Long Term Gene Expression in Human Induced Pluripotent Stem Cells and Cerebral Organoids to Model a Neurodegenerative Disease

Abstract

Human brain organoids (mini-brains) consist of self-organized three-dimensional (3D) neural tissue which can be derived from reprogrammed adult cells and maintained for months in culture. These 3D structures manifest substantial potential for the modeling of neurodegenerative diseases and pave the way for personalized medicine. However, as these 3D brain models can express the whole human genetic complexity, it is critical to have access to isogenic mini-brains that only differ in specific and controlled genetic variables. Genetic engineering based on retroviral vectors is incompatible with the long-term modeling needed here and implies a risk of random integration while methods using CRISPR-Cas9 are still too complex to adapt to stem cells. We demonstrate in this study that our strategy which relies on an episomal plasmid vector derived from the Epstein-Barr virus (EBV) offers a simple and robust approach, avoiding the remaining caveats of mini-brain models. For this proof-of-concept, we used a normal tau protein with a fluorescent tag and a mutant genetic form (P301S) leading to Fronto-Temporal Dementia. Isogenic cell lines were obtained which were stable for more than 30 passages expressing either form. We show that the presence of the plasmid in the cells does not interfere with the mini-brain differentiation protocol and obtain the development of a pathologically relevant phenotype in cerebral organoids, with pathological hyperphosphorylation of the tau protein. Such a simple and versatile genetic strategy opens up the full potential of human organoids to contribute to disease modeling, personalized medicine and testing of therapeutics.

Keywords: Alzheimer; IPS; cerebral organoid; disease modeling; fronto-temporal dementia; neurodegenerative disease; stem cells.

Figure 1

Generation of modified induced pluripotent stem cells (iPSCs) using an epstein-barr virus (EBV)-based plasmid. (A) Map of the EBV-based plasmid used in iPSCs, transgene expression under CAG promoter regulation, puromycin resistance under SV40 promoter regulation. (B) Workflow for EBV-based plasmid expressing iPSCs generation. (C) iPSCs colony after 30 passages post-electroporation expressing either wild type Tau 2N4R or mutated (P301S) form fused to YFP.

Figure 2

Pluripotency characterization of transgenic iPSCs. (A) Assessment of pluripotency by semi-quantitative PCR to confirm the expression of pluripotency markers in the control iPS line BJ and in EBV-based plasmid expressing lines. PCR analysis shows the expression of pluripotency markers OCT4, SOX2, NANOG and REX-1. BJ fibroblasts were used as a control and RPLP0 as a housekeeping gene. (B) H&E staining of a section of teratomas obtained from iPSCs expressing either Tau WT or P301S using an EBV-based plasmid showing the development of structures specific to the three germ layers.

Figure 3

MiniBrain generation with modified iPSCs for pathological modeling. (A) Diagram showing the major steps of the MiniBrain protocol from iPSCs to cerebral organoids. (B) Real-time PCR analysis shows the expression of different neural markers at different time points in the cerebral organoid formation protocol, MAP-2 for neuron evaluation, GFAP for astrocyte evaluation and Olig-2 for oligodendrocyte evaluation. (C) Tissue sections of a 30-day cerebral organoid showing the development of neural rosettes with a radial organization, as shown with vimentin staining. Sox-2 staining reveals a neural stem cell niche, from which a neuronal network is developing as can be seen with the Tuj1 staining.

Figure 4

FTD pathological modeling using modified iPSCs expressing an EBV-based plasmid. (A) Western Blot analysis of total tau protein (tTau, TAU5 antibody) and phosphorylated tau protein (pTau, AT8 antibody). The expression of a fusion protein enables the endogenously expressed tau protein to be distinguished from the exogeneous form expressed using the EBV-based plasmid. (B) Ratio of phosphorylated tau protein over total tau against GAPDH for endogenous and exogenous tau. Statistical analysis: one-way analysis of variance (ANOVA; multiple groups). On charts *p < 0.05.