Derivation of porcine extraembryonic endoderm‐like cells from blastocysts

Abstract Objectives Extraembryonic endoderm (XEN) cells are isolated from primitive endoderm (PrE) of blastocysts. Just like PrE, XEN cells have the ability to differentiate into parietal endoderm (PE) and visceral endoderm (VE), and therefore, they are useful tools for studying mechanisms of PrE cells development and differentiation. Pig is an ideal model for studying human cardiovascular and metabolic diseases and a potential organ source for allotransplantation, while no XEN cell has been obtained from porcine embryos. Materials and Methods Using a serum‐free culture system, we directly derived porcine extraembryonic endoderm‐like cells (pXEN‐like cells) from day 6‐7 blastocysts, which could maintain self‐renewal for at least 30 passages. Results The pXEN‐like cells resembled mouse XEN cells with large and flat clone morphology and expressed XEN marker genes but not pluripotent genes. Upon in vitro induction, the cells could differentiate into VE and PE. FGF/MEK signalling was not only essential for the maintenance of pXEN‐like cells, but also the induction of pXEN‐like cells from porcine embryonic stem (pES) cells. Conclusions We directly obtained cell lines with XEN characteristics from porcine embryos for the first time. The cells will be helpful tools for studying embryonic development and cell differentiation, which also represent promising cell sources for human regenerative medicine.

Sox2 and Nanog) or trophoblast (Cdx2). Since XEN cells have the same ability to differentiate into PE and VE as PrE, they are useful tools for studying the mechanisms of PrE cells development and differentiation. 7 As for mouse, we can get XEN cells via three different ways.
First, the cells can be derived directly from embryos. 8,9 Second, they can be converted from embryonic stem (ES) cells. 10 Third, they can be induced from fibroblasts by overexpression of pluripotent genes, which emerge during the transition of somatic state to pluripotent state. 11 Like mouse, rat XEN cells can be derived from embryos directly, 12 and canine XEN-like cells are induced from embryonic fibroblasts by introducing pluripotent genes. 13 Pigs are not only important farm animals, but also potential candidates as human disease models because of their similarities to humans in organ size and physiological characteristics. 14  cells, 17 while no report shows pXEN cells can be derived from embryos directly.
Mouse XEN cell lines, derived from the PrE of blastocysts, have unique characteristics. 7 Previous studies showed that Fgfr2 was enriched in mouse PrE cells. 18 In addition, the studies of intracellular signal transduction suggested that FGF/ERK signalling was a critical pathway to segregate PrE from epiblast. 19,20 In the study of mouse XEN cells, some results showed that the maintenance of XEN cells in vitro also needs FGF/ERK signalling activation, and during the differentiation of mouse ES cells into XEN cells, FGF/ ERK signalling is also required. 10 But as for rat XEN cells, their proliferation is dependent on LIF signalling. 21 Thus, the signalling pathways that regulate the growth of XEN cells have species specificity.
Here, using a serum-free culture system, we derived porcine extraembryonic endoderm-like cells (pXEN-like cells) from day 6-7 blastocysts. The cells expressed XEN marker genes Gata4, Gata6 and Sox17 and could differentiate into VE and PE upon induction.
The maintenance of pXEN-like cells depended on bFGF instead of LIF, and bFGF addition could induce pXEN-like cells from porcine ES cells. The pXEN-like cells will be a helpful tool for studying porcine embryonic development and represent a promising cell sources for contrasting human disease models.

| Animal
Porcine ovaries were collected from Guanglin slaughterhouse, and porcine spermatozoa were from Hongfu Pig Farm. All experiments involving animals were approved and conducted according to the guidelines of the Laboratory Animal Ethics Committee of Northeast Agricultural University, China.

