<figure><img src="image_1.png" /></figure>

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# Mammalian Stem Cells: from isolation to applications

Hervé Acloque

► To cite this version:

Hervé Acloque. Mammalian Stem Cells: from isolation to applications. Master. Mammalian Stem Cells: from isolation to applications, Master Biomedical Engineering (BME) Université Paris Cité, France. 2023. [hal-04454663](https://hal.inrae.fr/hal-04454663)

HAL Id: hal-04454663

https://hal.inrae.fr/hal-04454663

Submitted on 13 Feb 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

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destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
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publics ou privés.

Public Domain
# Mammalian Stem Cells: from isolation to applications

Hervé Acloque

► To cite this version:

Hervé Acloque. Mammalian Stem Cells: from isolation to applications. Master. Mammalian Stem Cells: from isolation to applications, Master Biomedical Engineering (BME) Université Paris Cité, France. 2023. [hal-04454663](https://hal.inrae.fr/hal-04454663v1)

HAL Id: hal-04454663

https://hal.inrae.fr/hal-04454663v1

Submitted on 13 Feb 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

Public Domain
# Mammalian Stem Cells
## from isolation to applications

Hervé Acloque

INRAE UMR1313, Jouy en Josas

herve.acloque@inrae.fr
# Mammalian Stem Cells
## from isolation to applications

Hervé Acloque

INRAE UMR1313, Jouy en Josas

herve.acloque@inrae.fr
# Mammalian stem cells

* Terminology and definition

* The quest for adult pluripotent stem cells

* Non rodent pluripotent stem cells: production and applications

* Interspecies chimera: why, why not, how
# Mammalian stem cells

* Terminology and definition

* The quest for adult pluripotent stem cells

* Non rodent pluripotent stem cells: production and applications

* Interspecies chimera: why, why not, how
# Terminology and definition
Terminology and definition
The concept of cellular potential is quite novel:
a biologically fixed cell fate ?

Au reſte, l'œuf n'est à pro-
prement parler que ce qu'on
appelle placenta, dont l'enfant,
après y avoir demeuré un cer-
tain temps tout courbé & com-
me en peloton, brisé en s'éten-
dant & en s'allongeant le plus
qu'il peut, les membranes qui le
couvroient, & poſant ſes pieds
contre le placenta, qui reſte atta-
ché au fond de la matrice, ſe
pouſſe ainſi avec la tête hors de
ſa prison ; en quoi il eſt aidé par
la mere, qui agitée par la dou-
leur qu'elle en ſent, pouſſe le
fond de la matrice en bas, &
donne par conſequent d'autant
plus d'occasion à cet enfant de

<figure><img src="image_2.png" /><figcaption>Human Fœtus into a spermatozoide: *The homunculus* (Nicolas Hartsoeker, 1694)</figcaption></figure>
The concept of cellular potential is quite novel:
a biologically fixed cell fate ?

Au reſte, l'œuf n'est à pro-
prement parler que ce qu'on
appelle placenta, dont l'enfant,
après y avoir demeuré un cer-
tain temps tout courbé & com-
me en peloton, brisé en s'éten-
dant & en s'allongeant le plus
qu'il peut, les membranes qui le
couvroient, & poſant ſes pieds
contre le placenta, qui reſte atta-
ché au fond de la matrice, ſe
pouſſe ainſi avec la tête hors de
ſa prison ; en quoi il eſt aidé par
la mere, qui agitée par la dou-
leur qu'elle en ſent, pouſſe le
fond de la matrice en bas, &
donne par conſequent d'autant
plus d'occasion à cet enfant de

<figure><img src="image_1.png" /><figcaption>Human Fœtus into a spermatozoide: *The homunculus* (Nicolas Hartsoeker, 1694)</figcaption></figure>
# The concept of pluripotency is quite novel
Experimental studies in aquatic species

Driesch experiments in 1891 on sea urchin blastomeres at the 2-cell and 4-cell stages: totipotency

<figure><img src="image_3.png" /></figure>

Plutei developed from
single cells of 4-cell embryo

Speeman's experiments in 1902 on triton's blastomeres blastomeres: totipotency and pluripotency

<figure><img src="image_4.png" /></figure>
# The concept of pluripotency is quite novel
Experimental studies in aquatic species

Driesch experiments in 1891 on sea urchin blastomeres at the 2-cell and 4-cell stages: totipotency

<figure><img src="image_2.png" /></figure>

Plutei developed from
single cells of 4-cell embryo

Speeman's experiments in 1902 on triton's blastomeres blastomeres: totipotency and pluripotency

<figure><img src="image_3.png" /></figure>
# Pluripotency in mammals: the mouse model

<figure><img src="image_5.png" /></figure>
# Pluripotency in mammals: the mouse model

<figure><img src="image_4.png" /></figure>
# Pluripotency in mammals: the mouse model
## Aggregation chimeras (Tarkowski 1962) and injection chimera (Gardner 1968)

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
# Pluripotency in mammals: the mouse model
## Aggregation chimeras (Tarkowski 1962) and injection chimera (Gardner 1968)

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
# Pluripotency in mammals: the mouse model

<figure><img src="image_11.png" /></figure>

**Totipotent:** sufficient to form entire organism.

**Pluripotent:** able to form all the body cell lineages, including germ cells.
# Pluripotency in mammals: the mouse model

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

**Totipotent:** sufficient to form entire organism.

**Pluripotent:** able to form all the body cell lineages, including germ cells.
# Pluripotency in mammals: the mouse model

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
# Pluripotency in mammals: the mouse model

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
# Pluripotency in mammals: the mouse model

The ultimate demonstration of pluripotency in mammals:
tetraploid complementation

<figure><img src="image_14.png" /></figure>
# Pluripotency in mammals: the mouse model

The ultimate demonstration of pluripotency in mammals:
tetraploid complementation

<figure><img src="image_14.png" /></figure>
Totipotency

Full differentiation potential
(ExtraEmb + Emb)
+ Self organisation

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

Expanded
pluripotency

Full differentiation potential
(ExtraEmb + Emb)
no Self organisation

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

Pluripotency

Full embryonic differentiation
potential
no Self organisation

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>
Totipotency

Full differentiation potential
(ExtraEmb + Emb)
+ Self organisation

<figure><img src="image_15.png" /></figure>

Expanded
pluripotency

Full differentiation potential
(ExtraEmb + Emb)
no Self organisation

<figure><img src="image_16.png" /></figure>

Pluripotency

Full embryonic differentiation
potential
no Self organisation

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>
## Totipotency

Full differentiation potential
+ Self organisation

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

## Multipotency

Multiple but restricted
differentiation potential
Self organisation: somehow
possible (ie organoids)

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /><figcaption>Bones</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>Skeletal muscle
and tendons</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>Pt</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>Hematopoietic
cells</figcaption></figure>

## Bipotency / Unipotency

Restricted differentiation potential

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

### Neural Crest Cells (NCCs)

### Hematopoietic stem cells (HSCs)

### Mesenchymal Stem Cells (MSCs)
## Totipotency

Full differentiation potential
+ Self organisation

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

## Multipotency

Multiple but restricted
differentiation potential
Self organisation: somehow
possible (ie organoids)

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /><figcaption>Bones</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>Skeletal muscle
and tendons</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>Pt</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>Hematopoietic
cells</figcaption></figure>

## Bipotency / Unipotency

Restricted differentiation potential

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

### Neural Crest Cells (NCCs)

### Hematopoietic stem cells (HSCs)

### Mesenchymal Stem Cells (MSCs)
# The quest for adult pluripotent stem cells
# The quest for adult pluripotent stem cells
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

The first description of adult multipotent stem cells: HSCs

In the 1960s, working on graft of bone marrow cells, Alexander Friedenstein discovered that some cells possess an osteogenic potential.

<figure><img src="image_39.png" /><figcaption>Alexander Friedenstein (1924-1998)
URSS</figcaption></figure>
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

The first description of adult multipotent stem cells: HSCs

In the 1960s, working on graft of bone marrow cells, Alexander Friedenstein discovered that some cells possess an osteogenic potential.

<figure><img src="image_39.png" /><figcaption>Alexander Friedenstein (1924-1998)
URSS</figcaption></figure>
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

J. Embryol. exp. Morph., Vol. 16, 3, pp. 581-390, December 1966
With 5 plates

Printed in Great Britain

381

### Osteogenesis in transplants of bone marrow cells

By A. J. FRIEDENSTEIN¹, I. I. PIATETZKY-SHAPIRO¹ & K. V. PETRAKOVA¹

From the Laboratory of Immunomorphology, Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the U.S.S.R., and Laboratory of Mathematical Methods in Biology, University of Moscow

<figure><img src="image_40.png" /></figure>
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

J. Embryol. exp. Morph., Vol. 16, 3, pp. 581-390, December 1966
*With 5 plates*

Printed in Great Britain

381

### Osteogenesis in transplants of bone marrow cells

By A. J. FRIEDENSTEIN¹, I. I. PIATETZKY-SHAPIRO¹ & K. V. PETRAKOVA¹

From the Laboratory of Immunomorphology, Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the U.S.S.R., and Laboratory of Mathematical Methods in Biology, University of Moscow

<figure><img src="image_40.png" /></figure>
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

<figure><img src="image_41.png" /></figure>

Friedenstein et al. 1987, Ashton et al. 1980, Bab et al. 1984 and others
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

<figure><img src="image_41.png" /></figure>

Friedenstein et al. 1987, Ashton et al. 1980, Bab et al. 1984 and others
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

## Many different stem cells with restricted potency ?

<figure><img src="image_42.png" /></figure>
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

## Many different stem cells with restricted potency ?

<figure><img src="image_42.png" /></figure>
# Adult pluripotent stem cells: do they exist ?

The discovery of mesenchymal stem cells (MSCs)

One unique multipotent stem cells with enlarged potency ?

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

Bone Marrow (Fibroblasts)
# Adult pluripotent stem cells: do they exist ?

The discovery of mesenchymal stem cells (MSCs)

One unique multipotent stem cells with enlarged potency ?

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

Bone Marrow (Fibroblasts)
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

## One unique multipotent stem cells with enlarged potency

# Multilineage Potential of Adult Human Mesenchymal Stem Cells

Mark F. Pittenger,¹* Alastair M. Mackay,¹ Stephen C. Beck,¹

Rama K. Jaiswal,¹ Robin Douglas,¹ Joseph D. Mosca,¹

Mark A. Moorman,¹ Donald W. Simonetti,¹ Stewart Craig,¹

Daniel R. Marshak¹,²
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

## One unique multipotent stem cells with enlarged potency

# Multilineage Potential of Adult Human Mesenchymal Stem Cells

Mark F. Pittenger,¹* Alastair M. Mackay,¹ Stephen C. Beck,¹

Rama K. Jaiswal,¹ Robin Douglas,¹ Joseph D. Mosca,¹

Mark A. Moorman,¹ Donald W. Simonetti,¹ Stewart Craig,¹

Daniel R. Marshak¹,²

Nature 1999
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

### One unique multipotent stem cells with enlarged potency
**CD73**

<figure><img src="image_45.png" /></figure>

A

<figure><img src="image_46.png" /></figure>

B

C

<figure><img src="image_47.png" /></figure>

D

<figure><img src="image_48.png" /></figure>

**CD105**
# Adult pluripotent stem cells: do they exist ?

## The discovery of mesenchymal stem cells (MSCs)

### One unique multipotent stem cells with enlarged potency
**CD73**

<figure><img src="image_45.png" /></figure>

A

<figure><img src="image_46.png" /></figure>

B

<figure><img src="image_47.png" /></figure>

C

<figure><img src="image_48.png" /></figure>

D

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

D

<figure><img src="image_51.png" /></figure>

### CD105
# Adult pluripotent stem cells: do they exist ?

The discovery of mesenchymal stem cells (MSCs)

One unique multipotent stem cells with enlarged potency

## Colonies from single cell

A

Donor
398

<figure><img src="image_49.png" /></figure>

Colony
3

<figure><img src="image_50.png" /></figure>

Colony
12

<figure><img src="image_51.png" /></figure>
# Adult pluripotent stem cells: do they exist ?

