最近读了一个文章，并做了一个汇报在学校的Journal club，如下：
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【原文：http://www.cell.com/cell-stem-cell/fulltext/S1934-5909(17)30456-3】
简单的翻译了一下。用于自己理解，肯定疏漏百出，还望见谅。
【Abstract】
Trophoblast cells play an essential role in the interactions between the fetus and mother.
滋养层细胞对于胎儿和母亲之间的相互作用起到了关键作用。
Mouse trophoblast stem (TS) cells have been derived and used as the best in vitro model for molecular and functional analysis of mouse trophoblast lineages, but attempts to derive human TS cells have so far been unsuccessful.
老鼠的滋养层干细胞已经可以获得，而且视作最佳的体外模型用来从分子和功能上分析老鼠滋养层的系谱。
Here we show that activation of Wingless/Integrated (Wnt) and EGF and inhibition of TGF-β, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells.
这里我们展示了如果激活 Wnt 和EGF信号通路，同时抑制TGF-β，HDAC和ROCH通路就有可能长期培养人的绒毛滋养层细胞。
The resulting cell lines have the capacity to give rise to the three major trophoblast lineages, which show transcriptomes similar to those of the corresponding primary trophoblast cells.
结果得到的细胞系具有分化成为三种主要滋养层细胞系谱的能力，而且与对应原细胞有相似的转录组水平。
【什么是Primary cells：原代细胞，是从活的组织里提取然后用于体外培养的细胞，这些细胞经历很少的分裂，所以能很大程度上代表原组织的功能。】
Importantly, equivalent cell lines can be derived from human blastocysts. Our data strongly suggest that the CT- and blastocyst-derived cell lines are human TS cells, which will provide a powerful tool to study human trophoblast development and function.
重要的是，相同的细胞系也可以从人囊胚获得。他们的数据支持绒毛滋养层细胞（CT）和囊胚导出的细胞系是人滋养层干细胞。这将提供一个非常有力的工具来研究人的滋养层细胞发育和功能。
【Introduction】
The placenta is a multifunctional organ essential for fetal development and survival.
胎盘是具有多重功能的器官，它对胎儿的生长生存至关重要。
Trophoblast cells are specialized cells in the placenta that mediate the interactions between the fetus and mother at the fetomaternal interface.
滋养层细胞是在胎盘中的母婴结合处进行调节母婴相互影响的专门细胞。
In the human placenta, there are three major trophoblast subpopulations: the cytotrophoblast (CT), extravillous cytotrophoblast (EVT), and syncytiotrophoblast (ST) (Bischof and Irminger-Finger, 2005; James et al., 2012).
在人的胚胎中有三种滋养层亚型：细胞滋养层（CT），绒毛外的细胞滋养层和合胞体滋养层。
CT cells are an undifferentiated and proliferative population that can give rise to EVT and ST cells.
CT细胞是没有分化的而且可以增殖后能够产生EVT和ST细胞。
CT cells aggregate into cell columns at the tips of villi, where they differentiate into EVT cells.
CT细胞聚集成一列在绒毛端，在那里他们分化称为EVT细胞。
EVT cells can be subdivided based on their anatomical locations (Cierna et al., 2016).
EVT细胞可以根据解剖学上的位置而再细分。
Those that invade the decidualized endometrium are called interstitial EVT cells.
侵袭到子宫内膜的叫做间质EVT细胞。
Those that invade and remodel the spiral arteries are known as endovascular EVTs.
那些侵袭并且改变动脉空间结构的叫做血管内EVT细胞。
Other subtypes likely exist because EVT cells have also been found in uterine glands, veins, and lymphatics (Moser et al., 2010; Windsperger et al., 2017).
其他亚型也有存在，因为EVT也在子宫腺，静脉以及淋巴中被发现。
Multinucleated ST cells are formed by fusion of CT cells and produce large quantities of placental hormones and other factors to maintain pregnancy.
多细胞核的ST细胞是由很多CT细胞融合产生，它们能够产生大量的孕激素和其他因子，来维持妊娠。
ST cells are directly in contact with maternal blood and mediate the exchange of gases and nutrients.
ST细胞是直接与母体血液连接的，而且能够调节气体和营养的交换。
All of the trophoblast lineages arise from the trophectoderm (TE) cells of the blastocyst, and their coordinated proliferation and differentiation is essential for a successful pregnancy.
所有的滋养层族系的细胞都是由胚囊里滋养外胚层的细胞长成的。它们协调的增殖和分化对于成功妊娠起到至关重要作用。
Impaired trophoblast development and function are thought to lead to various pregnancy complications, including miscarriage, preeclampsia, and intrauterine growth restriction (Moffett and Loke, 2006; Norwitz, 2006).
