doi: 10.1038/s41556-019-0333-2.
Epub 2019 Jun 3.
Establishment of porcine and human expanded potential stem cells
Affiliations
- PMID: 31160711
- PMCID: PMC7035105
- DOI: 10.1038/s41556-019-0333-2
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Establishment of porcine and human expanded potential stem cells
Nat Cell Biol.
2019 Jun.
Abstract
We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.
Figures
a. Doxycycline (Dox)-inducible expression of Yamanaka factors
OCT4, MYC, SOX2 and KLF4,
together with LIN28, NANOG,
LRH1 and RARG in porcine PFFs. Stable
genomic integration of cDNAs in PFFs was achieved by piggyBac transposition.
pOMSK: Porcine OCT4, MYC, SOX2 and
KLF4; pN+hLIN: porcine NANOG and human
LIN28; hRL: human RARG and
LRH1. The reprogrammed colonies were single-cell passaged
in the presence of Dox in M15 (15% fetal calf serum). b.
Co-expression of LIN28, NANOG,
LRH1 and RARG substantially increased the
number of reprogrammed colonies from 250,000 PFFs (n = 4 independent
experiments). c. Reprogramming of the porcine
OCT4-tdTomato knock-in reporter (POT) TAIHU and wide type
(WT) German Landrace PFFs to iPSCs. d. The iPSCs lines expressed
key pluripotency genes in RT-qPCR analysis. The iPSC lines #1 and
#2, and iPSC #3 and #4 were from WT German
Landrace and POT PFFs, respectively. e. RT-qPCR analysis of the
exogenous reprogramming factors in iPSCs either in the presence of Dox or 5 days
after its removal. f. POT iPSCs became Td-tomato negative 5 days
after Dox removal. g. RT-qPCR analysis of the expression of
endogenous pluripotency genes in iPSCs cultured with or without Dox.
h. Expression of lineage genes in porcine iPSCs 5-6 days after
DOX removal. Gene expression was measured by RT-qPCR. Relative expression levels
are shown with normalization to GAPDH. Experiments were
performed 3 times. i. Diagram depicting the screen strategy for
identifying culture conditions for porcine pluripotent stem cells using the
Dox-dependent iPSC. Small molecule inhibitors and cytokines were selected for
various combinations. Cell survival, cell morphology, and expression of
endogenous OCT4 and NANOG were employed as the
read-outs. j. Images of
OCT4-Tdtomato reporter (POT) iPSCs in
pEPSCM without Dox. In all RT-qPCR analysis, n=3 independent experiments. All
graphs represent the mean ± s.d. P values were computed
using a two-tailed t-test. For c, f and j, the experiments were repeated
independently three times with similar results. Source data are provided in
Supplementary Table
1. Scale bars, 100 μm.
a. Left: Schematic diagram of establishment of the pig (Sus
Scrofa) EPSCEmb lines from German Landrace day-5
in vivo derived blastocysts on STO feeder cells in pEPSCM,
and of pEPSCiPS lines by reprogramming German Landrace PFFs and China
TAIHU OCT4-Tdtomato knock-in reporter (POT) PFFs. Right panels:
images of established EPSC lines, and a fluorescence image of Td-tomato
expression in POT-pEPSCiPS. Three EPSCEmb lines (Male: K3
and K5; Female K1) and two pEPSCiPS lines (#10, #11) were extensively
tested in this study. These EPSC lines behaved similarly in gene expression and
differentiation. b. Immunostaining detection of pluripotency
factors and markers, SSEA-1 and SSEA-4, in pEPSCEmb and
pEPSCiPS. c. Bisulphite sequencing analysis of CpG
sites in the OCT4 and NANOG promoter regions
in PFFs, pEPSCiPS and pEPSCEmb. d. Gene
expression in embryoid bodies (EBs, day 7) of pEPSCsEmb. Expression
of genes of embryonic and extra-embryonic cell lineages were assessed by
RT-qPCR. Relative expression levels were normalized against
GAPDH. n=3 independent experiments. Data are mean ±
s.d. P values were calculated using a two-tailed t-test.
Statistical source data are provided in Supplementary table 10. e. Tissue composition
of pEPSCEmb teratoma sections (H&E staining): Examples of
glandular epithelium derived from endoderm (i), cartilage derived from mesoderm
(ii), immature neural tissue derived from ectoderm, which forms neuroepithelial
structures (iii), and large multinucleated cells reminiscent of trophoblasts
(arrows in iv). f. PL-1, KRT7 and SDC1 positive cells in
pEPSCEmb teratoma sections as revealed by immunostaining.
g. Detection of pEPSC descendants in the brain
(H2BmCherry+SOX2+) and the liver
(H2BmCherry+AFP+) in chimera #16.
H2B-mCherry and SOX2 are nuclear localised whereas AFP is a cytoplasmic protein.