The discovery of mesenchymal stem cells (MSCs)

One unique multipotent stem cells with enlarged potency

## Colonies from single cell

A

Donor
398

<figure><img src="image_52.png" /></figure>

Colony
3

<figure><img src="image_53.png" /></figure>

Colony
12

<figure><img src="image_54.png" /></figure>
# Adult pluripotent stem cells: do they exist ?

The discovery of mesenchymal stem cells (MSCs)

One unique multipotent stem cells with enlarged potency

* Adherent cells in 2D culture (Fibroblasts)

* Cells positives for the surface determinants CD73, CD90 and CD105

* Cells negative for hematopoietic markers :CD34-, CD45-, CD14-, CD79a-, HLA-DR-

* Multipotency in vitro and in vivo at least for osteoblastic, chondroblastic and adipocytic lineages (mesodermal)
# Adult pluripotent stem cells: do they exist ?

The discovery of mesenchymal stem cells (MSCs)

One unique multipotent stem cells with enlarged potency

* Adherent cells in 2D culture (Fibroblasts)

* Cells positives for the surface determinants CD73, CD90 and CD105

* Cells negative for hematopoietic markers :CD34-, CD45-, CD14-, CD79a-, HLA-DR-

* Multipotency in vitro and in vivo at least for osteoblastic, chondroblastic and adipocytic lineages (mesodermal)
# Adult pluripotent stem cells: do they exist ?

The discovery of mesenchymal stem cells (MSCs)

One unique multipotent stem cells with enlarged potency

* Originally discovered in the Bone Marrow

* Can be isolated from cord blood, placenta, circulating blood, bone marrow, dental pulp, cartilage, adipose tissue, skeletal muscle, blood vessel (pericytes) etc

* MSCs seem to be elsewhere in the organism but in extremely low proportion (0,01% - 0,001% in BM MNCs)
# Adult pluripotent stem cells: do they exist ?

The discovery of mesenchymal stem cells (MSCs)

One unique multipotent stem cells with enlarged potency

* Originally discovered in the Bone Marrow

* Can be isolated from cord blood, placenta, circulating blood, bone marrow, dental pulp, cartilage, adipose tissue, skeletal muscle, blood vessel (pericytes) etc

* MSCs seem to be elsewhere in the organism but in extremely low proportion (0,01% - 0,001% in BM MNCs)
# Adult pluripotent stem cells: do they exist ?

## The discovery of adult pluripotent stem cells

* Inspired by the discovery of adult multipotent stem cells in the bone marrow, laboratories worldwide demonstrated the existence of adult pluripotent stem cells in the 2000s
# Adult pluripotent stem cells: do they exist ?

## The discovery of adult pluripotent stem cells

* Inspired by the discovery of adult multipotent stem cells in the bone marrow, laboratories worldwide demonstrated the existence of adult pluripotent stem cells in the 2000s
# Adult pluripotent stem cells: do they exist ?

## The discovery of adult pluripotent stem cells

### Generalized Potential of Adult Neural Stem Cells

Diana L. Clarke,¹ Clas B. Johansson,¹,² Johannes Wilbertz,¹
Biborka Veress,¹ Erik Nilsson,¹ Helena Karlström,¹
Urban Lendahl,¹ Jonas Frisén¹*

### From Marrow to Brain: Expression of Neuronal Phenotypes in Adult Mice

Timothy R. Brazelton, Fabio M. V. Rossi, Gilmor I. Keshet, Helen M. Blau*

Science 2000

articles

## Pluripotency of mesenchymal stem cells derived from adult marrow

Yuehua Jiang*†, Balkrishna N. Jahagirdar*†‡, R. Lee Reinhardt§, Robert E. Schwartz*, C. Dirk Keene||, Xilma R. Ortiz-Gonzalez||, Morayma Reyes*, Todd Lenvik*, Troy Lund*, Mark Blackstad*, Jingbo Du*, Sara Aldrich*, Aaron Lisberg*, Walter C. Low||, David A. Largaespada¶ & Catherine M. Verfaillie*‡

<footnote>* Stem Cell Institute, ‡ Division of Hematology, Oncology and Transplantation, Department of Medicine, § Department of Microbiology, Center for Immunology, || Department of Neurosurgery, and ¶ Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA</footnote>

<footnote>† These authors contributed equally to this work</footnote>
# Adult pluripotent stem cells: do they exist ?

## The discovery of adult pluripotent stem cells

### Generalized Potential of Adult Neural Stem Cells

Diana L. Clarke,¹ Clas B. Johansson,¹,² Johannes Wilbertz,¹
Biborka Veress,¹ Erik Nilsson,¹ Helena Karlström,¹
Urban Lendahl,¹ Jonas Frisén¹*

### From Marrow to Brain: Expression of Neuronal Phenotypes in Adult Mice

Timothy R. Brazelton, Fabio M. V. Rossi, Gilmor I. Keshet, Helen M. Blau*

Science 2000

articles

## Pluripotency of mesenchymal stem cells derived from adult marrow

Yuehua Jiang*†, Balkrishna N. Jahagirdar*†‡, R. Lee Reinhardt§, Robert E. Schwartz*, C. Dirk Keene||, Xilma R. Ortiz-Gonzalez||, Morayma Reyes*, Todd Lenvik*, Troy Lund*, Mark Blackstad*, Jingbo Du*, Sara Aldrich*, Aaron Lisberg*, Walter C. Low||, David A. Largaespada¶ & Catherine M. Verfaillie*‡

<footnote>* Stem Cell Institute, ‡ Division of Hematology, Oncology and Transplantation, Department of Medicine, § Department of Microbiology, Center for Immunology, || Department of Neurosurgery, and ¶ Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA</footnote>

<footnote>† These authors contributed equally to this work</footnote>
# Adult pluripotent stem cells: do they exist ?

## The discovery of adult pluripotent stem cells

<figure><img src="image_52.png" /><figcaption>Figure 1. Evolving Concepts of Stem Cell Plasticity</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>Figure 8. Route Stem Cell</figcaption></figure>
# Adult pluripotent stem cells: do they exist ?

## The discovery of adult pluripotent stem cells

<figure><img src="image_55.png" /><figcaption>Figure 1. Evolving Concepts of Stem Cell Plasticity</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>Figure 8. Route Stem Cell</figcaption></figure>
# Adult pluripotent stem cells: do they exist ?

## The discovery of adult pluripotent stem cells

In all these studies, only very scarce cells show a pluripotent potential:

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /><figcaption>neurons</figcaption></figure>

<figure><img src="image_61.png" /><figcaption>Liver</figcaption></figure>

<figure><img src="image_62.png" /><figcaption>intestine</figcaption></figure>

<figure><img src="image_63.png" /><figcaption>lung</figcaption></figure>
# Adult pluripotent stem cells: do they exist ?

## The discovery of adult pluripotent stem cells

In all these studies, only very scarce cells show a pluripotent potential:

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /><figcaption>neurons</figcaption></figure>

<figure><img src="image_64.png" /><figcaption>Liver</figcaption></figure>

<figure><img src="image_65.png" /><figcaption>intestine</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>lung</figcaption></figure>
# Adult pluripotent stem cells: a scientific mistake

Adult pluripotent stem cells result from rare events of cell fusion

## Changing potency by spontaneous fusion

Qi-Long Ying*, Jennifer Nichols*, Edward P. Evans† & Austin G. Smith*

* Centre for Genome Research, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK

† Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

Nature 2002

## Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes

Manuel Alvarez-Dolado¹, Ricardo Pardal², Jose M. Garcia-Verdugo³,

John R. Fike¹, Hyun O. Lee², Klaus Pfeffer⁴, Carlos Lois⁵,

Sean J. Morrison² & Arturo Alvarez-Buylla¹

Nature 2003
# Adult pluripotent stem cells: a scientific mistake

Adult pluripotent stem cells result from rare events of cell fusion

## Changing potency by spontaneous fusion

Qi-Long Ying*, Jennifer Nichols*, Edward P. Evans† & Austin G. Smith*

* Centre for Genome Research, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK

† Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

Nature 2002

## Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes

Manuel Alvarez-Dolado¹, Ricardo Pardal², Jose M. Garcia-Verdugo³,

John R. Fike¹, Hyun O. Lee², Klaus Pfeffer⁴, Carlos Lois⁵,

Sean J. Morrison² & Arturo Alvarez-Buylla¹

Nature 2003
# Adult pluripotent stem cells: a scientific mistake

Adult pluripotent stem cells result from rare events of cell fusion

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>
# Adult pluripotent stem cells: a scientific mistake

Adult pluripotent stem cells result from rare events of cell fusion

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>
<figure><img src="image_67.png" /><figcaption>Hepatocytes</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>Purkinje cells</figcaption></figure>

<figure><img src="image_69.png" /><figcaption>Skeletal muscle</figcaption></figure>
<figure><img src="image_70.png" /><figcaption>Hepatocytes</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>Purkinje cells</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>Skeletal muscle</figcaption></figure>
Adult pluripotent stem cells: a scientific mistake

Adult pluripotent stem cells result from rare events of
cell fusion from MSCs with differentiated cells

Differentiation, cell fusion, and nuclear fusion during
ex vivo repair of epithelium by human adult stem
cells from bone marrow stroma

Jeffrey L. Spees*, Scott D. Olson*, Joni Ylostalo*, Patrick J. Lynch*, Jason Smith*, Anthony Perry*, Alexandra Peister*, Meng Yu Wang†, and Darwin J. Prockop‡

*Center for Gene Therapy, Tulane Health Sciences Center, New Orleans, LA 70112; and †Department of Tumor Biology, Institute for Cancer Research, Norwegian Radium Hospital, University of Oslo, 0310 Oslo, Norway

Contributed by Darwin J. Prockop, December 30, 2002

Fusion of MSCs with differentiated somatic cells is not a rare
events
Adult pluripotent stem cells: a scientific mistake

Adult pluripotent stem cells result from rare events of
cell fusion from MSCs with differentiated cells

Differentiation, cell fusion, and nuclear fusion during
ex vivo repair of epithelium by human adult stem
cells from bone marrow stroma

Jeffrey L. Spees*, Scott D. Olson*, Joni Ylostalo*, Patrick J. Lynch*, Jason Smith*, Anthony Perry*, Alexandra Peister*,
Meng Yu Wang†, and Darwin J. Prockop*‡

*Center for Gene Therapy, Tulane Health Sciences Center, New Orleans, LA 70112; and †Department of Tumor Biology, Institute for Cancer Research, Norwegian Radium Hospital, University of Oslo, 0310 Oslo, Norway

Contributed by Darwin J. Prockop, December 30, 2002

Fusion of MSCs with differentiated somatic cells is not a rare
events
Adult pluripotent stem cells: a scientific mistake

* MSCs are multipotent cells from adult tissues

* MSCs mostly act through paracrine actions on adjacent cells and tissues

* MSCs may contribute to tissue regeneration

* The existence of pluripotent adult stem cells has never been demonstrated to date
Adult pluripotent stem cells: a scientific mistake

* MSCs are multipotent cells from adult tissues

* MSCs mostly act through paracrine actions on adjacent cells and tissues

* MSCs may contribute to tissue regeneration

* The existence of pluripotent adult stem cells has never been demonstrated to date
# Non rodent pluripotent stem cells: Production and applications
# Non rodent pluripotent stem cells: Production and applications
Non rodent mammalian pluripotent stem cells
***
*the three main properties of true ESCs*

* Infinite self-renewal in culture with stable diploid caryotype and symmetrical division

* in vitro pluripotency: ability to differentiate toward cell types from the three embryonic germ-layers and the germ line.