滋养层发育和功能受到损害认为会导致不同的妊娠并发症，包括流产，子痫前期以及宫内生长抑制。
Mouse trophoblast stem (TS) cells, which were first derived from blastocysts and the extraembryonic ectoderm (ExE) of postimplantation embryos (Tanaka et al., 1998), are the best in vitro model for molecular and functional analysis of mouse trophoblast cells.
老鼠的滋养层干细胞，是最先由胚囊和着床后的外胚层诱导而来，是目前最佳的体外模型用来研究老鼠滋养层细胞分子和功能分析。
In the presence of fibroblast growth factor 4 (FGF4) and transforming growth factor b1 (TGF-b1)/Activin, mouse TS cells self-renew indefinitely without losing their ability to differentiate into all trophoblast lineages.
在FGF4和TGF-b1/Activin存在的情况下，老鼠的TS细胞就能持续自我增值而不失去分化称为其他滋养层细胞族系的功能。
A number of transcription factors, including Cdx2, Eomes, Elf5, Esrrb, and Gata3, have been identified as essential for maintaining the undifferentiated state of mouse TS cells (Latos and Hemberger, 2016).
一些转录因子，包括Cdx2，Eomes，Elf5，和Gata3都被证明了具有对老鼠TS细胞保持不分化起到关键作用。
Although it has been assumed that TE cells of human blastocysts and CT cells of early human placentas contain a stem cell population, attempts to derive human TS cells from these cells have so far been unsuccessful (Kunath et al., 2014; Soncin et al., 2015).
尽管之前被推断，人的囊胚里的滋养胚层以及早期胎盘的CT细胞都包含了干细胞群，企图从这细胞中引出人TS细胞，但是还未成功。
In this study, we analyzed the transcriptomes of primary trophoblast cells to infer how CT cells are maintained in their undifferentiated state in vivo.
在这个研究里，我们分析了早期滋养层细胞的转录组来推断CT细胞是如何可以维持在体内不分化。
Using this knowledge, we optimized the culture conditions and derived human TS cells from CT cells and blastocysts.
通过知道这些，我们优化培养的条件，并且从CT细胞中已经胚囊中引导出了人的TS细胞。
Our culture system will provide a powerful tool to study human trophoblast development and function.
我们的培养系统会提高强有力的工具用于研究人的培养层细胞发育和功能。
【Result】
- Transcriptome Analysis of Primary Trophoblast Cells
转录组分析原代滋养层细胞
We isolated CT, EVT, and ST cells from first-trimester placentas (Figures S1A–S1C) and performed RNA sequencing (RNA-seq) (Table S1).
我们分离第一阶段的胚胎得到了CT，EVT和ST细胞，然后执行了RNA-seq。【CT：ITGA6； EVT：HLA-G；ST：SDC1】
We identified 377, 228, and 289 genes that were predominantly expressed in CT, EVT, and ST cells, respectively (fragments per kilobase per million [FPKM] > 10 in the cell type with the highest expression, fold change > 4, adjusted p < 0.01) (Figure 1A).
我们鉴定出来在CT，EVT和ST细胞中显著表达的基因数量分别是，377，228和289个。【该细胞型里FPKM>10高表达的基因，倍数变化>4, FDR<0.01】
We confirmed that widely used lineage markers such as ITGA6 and TP63 (CT), ITGA5 and HLA-G (EVT), and CGB and CSH1 (ST) (Bischof and Irminger-Finger, 2005, Reis-Filho et al., 2003) were included in the gene lists.
我们确认了广泛运用的marker基因ITGA6和TP63（CT），ITGA5和HLA-G（EVT），和CGB和CSH1（ST）都在各自高表达的基因列表里。
We then conducted functional annotation of the gene lists using ConsensusPathDB (Herwig et al., 2016; Figure 1A).
我们然后对基因列表进行功能注视，使用ConsensusPathDB。
Intriguingly, genes related to the Wingless/Integrated (Wnt) and epidermal growth factor (EGF) signal transduction pathways (“regulation of FZD by ubiquitination” and “EGFR1”) were overrepresented in the CT highest (CThighest) gene list.
有趣的是，和wnt信号通路以及EGF信号通路有关的基因在CT里面高表达。
Wnt and EGF signaling are required for proliferation of various epithelial stem cells, including skin stem cells and intestinal stem cells (Fatehullah et al., 2016, Hsu et al., 2014).
Wnt和EGF信号通路在一些表皮干细胞的增殖起关键作用。包括皮肤干细胞，肠干细胞。
Consistently, the top-ranked pathway for the CT highest genes was “hair follicle development,” which included some genes important for the maintenance of hair follicle stem cells (TP63, FGFR2, and CTNNB1 [encoding β-catenin]).
