Gene Edited iPSC Lines

Genome-Edited iPSCs for Neuroscience

Genome engineering in induced pluripotent stem cells (iPSCs) offers the advantage of generating numerous cell line models in an isogenic background along with the parental cell line as an ideal control, where patient-derived primary cells are difficult to obtain. As well, genome editing to introduce and correct mutations in control and disease iPSC lines, is a powerful tool for comparative study of genetic mechanisms involved in cellular/organ development and disease pathology. 

The true potential of the iPSC technology lies in the directed differentiation of iPSCs into a desired cell line lineage, especially into neuronal lineage cells as it provides a more predictive source of CNS models. These cells are valuable tools for high throughput screening of neurotoxicological and neuroprotective compounds.   

Applied StemCell has engineered isogenic panels of extensively characterized, genome edited human iPSC lines that have been shown to recapitulate abnormal neuronal phenotypes seen in patient-derived iPSC. 

Key features:

  • Two isogenic panels, each from one “parental/master” male (ASE-9109) and one female (ASE-9110) control iPSC lines
  • The genome edited iPSC lines are fully characterized for pluripotency, differentiation capability, and functionality; and provide a fully encompassing variety of tissue models for various applications
  • Three panels of isogenic genome-edited iPSC lines:
    1. Neuronal disease-specific isogenic knockout lines
    2. Lineage-specific reporter gene knock-in lines
    3. iPSC lines with reporter gene inserted at a safe harbor locus

Applications:

We also have disease-specific iPSC lines derived from patients with ALS8, Parkinson’s Disease and Diabetes type II patient samples. Please inquire for details on differentiation of these genome-edited cell lines as well as custom differentiation services.

1. Neuronal Disease Specific Isogenic Knockout Lines: 

Isogenic Knock-out Lines

Associated Disease

Description

PARK2 -/-

PD

Biallelic KO

PARK7 -/-

PD

Biallelic KO

PINK1 -/-

PD

Biallelic KO

LRRK2 -/-

PD

Biallelic KO

Park2-/-; Park7-/-

PD

Biallelic KO

Park2-/-; Pink1-/-

PD

Biallelic KO

APOE -/-

Alzheimer's disease

Biallelic KO

SOD1 -/-

ALS

Biallelic KO

DICS1 -/-

Schizophrenia

Biallelic KO

CNTNAP2 -/-

Autism

Biallelic KO

BDNF -/-

CNS

Biallelic KO

2. Lineage-specific Reporter Gene Knock-in Lines:

Knock-in Lineage-specific Reporters

Description

MAP2-Nanoluc-Halotag KI

Neuron reporter

GFAP-Nanoluc-Halotag KI

Astrocyte reporter

MBP-Nanoluc-Halotag KI

Oligodendrocyte reporter

3. Safe Harbor Locus Reporter Gene Knock-in Lines:

Safe-harbor knock-in lines

Description

CAG-GFP, AAVS/Chr19

Ubiquitous reporter

DCXp-GFP

Neuron reporter

Technical Details

Examples of Genome Edited iPSC Lines:

Disease-specific Knockout iPSC Line: APOE Knockout (ASE-9405)

The ASE-9405 iPSC line is engineered with a bi-allelic (homozygous) knockout for APOE gene (APOE-/-) that has been implicated in Alzheimer’s disease etiology. The parental iPSC is ASE-9109 which is an integration-free, normal karyotype iPSC derived from male, CD34+ cord blood cells. The APOE knockout line can be further differentiated into an isogenic panel of neurons and glia for disease modeling and drug/ toxicity screening applications.

1. Genotyping of APOE-/- bi-allelic KO clone F4: APOE KO

technical-genomedited-ipsc-apoe-ko-1

technical-genomedited-ipsc-apoe-ko-2

Figure 1. Sequence alignment between parental/ control iPSC line (ASE-9109; male) and APOE-/- clone. The homozygous knockout clone shows a 4 bp deletion in allele 1, a 3 bp deletion in allele 2, and a 4bp insertion in allele 2, as compared to wild type (WT) sequence in parental iPSC line.

2. Expression of Pluripotency Markers

technical-genomeedited-ipsc-apoe-ko-3

Figure 2. Expression of pluripotency markers in APOE-/- iPSC line. The homozygous knockout iPSC line, APOE-/-, expresses pluripotency markers OCT4, NANOG, TRA-1-60, and TRA-1-81, indicating pluripotency of the iPSC line after genome editing. Nucleus stained with DAPI (blue).

3. Expression of APOE in neural stem cells differentiated from APOE-/- iPSC-line

technical-genomeedited-ipsc-apoe-ko-4

Figure 3. Expression of APOE in neural stem cells (NSC) differentiated from master/ parental iPSC line (ASE-9109) and APOE-/- iPSC line (ASE-9405) was quantified using qPCR. The APOE mRNA was significantly downregulated in the APOE-knockout NSC as compared to expression in the parental/ control iPSC line. 

WT APOE-/- 
1022 -4

Figure 4. APOE Expression in APOE knockout NSCs by microarray. APOE expression in the knockout NSC line was significantly lower than the wild type NSC line.

4. Differentiation of APOE -/- iPSCs into Neurons and GFAP

technical-genomeedited-ipsc-apoe-ko-5

Figure 5. Differentiation of APOE-/- iPSCs into cortical neurons and astrocytes. The APOE-/- line can be differentiated into cortical neurons (GABA; green) and astrocytes (GFAP; red) to model Alzheimer's disease. Other markers: Neuronal marker (MAP@; blue) and nucleus staining (DAPI; blue)


Lineage-specific Reporter Knock-in iPSC Line: Neuronal Lineage-specific Line (ASE-9500)

The ASE-9500 iPSC line is a mono-allelic (heterozygous) knock-in iPSC line engineered with a multiplexed reporter (Nanoluc-Halotag) inserted into the 3’ end of the endogenous MAP2 locus. The parental iPSC is ASE-9109 which is an integration-free, normal karyotype iPSC derived from male, CD34+ cord blood cells. The MAP2-Nanoluc-Halotag Knock-in iPSC line can be further differentiated into an isogenic panel of neurons and glia for disease modeling and drug/ toxicity screening applications.

