Establishment of a human induced pluripotent stem cell neuronal model for identification of modulators of A53T α-synuclein levels and aggregation

Inhibiting formation or promoting degradation of α-synuclein aggregates are among the therapeutical approaches under investigation as disease-modifying treatment strategies for Parkinson’s disease. To support these developments, several in vitro models based on seeded α-synuclein aggregation have been established in immortalized cell lines and murine primary neurons. Here, we report on a humanized model with a reproducibility and throughput that enables its use in supporting target identification and validation in pharmacological research. A human induced pluripotent stem cell (iPSC) line was genetically modified to express HA-tagged α-synuclein with the point mutation in position 53 from Alanine to Threonine (A53T) under an inducible system and differentiated into cortical neurons expressing neuronal markers and exhibiting spontaneous activity. Intracellular α-synuclein aggregation was triggered by exposure to exogenous added fibrillated recombinant wild-type human α-synuclein fibrils91 and demonstrated by several endpoints; the formation of Triton-insoluble SDS-soluble α-synuclein, biochemically in a fluorescence resonance energy transfer based aggregation assay and by immunocytochemistry of phosphorylated α-synuclein positive puncta. We demonstrate the feasibility of upscaling the iPSC neuron production for drug discovery and that the model has a suitable dynamic range allowing for both detection of increased and decreased α-synuclein aggregation. Moreover, gene modulation is feasible using siRNAs, making the model suitable for genetic screening for modulators of α-synuclein aggregation. Data on effects of USP8, USP13 and USP9X knockdown on α-synuclein expression and aggregation contradicts published data from immortalized cell lines and murine systems. This highlight the importance of including humanized neuronal models in the confirmation of biological mechanisms in specific variations of Parkinson’s disease.

Pituitary tumors and immortalized cell lines generated by cre-inducible expression of SV40 T antigen

Targeted oncogenesis is the process of driving tumor formation by engineering transgenic mice that express an oncogene under the control of a cell-type specific promoter. Such tumors can be adapted to cell culture, providing immortalized cell lines. To make it feasible to follow the process of tumorigenesis and increase the opportunity for generating cell lines, we developed a mouse strain that expresses SV40 T antigens in response to cre-recombinase. Using CRISPR/Cas9 we inserted a cassette with coding sequences for SV40 T antigens and an internal ribosome entry site with green fluorescent protein cassette (IRES-GFP) into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26LSL-SV40-GFP. These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. Both the Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice developed fully penetrant dwarfism and large tumors by 4 weeks. Tumors from both of these mouse lines were adapted to growth in cell culture. We have established a progenitor-like cell line (PIT-P1) that expresses Sox2 and Pitx1, and a thyrotrope-like cell line (PIT-T1) that expresses Pou1f1 and Cga. These studies demonstrate the utility of the novel, Rosa26LSL-SV40-GFP mouse line for reliable targeted oncogenesis and development of unique cell lines.

Generation and Characterization of Immortalized Mouse Cortical Astrocytes From Wildtype and Connexin43 Knockout Mice

We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1L1 (ALDH1L1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

GENERATION AND CHARACTERIZATION OF IMMORTALIZED MOUSE CORTICAL ASTROCYTES FROM WILDTYPE AND CONNEXIN43 KNOCKOUT MICE

ABSTRACTWe transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1 (ALDH1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

Cell Type-Specific Adhesion and Migration on Laser-Structured Opaque Surfaces

Cytocompatibility is essential for implant approval. However, initial in vitro screenings mainly include the quantity of adherent immortalized cells and cytotoxicity. Other vital parameters, such as cell migration and an in-depth understanding of the interaction between native tissue cells and implant surfaces, are rarely considered. We investigated different laser-fabricated spike structures using primary and immortalized cell lines of fibroblasts and osteoblasts and included quantification of the cell area, aspect ratio, and focal adhesions. Furthermore, we examined the three-dimensional cell interactions with spike topographies and developed a tailored migration assay for long-term monitoring on opaque materials. While fibroblasts and osteoblasts on small spikes retained their normal morphology, cells on medium and large spikes sank into the structures, affecting the composition of the cytoskeleton and thereby changing cell shape. Up to 14 days, migration appeared stronger on small spikes, probably as a consequence of adequate focal adhesion formation and an intact cytoskeleton, whereas human primary cells revealed differences in comparison to immortalized cell lines. The use of primary cells, analysis of the cell–implant structure interaction as well as cell migration might strengthen the evaluation of cytocompatibility and thereby improve the validity regarding the putative in vivo performance of implant material.

Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium. NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.

OR06-06 Pituitary Tumors and Immortalized Cell Lines Generated by Cre-Inducible Expression of SV40 T-Antigen

Pituitary and hypothalamic cell lines have been developed by targeted oncogenesis. This involved using cell-specific transcriptional regulatory sequences to drive expression of large and small SV40 T-antigens in transgenic mice. Invariably, tumors develop in some of the mice, and the cells in these tumors can sometimes be adapted to grow in culture into stable, immortalized cell lines that maintain some of the features of differentiated cells. Cell lines that represent pre-gonadotropes (αT3-1), gonadotropes (LβT2), precursors to the POU1F1 lineage (GHFT1, Pit1-zero), differentiated cells of the POU1F1 lineage (Pit1-triple, TaT1, and Pit1-PRL), and GnRH neurons (GT1-1) have been made by this approach. Tumors often develop early and cause infertility or death. To increase the opportunity for generating cell lines and to make it feasible to follow the process of tumorigenesis, we developed a mouse strain that expresses SV40 T-antigens in response to cre-recombinase. Using CRISPR/Cas9 we inserted an 8 kb cassette with coding sequences for SV40 T-antigens and IRES-GFP into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26LSL-SV40-GFP. 30% of the progeny born from hybrid zygotes injected with template DNA, CRISPR/Cas9, and sgRNA had correctly targeted the Rosa26 locus. These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. The majority of Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice developed dwarfism and large tumors by 4 wks. The pituitaries of Rosa26LSL-SV40-GFP/+; Tshb-cre mice appear grossly normal at birth, but they are enlarged and showing evidence of increased vascularization by 2 wks. Flow-sorted GFP-positive cells from Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice express Prop1 and TSH, respectively. Tumors from Rosa26LSL-SV40-GFP/+; Tshb-cre mice were adapted to growth in cell culture. We have established a thyrotrope-like cell line that expresses Cga and Pou1f1. These studies demonstrate the utility of the novel, Rosa26LSL-SV40-GFP mouse line for reliable targeted oncogenesis and development of unique cell lines. The authors have nothing to disclose.