PMCID stringclasses 30 values | sentence stringlengths 1 1.04k | entities listlengths 0 22 |
|---|---|---|
PMC10158546 | We therefore hypothesized that overexpressing different PrP versions in neural progenitor cells prior to differentiation would serve as a useful model for prion infection. | [] |
PMC10158546 | An interchangeable system like this could permit characterization of a wide range of prion strains and might serve as a platform to examine the effects of targeted genetic manipulations on prion infection. | [] |
PMC10158546 | Accordingly, here we describe, the lentivirus-delivered expression of PrP transgenes in an immortalized human neural progenitor cell line (ReN VM) that had been ablated for its endogenous PrP expression (ReN PRNP cells). | [
{
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"label": "CellLine",
"start": 139,
"text": "ReN VM"
},
{
"end": 212,
"label": "CellLine",
"start": 204,
"text": "ReN PRNP"
}
] |
PMC10158546 | We chose ReN VM cells because they have previously been shown to replicate misfolded amyloid-β and phosphorylated tau, serving as a model of Alzheimer’s disease [25–28]. | [
{
"end": 15,
"label": "CellLine",
"start": 9,
"text": "ReN VM"
}
] |
PMC10158546 | ReN cell lines were produced that express human, hamster and mouse version of PrP and were differentiated into TUBB3 neurons and GFAP astrocytes prior to challenge with four prion isolates. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Prion replication was assessed through repeated measurements of amyloid seeding activity in 6-week time course experiments. | [] |
PMC10158546 | Strikingly, these ReN cell-derived cultures were incapable of replicating multiple prion isolates despite the high level of PrP expression. | [
{
"end": 21,
"label": "CellLine",
"start": 18,
"text": "ReN"
}
] |
PMC10158546 | We explored the use of the ReN VM human neural progenitor cell line as a platform for modelling replication of human prions in vitro. | [
{
"end": 33,
"label": "CellLine",
"start": 27,
"text": "ReN VM"
}
] |
PMC10158546 | First, we characterized the wildtype ReN VM cell line (denoted here as ReN WT) that possesses the intact human PRNP gene with the MV polymorphism at codon 129. | [] |
PMC10158546 | Upon growth factor removal, ReN progenitor cells lose their ability to proliferate and instead differentiate into glial and neuronal cell types, coinciding with morphological changes as seen in the cultures [29–32]. | [] |
PMC10158546 | When differentiated as 2D monolayers, ReN cells take on a predominantly neuronal morphology with rounded cell bodies and increasing numbers of neuronal projections over at least 3 weeks in vitro (Figure 1a,b). | [
{
"end": 41,
"label": "CellLine",
"start": 38,
"text": "ReN"
}
] |
PMC10158546 | By embedding ReN cells within Matrigel, they can also be differentiated as thin-3D cultures. | [
{
"end": 16,
"label": "CellLine",
"start": 13,
"text": "ReN"
}
] |
PMC10158546 | We found ReN cells within these thin 3D cultures to self-arrange into spheroid-like clusters of cells (Figure 1c), similar to 3D neurospheroids produced by culturing ReN cells in low-attachment round-bottomed plates or microwell chips . | [
{
"end": 12,
"label": "CellLine",
"start": 9,
"text": "ReN"
},
{
"end": 169,
"label": "CellLine",
"start": 166,
"text": "ReN"
}
] |
PMC10158546 | The morphology of ReN progenitor cells was confirmed by immunofluorescence staining of NES at day 0 of differentiation (Figure 1d), while the morphology of ReN neurons and astrocytes was examined by staining for TUBB3 and GFAP at day 28 post-differentiation (Figure 1e). | [] |
PMC10158546 | Figure 1.In vitro characteristics of ReN human neural progenitor-derived cultures. ( | [] |
PMC10158546 | and (b) ReN neural progenitor cells differentiate into cultures with neuronal morphology over 21 DIV. ( | [
{
"end": 11,
"label": "CellLine",
"start": 8,
"text": "ReN"
}
] |
PMC10158546 | ReN cells can also be differentiated as thin 3D cultures embedded within Matrigel matrix. ( | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Representative Z-stacked projection of ReN neural progenitor cells stained with Nestin (NES; green) before differentiation. ( | [] |
