Detecting Tau Aggregates using iPSC-Derived Cortical Neurons

This article explains a powerful, scalable and disease-relevant model of tau aggregation using iPSC-derived cortical neurons that can be employed in drug discovery programs in neurodegeneration.

The ensuing assay is highly reproducible across users and operates in various commercially available iPSC-lines, offering a reliable tool for a deeper understanding of tau pathophysiology and for identifying new therapies against Alzheimer’s Disease (AD).

Abstract

Tau aggregation is the pathological feature that associates well with the advancement of AD. The occurrence of neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau, results in neuronal dysfunction and loss, and is directly related to the cognitive decline seen in AD patients.

The partial success in targeting β-amyloid pathologies has strengthened the hypothesis of inhibiting tau phosphorylation, aggregation and/or spreading as alternative therapeutic entry points to combat AD. Disease-relevant and scalable assays that can reproduce important features of the pathology in an in vitro setting are needed to recognize new treatments.

In this article, induced pluripotent stem cells (iPSCs) were used as a practically unlimited source of human cortical neurons to create a powerful and scalable tau aggregation model adaptable with high-throughput screening (HTS). The cell culture conditions were downscaled to 384-well plate format and diluted matrigel was used to introduce an additional physical barrier that protects against cell detachment and decreases stress associated with plate handling.

The assay was complemented with AlphaLISA technology for the identification of tau aggregates in a high-throughput-compatible format. The assay can be reproduced across users and works with various commercially available iPSC lines, representing a robust translational tool for discovering new therapies against tauopathies such as AD.

Methods

iPSC and Neural Differentiation

Two commercially available cell lines obtained from clinically unaffected donors were used—ChiPSC6b_m1 (Cellectis) and IPSC0028 (Sigma). Axol Biosciences differentiated iPSCs into Neuronal Progenitor Cells (NPCs) by following previously reported protocols (Shi et al., 2012).

Induction of Tau Aggregation

Tau aggregation was performed by adapting already reported protocol (Guo et al., 2011) with minor changes. NPCs were transduced with adenovirus driving expression of human P301L mutant tau type under the human synapsin promoter. Seeding with fibrils was carried out by incorporating recombinant truncated tau comprising four microtubule-binding repeats with P301L mutation (k18) at a concentration of 50 nM per 96-well/12-wells, and 133 nM per 384 well.

AlphaLISA

The non-wash ELISA alternative, AlphaLISA® technology, is a bead-based sensitive technique used for investigating biomolecular interactions. Highly specific antibodies (hTAU10, HT7) coated on beads mediate binding to the target analyte, that is, TAU.

To identify aggregated Tau, biotin, as well as acceptor beads, are conjugated to the same antibody (hTAU10). There should be a distance of ≤200 nm between the Donor and Acceptor bead so that energy transfer can occur and a quantifiable luminescent signal is produced (Calafate et al., 2015).

Results

Healthy Cortical Neurons can be Differentiated in 384-Well Plates for at Least 5 Weeks

The scheme in (A) represents the protocol employed for the amplification (first 30 days) and final differentiation of NPCs. After neuronal induction and expansion, NPCs are frozen. After thawing, NPCs are sub-plated in 384/96 well plates using diluted matrigel. Numbers depict days in culture (in brackets, the number of days after thawing.) (B) cortical identity (CTIP2) and early maturation markers (Tuj1) are expressed since day 7 of culture. Typical synaptic markers as synapsin (C) and VGlut1 (D) are present at 18 and 30 days, respectively. MAP2/TBR1 staining exhibits complex connectivity network of glutamatergic cortical neurons after 30 days of differentiation (E). (F) Cells maintained in culture for 39 days exhibit metabolic activity similar to cells cultured for 22 days (measured by quantifying ATP, CelltiterGlo), showing no alteration in cell viability (n=4 independent experiments). (G) no considerable difference was noticed in cell viability in the two cell lines tested in this study after 35 days in culture. Scale bars: 10 μm in B, 20 μm in C-D, and 40 nm in E. DIV: days in vitro.

The scheme in (A) represents the protocol employed for the amplification (first 30 days) and final differentiation of NPCs. After neuronal induction and expansion, NPCs are frozen. After thawing, NPCs are sub-plated in 384/96 well plates using diluted matrigel. Numbers depict days in culture (in brackets, the number of days after thawing.) (B) cortical identity (CTIP2) and early maturation markers (Tuj1) are expressed since day 7 of culture. Typical synaptic markers as synapsin (C) and VGlut1 (D) are present at 18 and 30 days, respectively. MAP2/TBR1 staining exhibits complex connectivity network of glutamatergic cortical neurons after 30 days of differentiation (E). (F) Cells maintained in culture for 39 days exhibit metabolic activity similar to cells cultured for 22 days (measured by quantifying ATP, CelltiterGlo), showing no alteration in cell viability (n=4 independent experiments). (G) no considerable difference was noticed in cell viability in the two cell lines tested in this study after 35 days in culture. Scale bars: 10 μm in B, 20 μm in C-D, and 40 nm in E. DIV: days in vitro. n=3 independent experiments, One-way Anova. ns, no significant ,*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Dunnett’s multiple comparison test versus non-seeded control).

