current results on ipsc differentiation in ad: readouts ... current results on ipsc differentiation...

Current results on iPSC differentiation in Alzheimer Disease

readouts, phenotypic assays

09-09-2014

Karolina Szczesna Biotalentum Gödöllö, Hungary

StemMad Project

human cortical neurons 2013

Alzheimer`s Disease (AD) is a neurodegenerative dissorder that represents, with a 70%, the main cause of dementia in the west countries (Seshardri 2002). AD affects the 11% of the people older than 65 years and the 50% of the people older than 85 (Rodríguez-Santiago 2001). The WHO stimates AD affects up to 20 millions of people around all the world, and acording to last previssions in only 25 years the number will be duplicate.

Dr. Alois Alzheimer

AD introduction


StemMad Project Karolina Szczesna Alzheimer Disease WS 09-09-2014

AD is a multifactorial disease with a combination of genetic and enviromental factors that can be deffined by the age of onset as: Early-onset (EOAD; <65 years)~(FAD) ~ 5% of the cases Familiar agregation High penetrance >90% APP, PSEN1 or PSEN2

Late-onset (LOAD; >65 years) ~ (EAD) ~ 95% of the cases Not familiar agregation Low penetrance



The hallmarks of AD starts in the Enthorrinal cortex and Hippocampus. Then, the rest of the cortex is also affected.

In AD not only more than 50% of the brain mass body is lose, but also the metabolism of the brain is strongly reduced.

AD progression



B-amyloid plaque Neurofibrillary tangle

• beta-amyloid plaques are dense deposits of protein and cellular material that accumulate outside and around nerve cells

• neurofibrillary tangles are twisted fibers that build up inside the nerve cell

AD hallmarks



Beta-amyloid Plaques

Amyloid precursor protein (APP) is the precursor to amyloid plaque.

1. APP sticks through the neuron membrane.

2. Enzymes cut the APP into fragments of protein, including beta-amyloid.

3. Beta-amyloid fragments come together in clumps to form plaques.

1.

2.

3.

In AD, many of these clumps form, disrupting the work of neurons. This affects the hippocampus and other areas of the cerebral cortex.





iPSC technology



iPSCs from fibroblasts of FAD patients with mutations in PS1 (A246E) and PS2 (N141I) Generation of PS1 and PS2 iPSC from patient fibroblasts. (A) Genotypic analysis of PS1 iPSC by PCR–RFLP and sequencing. (B) Genotypic analysis of PS2 iPSC by PCR–RFLP and sequencing. (C) Both PS1 and PS2 iPSC lines exhibit markers of pluripotency, all iPSCs express pluripotency markers including: Tra-1-60, Tra-1-81, SSEA3 and SSEA4. (D) RT–PCR analysis of the transgenes OCT3/4, SOX2, KLF4 and the endogenous hESC marker genes. • FAD-iPSC-derived differentiated neurons have increased

amyloid β42 secretion, recapitulating the molecular pathogenesis of mutant presenilins.

• secretion of amyloid β42 from these neurons sharply responds to γ-secretase inhibitors and modulators, indicating the potential for identification and validation of candidate drugs.

• FAD-iPSC-derived neuron is a valid model of AD (Based on Yagi et al., 2011)



Differentiation of PS1 and PS2 iPSC into neurons. Neural differentiation of control iPSC (201B7, PD01-25 and PD01-26), PS1 iPSC (PS1-2 iPSC and PS1-4 iPSC) and PS2 iPSC (PS2-1 iPSC and PS2-2 iPSC).

Representative pictures of immunocytochemistry for bIII-tubulin (A) and MAP-2 (B) after neural differentiation

Characterization of Aβ secretion in PS1 and PS2 iPSC-derived neurons. (A) The amount of A β 40 and Ab42 secreted from control iPSCderived neurons, PS1 iPSC (PS1-2 iPSC and PS1-4 iPSC) and PS2 iPSC (PS2-1 iPSC and PS2-2 iPSC)-derived neurons. (B) The ratio of Ab42/ Ab40 from control iPSC-derived neurons, PS1 iPSC-derived neurons and PS2 iPSC-derived neurons.

Note, the ratio of Ab42/Ab40 in both PS1 iPSC derived neurons and PS2 iPSC-derived neurons was significantly higher than that of control iPSC-derived neurons.

βAmyloid ELISA Kits (WAKO)

Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells (based on Israel et al., 2012)

• reprogrammed primary fibroblasts from two patients with familial Alzheimer’s disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APPDp), two with sporadic Alzheimer’s disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines

• iPSC-derived, purified neurons from the two APPDp patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1–40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β)



Increased amyloid-β, p-tau and aGSK-3β in sAD2 and APPDp neuronal cultures (based on Israel et al., 2012)

Establishment of Control and AD Patient-Specific iPSCs, and Derivation of Cortical Neurons Producing Aβs from iPSCs (based on Kondo et al., 2013)



(A) Established iPSCs from both controls and AD patients showed embryonic stem cell-like morphology (Phase) and expressed pluripotent stem cell markers NANOG (red) and TRA1-60 (green). (B) Genomic DNA sequences showed the presence of the homozygous genotype for E693 deletion and the heterozygous genotype for V717L mutation on the APP gene only in AD iPSCs. (C) Estimation of neuronal differentiation from control and AD-iPSCs. After 2 months of differentiation, neurons were immunostained with antibodies against the neuronal marker TUJ1 and the cortical neuron markers TBR1 and SATB2. The scale bar represents 30 mm. (D) Proportions of TUJ1-, TBR1-, and SATB2-positive cells in control and AD-iPSCs.