* in vivo pluripotency: production of chimerae, germ line transmission and tetraploid complementation
Non rodent mammalian pluripotent stem cells
***
*the three main properties of true ESCs*

* Infinite self-renewal in culture with stable diploid caryotype and symmetrical division

* *in vitro* pluripotency: ability to differentiate toward cell types from the three embryonic germ-layers and the germ line.

* *in vivo* pluripotency: production of chimerae, germ line transmission and tetraploid complementation
# Non rodent mammalian pluripotent stem cells

The work made to characterize distinct pluripotent states in mouse embryos helped to derive PSCs in other species

<figure><img src="image_70.png" /></figure>

## Totipotency

2Cs

EPSCs

ESC
Naive

LIF
MEKi
GS3Ki

ESC
Mixte

LIF
serum

EpiLC
FSCs

Activin low
Wnt/hippo inh.

## Pluripotency

EpiSC

Activin
FGF2

But it took decades to success!!!
# Non rodent mammalian pluripotent stem cells

The work made to characterize distinct pluripotent states in mouse embryos helped to derive PSCs in other species

<figure><img src="image_73.png" /></figure>

## Totipotency

2Cs

EPSCs

ESC
Naive

LIF
MEKi
GS3Ki

ESC
Mixte

LIF
serum

EpiLC
FSCs

Activin low
Wnt/hippo inh.

## Pluripotency

EpiSC

Activin
FGF2

But it took decades to success!!!
Non rodent mammalian pluripotent stem cells

BIOLOGY OF REPRODUCTION 55, 254-259 (1996)

Pluripotent Cell Lines Derived from Common Marmoset (Callithrix jacchus)
Blastocysts¹

Monkey

James A. Thomson,²,³ Jennifer Kalishman,³ Thaddeus G. Golos,³,⁴ Maureen Durning,³ Charles P. Harris,⁶ and John P. Hearn³,⁵

The Wisconsin Regional Primate Research Center,³ Departments of Obstetrics and Gynecology⁴ and Physiology,⁵
School of Medicine, and Cytogenetics Laboratory,⁶ State Hygiene Laboratory, University of Wisconsin, Madison,
Wisconsin 53715-1299

Embryonic Stem Cell Lines
Derived from Human
Blastocysts

Human

James A. Thomson,* Joseph Itskovitz-Eldor, Sander S. Shapiro, Michelle A. Waknitz, Jennifer J. Swiergiel, Vivienne S. Marshall, Jeffrey M. Jones

1998
Science

<figure><img src="image_71.png" /></figure>

Capture of Authentic Embryonic
Stem Cells from Rat Blastocysts

Rat

Mia Buehr,¹,² Stephen Meek,¹,² Kate Blair,³,⁴ Jian Yang,³,⁵ Janice Ure,¹ Jose Silva,³,⁴ Renee McLay,¹ John Hall,³,⁴ Qi-Long Ying,¹,⁶ and Austin Smith³,⁴*

2008
Science
Non rodent mammalian pluripotent stem cells

BIOLOGY OF REPRODUCTION 55, 254-259 (1996)

Pluripotent Cell Lines Derived from Common Marmoset (Callithrix jacchus)
Blastocysts¹

Monkey

James A. Thomson,²,³ Jennifer Kalishman,³ Thaddeus G. Golos,³,⁴ Maureen Durning,³ Charles P. Harris,⁶ and John P. Hearn³,⁵

The Wisconsin Regional Primate Research Center,³ Departments of Obstetrics and Gynecology⁴ and Physiology,⁵
School of Medicine, and Cytogenetics Laboratory,⁶ State Hygiene Laboratory, University of Wisconsin, Madison,
Wisconsin 53715-1299

Embryonic Stem Cell Lines
Derived from Human
Blastocysts

Human

1998
Science

James A. Thomson,* Joseph Itskovitz-Eldor, Sander S. Shapiro,
Michelle A. Waknitz, Jennifer J. Swiergiel, Vivienne S. Marshall,
Jeffrey M. Jones

<figure><img src="image_74.png" /></figure>

Capture of Authentic Embryonic
Stem Cells from Rat Blastocysts

Rat

2008
Science

Mia Buehr,¹,² Stephen Meek,¹,² Kate Blair,³,⁴ Jian Yang,³,⁵ Janice Ure,¹ Jose Silva,³,⁴ Renee McLay,¹ John Hall,³,⁴
Qi-Long Ying,¹,⁶ and Austin Smith³,⁴*
# Human embryonic stem cells are equivalent to mouse EpiSCs (epiblast stem cells)

* Non-human primate (1994) and human (1998) pluripotent stem cells have been derived from blastocysts

* Normal caryotype but unstable X inactivation

* Permanent cultures (no "crisis") but slow growth rate

* High telomerase activity

* Express specific "embryonic" antigens but relies on Activin and FGF signalling

* Formation of teratomas (injection into SCID mice) : tissues derived of all three embryonic germ layers

* *In vitro* differentiation

* No evidence for chimera
# Human embryonic stem cells are equivalent to mouse EpiSCs (epiblast stem cells)

* Non-human primate (1994) and human (1998) pluripotent stem cells have been derived from blastocysts

* Normal caryotype but unstable X inactivation

* Permanent cultures (no "crisis") but slow growth rate

* High telomerase activity

* Express specific "embryonic" antigens but relies on Activin and FGF signalling

* Formation of teratomas (injection into SCID mice) : tissues derived of all three embryonic germ layers

* *In vitro* differentiation

* No evidence for chimera
Rat embryonic stem cells are similar to mouse and
can be used for intra and interspecies chimerae

2i(+LIF) culture condition
allow ES cells derivation
from mice and rat

<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /></figure>

Buehr et al, Cell 2008

Li et al, Cell 2008
Rat embryonic stem cells are similar to mouse and
can be used for intra and interspecies chimerae

2i(+LIF) culture condition
allow ES cells derivation
from mice and rat

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

Buehr et al, Cell 2008

Li et al, Cell 2008
Chicken embryonic stem cells contribute poorly to the germline

<figure><img src="image_74.png" /></figure>
Chicken embryonic stem cells contribute poorly to the germline

<figure><img src="image_77.png" /></figure>
21 days of incubation

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

Embryo injection with ES cells from different feather colors

<figure><img src="image_77.png" /></figure>

Cross the chimeara

<figure><img src="image_78.png" /></figure>

Black chicks
derived from
ES-differentiated
germ cells

<figure><img src="image_79.png" /></figure>

Pain et al. 1996
21 days of incubation

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>

Embryo injection with ES cells from different feather colors

<figure><img src="image_80.png" /></figure>

Cross the chimeara

<figure><img src="image_81.png" /></figure>

Black chicks
derived from
ES-differentiated
germ cells

<figure><img src="image_82.png" /></figure>

Pain et al. 1996
# But it remains difficult to produce TRUE PSCs from many other mammalian species: a developmental issue ?

<figure><img src="image_80.png" /></figure>
# But it remains difficult to produce TRUE PSCs from many other mammalian species: a developmental issue ?

<figure><img src="image_83.png" /></figure>
# Challenges to produce TRUE mammalian PSCs: a developmental issue ?

<figure><img src="image_81.png" /></figure>

0.1mm

Early blastocyst
(EB)

<figure><img src="image_82.png" /></figure>

0.2mm

Hatched blastocyst
(HB)

<figure><img src="image_83.png" /></figure>

0.25mm

Spherical blastocyst (SB)

<figure><img src="image_84.png" /></figure>

0.4mm

Ovoid blastocyst (OB)

<figure><img src="image_85.png" /></figure>

<figure><img src="image_86.png" /></figure>

<figure><img src="image_87.png" /></figure>

<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>

<figure><img src="image_90.png" /></figure>

<figure><img src="image_91.png" /></figure>

6

7-8

9-10

11-12

16 = implantation

<figure><img src="image_92.png" /></figure>

6-7

8

9-10

11-13

20 = implantation

<figure><img src="image_93.png" /></figure>

3.5

4

5.5 = implantation

<figure><img src="image_94.png" /></figure>

5.5

6.5

8 = implantation
# Challenges to produce TRUE mammalian PSCs: a developmental issue ?

<figure><img src="image_84.png" /></figure>

Early blastocyst
(EB)

<figure><img src="image_85.png" /></figure>

Hatched blastocyst
(HB)

<figure><img src="image_86.png" /></figure>

Spherical blastocyst (SB)

<figure><img src="image_87.png" /></figure>

Ovoid blastocyst (OB)

<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>

<figure><img src="image_90.png" /></figure>

<figure><img src="image_91.png" /></figure>

<figure><img src="image_92.png" /></figure>

EB HB SB OB

Time post
fertilisation (days)

<figure><img src="image_93.png" /></figure>

6

7-8

9-10

11-12

16 = implantation

<figure><img src="image_94.png" /></figure>

6-7

8

9-10

11-13

20 = implantation

<figure><img src="image_95.png" /></figure>

3.5

4

5.5 = implantation

<figure><img src="image_96.png" /></figure>

5.5

6.5

8 = implantation
# How to tackle these developmental barriers ?

* A better knowledge of *in vivo pluripotency* :

* molecular characterization of the different pluripotent states

* Active signalling pathways in PSCs in vivo

* Startpoint of PSCs proliferation: when + how

* Composition of uterine fluids during preimplantatory development

* The production of faithfull reporter systems to efficiently track **endogenous** pluripotency

* Cell culture optimisation by

* Media optimization from transcriptomics and proteomics studies

* HTS screening of small molecules based using reporter systems
# How to tackle these developmental barriers ?

