Présentation réalisée par Shahragim Tajbakhsh durant le cours du réseau international des instituts Pasteur de "Médecine Génomique: du diagnostic à la thérapie " (17-21 octobre 2016)

1. Shahragim Tajbakhsh
Stem Cells & Development, Institut Pasteur
RIIP, Tunis 18Oct16
Stem cells in regenerative biology and medicine

2. Benefits of stem cell research

3. 1) Tissue specific stem cells (Multi ou Unipotent)
• In Embryo: organogenesis
• In adult: homeostasis and regeneration after injury or disease
2) Embryonic stem cells (Pluripotent)
• Derived from inner cell mass of preimplantation embryo
• Capable of generating all cell types in the embryo
3) Induced pluripotent stem cells, iPSCs (Pluripotent)
• Derived from reprogramming of specialized cells
• Capable of generating all cell types in the embryo
Different types of stem cells

4. Hematopoietic stem cells
Ø 1968: first bone marrow transplant
² used to treat severe combined immunodeficiency (SCID)
² since 1970's, BMT used to treat immunodeficiencies and leukemias
Ø Human bone marrow produces every day:
² 350 billion blood cells
²200 billion red blood cells
²100 billion platelets
²50 billion white blood cells

5. Stem cells and regeneration of cornea
Pellegrini et al. Trends Mol. Med, 2010

6. Pellegrini et al.
Trends Mol. Med, 2010
Stem cells and regeneration of cornea

7. De Luca et al. Brit J Derm. 2009
Stem cells and regeneration of skin
Epidermolysis bullosa

8. Mavilio et al. Nat Medicine 2006
Stem cells and regeneration of skin

9. Transcription factor-Induced Transdifferentation

10. WT Myf5-/-:Mrf4-/-:Myod-/- Pax3-/-:Myf5-/-(Mrf4-/-) Tbx1-/-:Myf5-/-
Modular design of skeletal muscle formation
Rudnicki et al. Cell 1993
Tajbakhsh et al. Cell 1997
Kassar-Duchossoy et al. Nature 2004
Kassar-Duchossoy et al. Genes & Dev. 2005
Sambasivan et al. Dev. Cell 2009
Cranial - HEAD
Somitic - TRUNK
- LIMBS
Mesoderm

11. Duchenne-type Emery-Dreifuss Limb-girdle Facioscapulohumeral Distal Oculopharyngeal
adapted from Emery 2002
Major Characteristics of some Muscular Dystrophies

12. Multiple cell types intervene asychronously during
skeletal muscle regeneration
➜ Notexin (snake venom) injection into Tibialis anterior muscle
3h 3 days 7 days 28 daysNormal
Macrophage waves
Fibrosis
‘M1’ ‘M2’ACUTE INJURY
Betzinger et al. 2013
Gayraud-Morel et al. 2009
CHRONIC INJURY
Mdx mouse model for human DMD
- Chronic degeneration/regeneration
- Less severe pathology than DMD

13. Ø Transcriptomics: microarrays to single cell RNAseq
Ø Epigenetic profiling
Ø Whole population – heterogeneity is a problem
Ø Single cell methylation and RNAseq
Ø Small and long non-coding RNAs
Ø Proteomics
Ø Metabolism and metabolomics
Strategies to investigate muscle stem cells

14. Transcriptome analysis and microRNAs

15. DCR-1
DCR-2 LOQS
LOQS
DCR-2 LOQS
DCR-2
Nature Reviews | Genetics
SAM
SAH
2 -OCH3
DCR-2
Antisense piRNA precursor
Transposon
mRNA
5 3
3 5
RNA
Pol II
Splicing
EXP-5
Branched
(pre-mirtron)
pri-miRNA
HEN1
SAM
SAH
HEN1
AGO2
AGO1
AGO3
R2D2
siRNA
duplex
RISC
loading
complex
Long dsRNA Structured loci
a siRNA pathway c piRNA pathwayb miRNA pathway
7mGppp
7mGppp
An...AAA
Pasha
Drosha
cleavage
Nucleus
Cytoplasm
Lariat
debranching
pre-miRNA
miRNA–miRNA duplex
Loading complex
AUB/Piwi
Exonuclease
H3CO-2
2 -OCH3
2 -OCH3
Sense piRISC
Antisense piRISC
AGO3
Drosha
Target cleavage
AGO1 RISC
AGO2
RISC
AGO2
pre-RISC
AGO1 pre-RISC
Translational repression mRNA destabilization
Figure 1 | Small RNA silencing pathways in Drosophila. The three small RNA silencing pathways in flies are the
REVIEWS Gene regulation by miRNAs