Establishment of Control and AD Patient-Specific iPSCs, and Derivation of Cortical Neurons Producing Abs from iPSCs (based on Kondo et al., 2013)

(E) A β 40 and A β 42 secreted from iPSC-derived neural cells into the medium (extracellular A β ) (F) A β 40 and A β 42 in cell lysates (intracellular A β ) (G) The amount of soluble APPβ was not altered in control and AD.

Basal forebrain cholinergic neurons (BFCNs) and AD

• An early substantial loss of basal forebrain cholinergic neurons (BFCNs) is a constant feature of Alzheimer’s disease (AD) and is associated with deficits in spatial learning and memory.

• Used BFCNs derived from iPSCs to model sporadic AD with a focus on patients with ApoE3/E4 genotypes (AD-E3/E4).

• BFCNs derived from AD-E3/E4 patients showed typical AD biochemical features evidenced by increased Aβ42/Aβ40 ratios.

(based on Duan et al., 2014)



AD-E3/E4 neurons exhibited altered responses to treatment with γ-secretase inhibitors

compared to control BFCNs or neurons derived from patients with familial AD.

AD-E3/E4 BFCNs have elevated Aβ42/Aβ40 ratios and altered responses to γ-secretase inhibitors. (A) Aβ42 levels were significantly elevated in familial AD line AG07872 and ApoE3/E4 lines AG04402 and AG11414 compared to the average of controls (B) The ratios of secreted Aβ42 to Aβ40 were compared to the average of controls ( (C, D) Quantification of Aβ40 secretion in response to different dosages of γ-secretase inhibitor compound E (C) or DAPT(D) treatment. (based on Duan et al., 2014)





Control and AD iPSCs-derived BFCNs express functional voltage-gated sodium and calcium channels. BFCNs from patients with AD-E3/E4 exhibited increased vulnerability to glutamate-mediated cell death which correlated with increased intracellular free calcium upon glutamate exposure. (based on Duan et al., 2014)

Neuronal Model for Alzheimer’s Disease - GSK company



• researchers at GSK used iCell Neurons to establish a cellular model of Alzheimer’s Disease. • neuronal loss was induced by exposure of the cells to an insult of Aβ1–42 aggregates

• using iPSC-derived neurons, beta amyloid (1-42) peptide was applied to mimic the undesired outcomes observed during AD • measurement of the neurotoxicity and decrease in neurite outgrowth by cell viability and high content imaging assays iPSC-derived neurons secrete relevant biomarkers and the release can be modulated with small-molecules CDK2-specific knockdown or inhibition with GW8510 rescues the neurotoxicity induced by the peptide Aβ (1–42)

Based on Xu, X., et al. 2013

ReproNeuro Glu™ Kit and Alzheimer’s Disease Model Cells first commercial human iPSC derived Glutamatergic neural cells and Alzheimer’s disease model neural cells

• Reliable source of human glutamatergic neurons and Transgenic Alzheimer’s disease (AD) model neurons for simple comparison with isogenic control

• Replicates disease phenotype in vitro (Amyloid β accumulation)

• Applicable to high throughput screening



βIII-tubulin were positive in more than 80% of the cells; most of the differentiated neurons express glutamatergic neuron marker

ReproNeuro Glu™ express synaptic terminal marker Synapsin1 as well as glutamatergic neuronal marker Vglut2

Amyloid β secretion (accumulation) can be quantified in vitro AlphaLISA Aβ Assay (PerkinElmer)



ReproNeuro acetylcholine (Ach) Kit and Alzheimer’s Disease Model Cells



• Reliable source of human cholinergic neurons

• The world’s first commercial human iPSC-derived Ach neurons and Alzheimer’s disease (AD) model neurons

• Transgenic AD model neurons for simple comparison with isogenic control

• Patient-derived AD model neurons (under development) for more complex analysis

• Replicates disease phenotype in vitro (Amyloid β accumulation)

• Applicable to high throughput screening



βIII-tubulin were positive in more than 80% of the cells.

Most of the differentiated neurons express cholinergic neuron markers, indicating the high purity of the product.

Thank you for your attention

Karolina Szczesna Biotalentum Gödöllö, Hungary

StemMad Project

human Alzheimer’s neurons in a dish 2012

current results on ipsc differentiation in ad: readouts ... current results on ipsc differentiation...

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