* A better knowledge of *in vivo pluripotency* :

* molecular characterization of the different pluripotent states

* Active signalling pathways in PSCs in vivo

* Startpoint of PSCs proliferation: when + how

* Composition of uterine fluids during preimplantatory development

* The production of faithfull reporter systems to efficiently track **endogenous** pluripotency

* Cell culture optimisation by

* Media optimization from transcriptomics and proteomics studies

* HTS screening of small molecules based using reporter systems
# Producing TRUE PSCs from many other mammalian species

Using these tools some research groups succeed in producing true PSCs in different species

## Producing rabbit ESCs

<figure><img src="image_95.png" /></figure>

### Stem Cell Reports

#### Article

ISSCR

<figure><img src="image_96.png" /></figure>

OPEN ACCESS

A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits

2017: empiric approach
# Producing TRUE PSCs from many other mammalian species

Using these tools some research groups succeed in producing true PSCs in different species

## Producing rabbit ESCs

<figure><img src="image_97.png" /></figure>

### Stem Cell Reports

#### Article

A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits

2017: empiric approach

ISSCR

<figure><img src="image_98.png" /></figure>

OPEN ACCESS
These rabbit ESCs poorly contribute to chimera

<figure><img src="image_97.png" /><figcaption>**Table 1. Colonization of Rabbit Blastocysts by AKSL-GFP and AKSgff-GFP Cells**</figcaption></figure>

<table><thead><tr><th></th><th>CKF</th><th>AKF</th><th>AKSF</th><th>AKSgff</th><th>AKSL</th></tr></thead><tbody><tr><td>Line no.</td><td>18</td><td>5</td><td>20</td><td>19</td><td>26</td><td>3</td><td>62</td><td>4</td><td>8</td></tr><tr><td>No. of injected embryos</td><td>87</td><td>82</td><td>84</td><td>78</td><td>82</td><td>61</td><td>56</td><td>75</td><td>71</td></tr><tr><td>No. of blastocysts</td><td>74</td><td>68</td><td>77</td><td>64</td><td>66</td><td>39</td><td>40</td><td>64</td><td>61</td></tr><tr><td>No. of blastocysts with GFP-positive cells</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>4</td><td>2</td><td>12</td><td>6</td></tr><tr><td>No. of blastocysts with colonized ICM</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>1</td><td>12</td><td>5</td></tr></tbody></table>

Only with LIF culture rbESCs

Osteil et al. 2017

A: Accutase

C: Collagenase

F: bFGF

L: LIF

S: SVF

K: KOSR
# These rabbit ESCs poorly contribute to chimera

<figure><img src="image_99.png" /><figcaption>**Table 1. Colonization of Rabbit Blastocysts by AKSL-GFP and AKSgff-GFP Cells**</figcaption></figure>

<table><thead><tr><th></th><th>CKF</th><th>AKF</th><th>AKSF</th><th>AKSgff</th><th>AKSL</th></tr></thead><tbody><tr><td>Line no.</td><td>18</td><td>5</td><td>20</td><td>19</td><td>26</td><td>3</td><td>62</td><td>4</td><td>8</td></tr><tr><td>No. of injected embryos</td><td>87</td><td>82</td><td>84</td><td>78</td><td>82</td><td>61</td><td>56</td><td>75</td><td>71</td></tr><tr><td>No. of blastocysts</td><td>74</td><td>68</td><td>77</td><td>64</td><td>66</td><td>39</td><td>40</td><td>64</td><td>61</td></tr><tr><td>No. of blastocysts with GFP-positive cells</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>4</td><td>2</td><td>12</td><td>6</td></tr><tr><td>No. of blastocysts with colonized ICM</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>1</td><td>12</td><td>5</td></tr></tbody></table>

Only with LIF culture rbESCs

Osteil et al. 2017

A: Accutase

C: Collagenase

F: bFGF

L: LIF

S: SVF

K: KOSR
<figure><img src="image_98.png" /></figure>

Article

# Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells

Toshihiro Kobayashi,<sup>1,2,12,*</sup> Aracely Castillo-Venzor,<sup>3,4,5</sup> Chris A. Penfold,<sup>4</sup> Michael Morgan,<sup>6,7</sup> Naoaki Mizuno,<sup>8</sup>

Walfred W.C. Tang,<sup>3,4</sup> Yasuyuki Osada,<sup>9</sup> Masao Hirao,<sup>9</sup> Fumika Yoshida,<sup>2</sup> Hideyuki Sato,<sup>8</sup> Hiromitsu Nakauchi,<sup>8,10</sup>

Masumi Hirabayashi,<sup>2,11,*</sup> and M. Azim Surani<sup>3,4,*</sup>

<figure><img src="image_99.png" /></figure>

2021: with reporters,
scRNAseq from embryos
Signalling pathway screening
<figure><img src="image_100.png" /></figure>

Article

# Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells

Toshihiro Kobayashi,<sup>1,2,12,*</sup> Aracely Castillo-Venzor,<sup>3,4,5</sup> Chris A. Penfold,<sup>4</sup> Michael Morgan,<sup>6,7</sup> Naoaki Mizuno,<sup>8</sup>

Walfred W.C. Tang,<sup>3,4</sup> Yasuyuki Osada,<sup>9</sup> Masao Hirao,<sup>9</sup> Fumika Yoshida,<sup>2</sup> Hideyuki Sato,<sup>8</sup> Hiromitsu Nakauchi,<sup>8,10</sup>

Masumi Hirabayashi,<sup>2,11,*</sup> and M. Azim Surani<sup>3,4,*</sup>

<figure><img src="image_101.png" /></figure>

2021: with reporters,
scRNAseq from embryos
Signalling pathway screening
<figure><img src="image_100.png" /></figure>

A

<figure><img src="image_101.png" /></figure>

Rabbit SOX17 locus

<figure><img src="image_102.png" /></figure>

Essential 8 medium (ITS + FGF2 + TGFb) WNT inhibition (IWP2 non canonical) + XAV939 (canonical)

C

<figure><img src="image_103.png" /></figure>

D

<figure><img src="image_104.png" /></figure>

E

<figure><img src="image_105.png" /></figure>

But no chimeras were produced
<figure><img src="image_102.png" /></figure>

A

<figure><img src="image_103.png" /></figure>

Rabbit SOX17 locus

<figure><img src="image_104.png" /></figure>

SOX17-T2A-H2BVenus

<figure><img src="image_105.png" /></figure>

Essential 8 medium (ITS + FGF2 + TGFb) WNT inhibition (IWP2 non canonical) + XAV939 (canonical)

C

<figure><img src="image_106.png" /></figure>

D

<figure><img src="image_107.png" /></figure>

E

<figure><img src="image_108.png" /></figure>

But no chimeras were produced
<figure><img src="image_106.png" /></figure>

PNAS 2018

# Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts

Yanina Soledad Bogliotti<sup>a,1</sup>, Jun Wu<sup>b,c,d,e,1,2</sup>, Marcela Vilarino<sup>a</sup>, Daiji Okamura<sup>d,f</sup>, Delia Alba Soto<sup>a</sup>, Cuiqing Zhong<sup>e</sup>, Masahiro Sakurai<sup>b,c,d,e</sup>, Rafael Vilar Sampaio<sup>a</sup>, Keiichiro Suzuki<sup>e</sup>, Juan Carlos Izpisua Belmonte<sup>e,2</sup>, and Pablo Juan Ross<sup>a,2</sup>

<sup>a</sup>Department of Animal Science, University of California, Davis, CA 95616; <sup>b</sup>Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; <sup>c</sup>Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; <sup>d</sup>Universidad Católica San Antonio de Murcia, 30107 Guadalupe, Murcia, Spain; <sup>e</sup>Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037; and <sup>f</sup>Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University, 631-8505 Nara, Japan

Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved January 3, 2018 (received for review September 13, 2017)

Canonical Wnt inhibition (IWR1) + bFGF2
<figure><img src="image_109.png" /></figure>

PNAS 2018

# Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts

Yanina Soledad Bogliotti<sup>a,1</sup>, Jun Wu<sup>b,c,d,e,1,2</sup>, Marcela Vilarino<sup>a</sup>, Daiji Okamura<sup>d,f</sup>, Delia Alba Soto<sup>a</sup>, Cuiqing Zhong<sup>e</sup>, Masahiro Sakurai<sup>b,c,d,e</sup>, Rafael Vilar Sampaio<sup>a</sup>, Keiichiro Suzuki<sup>e</sup>, Juan Carlos Izpisua Belmonte<sup>e,2</sup>, and Pablo Juan Ross<sup>a,2</sup>

<sup>a</sup>Department of Animal Science, University of California, Davis, CA 95616; <sup>b</sup>Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; <sup>c</sup>Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; <sup>d</sup>Universidad Católica San Antonio de Murcia, 30107 Guadalupe, Murcia, Spain; <sup>e</sup>Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037; and <sup>f</sup>Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University, 631-8505 Nara, Japan

Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved January 3, 2018 (received for review September 13, 2017)

# Canonical Wnt inhibition (IWR1) + bFGF2
<figure><img src="image_107.png" /></figure>

<figure><img src="image_108.png" /></figure>

<figure><img src="image_109.png" /></figure>

<figure><img src="image_110.png" /></figure>

Bovine primed ESCs

<figure><img src="image_111.png" /></figure>
<figure><img src="image_110.png" /></figure>

<figure><img src="image_111.png" /></figure>

<figure><img src="image_112.png" /></figure>

<figure><img src="image_113.png" /></figure>

Bovine primed ESCs

<figure><img src="image_114.png" /></figure>
# Recent progress for the production of ungulate PSCs

<figure><img src="image_112.png" /></figure>

Nat Cell Biol 2019

## Establishment of porcine and human expanded potential stem cells

Xuefei Gao<sup>1,2,23</sup>, Monika Nowak-Imialek<sup>3,4,23</sup>, Xi Chen<sup>1,2,23</sup>, Dongsheng Chen<sup>5,6</sup>, Doris Herrmann<sup>3,4</sup>, Degong Ruan<sup>1,7</sup>, Andy Chun Hang Chen<sup>8</sup>, Melanie A. Eckersley-Maslin<sup>9</sup>, Shakil Ahmad<sup>10</sup>, Yin Lau Lee<sup>8</sup>, Toshihiro Kobayashi<sup>11</sup>, David Ryan<sup>2</sup>, Jixing Zhong<sup>5,6</sup>, Jiacheng Zhu<sup>5,6</sup>, Jian Wu<sup>1</sup>, Guocheng Lan<sup>12</sup>, Stoyan Petkov<sup>3,4,20</sup>, Jian Yang<sup>2,21</sup>, Liliana Antunes<sup>2</sup>, Lia S. Campos<sup>2</sup>, Beiyuan Fu<sup>2</sup>, Shengpeng Wang<sup>5,6</sup>, Yu Yong<sup>2</sup>, Xiaomin Wang<sup>7</sup>, Song-Guo Xue<sup>13</sup>, Liangpeng Ge<sup>14</sup>, Zuohua Liu<sup>14</sup>, Yong Huang<sup>14</sup>, Tao Nie<sup>7</sup>, Peng Li<sup>17</sup>, Donghai Wu<sup>7</sup>, Duanqing Pei<sup>7,15</sup>, Yi Zhang<sup>16</sup>, Liming Lu<sup>17</sup>, Fengtang Yang<sup>2</sup>, Susan J. Kimber<sup>18</sup>, Wolf Reik<sup>9</sup>, Xiangang Zou<sup>12</sup>, Zhouchun Shang<sup>5,6</sup>, Liangxue Lai<sup>7</sup>, Azim Surani<sup>11</sup>, Patrick P. L. Tam<sup>19</sup>, Asif Ahmed<sup>10</sup>, William Shu Biu Yeung<sup>8</sup>, Sarah A. Teichmann<sup>10</sup>, Heiner Niemann<sup>3,4,22*</sup> and Pentao Liu<sup>1,2*</sup>

<figure><img src="image_113.png" /></figure>

PNAS 2021

## Establishment of bovine expanded potential stem cells

Lixia Zhao<sup>a,b,c,1</sup>, Xuefei Gao<sup>d,e,f,1</sup>, Yuxuan Zheng<sup>g,1</sup> <sup>1</sup>, Zixin Wang<sup>c</sup>, Gaoping Zhao<sup>c</sup>, Jie Ren<sup>g</sup>, Jia Zhang<sup>a,b</sup>, Jian Wu<sup>f</sup> <sup>1</sup>, Baojiang Wu<sup>a,b,c</sup> <sup>1</sup>, Yanglin Chen<sup>a,b</sup> <sup>1</sup>, Wei Sun<sup>b,c</sup>, Yunxia Li<sup>b,c</sup>, Jie Su<sup>c,h</sup>, Yulin Ding<sup>j</sup>, Yuan Gao<sup>c</sup>, Moning Liu<sup>h</sup>, Xiaochun Bai<sup>d,j</sup> <sup>1</sup>, Liangzhong Sun<sup>k</sup>, Guifang Cao<sup>h</sup>, Fuchou Tang<sup>g,l,m</sup>, Siqin Bao<sup>a,b</sup>, Pentao Liu<sup>f,n,2</sup> <sup>1</sup>, and Xihe Li<sup>a,b,c,2</sup>
# Recent progress for the production of ungulate PSCs