PMC10158546 | Representative Z-stacked projection of neurons and astrocytes in thin-3D ReN cells at day 28 post-standard differentiation. | [
{
"end": 76,
"label": "CellLine",
"start": 73,
"text": "ReN"
}
] |
PMC10158546 | Neurons were stained with beta-III-tubulin (TUBB3; red), astrocytes were stained with glial fibrillary acidic protein (GFAP; green) and nuclei were counterstained using DAPI (Blue). | [] |
PMC10158546 | Immunofluorescence images were acquired using the 63X oil immersion objective of a Zeiss LSM 700 instrument (scale bar = 10 µm). ( | [] |
PMC10158546 | ReN cells express markers of neurons (TUBB3) and astrocytes (GFAP), neural progenitors (NES) and PrP throughout standard differentiation. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | NeuN expression was also detected in ReN lysate at day 0 StdD. In vitro characteristics of ReN human neural progenitor-derived cultures. ( | [] |
PMC10158546 | and (b) ReN neural progenitor cells differentiate into cultures with neuronal morphology over 21 DIV. ( | [
{
"end": 11,
"label": "CellLine",
"start": 8,
"text": "ReN"
}
] |
PMC10158546 | ReN cells can also be differentiated as thin 3D cultures embedded within Matrigel matrix. ( | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Representative Z-stacked projection of ReN neural progenitor cells stained with Nestin (NES; green) before differentiation. ( | [] |
PMC10158546 | Representative Z-stacked projection of neurons and astrocytes in thin-3D ReN cells at day 28 post-standard differentiation. | [
{
"end": 76,
"label": "CellLine",
"start": 73,
"text": "ReN"
}
] |
PMC10158546 | Neurons were stained with beta-III-tubulin (TUBB3; red), astrocytes were stained with glial fibrillary acidic protein (GFAP; green) and nuclei were counterstained using DAPI (Blue). | [] |
PMC10158546 | Immunofluorescence images were acquired using the 63X oil immersion objective of a Zeiss LSM 700 instrument (scale bar = 10 µm). ( | [] |
PMC10158546 | ReN cells express markers of neurons (TUBB3) and astrocytes (GFAP), neural progenitors (NES) and PrP throughout standard differentiation. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | NeuN expression was also detected in ReN lysate at day 0 StdD. To examine the kinetics of ReN cell differentiation over time, we also tracked protein expression through western blotting of neuronal and astrocytic markers in lysate collected at days 0, 7, 14 and 21 post-differentiation (Figure 1f). | [
{
"end": 93,
"label": "CellLine",
"start": 90,
"text": "ReN"
}
] |
PMC10158546 | As anticipated, the expression of neuronal marker TUBB3 and astrocytic marker GFAP increased by day 7 in vitro. | [] |
PMC10158546 | We also found the expression of PrP to increase steadily upon differentiation, starting at day 7 in vitro. | [] |
PMC10158546 | Unexpectedly, we observed weak expression of NeuN (mature neuron marker) by the ReN progenitor cells at day 0, and this expression was quickly lost upon differentiation. | [] |
PMC10158546 | We also failed to detect expression of synaptic marker SYN1, or oligodendrocyte marker OLIG2 by ReN cells via western blotting at any time-point post differentiation (supplementary figure S1). | [
{
"end": 99,
"label": "CellLine",
"start": 96,
"text": "ReN"
}
] |
PMC10158546 | Taken together, while differentiated ReN cells expressed markers of neurons and astrocytes, in our hands they did not fully mature into neurons that express markers of synaptic signalling. | [
{
"end": 40,
"label": "CellLine",
"start": 37,
"text": "ReN"
}
] |
PMC10158546 | Consistent and stable overexpression of PrP is an important consideration for developing in vitro models of prion replication. | [] |
PMC10158546 | Lentivirus transduction proved effective for delivering PrP transgenes and producing ReN cell lines that express different versions of PrP. Prior to lentivirus transduction, we employed CRISPR-mediated knockout to disrupt the sequence of PRNP in the wildtype ReN cell line. | [