一致地，CT高表达的基因富集的通路中，排在最前面的是“毛囊发育”，这个通路里包括一些对于维持毛囊干细胞关键的基因（(TP63, FGFR2, and CTNNB1 [encoding β-catenin]）
These data imply that CT cells might be maintained under conditions similar to those of the other epithelial stem cells.
这些数据意味着CT细胞或许保持增殖的条件与其他的表皮干细胞类似。
- Establishment of Proliferative Human CT Cells in Culture 建立体外培养增殖的人CT细胞
Based on the results described above, we tried to culture CT cells in a medium containing CHIR99021 (a Wnt activator) and EGF, but the cells did not adhere to the culture plate and died within several days.
根据之前的结论，我们尝试在培养基里培养CT细胞。培养基包含CHIR99021（是Wnt激活物）和EGF，但是这些细胞并不贴壁在培养皿上生长，只能存活几天时间。
We then tested several inhibitors and growth factors (Figure 1B) that are known to enhance in vitro proliferation of various epithelial stem cells (Fatehullah et al., 2016).
我们接下来测试了一些抑制因子和生长因子。它们是已知的体外增殖培养表皮干细胞的因子。
In the presence of all of these inhibitors and growth factors, highly proliferative cell lines were derived from CT cells (condition 1 in Figure 1B and Figure S1D).
当所有的这些抑制因子和生长因子都存在时候，从CT细胞里面高度增殖的一个细胞系被诱导出来。（条件1）
Among the inhibitors and growth factors, Y27632 (a Rho-associated protein kinase [ROCK] inhibitor) was found to be essential for cell attachment and was added to all culture media in subsequent experiments.
在这些抑制因子与生长因子里面Y27632（ROCK抑制因子）被发现它对细胞能够贴壁增殖起到至关重要作用。所以在后续的实验中，ROCK抑制因子都被持续使用。
CHIR99021 was indispensable for cell proliferation, and its absence led to differentiation of CT cells into HLA-G-positive EVT-like cells (Figure S1E).
CHIR99021是细胞增殖不可或缺的因子，在缺少它的时候，会导致CT细胞分化称为HLA-G显阳性的EVT类似细胞。
EGF, A83-01 and SB431542 (TGF-b inhibitors) and valproic acid (VPA) (a histone deacetylase [HDAC] inhibitor) significantly enhanced proliferation of CT cells (Figure 1B).
EGF，A83-01和SB431542（TGF-b抑制剂）和丙戊酸（VPA，是组蛋白去乙酰化抑制剂）也明显的起到了增强CT细胞增殖的作用。
Eventually, we found that CHIR99021, EGF, TGF-b inhibitors, VPA, and Y27632 together were sufficient for long-term culture of CT cells (Figure 1C).
最终，我们发现CHIR99021, EGF, TGF-b inhibitors, VPA和Y27632一起能够有效的让CT细胞在体外培养。
We were able to derive proliferative CT cells from as few as 1,000 CT cells (five cell lines from five independent experiments) but failed to derive TS cells from single CT cells (n = 200).
我们能够从1000个CT细胞诱导增殖的CT细胞（做了5次同样的实验，得到了五个细胞系），但是没能得到TS从单独CT细胞里面（n=200）。
CHIR99021, EGF, TGF-b inhibitors, and VPA were all important for the long-term maintenance of proliferative CT cells (Figure S1F).
CHIR99021, EGF, TGF-b inhibitors, and VPA这些因子对于CT细胞长期保持增殖很关键。
VPA could be replaced by trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) (Figure S1G).
VPA可以有曲古抑菌素（TSA）或者异羟肟酸（SAHA）来代替。
Although either A83-01 or SB431542 could support the derivation of proliferative CT cells (Figure S1H), we retained both inhibitors in consideration of their different specificities (Vogt et al., 2011).
尽管A83-01 或者SB431542可以支持诱导和增殖CT细胞，我们保留了它们两个抑制剂，考虑到了它们的一些特异性。
The culture conditions tested in this study are summarized in Table S2.
培养的条件在Table S2里列出来了。
We successfully derived proliferative CT cell lines from all first- trimester placental samples tested (n = 8) (Table S3).
我们从八个不同个体的第一阶段胚胎组织中都成功的引导出了可以增殖的CT细胞系。
In contrast, we were unable to derive such cells from term placentas (placentas obtained after elective caesarean section, n = 5) under the same conditions.
相反，使用同样条件，我们不能在后面的胚胎（n=5，剖腹产得到的胚胎）中获取CT细胞系。
The proliferative CT cells had a normal karyotype (Figure S1I) and continued to proliferate for at least 5 months ($150 population doublings) (Figure 1D).
增殖的CT细胞有正常的染色体型，而且能持续增殖达到5个月。
These cells expressed a pan-trophoblast marker, KRT7 (Figure 1E), but HLA-ABC expression was very low (Figure 1F), which is a hallmark of CT cells (King et al., 2000).
