1 CELULES SOUCHES (Daniel Aberdam) 20129h-12h Je 8/3: cours 1: Définitions et généralités sur les CS Je 15/3: cours 2: CS embryonnaires: modèle et thérapie Je 22/3: cours 3: CS pluripotentes induites (iPS) Je 29/3: cours 4: CS adultes et physiopathologie Examen: 10/5

2 induced pluripotent stem cells (iPS)gènes de pluripotence (cocktails de 3-4 gènes) Shinya Yamanaka (2007) Fibroblast Pluripotent colonies oct-4 sox-2 nanog c-myc

3 Pas de problème d’éthiqueAutogreffe (?) oct-4 sox-2 Klf-4 c-myc neurogenerative disease patient 21-30 jours neurones cellules de patients iPS tératomes souris chimériques (trasnmission germinale) (Daley et al, Nature 2008)

4 (c-myc, Klf4) (Klf4) (oc-4/sox-2)

5 - + Reduced efficiency Delayed reprogramming - Increased specificityc-myc advantage and disadvantage Yamanaka, Nat. Biot, 2007 - + Reduced efficiency Delayed reprogramming - Increased specificity - No tumor

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7 (Yamanaka, Cell 2007)

8 Efficiency of reprogrammingLow efficiency of reprogramming 1- Differentiated hES cells (CD45+ cells, fibroblasts-like) (0.1%) 2- Fetal cells (MR90, Detroit551…) 3- Neonatal foreskin cells 4- Adult dermal fibroblasts (0-0.02%) Efficiency of reprogramming ≠ Mouse B cells (3.3%): quite efficient? (MEF: 0.5%)

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10

11 anémie à hématies falciformes, drépanocytose

12 HbA HbSS A/T 2/2 2/2S Science, 2007

13 2/2 2/2S HbA HbSS A/T Obtention of autologous iPSScience, 2007 Obtention of autologous iPS (2) Correction by homologous recombination

14 Recombinaison homologueloxP neoR mutation séquence homologue séquence homologue recombinase (cre) thérapie génique non insertionnelle ?

15 2/2 2/2S HbA HbSS A/T Obtention of autologous iPSScience, 2007 Obtention of autologous iPS (3) IPS differentiation (2) Correction by homologous recombination

16 Proof-of-principle of therapeutic potential of iPSHbA HbSS A/T 2/2 2/2S Science, 2007 Obtention of autologous iPS (4) transplantation (3) IPS differentiation (2) Correction by homologous recombination

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18 Prelavence: 1% worldwidehallucinations, delusions and cognitive deficits Heretability up to 80% Human schizophrenia brain tissue from postmortem samples (abnormal pH by hypoxia near death, patient drug and alcohol use and medication treatment Known (suggested): many neurotransmitter system affected? neurodevelomental disorder with onset in late adolescence. genetic studies suggest highly complex landscape (rare specific copy number variants, several loci statistically involved (ex. NRG1)

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20 Basic neural properties, measured with electrophysiology and calcium imaging were normal in patient-derived iPS.

21 In contrast, neuronal connectivity, measured by assessing the transneuronal transport of a modified Tomato rabies virus (that infect neurons and jump through synapses), was decreased in Schizo-iPS. Mechanism unknown but apparently due to decreased synaptic density (C); this in turn due to reduced neurites in culture (B)

22 Decreased neurites and synaptic prot. levelsNormal electrophysiological activity Normal Ca2+ transient activity

23 600 genes differentialy regulated in patient-iPSAmong them, NRG1 was elevated in neuron-derived cells but not in patient fibroblasts (cell spec)

24 600 genes differentialy regulated in patient-iPSAmong them, NRG1 was elevated in neuron-derived cells but not in patient fibroblasts (cell spec) Test 5 antipsochytic drugs to test potential rescue of neuronal connectivity:

25 Loxapine also revertes some of the gene expression changes in patient-iPS (ex. TCF4 to normal level)Model that will allow: to produce other cell types potentially involved in Schizo (oligodendrocytes,…) to screen for drugs avalaibility of many more fibroblasts from patients for large-scale studies (to reduce list of potential gene list) and design subgroup of patients for individualized medicine.

27 Cellular models: Human EEC-iPS (R304W)ED (R304W) fibroblasts iPS colony ED-iPS cell line Nanog Tra1-81 Alk. ph Ectodermal Mesodermal Endodermal teratoma PLURIPOTENCY embryoid bodies H. van Bokhoven Isabelle Petit

28 A phase Oct4 TRA-1-80 AP +/+ R204W/+ R304W/+ C +/+ R304W/+ R204W/+

29 cornea limbus Corneal progenitors (K3/K12) Limbal stem cells (K5/K14)Conjunctiva contains goblet cells that produce mucus as part of the tear films and rests on a vascularized stroma cornea: non-keratinized stratified epithelium that rests on a non vascularized collagenous stroma

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32 Could the phenotype be reverted ?CH2OH O S PRIMA-1 Klas Wiman APOPTOSIS PRIMA mutated pluripotent stem cells ectoderm (K8/K18) epidermal cells (K5/K14) ?

33 * * ** * A B C - - + - + - - + - + +/+ (-) R304W/+ (-) R304W/+ (+)1.2 1.0 0.8 Relative expression 0.6 0.4 0.2 K14 GJB6 GJA1 PCad Dlx5 Dlx6 K14 GJB6 GJA1 PCad Dlx5 Dlx6 KrtDap K14 GJB6 GJA1 PCad Dlx5 Dlx6 KrtDap K14 GJB6 GJA1 PCad Dlx5 Dlx6 KrtDap K14 GJB6 GJA1 PCad Dlx5 Dlx6 KrtDap KrtDap B C 120 40 100 Averaged expression of epithelial markers (% of iPSCWT) 80 % of K14+ cells 30 * 60 * 20 40 ** 10 * 20 APR-246: - - + - + APR-246: - - + - + +/+ R304W/+ R204W/+ +/+ R304W/+ R204W/+

34 injection intraperitoneally MSC implantation into brain huES

35 Ganciclovir treatmentimplantation into brain MSC implantation into brain huES reduce glioma Ganciclovir treatment injection into brain MSC-TK