Tau Aggregation Model Using Human Cortical Neurons

The scheme in (A) represents the protocol followed. 3R endogenous (c-) and 4R overexpressed (P301L) types of tau detected with 3R-specific (RD3) and total (HT7) tau antibodies, respectively. Insoluble aggregated tau is detected by IF after fixation with 4% PFA–1% Triton and staining with AT8 (C) and MC1 (E) only in seeded (+k18) samples. WB in (F) represents hyperphosphorylated forms of tau in total cell extracts detected by AT8. Observe the presence of typical high molecular weight band (>90 kDa) and smear corresponding to aggregated tau detected both with AT8 and HT7 antibodies after triton extraction (*). A 50% reduction in cell viability is noticed in transduced and seeded cells (H) but not in cells only overexpressing the transgene (G). RFU, relative fluorescence units. Scale bar: 20 µm. AAV: adenovirus,

The scheme in (A) represents the protocol followed. 3R endogenous (c-) and 4R overexpressed (P301L) types of tau detected with 3R-specific (RD3) and total (HT7) tau antibodies, respectively. Insoluble aggregated tau is detected by IF after fixation with 4% PFA–1% Triton and staining with AT8 (C) and MC1 (E) only in seeded (+k18) samples. WB in (F) represents hyperphosphorylated forms of tau in total cell extracts detected by AT8. Observe the presence of typical high molecular weight band (>90 kDa) and smear corresponding to aggregated tau detected both with AT8 and HT7 antibodies after triton extraction (*). A 50% reduction in cell viability is noticed in transduced and seeded cells (H) but not in cells only overexpressing the transgene (G). RFU, relative fluorescence units. Scale bar: 20 µm. AAV: adenovirus,

The scheme in (A) represents the protocol followed. 3R endogenous (c-) and 4R overexpressed (P301L) types of tau detected with 3R-specific (RD3) and total (HT7) tau antibodies, respectively. Insoluble aggregated tau is detected by IF after fixation with 4% PFA–1% Triton and staining with AT8 (C) and MC1 (E) only in seeded (+k18) samples. WB in (F) represents hyperphosphorylated forms of tau in total cell extracts detected by AT8. Observe the presence of typical high molecular weight band (>90 kDa) and smear corresponding to aggregated tau detected both with AT8 and HT7 antibodies after triton extraction (*). A 50% reduction in cell viability is noticed in transduced and seeded cells (H) but not in cells only overexpressing the transgene (G). RFU, relative fluorescence units. Scale bar: 20 µm. AAV: adenovirus, n=3 independent experiments, One-way Anova. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Dunnett’s multiple comparison test versus non seeded control).

AlphaLISA Technology to Quantify Tau Aggregation

Antibody validation to measure aggregated and total Tau using HEK cells (A, C, and D) and primary neurons (B, D, and F). hTAU10/hTAU10 and AT8/AT8 were selected for aggregate quantification based on the specific detection of aggregates in transduced and seeded cells (A–D, red). HT7/hTAU10 detected Tau overexpression but no difference between seeded and non-seeded (green) cells, making it more suitable for measurement of total Tau (E-F). (G–H) represent the dose-response curves acquired from the same sample by AlphaLISA of total extracts and western blot of the sarkosyl-insoluble fraction, respectively. The computation between both signals is plotted in I, representing a considerable correlation (Spearman

Antibody validation to measure aggregated and total Tau using HEK cells (A, C, and D) and primary neurons (B, D, and F). hTAU10/hTAU10 and AT8/AT8 were selected for aggregate quantification based on the specific detection of aggregates in transduced and seeded cells (A–D, red). HT7/hTAU10 detected Tau overexpression but no difference between seeded and non-seeded (green) cells, making it more suitable for measurement of total Tau (E-F). (G–H) represent the dose-response curves acquired from the same sample by AlphaLISA of total extracts and western blot of the sarkosyl-insoluble fraction, respectively. The computation between both signals is plotted in I, representing a considerable correlation (Spearman r=0.09429, p=0.0167). Total Tau levels are not impacted by the increase of K18 concentration as detected by western blot with AT8 and HT7 in total (LSS) and a sarkosyl-soluble fraction (HSS) (B). Only the sarkosyl-insoluble fraction (SP) increases (grey arrow). Dox: doxycicline. OD, optic density. n=3 technical replicates, Two-way Anova. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Dunnett’s multiple comparison test versus non-seeded control).

