Advances in cell reprogramming

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Advances in cell reprogramming

Marielle Afanassieff

To cite this version:

Marielle Afanassieff. Advances in cell reprogramming. 3. Biennal Seminar of CRB-Anim Infrastruc-ture, Centres de Ressources Biologiques pour les Animaux Domestiques (CRB-Anim). FRA., May 2017, Paris, France. 22 diapos. �hal-02784863�

Biennal Seminar 2017

Agenda

May 11

_-12

_{2017 – Paris}

Day One (11 May 2017)

Session 1: Achievements of CRB-Anim

9h30 Introduction of WP2&7 Elisabeth Blesbois

Developments in reproductive biotechnologies (WP2)

Advances in epigenetics: Hélène Jammes (INRA) New cryoconservation media for marine mollusk species:

Catherine Labbé (INRA) Progress on animal-free conservation media :

Lucie Gavin-Plagne (VetAgro Sup) Advances in cell reprogramming : Marielle Afanassieff (INSERM)

11h00 Prospects for exploitation of new achievements (WP7)

Non academic exploitation of the developments in reproductive biotechnologies: Xavier Vignon (INRA) Exploitation of DNA diagnostic tests : Anne Thomas (Antagene) Discussion on exploitation strategies

11h45 Collection enrichment and current balance for entry/exit (WP3/WP5)

Coralie Danchin-Burge (Idele) 12h30 : lunch buffet

Session 2: Launching the CRB-Anim portal

14h Functionalities of the portal and live demonstration

Sylvain Marthey (INRA) and Aurélie Delavaud (FRB) (WP4)

Session 3: Issues raised by genome editing for animal genetic resources and CRB-Anim

15h-16h30

State of the art, what can be done with genome editing (GE) Bertrand Pain (INRA) Science-policy challenges of synthetic biology Barbara Livoreil (FRB) at the Convention for Biological Diversity

Political challenges of GE and CBD Barbara Livoreil (FRB) Ethical challenges of GE Philippe Monget et coll. Questions for CRB-Anim Michèle Tixier-Boichard (INRA)

10’

16h30-17h30: roundtable with SAB and invited speakers “’

The SAB will have access to S217 room to prepare feedback for day 2.

Day two (12 May 2017)

Session 4: European landscape for animal biobanks

9h-10h30

Standard protocols for biobanking & COST action SALAAM Eckhard Wolf( LMU Munich) The Global Genome Biodiversity Network Michal Vinkler (Prague) Invited talk from a European infrastructure: AquaExcel

10h30 – 11h Coffee break

Survey of European Animal Biobanks for IMAGE project Anne-Sophie Passemard (idele) The EUGENA network S. Hiemstra (DLO)

Round-table with invited speakers chaired by Sipke Hiemstra : prospect for a European infrastructure

12h30 lunch buffet

Session 5: CRB-anim mid term evaluation and updated roadmap

14:00

Recommendations from INBS steering committee and actions to be taken Focus on the training strategy of CRB-Anim

By M. Tixier-Boichard and WP leaders Feedback from SAB

Concluding remarks by INRA and ANR representatives End by 16:00 www.crb-anim.fr WIMI ASIEM Salle de conférence 6 rue Albert de Lapparent 75007 Paris

CRB Anim

Centres de Ressources Biologiques

WP2.2 Developments in

Reproductive Biotechnologies

Marielle AFANASSIEFF - May 11th 2017

Advances in Cell Reprogramming

Marielle AFANASSIEFF

Stem cell and Brain Research Institute

Inserm U1208

Inra USC1361

Lyon, France

Reprogramming

Reprogramming

refers to erasure and remodeling of epigenetic marks,

during mammalian development or in cell culture, which results in a global

modification of genome expression

Discovered in 1962

by John Gurdon with

a nuclear transfer experiment using

intestine cell nucleus introduced into

enucleated frog eggs

Discovered in 2006

by Shinya Yamanaka with

the description of a small number of

genes responsible of reprogramming skin

cells into pluripotent stem cells

Nobel Prize in

Physiology and

Goals :

ü 

Cryobanking of somatic tissues (Skin, Blood, Bone Marrow)

ü 

Maintaining species biodiversity

ü 

Long term preservation of genetic resources

ü 

Rescue of endangered species

Reprogramming and species preservation

Advantages:

ü 

Alternative to gametes and embryos

ü 

Preservation of diploid genome

ü 

Large number of preserved samples

ü 

No reproductive induction required

ü 

Sampling does not require animal euthanasia

Cell reprogramming for species recovery

Nuclear cell transfer

Cloned Blastocyst Donor mouse Somatic tissues Somatic Cells Induced Pluripotent _{Stem Cells (iPSCs)}