<figure><img src="image_115.png" /></figure>

Nat Cell Biol 2019

## Establishment of porcine and human expanded potential stem cells

Xuefei Gao<sup>1,2,23</sup>, Monika Nowak-Imialek<sup>3,4,23</sup>, Xi Chen<sup>1,2,23</sup>, Dongsheng Chen<sup>5,6</sup>, Doris Herrmann<sup>3,4</sup>, Degong Ruan<sup>1,7</sup>, Andy Chun Hang Chen<sup>8</sup>, Melanie A. Eckersley-Maslin<sup>9</sup>, Shakil Ahmad<sup>10</sup>, Yin Lau Lee<sup>8</sup>, Toshihiro Kobayashi<sup>11</sup>, David Ryan<sup>2</sup>, Jixing Zhong<sup>5,6</sup>, Jiacheng Zhu<sup>5,6</sup>, Jian Wu<sup>1</sup>, Guocheng Lan<sup>12</sup>, Stoyan Petkov<sup>3,4,20</sup>, Jian Yang<sup>2,21</sup>, Liliana Antunes<sup>2</sup>, Lia S. Campos<sup>2</sup>, Beiyuan Fu<sup>2</sup>, Shengpeng Wang<sup>5,6</sup>, Yu Yong<sup>2</sup>, Xiaomin Wang<sup>7</sup>, Song-Guo Xue<sup>13</sup>, Liangpeng Ge<sup>14</sup>, Zuohua Liu<sup>14</sup>, Yong Huang<sup>14</sup>, Tao Nie<sup>7</sup>, Peng Li<sup>17</sup>, Donghai Wu<sup>7</sup>, Duanqing Pei<sup>7,15</sup>, Yi Zhang<sup>16</sup>, Liming Lu<sup>17</sup>, Fengtang Yang<sup>2</sup>, Susan J. Kimber<sup>18</sup>, Wolf Reik<sup>9</sup>, Xiangang Zou<sup>12</sup>, Zhouchun Shang<sup>5,6</sup>, Liangxue Lai<sup>7</sup>, Azim Surani<sup>11</sup>, Patrick P. L. Tam<sup>19</sup>, Asif Ahmed<sup>10</sup>, William Shu Biu Yeung<sup>8</sup>, Sarah A. Teichmann<sup>2</sup>, Heiner Niemann<sup>3,4,22*</sup> and Pentao Liu<sup>1,2*</sup>

<figure><img src="image_116.png" /></figure>

PNAS 2021

## Establishment of bovine expanded potential stem cells

Lixia Zhao<sup>a,b,c,1</sup>, Xuefei Gao<sup>d,e,f,1</sup>, Yuxuan Zheng<sup>g,1</sup> <sup>1</sup>, Zixin Wang<sup>c</sup>, Gaoping Zhao<sup>c</sup>, Jie Ren<sup>g</sup>, Jia Zhang<sup>a,b</sup>, Jian Wu<sup>f</sup> <sup>1</sup>, Baojiang Wu<sup>a,b,c</sup> <sup>1</sup>, Yanglin Chen<sup>a,b</sup> <sup>1</sup>, Wei Sun<sup>b,c</sup>, Yunxia Li<sup>b,c</sup>, Jie Su<sup>c,h</sup>, Yulin Ding<sup>j</sup>, Yuan Gao<sup>c</sup>, Moning Liu<sup>h</sup>, Xiaochun Bai<sup>d,j</sup> <sup>1</sup>, Liangzhong Sun<sup>k</sup>, Guifang Cao<sup>h</sup>, Fuchou Tang<sup>g,l,m</sup>, Siqin Bao<sup>a,b</sup>, Pentao Liu<sup>f,n,2</sup> <sup>1</sup>, and Xihe Li<sup>a,b,c,2</sup>
## ESCs with Expanded potential (EPSCs)

<figure><img src="image_114.png" /></figure>

LCDM:

- LIF

- GSK3b inh.

- DiM: Gprot-receptor

- MiH: PARP1 inh.

Yang et al. 2017 Cell
## ESCs with Expanded potential (EPSCs)

<figure><img src="image_117.png" /></figure>

**LCDM:**

- LIF

- GSK3b inh.

- DiM: Gprot-receptor

- MiH: PARP1 inh.

Yang et al. 2017 Cell
<figure><img src="image_115.png" /></figure>

# Producing pig ESCs (2019)

<figure><img src="image_116.png" /></figure>

<figure><img src="image_117.png" /></figure>
<figure><img src="image_118.png" /></figure>

# Producing pig ESCs (2019)

<figure><img src="image_119.png" /></figure>

<figure><img src="image_120.png" /></figure>
<figure><img src="image_118.png" /></figure>

# Producing pig ESCs (2019)

## Pluripotency markers

<figure><img src="image_119.png" /></figure>

## Chimera

<figure><img src="image_120.png" /></figure>

Liver
AFP
dTomato
<figure><img src="image_121.png" /></figure>

# Producing pig ESCs (2019)

## Pluripotency markers

<figure><img src="image_122.png" /></figure>

## Chimera

<figure><img src="image_123.png" /></figure>

Liver
AFP
dTomato
# Producing bovine ESCs (2021)

<figure><img src="image_121.png" /></figure>

## A

### Reprogramming

<figure><img src="image_122.png" /></figure>

### BFFs

PB-8F

<figure><img src="image_123.png" /></figure>

<figure><img src="image_124.png" /></figure>

### Primary Colony +Dox

<figure><img src="image_125.png" /></figure>

### Picking colony

### Expansion

<figure><img src="image_126.png" /></figure>

<figure><img src="image_127.png" /></figure>

### Medium testing -Dox

<figure><img src="image_128.png" /></figure>

bEPSC<sup>IPS</sup>

<figure><img src="image_129.png" /></figure>

<figure><img src="image_130.png" /></figure>

bEPSCM

Base:mTeSR™1

Essential components:

IWR-1/XAV939,

WH-4-023, ACTIVIN A

## H

<figure><img src="image_131.png" /></figure>

NANOG

<figure><img src="image_132.png" /></figure>

<figure><img src="image_133.png" /></figure>

E-CADHERIN

<figure><img src="image_134.png" /></figure>

## I

<figure><img src="image_135.png" /></figure>

Feeder free

<figure><img src="image_136.png" /></figure>

<figure><img src="image_137.png" /></figure>

## A

<figure><img src="image_138.png" /></figure>

Bovine blastocyst

<figure><img src="image_139.png" /></figure>

Bovine EPSCs medium

<figure><img src="image_140.png" /></figure>

Outgrowth

<figure><img src="image_141.png" /></figure>

TrypLE
digestion

<figure><img src="image_142.png" /></figure>

with feeder

<figure><img src="image_143.png" /></figure>

Remove
feeder cells

<figure><img src="image_144.png" /></figure>

p13

<figure><img src="image_145.png" /></figure>

without feeder

<figure><img src="image_146.png" /></figure>
<figure><img src="image_124.png" /></figure>

# Producing bovine ESCs (2021)

A

bOMSK+ pN-hLIN + hRL

## Reprogramming

<figure><img src="image_125.png" /></figure>

BFFs

PB-8F

<figure><img src="image_126.png" /></figure>

<figure><img src="image_127.png" /></figure>

## Primary Colony + Dox

<figure><img src="image_128.png" /></figure>

Picking colony

Expansion

<figure><img src="image_129.png" /></figure>

<figure><img src="image_130.png" /></figure>

## Medium testing - Dox

<figure><img src="image_131.png" /></figure>

bEPSC<sup>IPS</sup>

<figure><img src="image_132.png" /></figure>

<figure><img src="image_133.png" /></figure>

<figure><img src="image_134.png" /></figure>

bEPSCM

Base:mTeSR™1

Essential components:

IWR-1/XAV939,

WH-4-023, ACTIVIN A

H

<figure><img src="image_135.png" /></figure>

NANOG

<figure><img src="image_136.png" /></figure>

E-CADHERIN

<figure><img src="image_137.png" /></figure>

On feeder

<figure><img src="image_138.png" /></figure>

<figure><img src="image_139.png" /></figure>

<figure><img src="image_140.png" /></figure>

<figure><img src="image_141.png" /></figure>

<figure><img src="image_142.png" /></figure>

I

A

<figure><img src="image_143.png" /></figure>

Bovine blastocyst

<figure><img src="image_144.png" /></figure>

Bovine EPSCs medium

<figure><img src="image_145.png" /></figure>

Outgrowth

<figure><img src="image_146.png" /></figure>

Day 15

TrypLE
digestion

<figure><img src="image_147.png" /></figure>

with feeder

<figure><img src="image_148.png" /></figure>

Remove
feeder cells

<figure><img src="image_149.png" /></figure>

p13

<figure><img src="image_150.png" /></figure>

without feeder

<figure><img src="image_151.png" /></figure>
<figure><img src="image_147.png" /></figure>

# Producing bovine ESCs: chimera

<figure><img src="image_148.png" /></figure>

<figure><img src="image_149.png" /></figure>

Placenta

Smooth muscle
<figure><img src="image_152.png" /></figure>

# Producing bovine ESCs: chimera

<figure><img src="image_153.png" /></figure>

<figure><img src="image_154.png" /></figure>

<figure><img src="image_155.png" /></figure>

Placenta

Smooth muscle
Some applications of
mammalian ESCs
Some applications of
mammalian ESCs
# 1. Gene targeting (before CRISPR/Cas9)
(30 years of functional genomics in mice)

## The Nobel Prize in Physiology or Medicine 2007

<figure><img src="image_150.png" /></figure>

Photo: U. Montan
Mario R. Capecchi
Prize share: 1/3

<figure><img src="image_151.png" /></figure>

Photo: U. Montan
Sir Martin J. Evans
Prize share: 1/3

<figure><img src="image_152.png" /></figure>

Photo: U. Montan
Oliver Smithies
Prize share: 1/3

The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

<figure><img src="image_153.png" /></figure>
# 1. Gene targeting (before CRISPR/Cas9)
(30 years of functional genomics in mice)

## The Nobel Prize in Physiology or Medicine 2007

<figure><img src="image_156.png" /></figure>

Photo: U. Montan
Mario R. Capecchi
Prize share: 1/3

<figure><img src="image_157.png" /></figure>

Photo: U. Montan
Sir Martin J. Evans
Prize share: 1/3

<figure><img src="image_158.png" /></figure>

Photo: U. Montan
Oliver Smithies
Prize share: 1/3

The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

<figure><img src="image_159.png" /></figure>
## 2. Modelling disease from pluripotent stem cells

<figure><img src="image_154.png" /></figure>

Modelling chronic myeloid leukemia in a dish from PSCs

<figure><img src="image_155.png" /></figure>

**Impact on the differentiation**

**Identity and stem cell potential**

marker expression : FACS, qRT-PCR, immunofluorescence...
self-renewal / differentiation (in vitro/in vivo)
## 2. Modelling disease from pluripotent stem cells

<figure><img src="image_160.png" /></figure>

Modelling chronic myeloid leukemia in a dish from PSCs

<figure><img src="image_161.png" /></figure>

**Impact on the differentiation**

**Identity and stem cell potential**

marker expression : FACS, qRT-PCR, immunofluorescence...
self-renewal / differentiation (in vitro/in vivo)
3. Amplyfying virus for vaccine and screening
antiviral drugs

<figure><img src="image_156.png" /></figure>
3. Amplyfying virus for vaccine and screening
antiviral drugs

<figure><img src="image_162.png" /></figure>
# 3. Amplifying virus for vaccine and screening antiviral drugs