Quantification of Tau Aggregates in Human Neurons

(A) represents baseline values of the assay defined by the counts received in all negative control used (i.e. non transduced cells, EGFP transduced cells, cells transduced with P301L but not seeded with K18 and cells only seeded with K18). In (B), HT7/hTAU10 AlphaLISA confirming the overexpression of Tau in transduced cells. Optimal sampling time was defined at 35 days as hTAU10/hTAU10 signal offers the highest value in the shortest time after reaching a plateau (C). The levels of total tau do not increase after 28 days of differentiation (D). Tau aggregates are identified with hTAU10/hTAU10 (E) and AT8/AT8 (F) in a dose-dependent manner. The signal-to-basal ratio values obtained from 384-well assay plates after 35 days offer a Z’ factor of 0’52 (G). A shorter version of the protocol also offers important values for Tau aggregation as soon as 22 days post-thawing (H). (C) untreated cells; k18 cells seeded with P301L-mutant fibrils; EGFP, transduced cells overexpressing EGFP; EGFP+k18, cells transduced with EGFP and seeded with k18; P301L-EGFP+k18, cells overexpressing P301L and EGFP; P301L+k18, cells overexpressing P301L and seeded with K18 fibrils.

(A) represents baseline values of the assay defined by the counts received in all negative control used (i.e. non transduced cells, EGFP transduced cells, cells transduced with P301L but not seeded with K18 and cells only seeded with K18). In (B), HT7/hTAU10 AlphaLISA confirming the overexpression of Tau in transduced cells. Optimal sampling time was defined at 35 days as hTAU10/hTAU10 signal offers the highest value in the shortest time after reaching a plateau (C). The levels of total tau do not increase after 28 days of differentiation (D). Tau aggregates are identified with hTAU10/hTAU10 (E) and AT8/AT8 (F) in a dose-dependent manner. The signal-to-basal ratio values obtained from 384-well assay plates after 35 days offer a Z’ factor of 0’52 (G). A shorter version of the protocol also offers important values for Tau aggregation as soon as 22 days post-thawing (H). (C) untreated cells; k18 cells seeded with P301L-mutant fibrils; EGFP, transduced cells overexpressing EGFP; EGFP+k18, cells transduced with EGFP and seeded with k18; P301L-EGFP+k18, cells overexpressing P301L and EGFP; P301L+k18, cells overexpressing P301L and seeded with K18 fibrils. n=3 biological replicates in (A–D) and (H); n=6 technical replicates in E–F. One-way Anova. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Dunnett’s multiple comparison test versus non-seeded control).

Tau Aggregation Assay is Reproducible in Different Lines and Across Users

(A) illustrates positive Tau aggregation values when the protocol is applied to the ChiPSC6b iPSC line. In (B), hTAU10/hTAU10 normalized data acquired from different users using 96-well format and 20.000 cells per well.

(A) illustrates positive Tau aggregation values when the protocol is applied to the ChiPSC6b iPSC line. In (B), hTAU10/hTAU10 normalized data acquired from different users using 96-well format and 20.000 cells per well. n=3 biological replicates in (A); each plot in (B) represents a minimum of 3 technical replicates. (A) One-way Anova. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Tukey’s multiple comparison test).

Conclusions

  • An SOP has been created for measuring tau aggregation in human iPSC-derived cortical neurons differentiated in matrigel
  • The assay operates across users and in various cell lines, solving variability problems usually associated with iPSC technology
  • Due to its adaptability with biochemistry and imaging platforms, it is perfect for high-throughput and high-content screenings

Future Directions

  • Test tau aggregation potential of neurons differentiated from iPSCs obtained from patients carrying tauopathy-related mutations (for example, MAPT IVS10+3 G>A, and others).
  • Optimize protocol for speeding up maturation and for enhancing electrophysiological properties of the neurons.
  • Test alternative scaffolds (for example, chemically defined) with a goal to decrease possible sources of variability and costs.

References

  • Choi, S. H.; Kim, Y. H.; Hebisch, M., et al. A three-dimensional human neural cell culture model of Alzheimer's disease. Nature 2014, 515 (7526), 274-8.
  • Guo, J. L.; Lee, V. M. Seeding of normal Tau by pathological Tau conformers drives pathogenesis of Alzheimer-like tangles. The Journal of Biological Chemistry 2011, 286 (17), 15317-31.
  • Shi, Y.; Kirwan, P.; Smith, J., et al. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nature Neuroscience 2012, 15 (3), 477-86, S1.
  • Calafate, S.; Buist, A.; Miskiewicz, K., et al. Synaptic Contacts Enhance Cell-to-Cell Tau Pathology Propagation. Cell Reports 2015, 11 (8), 1176-83.

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Nov 12, 2019 at 5:40 AM

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