1. Mason A. Israel et. al. Nature 2012
Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells
Mason A. Israel et. al.
Nature 2012
Presentation by Airan Jansen
Program Administrator
CIRM Bridges to Stem Cell Research
California Polytechnic University, Pomona
California State University, Los Angeles

2. Alzheimer’s disease Common neurodegenerative disorder
6th leading cause of death in the US
More than 5 million Americans are living with the disease
1 in 3 seniors dies with Alzheimer’s or another dementia
Alzheimer’s is the only cause of death among the top 10 in America without a way to prevent it, cure it or even slow its progression.
Today, there are no survivors of Alzheimer’s. If you do not die from it, you die with it.
Alz.org
Graph of top 10 deaths

3. Alzheimer’s is defined post mortem by the increased presence of amyloid plaques and neurofibrillary tangles in the brain.
Amyloid plaques: extracellular deposits consisting primarily of amyloid-β peptides
Neurofibrillary tangles: intraneuronal aggregations of hyperphosphorylated tau
Tau: a microtubule-associated protein involved in microtubule stabilization

4. Sporadic (sAD) vs. Familial Alzheimer’s Disease (FAD)
FAD only involve 3 genes (APP, amyloid precursor protein; PSEN1 and 2, presenilin 1 and 2).
sAD involves many genes and affects many pathways.
Vast majority of Alzheimer’s Disease is sporadic and not familial
Studying the known mechanisms of FAD can lead to the development of appropriate directions for sAD research
The focus of this study is on developing an in vitro model using iPSCs to understand the differences between sAD and FAD.
Alzheimer disease: From inherited to sporadic AD—crossing the biomarker bridge
Harald Hampel & Simone Lista
Nature Reviews Neurology 8,   (November 2012)
doi: /nrneurol

5. Limits to understanding Alzheimer's
Disease Pathogenesis
Difficulties in obtaining live neurons from Alzheimer's patients
Inability to model the sAD form of the disease
To overcome these difficulties, the investigators reprogrammed Alzheimer’s Disease patient fibroblast cells to form induced pluripotent stem cells (iPSCs) which could be differentiated into neurons.

6. Induced Pluripotent Stem Cells (iPSCs)
ADULT CELL
iPSC Reprogramming Factors: Inserted into the nucleus (DNA) of the cell to reverse development of the cell
DEDIFFERENTIATION
iPSCs
Cardiac Muscle
Kidney Tubule
Cell
Smooth Muscle
Lung Cell
Pancreatic Cell
Skin Cell
Pigment Cell
Red Blood Cells
Red Blood Cells
Thyroid Cell
Neuron
Skeletal Muscle Cells

7. Questions to be addressed in this study
Can iPSC technology be used to produce neuronal cell phenotypes of patients with Alzheimer’s Disease?
Can iPSC technology be used to predict Alzheimer’s disease before a patient manifests the disease?
Is there a causative relationship between amyloid-β precursor protein (APP) processing and tau phosphorylation in the neurons?
Can neurons with the genome of an sAD patient exhibit phenotypes seen in an FAD patient?

8. Experimental Approach
sAD1
sAD2
NDC1
APPDp1
Fibroblasts
NDC2
APPDp2
Reprogramming with OSKM vectors
Supplementary Figure 1. Summary of main results. Primary cell cultures from 2 non-demented controls (NDC1, 2), 2 sporadic Alzheimer’s disease patients (sAD1, 2), and 2 familial Alzheimer’s disease patients (APPDp1, 2) were reprogrammed into patient-specic iPSC lines. Neurons were generated from iPSC lines by directed dierentiation and uorescence activated cell sorting (FACS) purication. Puried neurons from sAD2 and APPDp had signicantly higher levels of secreted Aß(1-40), active GSK3ß, phospho-tau, and large RAB5+ endosomes relative to NDC neurons. Pharmacologic inhibition of ß-secretase caused a signicant reduction in the levels of Aß(1-40), phospho-tau and active GSK3ß.
iPSCs
Directed neuronal differentiation and FACs purification
Purified Neurons

