurobiological changes influencing brain function and behavior. We investigated the functional maturation and dopaminergic specification of human cord blood-derived induced pluripotent stem cell (hCBiPSC)-derived small molecule neural precursor cells (smNPCs) after cultivation with the endogenous cannabinoid anandamide (AEA) and the exogenous THC, both potent agonists at the cannabinoid 1 receptor (CB1 R). Higher dosages of 10-µM AEA or THC significantly decreased functionality of neurons, indicated by reduced ion currents and synaptic activity. A lower concentration of 1-µM THC had no marked effect on neuronal and dopaminergic maturation, while 1-µM AEA significantly enhanced the frequency of synaptic activity. As there were no significant effects on DNA methylation in promotor regions of genes important for neuronal function, these cannabinoid actions seem to be mediated by another than this epigenetic mechanism. Our data suggest that there are concentration-dependent actions of cannabinoids on neuronal function in vitro indicating neurotoxic, dysfunctional effects of 10-µM AEA and THC during human neurogenesis.

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization.

Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function. SIGNIFICANCE STATEMENT: Chorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a human in vitro model using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2. Because Src kinases are involved in bridging the membrane to the actin cytoskeleton by membrane protein phosphorylation, our data suggest an actin-dependent mechanism of this dysfunctional phenotype and potential treatment targets in ChAc.

4-Aminopyridine Induced Activity Rescues Hypoexcitable Motor Neurons from Amyotrophic Lateral Sclerosis Patient-Derived Induced Pluripotent Stem Cells.

Despite decades of research on amyotrophic lateral sclerosis (ALS), there is only one approved drug, which minimally extends patient survival. Here, we investigated pathophysiological mechanisms underlying ALS using motor neurons (MNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying mutations in FUS or SOD1. Patient-derived MNs were less active and excitable compared to healthy controls, due to reduced Na(+) /K(+) ratios in both ALS groups accompanied by elevated potassium channel (FUS) and attenuated sodium channel expression levels (FUS, SOD1). ALS iPSC-derived MNs showed elevated endoplasmic reticulum stress (ER) levels and increased caspase activation. Treatment with the FDA approved drug 4-Aminopyridine (4AP) restored ion-channel imbalances, increased neuronal activity levels and decreased ER stress and caspase activation. This study provides novel pathophysiological data, including a mechanistic explanation for the observed hypoexcitability in patient-derived MNs and a new therapeutic strategy to provide neuroprotection in MNs affected by ALS. Stem Cells 2016;34:1563-1575.

Molecular and functional analyses of motor neurons generated from human cord-blood-derived induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have become the most promising candidates for in vitro modeling of motor neuron (MN) diseases, such as amyotrophic lateral sclerosis (ALS), and possibly for future therapeutic implementation in regenerative medicine. We here present for the first time the differentiation of human cord-blood-derived iPSCs (hCBiPSCs) into MNs, the cell type primarily affected in ALS. In contrast to iPSCs generated from adult tissue, the hCBiPSCs used in this study hold the promise of lower genetic mutation burden or epigenetic alterations, which makes them ideal candidates for transplantation studies. Small-molecule-derived neural precursor cells (smNPCs) were generated from hCBiPSCs and used for the following differentiation studies to substantially shorten MN differentiation time. Consequently, as early as 18 days of in vitro differentiation, the MNs stained positive for neuronal- and for MN-specific markers accompanied by respective gene expression patterns. To demonstrate that the hCBiPSC-derived neural precursor cells (smNPCs) can be differentiated into functional MNs, the cells were characterized by calcium imaging and patch-clamp analysis. Calcium imaging detected the expression of functional voltage-dependent calcium and ligand-gated channels of several important neurotransmitters. Using whole-cell patch-clamp recordings, we observed functional neuronal properties like sodium-inward currents and action potentials (APs). Some cells showed spontaneous APs and synaptic activity that are signs of essential functional maturation. Having established a rapid and efficient method to generate functional MNs from hCBiPSCs, we demonstrate the differentiation potential of genetically unbiased hCBiPSCs as promising source for transplantation studies and also create a framework for future in-vitro disease modeling.

Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells.

INTRODUCTION: Human induced pluripotent stem cells (hiPSCs) offer great promise for regenerative therapies or in vitro modelling of neurodegenerative disorders like Parkinson's disease. Currently, widely used cell sources for the generation of hiPSCs are somatic cells obtained from aged individuals. However, a critical issue concerning the potential clinical use of these iPSCs is mutations that accumulate over lifetime and are transferred onto iPSCs during reprogramming which may influence the functionality of cells differentiated from them. The aim of our study was to establish a differentiation strategy to efficiently generate neurons including dopaminergic cells from human cord blood-derived iPSCs (hCBiPSCs) as a juvenescent cell source and prove their functional maturation in vitro. METHODS: The differentiation of hCBiPSCs was initiated by inhibition of transforming growth factor-ß and bone morphogenetic protein signaling using the small molecules dorsomorphin and SB 431542 before final maturation was carried out. hCBiPSCs and differentiated neurons were characterized by immunocytochemistry and quantitative real time-polymerase chain reaction. Since functional investigations of hCBiPSC-derived neurons are indispensable prior to clinical applications, we performed detailed analysis of essential ion channel properties using whole-cell patch-clamp recordings and calcium imaging. RESULTS: A Sox1 and Pax6 positive neuronal progenitor cell population was efficiently induced from hCBiPSCs using a newly established differentiation protocol. Neuronal progenitor cells could be further maturated into dopaminergic neurons expressing tyrosine hydroxylase, the dopamine transporter and engrailed 1. Differentiated hCBiPSCs exhibited voltage-gated ion currents, were able to fire action potentials and displayed synaptic activity indicating synapse formation. Application of the neurotransmitters GABA, glutamate and acetylcholine induced depolarizing calcium signal changes in neuronal cells providing evidence for the excitatory effects of these ligand-gated ion channels during maturation in vitro. CONCLUSIONS: This study demonstrates for the first time that hCBiPSCs can be used as a juvenescent cell source to generate a large number of functional neurons including dopaminergic cells which may serve for the development of novel regenerative treatment strategies.