Células madre con pluripotencia inducida: posibles aplicaciones clínicas

Transcription

1 Células madre con pluripotencia inducida: posibles aplicaciones clínicas Angel Raya ICREA Research Professor Control of Stem Cell Potency Group Institute for Bioengineering of Catalonia Barcelona, Spain 13 de julio

2 The progressive loss of potency D E V E L O P M E N T POTENCY 2

3 The progressive gain of potency D E V E L O P M E N T POTENCY Cloning Induced reprogramming 3

4 Stem cells: self-renewal + potency Embryonic vs. adult Pluripotent vs. multipotent In vivo vs. in vitro 4

5 Adult stem cells Hematopoietic stem cells Epidermal stem cells Spermatogonial stem cells Intestinal stem cells Skeletal muscle stem cells Mammary gland stem cells Neural stem cells Cardiac stem cells Mesenchymal stem cells... 5

6 The plasticity of adult stem cells Leri A et al. Physiol Rev 2005;85:

7 Pluripotent stem cells Evans MJ, Kaufman MH! Establishment in culture of pluripotential cells from mouse embryos.! Nature Jul 9;292(5819):154-6.! 7

8 Publications on embryonic stem cells 2,500 2,000 1,500 1,

9 Derivation of human embryonic stem cells 9

10 The promise of stem cell therapies 10

11 Pluripotent stem cells Embryonic stem (ES) cells Parthenogenetic ES cells SCNT-derived ES-like cells Embryonic germ (EG) cells SSC-derived EG-like cells Cell fusion hybrids Induced pluripotent stem (ips) cells 11

12 Pluripotent stem cells Embryonic stem (ES) cells Parthenogenetic ES cells SCNT-derived ES-like cells Capture of pluripotency Embryonic germ (EG) cells SSC-derived EG-like cells Cell fusion hybrids Induced pluripotent stem (ips) cells Reprogramming to pluripotency 12

13 Induced pluripotent stem cells 13

14 Assessing pluripotency of stem cells 14

15 Induced pluripotent stem cells 15

16 Induced pluripotent stem cells!"#$%&$'(()*&+,'-.%$/+ KLF4 SOX2 cmyc OCT4 Retroviral supernatant ips cell colonies 4-6 weeks Somatic cells Dermal fibroblasts 16

17 Assessing pluripotency of stem cells 17

18 Criteria for defining bona fide ips cells Self-renewal!! > 20 passages!! Karyotypic stability Pluripotency!! Expression of pluripotency-associated markers!! In vitro differentiation!! Teratoma formation Molecular!! DNA fingerprinting!! Integration of reprogramming transgenes!! Silencing of reprogramming transgenes!! Reprogramming of gene expression profile!! Reprogramming of DNA methylation profile 18

19 Reprogramming of epidermal keratinocytes Aasen, Raya et al., Nat Biotechnol

20 In vitro differentiation of KiPS cells Aasen, Raya et al., Nat Biotechnol

21 Teratoma formation of KiPS cells Aasen, Raya et al., Nat Biotechnol

22 Potential uses of ips cell technology Understand reprogramming Acquisition of pluripotency Escaping senescence Autologous cell replacement therapies Degenerative diseases Monogenic diseases Modeling human disease Pathogenic mechanisms Gene-causing disease Disease onset & progression Drug screening 22

23 Proof-of-concept for ips-based cell therapy Raya et al., Nature

24 Shortcomings of ips-based cell therapy Generation of human ips cells Retroviral integrations Clinical-grade lines Genetic correction Silencing of correcting transgenes Control of cell proliferation Positive/negative selection Cancer predisposition? Specific differentiation protocols Directed differentiation Clinical-grade protocols 24

25 Modeling human disease with ips cells 25

26 Modeling human disease with ips cells 26

27 Modeling human disease with ips cells 27

28 Modeling human disease with ips cells 28

29 Modeling human disease with ips cells 29

30 Generation of vmda neurons from hes/ips cells A. Consiglio, IBUB 30

31 Dopaminergic differentiation of hes LMX1A cells 31

32 Dopaminergic differentiation of hes LMX1A cells Sanchez-Danes et al., Hum. Mol. Genet. in press 32

33 Dopaminergic differentiation of hes LMX1A cells M. Vila, VHRI 33

34 Modeling Parkinson s disease with ips cells 34

35 The challenges of modeling PD Most cases are idiopathic ~10% monogenic Incomplete penetrance Cell-type specific susceptibility Specific pathology Age-related progressive disease Spontaneous phenotypes? 35

36 Project outline Patients recruited (Eduard Tolosa, H. Clínic)! 4 G2019S LRRK2! 7 Sporadic PD! 4 Control Reprogramming! Research-grade ipsc lines! Retroviral delivery 3F (no c-myc)! Epidermal keratinocytes (fibroblasts)! 4 ipsc lines per patient! 2 ipsc fully characterized and selected Adriana Sànchez-Danés Generation of patient-specific vmda neurons (A. Consiglio, IBUB) Investigate spontaneous phenotypes (A. Consiglio; M. Vila, VHRI) Effect of PD-related toxins (M. Vila, VHRI) 36

37 Summary of PD-specific ips lines established Total # of ips lines: 50 Groups covered! 4 Control! 7 Sporadic PD! 4 G2019S LRRK2 Generation of patient-specific vmda neurons Sanchez-Danes et al., submitted 37

38 Synuclein aggregates in PD-iPS cells Sanchez-Danes et al., submitted 38

39 Modeling genetic PD with ips cells 39

40 In vitro aging of PD-iPS cells Sanchez-Danes et al., submitted 40

41 Impairment of autophagy in PD-iPS cells Sanchez-Danes et al., submitted 41

42 Modeling Parkinson s disease with ips cells 42

43 Potential uses of ips cell technology Understand reprogramming Acquisition of pluripotency Escaping senescence Autologous cell replacement therapies Degenerative diseases Monogenic diseases Modeling human disease Pathogenic mechanisms Gene-causing disease Disease onset & progression Drug screening 43

44 Acknowledgements Yvonne Richaud Senda Jiménez Alberto García Sergio Mora Claudia Di Guglielmo Eduard Sleep Isil Tekeli Bahaa Arefai IBEC Adriana Sánchez Antonella Consiglio IBUB Carles Gaig Eduard Tolosa Hospital Clínic Konstantin Levitsky Patricia Ortega José López-Barneo IBiS Iria Carballo-Carbajal Miquel Vila Vall d Hebron RI 44