16. Ø Distinct sets of miRNAs are associated with
different cell states
- quiescent
- activated
- differentiated
Ø Quiescent specific miRNAs are potential
regulators of the stem cell for its maintenance in
the niche
Summary

17. Dormancy and response to stress expose distinct
muscle stem cell states
Pax7Hi:
• lower mitochondrial activity
• lower ATP consumption
• lag in 1st mitosis in vivo and ex vivo
• higher expression of upstream markers
➜ Pax7Hi cells = a novel dormant cell state
Rocheteau et al. Cell, 2012
FITC-A
PE-A
Lathil et al. Nature Comm. 2012
Life after death:
Post-mortem muscle stem cells:
• survive 2-3 weeks for mouse and human
• properties similar to Pax7Hi
• severe hypoxia and low temperature
Collaboration Fabrice Chrétien, Pasteur

19. Transcriptomic and proteomics

20. Ø Muscle and blood stem cells survive for extended
periods post-mortem
Ø Stem cells can enter a deep state of quiescence
called dormancy
Summary

21. Stem cell properties during ageing
Carlson et al., 2008; Conboy et al., 2003; Mourikis et al., 2012; Bjornson et al., 2012;
Collins et al., 2007; Abou-Khalil et al., 2009; Nagata et al.,, 2006; Shea et al., 2010
Tajbakhsh, Cell Res., 2013

22. Differential histone signatures in Q and Act. stem cells
Liu et al. , Cell Reports, 2013

23. Differential histone signatures in young and old stem cells
Liu et al. , Cell Reports, 2013

24. Preclinical and clinical trials

25. Guiraud Ann Rev Genomics Hum Genet 2015

26. National Library of Medicine

27. Ø Corticosteroids could improve muscle strength with lasting benefits
Ø Side effect include weight gain, hypertension, diabetes, infections, etc.
Ø Correcting dystrophies (DMD) gene defects:
Ø Gene replacement (delivery vectors; shortened-Dystrophin gene)
Ø Exon skipping approach
Ø Genome editing CRISPR/Cas9 with AAV delivery
Ø IGF1, Folistatin (inhibits Myostatin), Myostatin inhibition(or combination of three)
Ø FDA rejection: AAV delivery (immune response); gentamycin treatment
(non-sense suppression); antisense oligonucleotides
Ø Advances in oligonucleotide strategies for exon skipping
Ø Next generation chemical and peptied modified oligos
Ø TricycloDNA Antisense Oligonucleotides for splice switching
Ø Specific force, respiratory and cardiac functions restored
(Goyenvalle et al , Nat Med 2015)
Overview of strategies to treat muscle dystrophies

28. Negroni et al. Human Gene Therapy 2016
Myogenic cell candidates for myopathies

29. Myogenic cell candidates for myopathies

30. Montarras et al. Science 2005
Ikemoto et al. Mol Therapy 2007
Culturing satellite cells diminishes transplantation efficiency

31. Maintaining muscle stem cell engraftment potential
after ex vivo expansion
Ø Substrate stiffness; suppression of p38 signaling; transient inhibition of
STAT3 promotes satellite cell expansion;
(Gilbert et al. Science 2010; Charville et al. Stem Cell Reports 2015;
Tierney et al. Nat. Med. 2014)
Ø Artificial niches can alter muscle stem cell proliferation kinetics
Ø Symmetric vs. Asymmetric cell divisions
(Yennek et al. Cell Reports 2014)

32. Negroni et al. Human Gene Therapy 2016
Strategies for delivery of stem cells in myopathies

33. Ø Skeletal muscles and their stem cells are distinct in
different anatomical locations
Ø Clinical consequences?
Ø Muscle and blood stem cells persist after death for extended
periods and remain functional
Ø Muscle stem cells decline in number during ageing as they
break quiescence; lose potency
Ø Epigenomic profiles altered in aged muscle stem cells
Ø Clinical trials involving myoblast transplantations for OPMD
and mesoangioblasts for DMD
Ø Exon-skipping strategies: oligonucleotides, etc.
Summary