## Process at Scale

* Grow cells

- Suspension efficient for scale-up

* Seed on scaffolds

* Use directly

* EB66 duck embryonic stem cells

- Bioreactor

- Contamination

* Personnel

<figure><img src="image_157.png" /></figure>

<figure><img src="image_158.png" /><figcaption>Valneva</figcaption></figure>

<figure><img src="image_159.png" /></figure>
# 3. Amplifying virus for vaccine and screening antiviral drugs

## Process at Scale

* Grow cells

- Suspension efficient for scale-up

* Seed on scaffolds

* Use directly

* EB66 duck embryonic stem cells

- Bioreactor

- Contamination

* Personnel

<figure><img src="image_163.png" /></figure>

<figure><img src="image_164.png" /><figcaption>Valneva</figcaption></figure>

<figure><img src="image_165.png" /></figure>
# 4. High-throughput phenotyping for genetic studies

## Use of cell populations instead of animals/patients

<figure><img src="image_160.png" /><figcaption>Bio-bank of PSCs</figcaption></figure>
# 4. High-throughput phenotyping for genetic studies

## Use of cell populations instead of animals/patients

<figure><img src="image_166.png" /></figure>

Bio-bank of PSCs
## 4. High-throughput phenotyping for genetic studies

### In vitro differentiation

1 cell line

<figure><img src="image_161.png" /></figure>

<figure><img src="image_162.png" /></figure>

Genome editing

<figure><img src="image_163.png" /></figure>

<figure><img src="image_164.png" /></figure>

<figure><img src="image_165.png" /></figure>

### Phenotyping:

- Biomechanics

- Gene expression

- Proteome

- Metabolome

- Absorption

- Endocytosis

- Exocytosis

- Phagocytosis

- Toxicity

- Infections

....

<figure><img src="image_166.png" /></figure>

X1000
lines

HTS

GWAS

Big data
## 4. High-throughput phenotyping for genetic studies

### In vitro differentiation

1 cell line

<figure><img src="image_167.png" /></figure>

<figure><img src="image_168.png" /></figure>

Genome editing

<figure><img src="image_169.png" /></figure>

<figure><img src="image_170.png" /></figure>

<figure><img src="image_171.png" /></figure>

<figure><img src="image_172.png" /></figure>

<figure><img src="image_173.png" /></figure>

### Phenotyping:

- Biomechanics

- Gene expression

- Proteome

- Metabolome

- Absorption

- Endocytosis

- Exocytosis

- Phagocytosis

- Toxicity

- Infections

....

<figure><img src="image_174.png" /></figure>

X1000
lines

HTS

GWAS

Big data
# 5. Using pluripotent stem cells for reproduction
## Producing functional gametes

*in vitro* differentiation: Male primordial germ cells

<figure><img src="image_167.png" /></figure>
# 5. Using pluripotent stem cells for reproduction
## Producing functional gametes

*in vitro* differentiation: Male primordial germ cells

<figure><img src="image_175.png" /></figure>
# 5. Using pluripotent stem cells for reproduction
*in vitro* differentiation: female primordial germ cells

Hayashi et al.
Science 2012

<figure><img src="image_168.png" /></figure>

<figure><img src="image_169.png" /></figure>

<figure><img src="image_170.png" /></figure>

<figure><img src="image_171.png" /></figure>

<figure><img src="image_172.png" /></figure>

<figure><img src="image_173.png" /></figure>

<figure><img src="image_174.png" /></figure>

<figure><img src="image_175.png" /></figure>
# 5. Using pluripotent stem cells for reproduction
*in vitro* differentiation: female primordial germ cells

<figure><img src="image_176.png" /></figure>

Hayashi et al.
Science 2012

<figure><img src="image_177.png" /></figure>
# 5. Using pluripotent stem cells for reproduction

## *in vitro* differentiation: fully derived oocytes from PSCs

<figure><img src="image_176.png" /></figure>
# 5. Using pluripotent stem cells for reproduction

## *in vitro* differentiation: fully derived oocytes from PSCs

<figure><img src="image_178.png" /></figure>
Producing functional gametes in host gonads

Nanos mutant and testis complementation

Generation of germline ablated
male pigs by CRISPR/Cas9 editing
of the NANOS2 gene

Ki-Eun Park¹,²,³,*, Amy V. Kaucher⁴,*, Anne Powell², Muhammad Salman Waqas⁴,  
Shelley E.S. Sandmaier¹,², Melissa J. Oatley⁴, Chi-Hun Park¹,², Ahmed Tibary⁴,  
David M. Donovan², Le Ann Blomberg², Simon G. Lillico⁵, C. Bruce A. Whitelaw⁵,  
Alan Mileham⁶, Bhanu P. Telugu¹,²,³ & Jon M. Oatley⁴

<figure><img src="image_177.png" /><figcaption>Boar #146-/-</figcaption></figure>

<figure><img src="image_178.png" /></figure>

Also Dazl mutant in chicken
Producing functional gametes in host gonads

Nanos mutant and testis complementation

Generation of germline ablated
male pigs by CRISPR/Cas9 editing
of the NANOS2 gene

Ki-Eun Park¹,²,³,*, Amy V. Kaucher⁴,*, Anne Powell², Muhammad Salman Waqas⁴,  
Shelley E.S. Sandmaier¹,², Melissa J. Oatley⁴, Chi-Hun Park¹,², Ahmed Tibary⁴,  
David M. Donovan², Le Ann Blomberg², Simon G. Lillico⁵, C. Bruce A. Whitelaw⁵,  
Alan Mileham⁶, Bhanu P. Telugu¹,²,³ & Jon M. Oatley⁴

<figure><img src="image_179.png" /><figcaption>Boar #146-/-</figcaption></figure>

<figure><img src="image_180.png" /></figure>

Also Dazl mutant in chicken
# 5. Using pluripotent stem cells for reproduction

## Producing functional gametes in host gonads

<figure><img src="image_179.png" /></figure>
# 5. Using pluripotent stem cells for reproduction

## Producing functional gametes in host gonads

<figure><img src="image_181.png" /></figure>
# 5. Using pluripotent stem cells for reproduction

## In vitro breeding to speed-up genomic selection in livestock

<figure><img src="image_180.png" /></figure>
# 5. Using pluripotent stem cells for reproduction

## In vitro breeding to speed-up genomic selection in livestock

<figure><img src="image_182.png" /></figure>
6. To produce organs

Chimera and interspecies chimera
6. To produce organs

Chimera and interspecies chimera
# Chimera: a powerful tool to model disease and organs in mammalian species (except human)

<figure><img src="image_181.png" /></figure>
# Chimera: a powerful tool to model disease and organs in mammalian species (except human)

<figure><img src="image_183.png" /></figure>
# Chimera: a powerful tool to model disease and organs in mammalian species (except human)

<figure><img src="image_182.png" /></figure>
# Chimera: a powerful tool to model disease and organs in mammalian species (except human)

<figure><img src="image_184.png" /></figure>
That's why studies on chimera and interspecies chimera are mostly done on rodents
That's why studies on chimera and interspecies chimera are mostly done on rodents
Producing organs using chimera:
Organ complementation by interspecies chimeras

<figure><img src="image_183.png" /></figure>

<figure><img src="image_184.png" /></figure>
# Producing organs using chimera: Organ complementation by interspecies chimeras

<figure><img src="image_185.png" /></figure>

<figure><img src="image_186.png" /></figure>
Organ complementation by interspecies chimeras

<figure><img src="image_185.png" /></figure>

A

<figure><img src="image_186.png" /></figure>

C

<figure><img src="image_187.png" /></figure>
Organ complementation by interspecies chimeras

<figure><img src="image_187.png" /></figure>

A

<figure><img src="image_188.png" /></figure>

C

<figure><img src="image_189.png" /></figure>
Organ complementation by interspecies chimeras

<figure><img src="image_188.png" /></figure>
Organ complementation by interspecies chimeras

<figure><img src="image_190.png" /></figure>
# Organ complementation by interspecies chimeras
## Pancreas

<figure><img src="image_189.png" /></figure>

Kobayashi et al. 2010

<figure><img src="image_190.png" /></figure>

<figure><img src="image_191.png" /></figure>
# Organ complementation by interspecies chimeras
## Pancreas

<figure><img src="image_191.png" /></figure>

Kobayashi et al. 2010

<figure><img src="image_192.png" /></figure>

<figure><img src="image_193.png" /></figure>
# Organ complementation by interspecies chimeras
## Heart and eyes

Nkx2.5/- + rPSCs

<figure><img src="image_192.png" /></figure>

Heart

D

<figure><img src="image_193.png" /></figure>

Retina
# Organ complementation by interspecies chimeras
## Heart and eyes

Nkx2.5/- + rPSCs

D

<figure><img src="image_194.png" /></figure>

<figure><img src="image_195.png" /></figure>

Heart

WT

P0

<figure><img src="image_196.png" /></figure>

WT + rPSCs

P0

<figure><img src="image_197.png" /></figure>

Pax6/- + rPSCs

P0

<figure><img src="image_198.png" /></figure>

<figure><img src="image_199.png" /></figure>

<figure><img src="image_200.png" /></figure>

<figure><img src="image_201.png" /></figure>

Retina
# Producing human organs in non human species: Xenografts and organ complementation

## The interest for the pig species

<figure><img src="image_194.png" /></figure>

<figure><img src="image_195.png" /></figure>
# Producing human organs in non human species: Xenografts and organ complementation

## The interest for the pig species

<figure><img src="image_202.png" /></figure>

<figure><img src="image_203.png" /></figure>
Grafting pig organs in human :
Xenografts and organ complementation

The interest for the pig species

BRIEF REPORT

Genetically Modified Porcine-to-Human
Cardiac Xenotransplantation

Bartley P. Griffith, M.D., Corbin E. Goerlich, M.D., Ph.D.,
Avneesh K. Singh, Ph.D., Martine Rothblatt, Ph.D., Christine L. Lau, M.D.,
Aakash Shah, M.D., Marc Lorber, M.D., Alison Grazioli, M.D.,
Kapil K. Saharia, M.D., Susie N. Hong, M.D., Susan M. Joseph, M.D.,
David Ayares, Ph.D., and Muhammad M. Mohiuddin, M.D.
Grafting pig organs in human :
Xenografts and organ complementation

The interest for the pig species

BRIEF REPORT

Genetically Modified Porcine-to-Human
Cardiac Xenotransplantation

Bartley P. Griffith, M.D., Corbin E. Goerlich, M.D., Ph.D.,
Avneesh K. Singh, Ph.D., Martine Rothblatt, Ph.D., Christine L. Lau, M.D.,
Aakash Shah, M.D., Marc Lorber, M.D., Alison Grazioli, M.D.,
Kapil K. Saharia, M.D., Susie N. Hong, M.D., Susan M. Joseph, M.D.,
David Ayares, Ph.D., and Muhammad M. Mohiuddin, M.D.
Producing human organs in non human species:
Organ complementation by interspecies chimearas

<figure><img src="image_196.png" /></figure>
# Producing human organs in non human species:
Organ complementation by interspecies chimearas

<figure><img src="image_204.png" /></figure>
# Producing human organs in non human species:
## Optimisation of interspecies chimearas

### Human EPSCs in mouse embryo

<figure><img src="image_197.png" /></figure>
# Producing human organs in non human species:
## Optimisation of interspecies chimearas

### Human EPSCs in mouse embryo

<figure><img src="image_205.png" /></figure>
# Producing human organs in non human species:
## Optimisation of interspecies chimearas

