Testing Porcine Induced Pluripotent Stem Cells Chimeric Embryo Production

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1 Testing Porcine Induced Pluripotent Stem Cells Chimeric Embryo Production Jan Secher, DVM, PhD. PostDoc. Department of Veterinary and Animal Sciences Section for Anatomy & Biochemistry Faculty of Health and Medical Science, University of Copenhagen Dias 1

2 Outline Stem cells Stem cell potency Cell reprogramming ipsc Current status of pipsc Our findings Dias 2

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4 Donovan and Gerhart, 2001 Dias 4

5 Cell reprogramming/ pluripotency induction Dolly 1996 (Wilmut et al., 1997). A somatic cell reprogrammed to a totipotent state by injecting it into an enucleated oocyte + = Dias 5

6 Cell reprogramming/ pluripotency induction Murine induced pluripotent stem cells 2006 (Takahashi and Yamanaka,2006). mipsc from murine fibroblasts by introducing 4 reprogramming factors (oct4,c-myc, sox2 and klf4) Human ipsc 2007 (Takahashi et al., 2007) Porcine ipsc (Esteban et al.,2009; Ezashi et., 2009; Wu et al., 2009) omsk Dias 6

7 PSC Putative PSC? 4n 4n 4n In vitro differentiation Teratoma assay Chimeric contribution Tetraploid complementation Dias 7

8 Great potential Research tool Cell therapy Organ generation (Wu et al., 2016) Drug development Stadtfeld & Hochedlinger, 2010 Dias 8

9 Why porcine ipsc? No bona fide PSC available in ungulates Could increase the efficiency of producing genetically modified pigs Functional testing of human ipsc (not possible) A valuable model in addition to murine and rat ipsc for testing human ipsc Dias 9

10 Challenges Ability to produce germ line chimeras is a test for true pluripotency (West et., al 2010) Only reproducible in mice and rats (Okita et al., 2007 and Hamanaka et al., 2011) Dependent on continuous transgene expression X (West et., al 2010) Dias 10

11 Naive and primed pluripotent states Definition in mice ESC (Nichols and Smith 2009) The naive pluripotent state is isolated from the ICM of the mature blastocyst before implantation The primed pluripotent state is isolated from the epiblast shortly after implantation Dias 11

12 !!!!! From Nichols and Smith 2009 Dias 12

13 Naive type available in pigs? The mipsc Yamanaka created in 2006 resembles the Naïve pluripotent stem cells First porcine ipsc from 2009 resembles the Primed pluripotent stem cell (Ezashi 2012) pipsc that resembles the naive type stem cells have been derived (Telugu et al., 2010, 2011; Fujishiro et al. 2013) Dias 13

14 Reprogramming of Porcine fetal fibroblasts to Naive ipsc and Primed ipsc (Ezashi et al., 2012) Dias 14

15 Contribution to fetal development Fujishiro et al., 2013 reported that their LIF-dependent naive type ipsc contributed to fetal development Constitutive promoter No live offspring No germline contribution Du et al., 2015 similar results Kawaguchi et al., 2015 in cattle. Dias 15

16 Cell reprogramming basic developmental studies in the pig IVP Evaluate different methods to introduce pipsc into preimplantory porcine embryos Investigate if pipsc can survive and proliferate the IVP embryos Investigate if pipsc can survive and proliferate in the in vivo proeduced embryos. Dias 16

17 Cell reprogramming basic developmental studies in the pig Two Yorkshire male neonatal fibroblast cell lines. One tagged with RFP (Cherry) and one with Venus (Venus) Venus transduced with porcine specific OMSK lentiviral construct growing on bfgf and LIF Cherry transduced with porcine specific OMSK lentiviral construct growing on bfgf and LIF + 2i, LIF Naïve type pipsc Dox pomsk 2i, bfgf Primed type pipsc JS Dias 17

18 Fibroblast Reprogramming Method In Vitro characterization Culture Condition AP staining Q-RT ICC EB formation Spontaneous Differentiation Karyotyping P No Venus Primed type pomsk polycistronic LV 2i FGF + Nanog ; Oct4; Sox2; Lin28; c-myc; Rex1 Nanog SSEA3 + Poor outgrowth of EB s. Only ectoderm and mesoderm no abnormal chromosome 9 with additional endodermal cells material at its (AFP negative) distal long arm. P25 Venus Naïve type pomsk polycistronic LV 2i LIF + Nanog ; Oct4; Sox2; Lin28; c-myc; Rex1 Nanog SSEA4 SSEA3 + Very good EB attachment and outgrowth into all 3 lineages XXY P30 Positive for B3 tub, SMA and AFP Dias 18