10. Characterization of patient fibroblasts
Suplementary Figure 2. Additional characterization of patient fibroblasts. a, Representative brightfield image of fibroblast cultures (line NDC1 shown). Scale bar marks 100 m. b, Familial Alzheimer’s disease samples contained 3 copies of the APP locus, while other samples were diploid. Down’s, Down’s syndrome fibroblasts. Quantitative PCR for two regions of the APP gene was performed on genomic DNA preparations and normalized to -globin levels. c, Familial Alzheimer’s disease fibroblasts expressed higher levels of APP mRNA relative to NDC and sAD samples. APP expression levels were normalized to expression levels of the housekeeping gene NONO. d, APPDp1 and 2 fibroblasts secrete increased levels of amyloid-ß(1-40) compared to NDC cells (P = 0.01 and , respectively, n = 3). e, No significant diference in amyloid-ß- (1-42/1-40) or (1-38/1-40) between patients.
Get rid of Figures a,b and e
Familial Alzheimer’s disease fibroblasts (APP) expressed higher levels of APP mRNA relative to NDC and sAD samples.
APP Dp1 and APP Dp2 fibroblasts secrete increased levels of amyloid-ß(1-40) compared to NDC cells

11. Dedifferentiation (Patient fibroblasts) OSKM (OCT4, SOX2, KLF4, c-MYC)
Maintain embryonic stem cell like morphology
Express pluripotent-associated proteins (NANOG and TRA1-81)
Can differentiate into cells of ectodermal, mesodermal and endodermal lineages under in vitro conditions
Form teratomas when injected into nude rats

12. Teratoma formation shows pluripotency of iPSCs
One iPSC line per individual plus an ESC line (HUES-9) was tested for pluripo-tency in vivo by ten bilateral injections into lumbar spinal cords of nude rats.
Scale bar, 2 mm
Scale bars, 50 µm
Supplementary Figure 5. Teratoma formation. One iPSC line per individual plus an ESC line (HUES-9) was tested for pluripo-tency in vivo by ten bilateral injections into lumbar spinal cords of nude rats (see methods). a, H&E stained horizontal section of a whole spinal cord showing the formation of multiple teratomas in individual injection sites per animal (line sAD1.1 shown). Arrow marks one injection site. Scale bar, 2 mm. b, Higher magnication images showing the presence of ectodermal, mesodermal and endoder-mal lineages for each iPSC line. Scale bars, 50 µm
H&E stained horizontal section of a whole spinal cord showing the formation of multiple teratomas.
Higher magnication images showing the presence of ectodermal, mesodermal and endoder-mal lineages for each iPSC line.

13. Fluorescence-activated cell sorting (FACS) for
purification of neurons derived from iPSCs
Fibroblast culture
iPSC culture showing
human Embryonic stem
cell (hESC)-like
morphology

14. Nucleated FACS-purified neurons express MAP2 and βIII-tubulin
Fluorescence-activated cell sorting (FACS) for purification of neurons derived from iPSCs
Neural progenitor
cells (NPCs)
differentiated NPCs
Nucleated FACS-purified neurons express MAP2 and βIII-tubulin

15. Almost all neurons tested generated voltage-dependent
action potentials and currents indicating true neuronal phenotype

16. Purified neurons from sAD2, APPDp1 and APPDp2 patients secrete increased amyloid-β(1–40) (Aβ(1–40)) compared to NDC patient samples.
Increased amyloid-β, p-tau and aGSK-3β in sAD2 and APPDp neuronal culturesa, Purified neurons from sAD2, APPDp1 and APPDp2 secrete increased amyloid-β(1–40) (Aβ(1–40)) compared to NDC samples (P = , and < , respectively). b, Amyloid-β differences between patients and controls are larger in neurons versus fibroblasts. Data sets are relative to NDC mean. c, d, Neurons from sAD2, APPDp1 and APPDp2 have increased aGSK-3β (percentage non-phospho-Ser 9) and p-tau/total tau (p-tau/t-tau) compared to NDC samples (aGSK-3β, P < , and ; p-tau/total tau, P < , and ). In a–d, n values on graphs indicate the number of biological replicates per patient, contributed equally by three iPSC lines. e, sAD2 findings verified in two additional iPSC lines (sAD2.4 and sAD2.5). sAD2(1-3) indicates findings from initial sAD2 iPSC lines. For amyloid-β, aGSK-3β and p-tau/total tau, sAD2 remained significantly higher than controls (P< ). No significant difference was found between original and secondary sAD2 lines (P = 0.14, 0.44, 0.63). f, Strong positive correlations between amyloid-β(1–40), aGSK-3β and p-tau/total tau in purified neurons. Pearson R = 0.94, 0.91 and 0.83, respectively.g, Twenty-four hour treatment with β- and γ-secretase inhibitors reduced secreted amyloid-β(1–40) compared to control treatment. β-Secretase inhibitors partially rescued aGSK-3β and p-tau/total tau in sAD2 and APPDp2 neurons (P < 0.01 for aGSK-3β, P < 0.03 for p-tau). γ-Secretase inhibition did not significantly affect aGSK-3β and p-tau/total tau. In g, number of treatment sets is indicated on the graph (n), NDCs are represented by two iPSC lines each and sAD2 and APPDp2 are represented by three. Error bars indicate s.e.m.