1. Schematic representation of MAP2-Nanoluc-Halotag reporter construct

 schematic-genomeedited-ipsc-map2-ki-1

Figure 1. The dual reporter, Nanoluc-Halotag construct is expressed by neuronal lineage-specific promoter, MAP2. This construct is inserted into the endogenous MAP2 locus of the iPSC line.

2. Validation of MAP2-Nanoluc-Halotag knock-in clone by PCR

 technical-genomeedited-ipsc-map2-reporter-ki-2

Figure 2. PCR was used to confirm the insertion of reporter genes to the MAP2 locus in ASE-9500 MAP2-Nanoluc-Halotag knock-in (KI) heterozygous iPSC line. Three sets of primers were used to verify insertion of the reporter gene at the MAP2 locus: 3’, right arm integration; 5’, left arm integration; wild type (WT) open reading frame (ORF) confirmed reporter gene KI at the correct locus. Sequence validation for ASE-9500 MAP2-Nanoluc-Halotag knock-in (KI) heterozygous iPSC line.

3. Pluripotency marker validation and karyotype analysis of MAP2-Nanoluc-Halotag reporter knock-in iPSC line

technical-genomeedited-ipsc-map2-reporter-ki-3      technical-genomeedited-ipsc-map2-reporter-ki-4

Figure 3. Pluripotency characterization and karyotype analysis of ASE-9500 Lineage-specific reporter knock-in iPSC line, MAP2-Nanoluc-Halotag heterozygous knock-in line. The MAP2-Nanoluc-Halotag line expresses pluripotency markers OCT4, NANOG, TRA-1-60, and TRA-1-81, indicating pluripotency of the iPSC line after genome editing. Nucleus stained with DAPI (blue). Karyotype Analysis of MAP2-Nanoluc-Halotag iPSC line indicates normal karyotype and no clonal abnormalities in the iPSC line at the stated band level of resolution, after genome editing.

4.  Functional validation of neuronal lineage-specific reporter line, ASE-9500 MAP2-Nanoluc-Halotag knock-in iPSC line.

 technical-genomeedited-ipsc-map2-reporter-ki-5      technical-genomeedited-ipsc-map2-reporter-ki-6

Figure 4. Functional validation of ASE-9500 Lineage-specific reporter knock-in iPSC line, MAP2-Nanoluc-Halotag heterozygous knock-in line by luciferase activity assay measured over 18 days from the neural stem cell to neuronal maturation.  Immunohistochemical staining for MAP2 (green) and Halotag (red) showed co-localization (yellow) of reporter gene in neuronal cells.


iPSC lines with reporter gene inserted at a safe harbor locus: AAVS1-DCXp-GFP iPSc line (ASE-9502)

ASE-9502 iPSC line is a mono-allelic (heterozygous) knock-in iPSC line engineered with a GFP reporter tag driven by a neuronal-lineage-specific promoter, Doublecortin (DCX) inserted into the AAVS safe harbor locus. The parental iPSC is ASE-9109 which is an integration-free, normal karyotype iPSC derived from male, CD34+ cord blood cells. The AAVS-DCX-GFP Knock-in iPSC line can be further differentiated into an isogenic panel of neurons and glia for disease modeling and drug/ toxicity screening applications.

1. Schematic representation of AAVS-DCXp-GFP reporter construct

SCHEMATIC-genomeedited-ipsc-aavs-dcxp-gfp-ki-1

Figure 1. Schematic representation of iPSC line with reporter gene inserted in AAVS safe harbor locus, AAVS1-DCXp-GFP (ASE-9502). The reporter gene expression is driven by a neuronal-lineage (neuronal development) specific promoter, Doublecortin (DCXp). The ASE-9502 cell line is derived from master cell line with a ubiquitous promoter expressiong a CopGFP reporter gene. After Cre-recombinase mediated cassette exchange (RMCE), a single copy of the DCXp-TagGFP cassette is inserted into the AAVS1 locus.

2. Validation of neuronal-lineage specific reporter cassette knock-in in AAVS1 safe harbor locus

technical-genomeedited-ipsc-aavs-dcxp-gfp-ki-2

Figure 2. Validation of the DCXp-GFP reporter gene insertion in AAVS1 locus in iPSC line, ASE-9502.  In the AAVS-CAG-GFP master cell line, GFP expression is driven by the ubiquitous promoter, CAG (all cells are stained green for GFP). After RMCE, there is no GFP expression in the iPSCs confirming the absence of a ubiquitous CAG promoter after cassette exchange. When these iPSCs are differentiated into neurons, immunohistochemical characterization showed the co-localization (yellow) of GFP (green) and DCXp-postiive neurons (red). The GFP is expressed only in neuronal cells confirming the insertion of the reporter gene in the correct locus. 

 

 

 

 

FAQ

Frequently Asked Questions

1. How specific is the GFP reporter driven by the DCXp promoter in the AAVS1-DCXp-GFP knock-in iPSC line?
The expression of the tagGFP reporter under control of the DCXp promoter is observed only in differentiated neuronal cells (please refer to technical details section of this page). All GFP positive cells (green) co-localized with DCXp positive cells (using anti-DCX antibody; red). We did not observe GFP positive cells that were negative for DCX staining. As well, we did not observe DCX positive cells that did not have GFP staining.

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