{
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"label": "CellLine",
"start": 85,
"text": "ReN"
},
{
"end": 262,
"label": "CellLine",
"start": 259,
"text": "ReN"
}
] |
PMC10158546 | This resulted in a ReN VM PRNP cell line (denoted here ReN KO) that is deficient in endogenous PrP expression and was subsequently transduced with different lentivirus constructs. | [
{
"end": 25,
"label": "CellLine",
"start": 19,
"text": "ReN VM"
}
] |
PMC10158546 | Puromycin antibiotic selection was next used to enrich for successfully transduced cells and establish stable cell lines (Figure 2a,b). | [] |
PMC10158546 | Finally, successful transduction was confirmed via fluorescence microscopy of GFP expression in lentivirus-transduced ReN cells (Figure 2c). | [
{
"end": 121,
"label": "CellLine",
"start": 118,
"text": "ReN"
}
] |
PMC10158546 | According to this method, we produced ReN cell lines that express the mouse, hamster, human-129 M and human-129 V versions of PrP (Table 1). | [] |
PMC10158546 | To confirm expression of the appropriate version of PrP, we western blotted lysate from each cell line (grown as proliferating neural progenitor cells) using the 6H4 and 3F4 monoclonal antibodies, which recognize human/mouse PrP and human/hamster PrP, respectively (Figure 3A). | [] |
PMC10158546 | We found that the monoclonal antibodies recognized PrP from the appropriate cell lines and that the transduced cells overexpressed PrP compared to the WT cells. | [] |
PMC10158546 | The ReN 129 M and 129 V cell lines had very similar levels of PrP expression (Figure 3a). | [] |
PMC10158546 | We attributed differences in signal between the mouse, hamster and human PrP overexpressing cells to differences between antibody-epitope binding affinities, and could not distinguish true differences in protein abundance. | [] |
PMC10158546 | Figure 2.Enrichment of GFP expressing transduced ReN cells using puromycin antibiotic selection. ( | [
{
"end": 52,
"label": "CellLine",
"start": 49,
"text": "ReN"
}
] |
PMC10158546 | Kill curve with puromycin antibiotic on non-treated ReN cells revealed a concentration of 0.5 µg/mL was sufficient to kill all cells after 48 hours. ( | [
{
"end": 55,
"label": "CellLine",
"start": 52,
"text": "ReN"
}
] |
PMC10158546 | Lentiviral-transduced ReN cells can be selected with puromycin antibiotic treatment. | [
{
"end": 25,
"label": "CellLine",
"start": 22,
"text": "ReN"
}
] |
PMC10158546 | ReN cells were treated with 1 μL of mouse PrP expressing lentiviral prep (LV-prep) for 72 hours, with or without TransDux enhancer, at which point the cells were treated with 0.5 μg/mL puromycin. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Images were taken with a phase contrast microscope 3 days following puromycin treatment. ( | [] |
PMC10158546 | GFP expression in ReN cells before and after lentiviral transduction. | [
{
"end": 21,
"label": "CellLine",
"start": 18,
"text": "ReN"
}
] |
PMC10158546 | Cells were fixed and stained with DAPI before imaging for GFP via fluorescence microscopy. | [] |
PMC10158546 | Images were acquired using the 40X oil objective of a Zeiss LSM 700 instrument (scale bar = 50 µm). | [] |
PMC10158546 | Figure 3.Lentiviral-transduced ReN cell lines overexpress PrP and differentiate into neurons and astrocytes. ( | [
{
"end": 34,
"label": "CellLine",
"start": 31,
"text": "ReN"
}
] |
PMC10158546 | Lentiviral-transduced ReN cell lines were produced that overexpress the mouse, hamster and human 129M and 129V versions of PrP. Lysate from proliferating ReN progenitor cell lines was western blotted for PrP using the 6H4 (recognizes mouse and human PrP) and 3F4 (recognizes hamster and human PrP) monoclonal antibodies. | [] |
PMC10158546 | Total protein signal was measured in Bio-Rad TGX stain-free gels according to manufacturer’s instructions. ( | [] |
PMC10158546 | the 129M and WT ReN cell lines exhibit a similar pattern of protein expression for PrP, TUBB3 and GFAP throughout the process of differentiation into neurons and astrocytes. | [] |