17. Background: Tau forms neurofibrillary tangles (NFTs) and adds to Alzheimer’s Disease severity
Kinase GSK-3β phosphorylates tau at Thr231 (p-tau(Thr231). P-tau(Thr231) regulates microtubule stability and correlates with:
1. neurofibrillary tangle number
2. degree of cognitive decline

18. Neurons from sAD2, APPDp1 and APPDp2 patients had significantly higher p-tau/total tau (p-tau/t-tau) compared to NDC patient samples

19. Neurons from sAD2, APPDp1 and APPDp2 patients had significantly higher active GSK-3β compared to NDC patient samples
GSK-3β is thought to be constitutively active but is inactivated when phosphorylated at Ser 9. Therefore, the proportion of aGSK-3β in purified neurons was calculated by measuring the amount of GSK-3β lacking phosphorylation at Ser 9 relative to total GSK-3β levels

20. Two additional iPSC lines from the sAD2 patient were analyzed to confirm elevated levels of amyloid-β, aGSK-3β and p-tau/t-tau compared to NDC controls

21. There are strong positive correlations between amyloid-β(1–40), aGSK-3β and p-tau/total tau in purified neurons from FAD and sAD patients 

22. Twenty-four hour treatment with β- and γ-secretase inhibitors reduced secreted amyloid-β(1–40) compared to control DMSO treatment. β-secretase inhibitors partially rescued aGSK-3β and p-tau/total tau in sAD2 and APPDp2 neurons 

23. Alzheimer’s Disease patient autopsy samples (not shown)
 Neurons from both sAD2 and APPDp2 patients frequently had Rab5+ early endosomes similar in volume, morphology and localization to that observed in neurons from
Alzheimer’s Disease patient autopsy samples (not shown)
Accumulation of large RAB5+ early endosomes in neurons has been observed in autopsies from sporadic Alzheimer’s disease and some forms of familial Alzheimer’s disease24,25. As β-secretase is localized to endosomes and has an acidic pH optimum, it has been proposed that early endosomes potentially mediate the effects of APP processing on downstream pathologies such as increased p-tau, neurofibrillary tangles, synaptic loss and apoptosis26; however, these hypotheses have been difficult to test directly without live, patient-specific neurons.
Add arrows to indicate endosome

24. The neurons from both sAD2 and APPDp2 patients had significantly increased numbers of both large and very large early endosomes relative to NDC controls 

25. No significant difference in the number of synapsin I+ puncta per μm MAP2+dendrite was observed between NDC and either sAD2 or APPDp2 patients
In Alzheimer’s disease autopsies, synaptic loss is one of the strongest pathological correlates with dementia severity, and in regions of the brain affected by Alzheimer’s disease, the presynaptic marker synapsin I is decreased in patients versus controls27,28. 
 Extended culture periods may be required to study Alzheimer’s disease-associated loss of synaptic proteins.

26. Summary of Results Fibroblasts iPSCs Purified Neurons sAD1 sAD2 NDC1
Reprogramming with OSKM vectors
Directed neuronal differentiation and FACs purification
NDC1
NDC2
sAD1
sAD2
APPDp1
APPDp2 26

27. iPSC technology can be used to study early pathogenesis and drug response in both Sporadic and Familial Alzheimer’s disease
SUMMARY
There were significantly increased levels of three major biochemical markers of Alzheimer’s disease ( amyloid-β(1–40), aGSK-3β and p-tau/total tau) in neurons from one Sporadic Alzheimer’s disease and two Familial Alzheimer’s disease patients.
These studies suggest that the APP processing pathway has a causative role in tau Thr 231 phosphorylation in human neurons.
Products of APP processing other than amyloid-β may have a role in induction of GSK-3β activity and p-tau. 
Early endosome phenotypes have been found in neurons from sAD2 (Sporadic Alzheimer’s) and APPDp2 (Familial Alzheimer’s) patients.