19 IVP and methods to introduce pipsc into the embryos (Fujishiro et al., 2012) (West et., al 2010) Dias 19

20 Testing Porcine Induced Pluripotent Stem Cells Chimeric Embryo Production In vitro experiments to ascertain if we can produce chimeric blastocysts using parthenotes Injection of Venus-tagged ipsc into day 5 embryos and then follow them until day 7 in time-lapse analyses TEM Dias 20

21 ? Dias 21

22 Confocal Gao et al., 2013 (Fujishiro et al., 2012) (Telegu et al., 2011) Dias 22

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25 Freude et al., IPH,VCS 25

26 Cell reprogramming/chimeric embryo production In vivo experiments Teratomas In vivo embryos. Chimeras generated with Venus-tagged ipsc Embryos collected at day 32 Dias 26

27 Teratoma 6 mice with Venus 2i LIF in PBS. 6mice Venus 2iLIF, 6mice Venus 2iFGF, 4Venus pnf 2mg/ml dox, 5% succrose 45 days 14 days water HE-staining and immunohistochemistry Dias 27

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29 Venus 2i LIF pipsc Litterature (Telugu et al., 2010, Ezashi et al., 2012 Cheng et al., 2012) Higher in Lin28 SSEA3 and SSEA4 Better survival in in vitro chimeras when cultured with doxycycline Teratoma formation Dias 29

30 Chimeric embryo production Donor Donor AI 5 and 6 days after weaning Slaughtered and flushed 4 days after last AI Each blastocyst injected With 15 ipsc and incubated overnight recipient Chimeric embro Transfer to a sow 5 days after heat After 32 day sow is slaught. and utrus Removed en block. Fetal, uterine and placental samples Dias 30

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32 Analysis of embryos Macro Venus AU: Liver Brain Corpus FACS, Culture SUND: Mesonephros Gonads Liver Intestine Heart Lungs Head Corpus Placenta Genome (PCR) Transcriptome (RT-PCR) Culturing PFA for ICC. Dias 32

33 FACS hepatocytes negative No macroscopically-detectable Venus Venus positive fetal membranes from 2 embryos Ezashi et al., 2012 (trophoballs) Dias 33

34 Chimeric embryo production II Donor Donor AI 5 and 6 days after weaning Slaughtered and flushed 4 days after last AI Each blastocyst injected With 15 ipsc and incubated overnight with doxycycline recipient Chimeric embryos Transferred to a sow 5 days after heat. Sow feed doxycycline 2 days before and 5 days after ET After 32 days sow is slaught. and utrus Removed en block. Embryos collected Dias 34

35 Clonogenicity Fan et al reported in 2013 that the first pipsc clones were born. Only live piglets after spontaneous differentation Du et al., 2015 obtained similar results Dias 35

36 Fibroblast clones Dias 36

37 Venus fibroblasts Global gene expression analysis: Principal componenet analysis Epiblast from 7-8 days old in vivo embryo (ICM) Embryonic disc from days old in vivo embryo (Epi) Whole transcriptome RNA sequencing Venn diagram Gene expression only found in pluripotent cells. Embryonic disc from days old in vivo embryo (GE) Dias 37 Unsupervised hierarchical clustering

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43 Summary results Property Venus 2i LIF Venus 2i FGF Venus pnf Mouse naïve PSC Colony shape Dome Flat Fibroblasts Dome Pluripotency factors Nanog, Oct4, (low klf4) Sox2 Oct4, KLF4, KLF2, SOX4, Nanog Naive marker Lin28, REX1 REX1,FGF4,NrOb1 EB formation Yes Yes Yes Spontaneous differentation 3 germlayer Poor only ecto and meso. Teratoma Yes (dox) Yes Blastocyst chimeras No (fetal membranes) Clonogenicity Poor Possible good Respons to 2i Self reneval Self reneval Self reneval Respons to LIF Self reneval Self reneval Yes Respons to FGF Self reneval Differentiation Karyotype XXY abnormal chromosome 9 with additional material at its distal long arm. JS Dias 43

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45 Thank You Mette Schmidt (KU) Dr. Hernik Callesen (AU) Dr. Poul Hyttel (KU) Kristine Freude (KU) Dr. Torben Greve Dr. Randalf Prather (MU) Dr. Michael Roberts (MU) Rong Li (AU) Ying Li (AU) Kaveh Mashayekhi (KU) Gianluca Mazzoni (KU) Wilfried Kues (FLI) Stoyan Petkov (FLI) Dias 45 Technical assistance Anne Dorthe V. Roed (KU) Tina Christoffersen (KU) Anitha Pacht (KU) Hanne M. Holm (KU) Helle A. Ruby (KU) Jane Adamsen (AU) Klaus Villemoes (AU) Annette M. Pedersen Lars Fabricius VRO CFIM Funding FNU Fonden til Veterinærvidenskabens Fremme