PMC10158546 | ReN cells were differentiated as thin-3D cultures and lysate collected at weekly timepoints post-differentiation was western blotted for PrP (via 3F4 mAb), TUBB3 and GFAP. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Signal was normalized to the most prominent band in the corresponding total protein image (see arrow). ( | [] |
PMC10158546 | Normalized protein expression of PrP, TUBB3 and GFAP in ReN WT and 129M cells throughout standard differentiation. | [
{
"end": 59,
"label": "CellLine",
"start": 56,
"text": "ReN"
},
{
"end": 71,
"label": "CellLine",
"start": 67,
"text": "129M"
}
] |
PMC10158546 | Signal intensity was quantified using imageJ and normalized to the signal from the strongest band identified in total protein images. ( | [] |
PMC10158546 | qPCR-based quantification of GFAP, TUBB3 and PRNP of ReN 129M, WT and Empty cells over 4 weeks of differentiation. | [
{
"end": 61,
"label": "CellLine",
"start": 53,
"text": "ReN 129M"
}
] |
PMC10158546 | PRNP was quantified using PCR primers that bind the non-coding mRNA region (PRNP) and protein-coding region of the mRNA (PRNP_CDS). * | [] |
PMC10158546 | p-value<0.05 (calculated using one-way ANOVAs). | [] |
PMC10158546 | Table 1.Summary of ReN cell lines. | [] |
PMC10158546 | WT – wild type; KO – PRNP knocked out; NPC – neural progenitor cell; PuroR – puromycin resistance; GFP – green fluorescence protein. | [] |
PMC10158546 | ReN Cell lineParent cell linePrP sequencePrP expressionTransgeneWTPrimary NPCsHuman 129MVPrPNoneKOWTN/APrPNoneEmptyKON/APrPEF1-(empty)-PGK-GFP-T2A-PuroRMmuKOMousePrPEF1-(mmu)PRNP-PGK-GFP-T2A-PuroRHamKOHamsterPrPEF1-(ham)PRNP-PGK-GFP-T2A-PuroR129MKOHuman 129MPrPEF1-(129M)PRNP-PGK-GFP-T2A-PuroR129VKOHuman 129VPrPEF1-(129V)PRNP-PGK-GFP-T2A-PuroR Enrichment of GFP expressing transduced ReN cells using puromycin antibiotic selection. ( | [] |
PMC10158546 | Kill curve with puromycin antibiotic on non-treated ReN cells revealed a concentration of 0.5 µg/mL was sufficient to kill all cells after 48 hours. ( | [
{
"end": 55,
"label": "CellLine",
"start": 52,
"text": "ReN"
}
] |
PMC10158546 | Lentiviral-transduced ReN cells can be selected with puromycin antibiotic treatment. | [
{
"end": 25,
"label": "CellLine",
"start": 22,
"text": "ReN"
}
] |
PMC10158546 | ReN cells were treated with 1 μL of mouse PrP expressing lentiviral prep (LV-prep) for 72 hours, with or without TransDux enhancer, at which point the cells were treated with 0.5 μg/mL puromycin. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Images were taken with a phase contrast microscope 3 days following puromycin treatment. ( | [] |
PMC10158546 | GFP expression in ReN cells before and after lentiviral transduction. | [
{
"end": 21,
"label": "CellLine",
"start": 18,
"text": "ReN"
}
] |
PMC10158546 | Cells were fixed and stained with DAPI before imaging for GFP via fluorescence microscopy. | [] |
PMC10158546 | Images were acquired using the 40X oil objective of a Zeiss LSM 700 instrument (scale bar = 50 µm). | [] |
PMC10158546 | Lentiviral-transduced ReN cell lines overexpress PrP and differentiate into neurons and astrocytes. ( | [
{
"end": 25,
"label": "CellLine",
"start": 22,
"text": "ReN"
}
] |
PMC10158546 | Lentiviral-transduced ReN cell lines were produced that overexpress the mouse, hamster and human 129M and 129V versions of PrP. Lysate from proliferating ReN progenitor cell lines was western blotted for PrP using the 6H4 (recognizes mouse and human PrP) and 3F4 (recognizes hamster and human PrP) monoclonal antibodies. | [] |
PMC10158546 | Total protein signal was measured in Bio-Rad TGX stain-free gels according to manufacturer’s instructions. ( | [] |
PMC10158546 | the 129M and WT ReN cell lines exhibit a similar pattern of protein expression for PrP, TUBB3 and GFAP throughout the process of differentiation into neurons and astrocytes. | [] |
PMC10158546 | ReN cells were differentiated as thin-3D cultures and lysate collected at weekly timepoints post-differentiation was western blotted for PrP (via 3F4 mAb), TUBB3 and GFAP. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Signal was normalized to the most prominent band in the corresponding total protein image (see arrow). ( | [] |