Human EPSCs in mouse embryo

Embryonic tissue

<figure><img src="image_198.png" /></figure>

<figure><img src="image_199.png" /></figure>
# Producing human organs in non human species:
## Optimisation of interspecies chimearas

Human EPSCs in mouse embryo

Embryonic tissue

<figure><img src="image_206.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_207.png" /></figure>
# Producing human organs in non human species:
## Optimisation of interspecies chimearas

Human EPSCs in mouse embryo

<figure><img src="image_200.png" /></figure>

<figure><img src="image_201.png" /></figure>

Extra- Embryonic tissue
# Producing human organs in non human species:
## Optimisation of interspecies chimearas

Human EPSCs in mouse embryo

<figure><img src="image_208.png" /></figure>

<figure><img src="image_209.png" /></figure>

Extra- Embryonic tissue
# Producing human organs in non human species:
## Optimisation of interspecies chimeras

<figure><img src="image_202.png" /></figure>

### Monkey cells in pig embryos

updates

## RESEARCH ARTICLE

# Domesticated cynomolgus monkey embryonic stem cells allow the generation of neonatal interspecies chimeric pigs

Rui Fu¹,², Dawei Yu¹,², Jilong Ren¹,², Chongyang Li¹,²,³, Jing Wang¹,²,³, Guihai Feng¹,², Xuepeng Wang¹,², Haifeng Wan¹,², Tianda Li¹,², Libin Wang¹,², Ying Zhang¹,²,³, Tang Hai¹,², Wei Li¹,²,³, Qi Zhou¹,²,³

<figure><img src="image_203.png" /></figure>
# Producing human organs in non human species:
## Optimisation of interspecies chimeras

<figure><img src="image_210.png" /></figure>

### Monkey cells in pig embryos

updates

## RESEARCH ARTICLE

# Domesticated cynomolgus monkey embryonic stem cells allow the generation of neonatal interspecies chimeric pigs

Rui Fu¹,², Dawei Yu¹,², Jilong Ren¹,², Chongyang Li¹,²,³, Jing Wang¹,²,³, Guihai Feng¹,², Xuepeng Wang¹,², Haifeng Wan¹,², Tianda Li¹,², Libin Wang¹,², Ying Zhang¹,²,³, Tang Hai¹,², Wei Li¹,²,³, Qi Zhou¹,²,³

<figure><img src="image_211.png" /></figure>
Monkey cells in pig embryos > chimera

D

<figure><img src="image_204.png" /></figure>

<figure><img src="image_205.png" /></figure>

B

<figure><img src="image_206.png" /></figure>

<figure><img src="image_207.png" /></figure>
Monkey cells in pig embryos > chimera

D

<figure><img src="image_212.png" /></figure>

<figure><img src="image_213.png" /></figure>

B

<figure><img src="image_214.png" /></figure>

<figure><img src="image_215.png" /></figure>
# Producing human organs in non human species:
First step: human cells in pig embryos > chimera

A

<figure><img src="image_208.png" /></figure>

<figure><img src="image_209.png" /></figure>

BL injection
(3-10 cells)

ET

<figure><img src="image_210.png" /></figure>

Female recipient

<figure><img src="image_211.png" /></figure>

2iLD-hiPSCs

<figure><img src="image_212.png" /></figure>

4i-hiPSCs

<figure><img src="image_213.png" /></figure>

NHSM-hiPSCs

<figure><img src="image_214.png" /></figure>

FAC-hiPSCs

<figure><img src="image_215.png" /></figure>

<figure><img src="image_216.png" /></figure>

Pig embryo
(Day21-28)

1. Check fluorescence

2. IHC analyses

3. Genomic PCR analyses

<figure><img src="image_217.png" /></figure>

<figure><img src="image_218.png" /></figure>

<figure><img src="image_219.png" /></figure>

<figure><img src="image_220.png" /></figure>

<figure><img src="image_221.png" /></figure>

<figure><img src="image_222.png" /></figure>

<figure><img src="image_223.png" /></figure>

<figure><img src="image_224.png" /></figure>

<figure><img src="image_225.png" /></figure>

<figure><img src="image_226.png" /></figure>

<figure><img src="image_227.png" /></figure>

<figure><img src="image_228.png" /></figure>

<figure><img src="image_229.png" /></figure>

<figure><img src="image_230.png" /></figure>

<figure><img src="image_231.png" /></figure>

<figure><img src="image_232.png" /></figure>

<figure><img src="image_233.png" /></figure>

<figure><img src="image_234.png" /></figure>
# Producing human organs in non human species:
First step: human cells in pig embryos > chimera

A

<figure><img src="image_216.png" /></figure>

<figure><img src="image_217.png" /></figure>

BL injection
(3-10 cells)

ET

<figure><img src="image_218.png" /></figure>

Female recipient

<figure><img src="image_219.png" /></figure>

2iLD-hiPSCs

<figure><img src="image_220.png" /></figure>

4i-hiPSCs

<figure><img src="image_221.png" /></figure>

NHSM-hiPSCs

<figure><img src="image_222.png" /></figure>

FAC-hiPSCs

<figure><img src="image_223.png" /></figure>

<figure><img src="image_224.png" /></figure>

Pig embryo
(Day21-28)

1. Check fluorescence

2. IHC analyses

3. Genomic PCR analyses

<figure><img src="image_225.png" /></figure>

<figure><img src="image_226.png" /></figure>

<figure><img src="image_227.png" /></figure>

GFP

GFP

GFP

GFP

<figure><img src="image_228.png" /></figure>

<figure><img src="image_229.png" /></figure>

<figure><img src="image_230.png" /></figure>

SMA

CK8

TUJ1

HUMAN

Merge

Merge

Merge

Merge
nature
biotechnology

LETTERS

https://doi.org/10.1038/s41587-019-0373-y

# Generation of human endothelium in pig embryos deficient in ETV2

Satyabrata Das¹,⁶, Naoko Koyano-Nakagawa¹,²,⁶, Ohad Gafni¹, Geunho Maeng ID ¹, Bhairab N. Singh¹, Tara Rasmussen¹, Xiaoyan Pan¹, Kyung-Dal Choi¹, Daniel Mickelson¹, Wuming Gong ID ¹, Pruthvi Pota¹, Cyprian V. Weaver¹, Stefan Kren¹, Jacob H. Hanna ID ³, Demetris Yannopoulos¹,⁴, Mary G. Garry ID ¹,⁴,⁵,⁷* and Daniel J. Garry ID ¹,²,⁴,⁵,⁷*
nature
biotechnology

LETTERS

https://doi.org/10.1038/s41587-019-0373-y

# Generation of human endothelium in pig embryos deficient in ETV2

Satyabrata Das¹,⁶, Naoko Koyano-Nakagawa¹,²,⁶, Ohad Gafni¹, Geunho Maeng ID ¹, Bhairab N. Singh¹, Tara Rasmussen¹, Xiaoyan Pan¹, Kyung-Dal Choi¹, Daniel Mickelson¹, Wuming Gong ID ¹, Pruthvi Pota¹, Cyprian V. Weaver¹, Stefan Kren¹, Jacob H. Hanna ID ³, Demetris Yannopoulos¹,⁴, Mary G. Garry ID ¹,⁴,⁵,⁷* and Daniel J. Garry ID ¹,²,⁴,⁵,⁷*
Organ complementation by interspecies pig/human chimearas

<figure><img src="image_235.png" /><figcaption>Figure S3. Das et al.</figcaption></figure>
Organ complementation by interspecies pig/human chimearas

<figure><img src="image_231.png" /><figcaption>Figure S3. Das et al.</figcaption></figure>
Characterization of ETV2-/- pig fetuses

VWF/NKX2.5/SMA

<figure><img src="image_236.png" /></figure>

<figure><img src="image_237.png" /></figure>

<figure><img src="image_238.png" /></figure>

<figure><img src="image_239.png" /></figure>

<figure><img src="image_240.png" /></figure>

<figure><img src="image_241.png" /></figure>
Characterization of ETV2-/- pig fetuses

VWF/NKX2.5/SMA

<figure><img src="image_232.png" /></figure>

<figure><img src="image_233.png" /></figure>

<figure><img src="image_234.png" /></figure>

<figure><img src="image_235.png" /></figure>

<figure><img src="image_236.png" /></figure>

<figure><img src="image_237.png" /></figure>
<figure><img src="image_242.png" /><figcaption>Complementation of ETV2-/- pig fetuses by pig embryonic cells
Intra-species chimera</figcaption></figure>
<figure><img src="image_238.png" /><figcaption>Complementation of ETV2-/- pig fetuses by pig embryonic cells
Intra-species chimera</figcaption></figure>
# Complementation of ETV2-/- pig fetuses with human iPSCs
## Inter-species chimera

### Blastocyst colonization

<figure><img src="image_243.png" /></figure>

<figure><img src="image_244.png" /></figure>

<figure><img src="image_245.png" /></figure>

<figure><img src="image_246.png" /></figure>

<figure><img src="image_247.png" /></figure>

<figure><img src="image_248.png" /></figure>

<figure><img src="image_249.png" /></figure>

<figure><img src="image_250.png" /></figure>

n=400

n=300

The best pictures you can get from 400 injected embryos
# Complementation of ETV2-/- pig fetuses with human iPSCs
## Inter-species chimera

### Blastocyst colonization

<figure><img src="image_239.png" /></figure>

<figure><img src="image_240.png" /></figure>

<figure><img src="image_241.png" /></figure>

<figure><img src="image_242.png" /></figure>

<figure><img src="image_243.png" /></figure>

<figure><img src="image_244.png" /></figure>

<figure><img src="image_245.png" /></figure>

<figure><img src="image_246.png" /></figure>

n=400

n=300

The best pictures you can get from 400 injected embryos
# Complementation of ETV2-/- pig fetuses with human iPSCs
## Inter-species chimera

<figure><img src="image_251.png" /><figcaption>### Fetus colonization</figcaption></figure>

Top 10 over 1700 embryos
# Complementation of ETV2-/- pig fetuses with human iPSCs
## Inter-species chimera

<figure><img src="image_247.png" /><figcaption>### Fetus colonization</figcaption></figure>

Top 10 over 1700 embryos
Complementation of ETV2-/- pig fetuses with human iPSCs
Inter-species chimera

Low chimera efficiency due to high apoptosis levels

<figure><img src="image_252.png" /></figure>
Complementation of ETV2-/- pig fetuses with human iPSCs
Inter-species chimera

Low chimera efficiency due to high apoptosis levels

<figure><img src="image_248.png" /></figure>
# Complementation of ETV2-/- pig fetuses with human iPSCs
## Inter-species chimera

OE of BCL2 in human iPSCs to decrease apoptosis levels
and increase colonization

q

RhiPSC11 cGFP

<figure><img src="image_253.png" /></figure>

RhiPSC11 cGFP-BCL2

<figure><img src="image_254.png" /></figure>

r

RhiPSC11 cGFP

RhiPSC11 cGFP-BCL2

GFP+ cells parthenote

<figure><img src="image_255.png" /></figure>

s

Human ALU fold change

<figure><img src="image_256.png" /></figure>
# Complementation of ETV2-/- pig fetuses with human iPSCs
## Inter-species chimera

OE of BCL2 in human iPSCs to decrease apoptosis levels
and increase colonization

q

RhiPSC11 cGFP

<figure><img src="image_249.png" /></figure>

RhiPSC11 cGFP-BCL2

<figure><img src="image_250.png" /></figure>

r

GFP+ cells parthenote

RhiPSC11 cGFP

RhiPSC11 cGFP-BCL2

<figure><img src="image_251.png" /></figure>

s

Human ALU fold change

<figure><img src="image_252.png" /></figure>
# Complementation of ETV2-/- pig fetuses with human iPSCs
## Inter-species chimera