Cryobanking Cryobanking Cryobanking Reprogramming

Derivation Sampling

Reprogramming in farming animals

Mammals

Rabbit

Fishes

Goldfish

Birds

Chicken

Usual cryobanking:

ü  Sperm

Oocytes

Embryos

ü  Primordial Germ Cells

Usual cryobanking:

ü  Sperm

Oocytes

Embryos

ü  Primordial Germ Cells

Classical cryobanking:

ü  Sperm

Oocytes

ü  Embryos

Primordial Germ Cells

Reprogramming in farming animals

Mammals

Rabbit

Fishes

Goldfish

Birds

Chicken

Fibroblast derivation

Sampling of fin biopsies

Cloning by nuclear

cell transfer

In vitro

reprogramming

Adult goldfish

Alexandra Depincé

Nathalie Chenais

Catherine Labbé

INRA

Rennes

Sampling and cryobanking

of skin biopsies

In vitro reprogramming into iPSCs

Embryo

colonization

Cloning by nuclear

cell transfer

Fibroblast derivation

Adult rabbit

Marielle Afanassieff

Pierre Savatier

SBRI

INSERM 1208

Lyon

Nathalie Daniel

Véronique Duranthon

BDR

INRA

Jouy-En-Josas

Derivation cESCs

Reprogramming

into iPSCs

Embryo colonization

Reprogramming

Into PGCs

Chicken embryos

Aurélie Fuet

Bertrand Pain

SBRI

INSERM U1208

Lyon

Derivation of CEFs

Reprogramming of rabbit cells

Donor rabbit Skin biopsy Rb Fibroblasts RbiPSCs

Reprogramming using retroviral vectors expressing human OSKM factors Derivation

Sampling

Optimizing cryobanking conditions of skin biopsies

Enhancing pluripotency of rbiPSCs by overexpressing

human Klf2 and Klf4 factors

RbEKA cells

Optimizing reprogramming with Sendaï vectors

Microinjection into 4- to 8-cell embryos Colonization of rabbit gastrula (1.4%) Microinjection into 4- to

Reprogramming of rabbit somatic cells

Nuclear cell transfer Rb Fibroblasts

Rb Cumulus cells

Cloned Blastocyst Cloned Rabbits

30-50% 2-3%

Reprogramming of rabbit somatic cells

Rb Fibroblasts RbiPSCs

Reprogramming using retroviral vectors expressing human OSKM factors Rb Blastocyst RbESCs

Derivation

Nuclear cell transfer

Cell fusion with enucleated rabbit oocyte

Direct microinjection of the cell into an

enucleated rabbit oocyte Developmental arrest 8-cell embryo Morula Blastocyst 7% Developmental arrest after implantation (92 embryos into 5 femals) Cell fusion with

enucleated rabbit oocyte

Reprogramming of goldfish cells

Donor goldfish Fin biopsy

Cryobanking Derivation

Sampling

Optimizing derivation and cryobanking conditions of fin fibroblasts

Microinjection under the cytoplasmic membrane

though the micropyle

In vivo reprogramming by somatic cell transfer

into oocytes Water activation 60-80% Blastula 20% 24h-Gastrula <10% Hatching 1-10% Abnormal development

Reprogramming defect

Genome hypermethylation

of cloned embryos

Reprogramming of goldfish cells

Donor goldfish Fin biopsy

Cryobanking Derivation

Sampling

Optimizing derivation and cryobanking conditions of fin fibroblasts

Microinjection under the cytoplasmic membrane

though the micropyle

In vivo reprogramming by somatic cell transfer

into oocytes Water activation 60-80% Blastula 20% 24h-Gastrula In vitro reprogramming by extracts from Xenopus laevis metaphase oocytes Study of

- Xenopus oocyte stage - Extract quality

- Fin cell permeabilisation - Fin cell reprogramming

Reprogramming of chicken cells

Fertilized Egg 2-cell stage Stage X Stage XV 48h Embryo

Blastoderm Isolation

Blastoderm cells (cBCs) Embryonic Stem Cells (cESCs) Microinjection into stage X embryo Chick Somatic chimeric Chicks (20-50%) Derivation Germline chimeric Chicks (20-50%) Blood sampling Circulating Primordial Germ Cells (PGCs) Isolation

Reprogramming of chicken cells

Reprogramming of chicken cells

Reprogramming

ü 

Alternative solution for species preservation

ü 

Several reprogramming strategies depending on species

ü 

Encouraging results in all analyzed species

ü 

Not yet available in farm animals

Conclusions

Thanks to

ü 

Team of Véronique Duranthon (INRA, Jouy-en-Josas)

ü 

Team of Catherine Labbé (INRA, Rennes)

ü 

Team of Bertrand Pain (SBRI, INSERM, Lyon)

Labogena Labogena