Boxed areas are shown in higher magnification. Arrows indicate representative
cells that were donor cell descendants (mCherry+). DAPI stained
nuclei. Additional chimera analyses are presented in Extended Data Fig.
3e-3f. For a-b and e-g, the
experiments were repeated independently three times with similar results. Scale
bars, 100 μm.
a. Induction of pPGCLC by transiently expressing
SOX17 in
NANOS3-H2BmCherry reporter pEPSCs. The
presence of H2BmCherry+TNAP+ cells in embryoid bodies
(EBs) was analysed by FACS. The experiments were repeated independently three
times with similar results. b. RT-qPCR analysis of PGC genes in day
3 EBs following pPGCLC induction. Relative expression levels werenormalized
against GAPDH. n=3 independent experiments. Data are mean
± s.d. P values were calculated using a two-tailed
t-test. Statistical source data are provided in Supplementary table 10.
c. Immunofluorescence analysis of PGC factors in the sections
of EBs at day 3-4 following pPGCLC induction. The H2BmCherry+ cells
co-expressed NANOG, OCT4, BLIMP1, TFAP2C and SOX17. DAPI stained nuclei.
Experiments were performed three times. d. RNAseq analysis (Heat
map) of sorted H2BmCherry+ of pPGCLC induction shows expression of
genes associated with PGCs, pluripotency or somatic lineages (mesoderm,
endoderm, and gonadal somatic cells). e. Pair-wise gene expression
comparison between pEPSCsEmb and pPGCLCs. Key up-regulated (red) and
down-regulated (blue) genes are highlighted. f. Bar plot shows
expression of genes related to DNA methylation in pPGCLCs and the parental
pEPSCsEmb. Data were from RNAseq of sorted H2BmCherry+ of pPGCLC
induction. Scale bars, 100 μm.
a. Images of the established H1-EPSCs or M1-EPSCs (passage 25). The
experiments were repeated independently three times with similar results.
b. Expression of pluripotency genes in H1-ESCs, H1-naïve
ESCs (5i), H1-EPSCs and iPSC-EPSCs. Data are mean ± s.d. (n = 3).
P values were computed for two-tailed t-test.
c. EBs of H1-EPSCs to PGCLCs immunostained for SOX17, BLIMP1
and OCT4. Scale bar: 100 μm. d. Hierarchical clustering of
gene expression (bulk RNAseq) of EPSCs, and other human pluripotent stem cells.
Correlation matrix was clustered using Spearman correlation and complete
linkage. Data sets are: pEPSCPar: porcine parthenogenetic EPSCs. E14
and AB2-EPSCs: mouse EPSCs (ref. 1); Human
primed ESCs (WIBR1, iPS_NPC_4 and iPS_NPC_13) and naïve ESCs (WIBR2,
WIBR3_cl_12, WIBR3_cl_16, WIN1_1 and WIN1_2) (Ref. and 35); Human
primed H1 ES cell (H1-rep1 and H1-rep2) and extended pluripotent stem (EPS)
cells (H1_EPS_rep1, H1_EPS_rep2, ES1_EPS_rep1 and ES1_EPS_rep2) (ref. 36). e. Principal component
analysis (PCA) of bulk RNA-seq data of EPSCs, human primed and naïve
ESCs, and PFFs. Human naïve (n=5), human primed (n=3), H1-EPSC (n=2),
hiPSC-EPSC (n=2), pEPSCEmb (n=2), pEPSCiPS (n=2),
pEPSCPar (n=2), PFF (n=2), E14_EPSC (n=2) and AB2_EPSC (n=1). n=
biologically independent experiments. f. Pair-wise comparison of gene expression
between H1-ESCs and H1-EPSCs, showing the highly expressed genes (>8
folds) in hEPSCs (total 76, red dots) and representative histone genes (blue
dots). g. Heatmap showing expression of selected histone genes in
human ESCs, EPSCs and preimplantation embryos. RNAseq data of human ESCs were
from ref. , whereas embryo cell data
were from ref. . h.
RT-qPCR analysis of four histone 1 cluster genes in seven human ESC or iPSC
lines cultured under three conditions. hiPSC lines were from the HIPSC project
(http://www.hipsci.org ): #1, HPSI1113i-bima_1;
#2, HPSI1113i-qolg_3; #3, HPSI1113i-oaaz_2;
#4, HPSI1113i-uofv_1. Relative expression levels are shown with
normalization to GAPDH. n = 3 independent experiments. Data are
mean ± s.d. *P <0.01 compared with the FGF
condition cultured cells. #P <0.01 compared
with 5i condition cultured cells. Experiments were performed three times.