OE of BCL2 in human iPSCs favors complementation of
ETV2-/- pig embryos

t

<figure><img src="image_257.png" /></figure>

<figure><img src="image_258.png" /></figure>

<figure><img src="image_259.png" /></figure>

<figure><img src="image_260.png" /></figure>

<figure><img src="image_261.png" /></figure>

<figure><img src="image_262.png" /></figure>

<figure><img src="image_263.png" /></figure>
# Complementation of ETV2-/- pig fetuses with human iPSCs
## Inter-species chimera

OE of BCL2 in human iPSCs favors complementation of
ETV2-/- pig embryos

t

<figure><img src="image_253.png" /></figure>

<figure><img src="image_254.png" /></figure>

<figure><img src="image_255.png" /></figure>

<figure><img src="image_256.png" /></figure>

<figure><img src="image_257.png" /></figure>

<figure><img src="image_258.png" /></figure>

<figure><img src="image_259.png" /></figure>
<figure><img src="image_264.png" /></figure>

# Humanized skeletal muscle in
MYF5/MYOD/MYF6-null pig embryos

Geunho Maeng ID ¹,⁶, Satyabrata Das ID ¹,⁶, Sarah M. Greising², Wuming Gong ID ¹, Bhairab N. Singh¹, Stefan Kren¹, Daniel Mickelson¹, Erik Skie¹, Ohad Gafni¹, Jacob R. Sorensen ID ², Cyprian V. Weaver¹, Daniel J. Garry ¹,³,⁴,⁵ ☒ and Mary G. Garry ID ¹,⁴,⁵ ☒
<figure><img src="image_260.png" /></figure>

# Humanized skeletal muscle in
MYF5/MYOD/MYF6-null pig embryos

Geunho Maeng ID ¹,⁶, Satyabrata Das ID ¹,⁶, Sarah M. Greising², Wuming Gong ID ¹, Bhairab N. Singh¹, Stefan Kren¹, Daniel Mickelson¹, Erik Skie¹, Ohad Gafni¹, Jacob R. Sorensen ID ², Cyprian V. Weaver¹, Daniel J. Garry ¹,³,⁴,⁵ ☑ and Mary G. Garry ID ¹,⁴,⁵ ☑
Balancing cell competition to improve niche receptivity in inter-species chimera

<figure><img src="image_265.png" /></figure>
# Balancing cell competition to improve niche receptivity in inter-species chimera

<figure><img src="image_261.png" /></figure>
<figure><img src="image_266.png" /><figcaption>Balancing cell competition to improve niche receptivity:
Mouse in mouse chimera</figcaption></figure>
Balancing cell competition to improve niche receptivity:
Mouse in mouse chimera

<figure><img src="image_262.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_263.png" /></figure>

<figure><img src="image_264.png" /></figure>
Balancing cell competition to improve niche receptivity:
Rat in mouse chimera

<figure><img src="image_267.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_268.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_269.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_270.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_271.png" /></figure>

Kidney

<figure><img src="image_272.png" /><figcaption>F</figcaption></figure>

Lung

<figure><img src="image_273.png" /><figcaption>G</figcaption></figure>

Heart

<figure><img src="image_274.png" /><figcaption>H</figcaption></figure>

Thymus

<figure><img src="image_275.png" /><figcaption>I</figcaption></figure>

CT
Balancing cell competition to improve niche receptivity:
Rat in mouse chimera

<figure><img src="image_265.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_266.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_267.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_268.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_269.png" /></figure>

Kidney

<figure><img src="image_270.png" /><figcaption>F</figcaption></figure>

Lung

<figure><img src="image_271.png" /><figcaption>G</figcaption></figure>

Heart

<figure><img src="image_272.png" /><figcaption>H</figcaption></figure>

Thymus

<figure><img src="image_273.png" /><figcaption>I</figcaption></figure>

CT
Cell Stem Cell

# Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation

**Graphical abstract**

<figure><img src="image_276.png" /></figure>

**Authors**

Jiaowei Wang, Wenguang Xie,
Nan Li, ..., Miguel A. Esteban, Zhen Dai,
Liangxue Lai

**Correspondence**

pan_guangjin@gibh.ac.cn (G.P.),
miguel@gibh.ac.cn (M.A.E.),
dai_zhen@gibh.ac.cn (Z.D.),
lai_liangxue@gibh.ac.cn (L.L.)

**In brief**

Wang and colleagues show that 4CL medium and MYCN/BCL2 overexpression generate human iPSCs with superior interspecies chimeric potential in pig embryos. This led to the successful formation of a humanized mesonephros in nephric-defective pig embryos via early embryo complementation, paving the way for growing a human kidney in pigs.
Cell Stem Cell

# Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation

**Graphical abstract**

<figure><img src="image_274.png" /></figure>

**Authors**

Jiaowei Wang, Wenguang Xie,
Nan Li, ..., Miguel A. Esteban, Zhen Dai,
Liangxue Lai

**Correspondence**

pan_guangjin@gibh.ac.cn (G.P.),
miguel@gibh.ac.cn (M.A.E.),
dai_zhen@gibh.ac.cn (Z.D.),
lai_liangxue@gibh.ac.cn (L.L.)

**In brief**

Wang and colleagues show that 4CL medium and MYCN/BCL2 overexpression generate human iPSCs with superior interspecies chimeric potential in pig embryos. This led to the successful formation of a humanized mesonephros in nephric-defective pig embryos via early embryo complementation, paving the way for growing a human kidney in pigs.
Test chimera contribution of hPSCs with different naive human
PSC media + OE MYCN/BCL2

<figure><img src="image_277.png" /></figure>

<figure><img src="image_278.png" /></figure>

<figure><img src="image_279.png" /></figure>

<figure><img src="image_280.png" /></figure>
2023: Amazing progress toward human organs in pigs

Test chimera contribution of hPSCs with different naive human
PSC media + OE MYCN/BCL2

<figure><img src="image_275.png" /></figure>

<figure><img src="image_276.png" /></figure>

<figure><img src="image_277.png" /></figure>

<figure><img src="image_278.png" /></figure>
2023: Amazing progress toward human organs in pigs

Pigs mutated for SIX1 and SALL1: no kidney

<figure><img src="image_281.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_282.png" /></figure>
2023: Amazing progress toward human organs in pigs

Pigs mutated for SIX1 and SALL1: no kidney

<figure><img src="image_279.png" /></figure>

<figure><img src="image_280.png" /></figure>
2023: Amazing progress toward human organs in pigs

Complementation of Pig kidney with human PSCs

A

<figure><img src="image_283.png" /></figure>

SCNT

<figure><img src="image_284.png" /></figure>

CBE / SALL1 sgRNA

<figure><img src="image_285.png" /></figure>

<figure><img src="image_286.png" /></figure>

<figure><img src="image_287.png" /></figure>

S/X1/SALL1
-null morula

<figure><img src="image_288.png" /></figure>

<figure><img src="image_289.png" /></figure>

S/X1/SALL1
-null morula

<figure><img src="image_290.png" /></figure>

<figure><img src="image_291.png" /></figure>

4CL/N/B iPSCs

<figure><img src="image_292.png" /></figure>

<figure><img src="image_293.png" /></figure>

Surrogate sow

<figure><img src="image_294.png" /></figure>

<figure><img src="image_295.png" /></figure>

Nephric-defective embryo

<figure><img src="image_296.png" /></figure>

<figure><img src="image_297.png" /></figure>

Chimeric embryo
2023: Amazing progress toward human organs in pigs

Complementation of Pig kidney with human PSCs

<figure><img src="image_281.png" /></figure>
2023: Amazing progress toward human organs in pigs

Complementation of Pig kidney with human PSCs

D

WT (E28)

<figure><img src="image_298.png" /></figure>

Phase

Stereo
fluorescence

<figure><img src="image_299.png" /></figure>

E

DAPI

<figure><img src="image_300.png" /></figure>

DsRed

<figure><img src="image_301.png" /></figure>

E25 embryo-1

<figure><img src="image_302.png" /></figure>

<figure><img src="image_303.png" /></figure>

<figure><img src="image_304.png" /></figure>

<figure><img src="image_305.png" /></figure>

E28 embryo-3

<figure><img src="image_306.png" /></figure>

<figure><img src="image_307.png" /></figure>

<figure><img src="image_308.png" /></figure>

<figure><img src="image_309.png" /></figure>
# 2023: Amazing progress toward human organs in pigs

## Complementation of Pig kidney with human PSCs

D

WT (E28)

<figure><img src="image_282.png" /></figure>

Stereo
fluorescence

<figure><img src="image_283.png" /></figure>

E

DAPI

<figure><img src="image_284.png" /></figure>

DsRed

<figure><img src="image_285.png" /></figure>

E25 embryo-1

<figure><img src="image_286.png" /></figure>

<figure><img src="image_287.png" /></figure>

<figure><img src="image_288.png" /></figure>

<figure><img src="image_289.png" /></figure>

E28 embryo-3

<figure><img src="image_290.png" /></figure>

<figure><img src="image_291.png" /></figure>

<figure><img src="image_292.png" /></figure>

<figure><img src="image_293.png" /></figure>
# 2023: Amazing progress toward human organs in pigs

But still with really low efficiency....

C

normal embryos

<table><thead><tr><th>Cell line</th><th>Transferred embryos</th><th>Transferred sows</th><th>Pregnant sows</th></tr></thead><tbody><tr><td>4CL/N/B</td><td>1820</td><td>13</td><td>6</td></tr><tr><td colspan="2">Total embryos</td><td colspan="2">DsRed+</td></tr><tr><td>Normal</td><td>Retarded</td><td>Normal</td><td>Retarded</td></tr><tr><td>E25</td><td>E28</td><td>E25</td><td>E28</td></tr><tr><td>2</td><td>3</td><td>4</td><td>0</td></tr><tr><td></td><td></td><td>2</td><td>3</td></tr><tr><td></td><td></td><td></td><td>1</td></tr><tr><td></td><td></td><td></td><td>0</td></tr></tbody></table>
# 2023: Amazing progress toward human organs in pigs

But still with really low efficiency....

C

normal embryos

<table><thead><tr><th>Cell line</th><th>Transferred embryos</th><th>Transferred sows</th><th>Pregnant sows</th></tr></thead><tbody><tr><td>4CL/N/B</td><td>1820</td><td>13</td><td>6</td></tr><tr><td colspan="2">Total embryos</td><td colspan="2">DsRed+</td></tr><tr><td colspan="2">Normal</td><td colspan="2">Normal</td></tr><tr><td>E25</td><td>E28</td><td>E25</td><td>E28</td></tr><tr><td>2</td><td>3</td><td>4</td><td>0</td></tr><tr><td colspan="2">Retarded</td><td>E25</td><td>E28</td></tr><tr><td></td><td></td><td>2</td><td>3</td></tr><tr><td colspan="2"></td><td>E25</td><td>E28</td></tr><tr><td></td><td></td><td>1</td><td>0</td></tr></tbody></table>
Non rodent mammalian pluripotent stem cells

* All non rodent PSCs are close to a primed state of pluripotency

* Until recently, few true PSCs from non primate species

* Intraspecies chimera production remains complicated with this cells:

* Interspecies chimera in non-rodent species are at their beginning > close to Science Fiction but ...
Non rodent mammalian pluripotent stem cells

* All non rodent PSCs are close to a primed state of pluripotency

* Until recently, few true PSCs from non primate species

* Intraspecies chimera production remains complicated with this cells:

* Interspecies chimera in non-rodent species are at their beginning > close to Science Fiction but ...