| Production of porcine blastocysts
Porcine blastocysts were got by in vitro oocyte maturation (IVM) and in vitro fertilization (IVF) as previously reported. 16 Briefly, after 42 hours of cultivation in the mature medium, we selected highquality oocytes with the first polar body for IVF. 22 Then, the fertilized embryos were cultured in porcine zygote 3 (PZM-3) medium at 39°C in 5% CO 2 atmosphere for 6-7 days.
The outgrowths tore into small pieces and transferred onto fresh feeder cells for subculture. pXEN-like cells were passaged every 4-5 days using 1 mg/mL collagenase IV and were cultured in humidified conditions with 5% O 2 , 5% CO 2 and 90% N 2 at 39°C.

| Culture of porcine pluripotent stem cells
pES cells were derived from porcine embryos and maintained on mitomycin-treated mouse embryonic fibroblasts. EPSCM medium was used to culture pES cells, which was changed daily. The composition of EPSCM medium is consistent with previous publication. 23 The porcine induced pluripotent stem (piPS) cells are from Pengtao Liu's group by a gift, which was cultured in pEPSCM medium. The medium was changed every day. 24

| Construction of TE-labelled EGFP embryos
The lenti-EGFP plasmid was a gift from Prof. Jiaqiang Wang (Northeast Agricultural University). Briefly, EGFP gene was promoted by elongation factor 1α short promoter and cloned into a lentiviral backbone. 293T cells were transfected with 24 μg of plasmids, 48 μL of LTX and 24 μL of PLUS regents, and the proportions of PMD2.G (Addgene#12259), PSPAX (Addgene#12260) and lenti-EGFP were 1:2:3. The supernatants were collected at 24 hours and 48 hours after transfection, filtered by 0.45 μm filters and concentrated by centrifugal filters (Millipore) at 4°C and 4000 g for 30 minutes.
The 5.5 days of small cavity blastocysts were collected, and their zona pellucida was removed by Protease K (Promega, V302B). Then, the embryos were incubated in PZM-3 medium containing lentivirus carrying EGFP gene. After 3 hours, the embryos were washed 3-4 times with washing solution, and then, the infected embryos were cultured in PZM-3 medium at 39°C in 5% CO 2 atmosphere for 12-24 hours.

| Isolation and culture of trophectoderm
We isolated trophectoderm from day 6-7 embryos transfected with EGFP gene using syringe needles ( Figure S1), and then, the TE was seeded on feeders and cultured with PXEN medium. The medium was changed every day.

| Embryoid body formation piPS cells and pXEN-like cells were digested into single cells and sus-
pended in substrate-free culture dishes with 15% FBS medium. The

Gene name Forward primers Reverse primers
Oct4 medium was changed every other day. After 3-5 days of cultivation, embryoid bodies (EBs) formed.

| VE and PE differentiation
When pXEN-like cells reached 80% confluency, they were passaged with collagenase IV and seeded on Matrigel (Corning, 35423). The culture medium was changed to PXEN without bFGF and LIF but supplemented with 10 ng/mL BMP4 or 2 mM dorsomorphin (DM).
After 5 days, VE and PE differentiation ability was evaluated.

| Trypan Blue staining
Cells were digested into single cells and washed two times with DPBS. After centrifugation, the cells were resuspended and mixed with 0.4% Trypan Blue solution at a ratio of 9:1 in volume. After dyeing at room temperature for 3-5 minutes, the numbers of live and dead cells were counted under the microscope.  presented as mean ± SD P < .05 was considered statistically significant (*), and P < .01 was considered extremely significant (**).

| Derivation of pXEN-like cells from blastocysts
Day 6-7 porcine blastocysts were cultured in PXEN medium, and 3-5 days later, the outgrowth with cubical cells appeared ( Figure 1A, Table 2). Then, the outgrowth was mechanically iso-

| pXEN-like cells are derived from ICM but not TE
Generally, XEN cells are derived from the PrE of the ICM of blastocysts. 8,13 To explore the source of pXEN-like cells, we used TE-labelled EGFP embryos for cell derivation (Figure 2A). After 16 days of cultivation, the EGFP-labelled TE cells disappeared ( Figure 2B). When we separated TE cells from the embryos and cultured them alone, they did not survive more than 7 days ( Figure S1).
Immunofluorescence staining showed that the obtained cells expressed PrE marker GATA4, but not the epiblast marker SOX2 or trophoblast marker CDX2 ( Figure 2C). These results confirmed that pXEN-like cells were derived from ICM but not TE.