Statistical source data and precise P values are provided in
Supplementary table
10.
a. Violin plots show the distribution and the probability density
the scRNAseq expression of pluripotency genes in pEPSCsEmb (top
panel, n=150) and human H1-EPSCs (lower panel, n=96). n represents the number of
cells in each plot. b. PCA of global gene expression pattern (by
scRNAseq) of pEPSCsEmb (left panel, n=150) and H1-EPSCs (right panel,
n=96). n represents the number of cells in each plot. c. PCA and
comparison of gene expression assessed by scRNAseq of human H1-EPSCs and human
preimplantation embryos (ref. 39).
H1-EPSCs (n=96), oocyte (n=3), zygote (n=3), 2 cell (n=6), 4 cell (n=12), 8 cell
(n=20), morulae (n=16), Late blastocyst (n=30). d. ChIP-seq
analysis of H3K27me3 and H3K4me3 marks at pluripotency gene loci in
pEPSCsEmb and human H1-EPSCs. e. Histone
modifications (H3K4me3 and H3K27me3) at the loci for genes encoding enzymes
involved in DNA methylation and demethylation and for cell lineage genes. For
d and e, experiments were performed three times
with similar results.
a-b. Gene expression in pEPSCsEmb (a) and
H1-EPSCs (b) analysed by RT-qPCR. “-SRCi, -XAV939, -ACTIVIN,
-Vc, -CHIR99”: removing individually; “+TGFBi, +H-CHIR99,
+PD03”: adding SB431542, 3.0 μM CHIR99021, or MEK1/2 inhibitor
PD0325901, respectively. “WH04/A419”: replacing A419259 with
another SRC inhibitor, “WH-4-23. +L-CHIR99”: 0.2 μM in
hEPSCM. Porcine and human EPSC media contain 0.2 μM and 1.0 μM
CHIR99021, respectively. Red triangle: no cell survived. c.
Targeting the H2B-Venus cassette to the OCT4
last coding exon in H1-EPSCs with the stop codon TGA being deleted. Five of 19
colonies genotyped were correctly targeted. d. The effects of
removing WH-4-023 (-SRCi) or XAV939 (-XAV) for 7 days assessed by
Venus+ reporter by fluorescence microscopy and flow cytometry.
e. Western blot analysis of AXIN1 and phosphorylation of
SMAD2/3 in EPSCs. EPSCs had higher levels of AXIN1 and pSMAD2/3 (for TGFβ
signalling) than the differentiated (D) EPSCEmb or primed H1-ESCs.
f. TOPflash analysis for canonical Wnt signalling activity in
EPSCs. Removing XAV939 (pEPSCM-X and hEPSCM-X) for 5 days increased TOPflash
activity. g. Bright-field and immunofluorescence images showing
pEPSCsEmb cultured with the indicated changes in medium
component. Cells were stained for OCT4 and DAPI. h-i. Quantitation
of AP+ colonies formed from 2,000 pEPSCsEmb
(h) or H1-EPSCs (i) cultured on STO feeders with
different medium components. The colonies were scored for 5 consecutive
passages. -ROCKi: passaging EPSCs without the ROCK inhibitor Y27632.
j-k. RT-qPCR analysis of the expression of lineage genes in
pEPSCsEmb (j) or hEPSCs (k) following
removal of XAV939 or ACTIVIN A, inhibition of TGFβ signalling by
SB431542, or treatment with 3.0 μM CHIR99021. l. The effects
of supplementing 5.0 ng/ml ACTIVIN A on gene expression in EBs generated from
H1-EPSCs. m-n. Effects of 5.0ng/ml ACTIVIN A on PGCLC
(Tdtomato+) production from the NANOS3-Tdtomato reporter EPSCs
assessed by FACS (m) and RT-qPCR (n). Relative
expression levels were normalized to GAPDH. All graphs
represent the mean ± s.d. For a-b, j-l and
n, n = 3 independent experiments. For f,
h-i and m, n = 4 independent experiments. For
d-e and g, the experiments were repeated
independently three times with similar results. P values were
computed by two-tailed t-test. Statistical source data are presented in Supplementary table 10.
Scale bars: 100 μm.
a. Left panel: differentiation of hEPSCs to trophoblasts under
TGFβ inhibition. Right panel: flow cytometry analysis of trophoblast
differentiation of CDX2-H2B-Venus reporter EPSCs, collected 4
days after TGFβ inhibition. The CDX2-H2B-Venus reporter
EPSCs were also cultured in conventional FGF-containing hESCs medium or
5i-naïve medium and differentiated under TGFβ inhibition and
examined by flow cytometry. The experiments were repeated independently three
times with similar results. b. The dynamic changes in the
expression of trophoblast genes during hEPSC differentiation (sampled at 2-day
intervals for 12days) were assayed by RT-qPCR. Relative expression levels were
normalized against GAPDH. n = 3 independent experiments. Data
are mean ± s.d. *P <0.01 compared with H1-ESC
cells. #P <0.01 compared with H1-5i cells.
