Gene Editing and iPSC Differentiation as Powerful Tools in Modeling Neurological Disease

Neurodegenerative diseases such as Alzheimer’s, Huntington’s and Parkinson’s, as well as other age-associated dementias are incurable and incapacitating illnesses, with Alzheimer’s disease alone responsible for ~60%–70% of cases.

Induced pluripotent stem cells (iPSCs) and gene-editing technology provide an unparalleled biomedical prospect for disease modeling, development of therapeutic strategies, and high-throughput drug screening for such diseases. AXOL Biosciences has produced stable human iPSC lines from healthy human dermal fibroblasts as well as patient-derived fibroblasts.

The fibroblasts were reprogrammed using a non-integrating episomal technique coding for Yamanaka factors (license agreement with iPS Academia Japan) and then separated into neural stem cells (NSCs) to deliver a total modeling solution in a dish.

Using the CRISPR-Cas9 genome-editing technology, AXOL Biosciences has created patient-relevant disease models transporting microtubule-associated protein TAU (MAPT) mutations. Microtubules are important for neuron function, as they facilitate the growth and reliability of axons and dendrites, and transport between the cell body and distant dendrites. Clinically identified missense mutations lower the ability of TAU to boost microtubule assembly, causing neuronal cell death and subsequent disease phenotype.

These renewable and biologically pertinent resources will additionally enable the study of the mechanisms of disease development, with more models applicable to Alzheimer’s disease, Huntingdon’s disease, Parkinson’s disease, and epilepsy being produced to support in the identification of unique drug discovery targets.

iPSC Gene-Editing Using CRISPR

Approach to engineer the three MAPT mutations; MAPT P301L, MAPT V337M, and MAPT R406W. (A) An iPSC line known to differentiate well to NSCs was transfected with plasmids expressing Cas9 nuclease (either wild-type or nickase), a validated gRNA and a donor template containing a selection cassette. Single cell clones were derived and screened for the incorporation of the mutation of interest. The genotype of the resultant iPSC clone was validated by Sanger sequencing and ddPCR. (B) Schematic representation of a donor template vector used for engineering the different MAPT mutations (for example, MAPT P301L). The protospacer adjacent motif (PAM) site was disrupted in all plasmid donor templates to prevent re-cutting by the gRNA.

Approach to engineer the three MAPT mutations; MAPT P301L, MAPT V337M, and MAPT R406W. (A) An iPSC line known to differentiate well to NSCs was transfected with plasmids expressing Cas9 nuclease (either wild-type or nickase), a validated gRNA and a donor template containing a selection cassette. Single cell clones were derived and screened for the incorporation of the mutation of interest. The genotype of the resultant iPSC clone was validated by Sanger sequencing and ddPCR. (B) Schematic representation of a donor template vector used for engineering the different MAPT mutations (for example, MAPT P301L). The protospacer adjacent motif (PAM) site was disrupted in all plasmid donor templates to prevent re-cutting by the gRNA.

Figure 1. Approach to engineer the three MAPT mutations; MAPT P301L, MAPT V337M, and MAPT R406W. (A) An iPSC line known to differentiate well to NSCs was transfected with plasmids expressing Cas9 nuclease (either wild-type or nickase), a validated gRNA and a donor template containing a selection cassette. Single cell clones were derived and screened for the incorporation of the mutation of interest. The genotype of the resultant iPSC clone was validated by Sanger sequencing and ddPCR. (B) Schematic representation of a donor template vector used for engineering the different MAPT mutations (for example, MAPT P301L). The protospacer adjacent motif (PAM) site was disrupted in all plasmid donor templates to prevent re-cutting by the gRNA.

Genotyping Validation and Targeting Efficiency

Genetic validation of generated mutant lines. (A) Schematic representation of genotyping primers used. B) Table indicates the targeting efficiencies of all desired genotypes. (B-D) Sanger sequencing data indicated that six genotypes were achieved, including (C) MAPT (P301L/P301L) and MAPT (P301L/+), (D) MAPT (V337M/V337M) and MAPT (V337M/+), and (E) MAPT (R406W/R406W) and MAPT (R406W/+). Silent mutations added to disrupt binding of the gRNA to the donor sequences are highlighted in red. Copy number variation assessment by digital droplet PCR revealed no off-target integration of the plasmid donor template in any of the validated clones (data not shown).

(B)

Mutation % modified clones* % modified clones sequenced % KI/KI$ % KI/WT$ % KI/KO$
MAPT P301L 87.5 63 33 4 43
MAPT V337M 50 64 16 29 0
MAPT R406W 83 43 29 0 44

 

* Modified clones refers to clones identified by PCR that have incorporated a selection cassette at the right locus.
$ Calculated relative to the number of modified clones sequenced.

Genetic validation of generated mutant lines. (A) Schematic representation of genotyping primers used. B) Table indicates the targeting efficiencies of all desired genotypes. (B-D) Sanger sequencing data indicated that six genotypes were achieved, including (C) MAPT (P301L/P301L) and MAPT (P301L/+), (D) MAPT (V337M/V337M) and MAPT (V337M/+), and (E) MAPT (R406W/R406W) and MAPT (R406W/+). Silent mutations added to disrupt binding of the gRNA to the donor sequences are highlighted in red. Copy number variation assessment by digital droplet PCR revealed no off-target integration of the plasmid donor template in any of the validated clones (data not shown).