PMC10158546 | Normalized protein expression of PrP, TUBB3 and GFAP in ReN WT and 129M cells throughout standard differentiation. | [
{
"end": 59,
"label": "CellLine",
"start": 56,
"text": "ReN"
},
{
"end": 71,
"label": "CellLine",
"start": 67,
"text": "129M"
}
] |
PMC10158546 | Signal intensity was quantified using imageJ and normalized to the signal from the strongest band identified in total protein images. ( | [] |
PMC10158546 | qPCR-based quantification of GFAP, TUBB3 and PRNP of ReN 129M, WT and Empty cells over 4 weeks of differentiation. | [
{
"end": 61,
"label": "CellLine",
"start": 53,
"text": "ReN 129M"
}
] |
PMC10158546 | PRNP was quantified using PCR primers that bind the non-coding mRNA region (PRNP) and protein-coding region of the mRNA (PRNP_CDS). * | [] |
PMC10158546 | p-value<0.05 (calculated using one-way ANOVAs). | [] |
PMC10158546 | Summary of ReN cell lines. | [] |
PMC10158546 | WT – wild type; KO – PRNP knocked out; NPC – neural progenitor cell; PuroR – puromycin resistance; GFP – green fluorescence protein. | [] |
PMC10158546 | To identify any effects of lentiviral transduction on ReN cell differentiation, we also compared protein and RNA expression of PrP, TUBB3 and GFAP longitudinally between ReN WT and 129 M cells throughout 4 weeks of differentiation (Figure 3c,d). | [
{
"end": 57,
"label": "CellLine",
"start": 54,
"text": "ReN"
}
] |
PMC10158546 | In the case of qPCR, we additionally examined the ReN Empty cell line. | [] |
PMC10158546 | Semi-quantitative analysis of western blot and qPCR data revealed increased TUBB3 abundance in all cell lines beginning at day 7 post differentiation (Figure 3c,d), consistent with differentiation into neurons and astrocytes. | [] |
PMC10158546 | The overall pattern of TUBB3 and GFAP expression was similar between the WT and 129 M cell lines throughout differentiation, although we noted that GFAP abundance decreased consistently in 129 M cells compared to WT (Figure 3c,d). | [] |
PMC10158546 | We also confirmed continuous PrP overexpression throughout differentiation in ReN 129 M cells and found that PrP expression increased over time post-differentiation in ReN 129 M and WT cells (Figure 3c,d). | [
{
"end": 87,
"label": "CellLine",
"start": 78,
"text": "ReN 129 M"
},
{
"end": 177,
"label": "CellLine",
"start": 168,
"text": "ReN 129 M"
}
] |
PMC10158546 | To characterize PrP localization in lentivirus-transduced ReN neurons and astrocytes, we stained ReN WT, KO and 129 M cells for PrP, TUBB3 and GFAP and visualized them via immunofluorescence at day 7 and day 21 post-differentiation (Figure 4). | [
{
"end": 61,
"label": "CellLine",
"start": 58,
"text": "ReN"
},
{
"end": 100,
"label": "CellLine",
"start": 97,
"text": "ReN"
}
] |
PMC10158546 | Due to the weak expression of PrP in the WT cells, we failed to detect any PrP signal in these cells via immunofluorescence. | [] |
PMC10158546 | However, we detected PrP in the 129 M cells and observed increased expression on day 21 compared to day 7 of differentiation, consistent with the western blot and qPCR data. | [
{
"end": 35,
"label": "CellLine",
"start": 32,
"text": "129"
}
] |
PMC10158546 | When the thin-3D method of differentiation was employed (Figure 1c), we noticed that the ReN cells arranged into 3D spheroid-like clusters of TUBB3 neurons separated by a monolayer of GFAP glial cells (assuming enough cells are seeded on the coverslip). | [] |
PMC10158546 | PrP staining was highly associated with these spheroids of TUBB3 neurons in the 129 M cells, and the staining pattern suggested localization to the cell membrane (Figure 4). | [] |
PMC10158546 | Conversely, little PrP signal was detected in GFAP astrocytes. | [] |
PMC10158546 | Thus, we concluded that lentiviral transduction primarily resulted in PrP overexpression within TUBB3 neurons of differentiated ReN cultures. | [
{
"end": 131,
"label": "CellLine",
"start": 128,
"text": "ReN"
}
] |
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