| pXEN-like cells undergo PE and VE differentiation
In low-adherent culture condition, the ES cells aggregate and form EBs, which recapitulate important aspects of early embryogenesis. 10,25 Using piPS cells, we successfully obtained EBs ( Figure 3Ab). But pXEN-like cells were difficult to generate EBs under the same condition (Figure 3Aa). Previous studies showed that XEN cells can differentiate into VE and PE of the yolk sac. 13,17 So, we used DM and BMP4 to treat the cells for PE and VE induction. 26 showed that spontaneously differentiated cells were positive for AFP ( Figure 3C). Taken together, we concluded that the pXEN-like cells had the capacity to differentiate into VE and PE.

| RNA-seq analysis of pXEN-like cells
Multi-sample cluster analysis of the RNA-seq data indicated highly reproducible gene expression patterns in piPS cells or pXEN-like cells; however, the gene expression patterns of these two cell lines were significantly different ( Figure 4A). pXEN-like cells expressed typical XEN marker genes, but lacking pluripotent marker genes, such as Pou5f1, Sox2 and C-myc ( Figure 4B). Compared with piPS, pXEN-like cells had 1776 upregulated genes and 1988 downregulated genes ( Figure 4C, Tables S2 and S3). Signalling pathway analysis showed that the upregulated genes were mostly enriched in disease related bioprocess, while the downregulated genes were mostly enriched in biosynthesis and metabolism relation ( Figure 4D-E).

| Both derivation and maintenance of pXEN-like cells are dependent on FGF/MEK signalling
Since the culture medium contained only two cytokines, LIF and bFGF, we studied their effects on the maintenance of pXEN-like Then, we tried to derive new cell lines from porcine blastocysts using PXEN culture medium without bFGF or addition of PD0325901. As a result, neither of the culture system supported new cell derivation (Table 2). These results demonstrated that both derivation and maintenance of pXEN-like cells were dependent on FGF/MEK signalling.

| Conversion of pig embryonic stem cells to pXEN-like cells by bFGF
We then tried to establish pES cells with expanded pluripotency from porcine embryos using EPSCM as culture medium and finally obtained cells expressing pluripotent marker genes ( Figure S3, Table   S1). When we added bFGF to the medium, the cells exhibited XENlike clone morphology ( Figure 6A) and expressed XEN marker genes Gata4, Gata6 and Sox17 but not pluripotent genes Nanog, Oct4 and Sox2 and TE marker gene Cdx2 at both RNA and protein levels ( Figure 6B-C). Besides, these cells could differentiate into PE and VE upon appropriate induction ( Figure 6D-E). These results indicated that bFGF adding induced pESCs to turn to pXEN-like cells.

| D ISCUSS I ON
The extraembryonic lineage of mammals is essential for the nutritive support of foetus and the patterning of early embryos As a major extraembryonic lineage, PrE is the secondary formed tissue during embryogenesis in mammals. 7 15,16,33 However, the pXEN-like cells expressed XEN marker genes but not pluripotent markers, and upon in vitro induction, they could only differentiate into extraembryonic endoderm.
In summary, we directly obtained cell lines with XEN cell characteristics from porcine blastocysts for the first time. The pXEN-like cells are useful tools to study porcine embryo development and cell differentiation, which also represent a promising cell source for human regenerative medicine. Our study may also provide some clues for deriving authentic porcine ES cells.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

AUTH O R CO NTR I B UTI O N S
Yan Li conceived the study, designed and carried out the experi-