Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Summary

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Abstract

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Introduction

Recently, a study comparing several approaches of mouse embryonic stem cell to trophoblast stem cells conversion has revealed that in all analyzed systems, lineage conversion remained incomplete. Instead of induced trophoblast stem cells (iTSCs) so called trophoblast stem cell-like cells have been generated retaining a memory of the cell fate of origin¹. Here, we followed a different approach of iTSC generation. Similar to the direct induction of pluripotent stem cells from murine embryonic fibroblasts (MEFs) ², iTSCs have been directly converted from differentiated somatic tissue. First, we identified 12 candidate factors inducing TSC fate when ....

Protocol

All mouse experiments were conducted according to the German law of animal protection and in agreement with the approval of the local institutional animal care committees (Landesamt fuer Natur, Umwelt und Verbraucherschutz, North Rhine-Westphalia [approval ID number: AZ 84-02.04.2013.A428]).

1. Media Preparation

1. Prepare 293T medium: Dulbecco's Modified Eagle Medium (DMEM), 10% (v/v) FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin/streptomycin (1x).
2. Prepare MEF medium: DMEM, 10% (v/v) FBS, L-glutamine (2 mM), 1x non-essential amino acids, penicillin/streptomycin (1x).
3. Prepare TS medium (w/o FGF....

Results

On the dish where transgene expression of the 4F is activated, cells rapidly change morphology (compare Figure 2A and B). Around day 14 - 21 distinct transdifferentiated areas emerge (two examples are given in Figure 2B and C). These primary colonies lack typical TSC morphology; however once they are sub-cultured, characteristic epithelial morphology with tight edges and bright boundaries highly reminiscent of bona fide TSCs emerges (Figure 2D

Discussion

The 4 factor (Tfap2c, Gata3, Eomes, Ets2) based transdifferentiation protocol presented here offers a reliable method to generate faithfully converted iTSCs from mouse embryonic fibroblasts. Further, the method is also applicable for post-natal tail fibroblasts, although with a drop in efficiency compared to embryonic fibroblasts ³. In general, quality of primary fibroblasts is a critical factor of transdifferentiation outcome and care should be taken to use early passage cells (passage two to three).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Doxycycline hyclate	Sigma-Aldrich	D9891-10G	Dissolve in sterile PBS, prepare 2 mg/ml stock solution. Store aliquots at -20 °C
Chloroquine diphosphate salt	Sigma-Aldrich	C6628-25G	Prepare 25 mM stock solution in sterile cell culture grade water. Store aliquots at -20 °C
Polybrene (Hexadimethrine bromide)	Sigma-Aldrich	107689-10G	Prepare 8 mg/ml stock solution in sterile cell culture grade water. Store aliquots at -20 °C
human recombinant Fibroblast Growth Factor 4	ReliaTech	300-131L	Dissolve in sterile 0.1% BSA in PBS, prepare 25 µg/ml stock solution. Store aliquots at -80 °C
Heparin sodium salt	Sigma-Aldrich	H3149-10KU	Prepare 1 mg/ml stock solution by dissolving in sterile PBS. Store aliquots at -80°C
ProFection Mammalian Transfection System	Promega	E1200
PBS, no calcium, no magnesium	ThermoFisher Scientific	1419-094
DMEM (1x) + GlutaMAX	ThermoFisher Scientific	31966-021
RPMI 1640, no glutamine	ThermoFisher Scientific	31870-025
Advanced DMEM (1x)	ThermoFisher Scientific	12491-015
Trypsin-EDTA (0.05%), phenol red	ThermoFisher Scientific	25300-054
Poly-L-lysine	Sigma-Aldrich	P4707
Fetal bovine serum	Hyclone	SH30071.03IR
Surfactant-free cellulose acetate-membrane filter	Corning	431220
Non-essential amino acids	ThermoFisher Scientific	11140-035
Penicillin Streptomycin	ThermoFisher Scientific	15140-122
Sodium Pyruvate	ThermoFisher Scientific	11360-039
L-glutamine	ThermoFisher Scientific	25030-24
2-Mercaptoethanol	ThermoFisher Scientific	31350-010
Dimethyl sulfoxide (DMSO), cell culture grade	Panreac AppliChem GmbH	A3672,0100
pLV-tetO-Tfap2c	Addgene	70269
pLV-tetO-Gata3	Addgene	70270
pLV-tetO-Eomes	Addgene	70271
pLV-tetO-Ets2	Addgene	70272
pLV-tetO-mCherry	Addgene	70273
psPAX2	Addgene	12260
pMD2.G	Addgene	12259
FUdeltaGW-rtTA	Addgene	19780
Mice B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J 7	JAX Mice	6965
129S2SV	Charles River	129