The precise P values are presented in Supplementary table 10.
c. tSNE analysis of RNA-seq data of the differentiating human
ESCs (n = 2) and iPSC-EPSCs (n = 4) treated with TGFβ inhibitor SB431542.
RNAs were sampled from cells at Day 0-12 of differentiation. The of H1-EPSCs and
hiPSC-EPSCs showed different trajectory of differentiation from H1-ESCs.
d. Heatmap shows changes in the expression of
trophoblast-specific genes in differentiating H1-ESCs (green), H1-EPSCs (red)
and iPSC-EPSCs (blue) collected at several time points of culture for RNAseq
analysis. e. DNA demethylation at the promoter region of the
ELF5 locus in differentiating H1-EPSCs and other cell types
following 6 days of SB431542 treatment. Cells from H1-ESCs, H1-naïve ESCs
(5i) showed no discernible DNA demethylation at the ELF5
promoter. f. Secreted hormones from trophoblasts derived from
H1-EPSCs induced by TGFβ inhibition (SB431542). VEGF, PLGF, sFlt-1and
sEng were measured in the conditioned media for culturing the differentiating
EPSCs or ESCs for 16 days following a 48-h SB431542 treatment. g.
hCG produced by trophoblasts from SB431542-treated EPSCs or ESCs at day 10 of
differentiation, measured by ELISA. n = 4 independent experiments. Data are mean
± s.d. P values were calculated using a two-tailed
t-test. Statistical source data are presented in Supplementary table
10.
a. Phase-contrast images of primary TSC colonies formed from
individual hEPSCs (left) and of TSCs at passage 7 (right). b.
Expression of trophoblast markers GATA3, TFAP2C and KRT7 in EPSC-TSCs detected
by immunostaining. Nuclei were stained with DAPI. Similar results were obtained
with four independent EPSC-TSC lines. c. RT-qPCR analysis of
pluripotency and TSC genes in four EPSC-derived TSC lines and their parental
hEPSCs. JEG3 and JAR are trophoblast cell lines. n = 3 independent experiments.
Data are mean ± s.d.. *p < 0.01 compared to TSCs.
The precise P values are presented in Supplementary table 10.
d. PCA of gene expression of hTSCs (n = 3) and of cells
differentiated from human EPSCs (n = 2) under TGFβ inhibition at several
time points. hTSCs showed enriched transcriptomic features of day-4
differentiated EPSCs. e. Immunostaining of SDC1 and CGB in
hTSC-dervied syncytiotrophoblasts. DAPI stained the nucleus. f.
Phase-contrast and Hoechst staining images of multinucleated hTSC-derived
syncytiotrophoblasts. g. The fusion index of forming
syncytiotrophoblasts from hTSCs calculated as the number of nuclei in
syncytia/total number of nuclei. n = 4 independent experiments. Data are mean
± s.d. h. RT-qPCR analysis of trophoblast-specific genes in
syncytiotrophoblast (ST) and extravillous trophoblast (EVT) derived from three
hTSC lines. Expression levels were normalized against GAPDH. n
= 3 independent experiments. Data are mean ± s.d. i. Flow
cytometry detection of HLA-ABC and HLA-G in hESCs, hEPSCs, hTSCs, and
hTSC-derived EVT cells (protocol of ref. 46). The choriocarcinoma cells JEG-3 and JAR represent the
extravillous and villous trophoblast cells, respectively. JEG-3 cells expressed
HLA-G, HLA-C and HLA-E, but not JAR cells. j. H&E staining
of lesions formed by hTSCs engrafted subcutaneously in NOD-SCID mice.
k. Confocal images of immunostaining for SDC1- or KRT7-positive
cells in hTSC-derived lesions. DAPI stained the nucleus. l. Serum
hCG levels in six NOD-SCID mice 7 days after hTSC engraftment (n = 3) or
injection of vehicle only (n = 3). For a-b, e-f and
h-l, the experiments were repeated independently three times
with similar results. Statistical source data are presented in Supplementary table 10.
Scale bars: 100 μm.
Comment in
-
A boost towards totipotency for stem cells.Nat Cell Biol. 2019 Jun;21(6):671-673. doi: 10.1038/s41556-019-0340-3. Nat Cell Biol. 2019. PMID: 31160707 No abstract available.
-
Stem cells with potential.Nat Methods. 2019 Jul;16(7):578. doi: 10.1038/s41592-019-0487-7. Nat Methods. 2019. PMID: 31249416 No abstract available.
-
Modeling the Placenta with Stem Cells.N Engl J Med. 2019 Oct 24;381(17):1681-1683. doi: 10.1056/NEJMcibr1907773. N Engl J Med. 2019. PMID: 31644851 No abstract available.
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