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Reprogramming to pluripotency does not require

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Prof. Dr. Stefan Liebau. Email: stefan.liebau@uni-tuebingen.de. Phone: +49-7071-2978233. Fax: +49-7071-294014. Institute of Neuroanatomy & Developmental ...
Reprogramming to pluripotency does not require transition through a primitive streak-like state Stefanie Raab1#, Moritz Klingenstein1#, Anna Möller2#, Anett Illing2, Jelena Tosic3,4,5, Markus Breunig2, Georg Kuales3, Leonhard Linta1, Thomas Seufferlein2, Sebastian J. Arnold3,6$, Alexander Kleger2$, Stefan Liebau1$

1

Institute of Neuroanatomy & Developmental Biology (INDB), Eberhard Karls University Tuebingen, Oesterbergstr. 3, 72074 Tuebingen,

Germany 2

Department of Internal Medicine I, University Medical Center Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany

3

Institute of Experimental and Clinical Pharmacology and Toxicology II, Faculty of Medicine, University of Freiburg, Freiburg, Germany

4

Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, Freiburg, Germany

5

Faculty of Biology, Schänzlestraße 1, Albert-Ludwigs-University Freiburg, Freiburg, Germany

6

BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany.

#

contributed equally

$

contributed equally

Corresponding authors: Dr. Sebastian J Arnold Email: [email protected] Phone: +49-761-203-96819 Fax: +49-731-203-96820 Institute of Clinical and Experimental Pharmacology and Toxicology Albertstr. 25 79104 Freiburg, Germany Prof. Dr. Alexander Kleger Email: [email protected] Phone: +49-731-500-44728 Fax: +49-731-500-44665 Department of Internal Medicine I, University Medical Center Ulm Albert-Einstein-Allee 23 89081 Ulm, Germany Prof. Dr. Stefan Liebau Email: [email protected] Phone: +49-7071-2978233 Fax: +49-7071-294014 Institute of Neuroanatomy & Developmental Biology (INDB), Eberhard Karls University Tübingen Österbergstr. 3 72074 Tübingen

Supplemental figure legend Supplemental figure 1: Expression profiles of human somatic cells during reprogramming display primitive streak and mesendoderm signatures (A) Schematic overview of the epithelial-to-mesenchymal transition (EMT) and the early steps of human embryonic development. The blastocyst includes the first differentiated cells in human development – the pluripotent inner cell mass or embryoblast and the trophoblast. The pluripotent embryoblast cells differentiate to the so called germinal disc, composed of epiblast and hypoblast. Gastrulation starts when cells of the epiblast form the primitive streak undergo EMT and migrate between hypo- and epiblast. The cells integrating and exchanging the former hypoblast give rise to the endoderm. The cells between the two disks differentiate to mesoderm, while the remaining cells in the epiblast have ectodermal fate. Keratinocytes are known to differentiate from the ectoderm while fibroblasts are of mesoderm origin. (B,C) Expression patterns during reprogramming of fibroblasts (B) keratinocytes (C). Single marker gene profiles for genes expressed during primitive streak and mesendoderm formation in reprogramming keratinocytes and fibroblasts towards pluripotency are shown. Supplemental figure 2: Expression profile for Eomesodermin (Eomes) during reprogramming of mouse embryonic fibroblasts. Time points as indicated.

Supplemental Figure 1

A Blastocyst

Germinal Disc Embryoblast

Epibast

Gastrulation/EMT

Amnion Cavity

Germlayers

Epiblast Hypoblast Ectoderm

B

mRNA levels relative to HMBS

Trophoblast

80

T

1.6

60 40 20 0

K

2

4

6

9 12 15 18 21 iPS

Hypoblast Trophoblast

CER1

8

1.2

6

0.8

4

0.4

2

0 K

2

4

6

Endoderm Mesoderm Mesendoderm

9 12 15 18 21 iPS

0

Keratinocytes

Fibroblasts

Definitive Endoderm Primitive Streak

LHX1

2.0 FGF8

FGF4

60 40

1.0

20

K

2

4

6

9

12 15 18 21 iPS

0

K

2

4

6

9 12 15 18 21 iPS

0

K

2

4

6

9 12 15 18 21 iPS

C

mRNA levels relative to HMBS

Days of fibroblast reprogamming (d)

120 FGF4 100 600 80 400 400 60 400 300 40 200 200 200 20 0 0 0 0 F 3 6 8 10 12 14 16 18 20 22 24 28 iPS F 3 6 8 10 12 14 16 18 20 22 24 28 iPS F 3 6 8 10 12 14 16 18 20 22 24 28 iPS F 3 6 8 10 12 14 16 18 20 22 24 28 iPS 600

T

700 CER1

500

800 LHX1

Days of keratinocyte reprogamming (d)

8 FGF8 6 4 2 0

F 3 6 8 10 12 14 16 18 20 22 24 28 iPS

Supplemental Figure 2

mRNA levels relative to HMBS

0.15

Eomes

0.10

0.05

0.00

3

6

9

12

15

18

21