Figure 2. Genetic validation of generated mutant lines. (A) Schematic representation of genotyping primers used. B) Table indicates the targeting efficiencies of all desired genotypes. (B-D) Sanger sequencing data indicated that six genotypes were achieved, including (C) MAPT (P301L/P301L) and MAPT (P301L/+), (D) MAPT (V337M/V337M) and MAPT (V337M/+), and (E) MAPT (R406W/R406W) and MAPT (R406W/+). Silent mutations added to disrupt binding of the gRNA to the donor sequences are highlighted in red. Copy number variation assessment by digital droplet PCR revealed no off-target integration of the plasmid donor template in any of the validated clones (data not shown).

AXOL iPSC-Derived Neural Stem Cells

The foundational platform for the CRISPR-Cas9-based MAPT isogenic lines lies with AXOL’s strong and reliable human iPSC-derived NSCs and cerebral cortical neurons (CCNs) technology and procedures. The axolGEMs (Axol Genetically Edited Models) iPSC-Derived NSCs express standard markers such as FOXG1 and PAX6, and automatically develop polarized neural tube-like rosette structures when plated as a monolayer culture or in 3D (Figure 3).

Moreover, AXOL iPSC-Derived NSCs can yield a range of cortical neurons that usually express CTIP2, BRN2, TBR1, and CUX1 (Figure 3), also being electrically active and having the capacity to develop functional synapses and circuits in vitro (Figure 4).

Differentiation and Validation of iPSC-Derived Neural Stem Cells

Differentiation of iPSC-Derived Neural Stem Cells. (A) Neural stem cell and cerebral cortical neuron marker expression. (B) On culturing human iPSC-derived NSCs on top of the RAFT™ collagen matrix (TAP Biosystems/Lonza), cell migration into the matrix was observed. The cells form the 3D structure of commonly seen neural rosettes. Outside of the rosette, there is a matrix of cells ordered in a non-uniform manner (FOXG1, red; nestin, green). (C) On culturing human iPSC-derived CCNs on top of the collagen gel, they formed a uniform static layer of cell bodies. Neurites projected out of these cells and grew downwards creating a network of interconnected neurites (TBR1, red; TUJ1, green).

Figure 3. Differentiation of iPSC-Derived Neural Stem Cells. (A) Neural stem cell and cerebral cortical neuron marker expression. (B) On culturing human iPSC-derived NSCs on top of the RAFT™ collagen matrix (TAP Biosystems/Lonza), cell migration into the matrix was observed. The cells form the 3D structure of commonly seen neural rosettes. Outside of the rosette, there is a matrix of cells ordered in a non-uniform manner (FOXG1, red; nestin, green). (C) On culturing human iPSC-derived CCNs on top of the collagen gel, they formed a uniform static layer of cell bodies. Neurites projected out of these cells and grew downwards creating a network of interconnected neurites (TBR1, red; TUJ1, green).

Whole cell patch clamp recordings. (A) Number of cells recorded that showed evoked action potentials compared to the number of total cells recorded. Three different development stages were analyzed: 10-15 days after plating (DAP) on coverslips, 25-30 DAP and 40-45 DAP. (B) Representative traces of evoked action potentials. (C) Developmental profile of the spike properties of neurons derived from human NSCs. (D) Voltage clamp recording at -70 mV from NSCs at 10-15 DAP – no synaptic currents were detected. (E) 25-30 DAP, some synaptic currents were excitatory postsynaptic currents (EPSCs) and were blocked by the AMPA and kainate receptors blocker CNQX (10 mM). (F) Fully mature neurons at 40-45 DAP showed both EPSCs and inhibitory post-synaptic currents (iPSCs), which could be blocked using a GABAA receptor blocker Gabazine (2 µM).

Figure 4. Whole cell patch clamp recordings. (A) Number of cells recorded that showed evoked action potentials compared to the number of total cells recorded. Three different development stages were analyzed: 10-15 days after plating (DAP) on coverslips, 25-30 DAP and 40-45 DAP. (B) Representative traces of evoked action potentials. (C) Developmental profile of the spike properties of neurons derived from human NSCs. (D) Voltage clamp recording at -70 mV from NSCs at 10-15 DAP – no synaptic currents were detected. (E) 25-30 DAP, some synaptic currents were excitatory postsynaptic currents (EPSCs) and were blocked by the AMPA and kainate receptors blocker CNQX (10 mM). (F) Fully mature neurons at 40-45 DAP showed both EPSCs and inhibitory post-synaptic currents (iPSCs), which could be blocked using a GABAA receptor blocker Gabazine (2 µM).

(Whole cell patch clamp recordings were carried out in collaboration with Ana González Rueda, Ole Paulsen Lab, University of Cambridge)

Summary

The joint technology platforms of both AXOL Bioscience and Horizon Discovery offer an exclusive chance to derive a limitless amount of patient-specific, disease-applicable neural stem cells, which can subsequently produce functional brain tissue for neurological study, drug discovery and screening.

  • Matched set of wild-type and MAPT mutation in the same genetic background permits accurate examination of gene function and the fundamental role in disease establishment or advancement
  • Can be utilized as controls for disease modeling together with patient-centric iPSC-derived neural stem cells and cerebral cortical neurons

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. 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 14, 2019 at 7:09 AM

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