这些细胞有一个凡滋养层marker，KRT7。但是，HLA-ABC表达很低。
They also expressed TP63 and TEAD4 (CT markers) and GATA3 (a mononuclear trophoblast marker) (Figure 1G).
它们也表达TP63和TEAD4（CT的marker）和GATA3（是单核滋养层细胞的marker）
The proliferative CT cells were designated CT-derived TS cells (TSCT cells) because they had the ability to differentiate into EVT- and ST-like cells as detailed below.
可以增殖的CT细胞被指定为CT-诱导的滋养层干细胞，因为它们有能力分化称为EVT和ST样子的细胞，如下所述。
Directed Differentiation of TSCT Cells into EVT- and ST-like Cells
TSCT定向分化为EVT和ST类似的细胞
To analyze the differentiation potential of TSCT cells, we first cultured TS cells in a basal medium containing only Y27632.
为了分析TSCT细胞具有分化的潜能，我们首先培养TS细胞，在只含有Y27632的培养基里。
Most of the cells differentiated into multinucleated ST-like cells, but some cells remained mononucleated (Figure S2A).
绝大多数细胞分化成了多细胞核的ST-like 细胞，但是一些细胞仍然是单核细胞
The culture conditions did not support the survival of the differentiated cells, and most of them died within 5 days.
培养条件不能够支持分化细胞的存活，绝大多数的细胞在5天内死亡。
Therefore, additional factor(s) may be required for the efficient and directed differentiation of TSCT cells.
因此，附近的因子或许需要TSCT细胞的定向分化。
Matrigel is widely used to induce outgrowth of EVT cells from placental explants (Miller et al., 2005).
人工基质胶体被广泛用于诱导胚胎移植得到EVT细胞衍生物。
A recent study also revealed that decidua-derived NRG1 promotes EVT formation in placental explant cultures (Fock et al., 2015).
最近的研究也显示经期脱膜得到的NRG1能够推动EVT形成在胚胎移植培养中。
In addition, CT cells preferentially differentiated into EVT-like cells under condition 2 shown in Figure 1B (see also Figure S1E).
另外，CT细胞有潜力分化为EVT-like细胞在条件二中。
Among the inhibitors and growth factors contained in condition 2, the TGF-β inhibitors were found to promote differentiation of CT cells into EVT-like cells, and A83-01 was more potent than SB431542 (Figure S2B).
在条件二里的抑制剂和生长因子中TGF-β抑制剂被发现能够促进CT细胞的分化称为EVT-like细胞。而且A83-01比SB431542更有效。
In a culture system containing NRG1, A83-01, and Matrigel (Figure 2A), TSCT cells underwent epithelial-mesenchymal transition (Figure 2B; Figure S2C) and gave rise to EVT-like cells that strongly expressed HLA-G (Figure 2C).
在包含了NRG1，A83-01，和人工基质胶的培养系统中，TSCT细胞经历了EMT转化形成了EVT-like 细胞。而且这些细胞高表达HLA-G蛋白。
The resulting cells were named EVT-TSCT cells.
结果得到的细胞被称为EVT-TSCT细胞。
We confirmed that NRG1, A83-01, and Matrigel were all important for the induction of EVT-TSCT cells (Figure S2D).
我们确认了NRG1，A83-01，和人工基质胶对于诱导EVT-TSCT细胞都很关键。
ITGA6 and CDH1 (CT markers), SDC1 (an ST marker), and VIM (a stromal marker) expression was low or undetectable in EVT-TSCT cells (Figure 2G).
ITGA6和CDH1 （CT 细胞的marker），SDC1（ST 细胞的marker），VIM（基质marker？）在EVT-TSCT细胞里面表达很低或者没有检测到。
CGB (an ST marker) is expressed at low levels in EVT cells (Pröll et al., 2000) and, consistently, was detectable in EVT-TSCT cells (Figure 2G).
已知CGB（ST细胞的marker）在EVT细胞里面表达很低，EVT-TSCT同样也没有检测到CGB的表达。
Previous studies on choriocarcinoma cell lines revealed that cyclic AMP (cAMP) enhances ST formation (Strauss et al., 1992).
以往的研究绒毛癌细胞系揭示了cAMP能够增强ST形成。
Thus, we treated TSCT cells with forskolin, a cAMP agonist (Figure 2A).
因此，我们用forskolin细胞去处理TSCT。forskolin是cAMP的增筋剂。
In the presence of forskolin, the cells started to make aggregates and efficiently fused to form large syncytia (Figures 2D and 2E; Figure S2E).
在有forskolin存在的情况下这些细胞开始聚集并且高效的成为合胞体。
The ST markers CGB and SDC1 were highly expressed in these syncytia, whereas ITGA6, CDH1, HLA-G, and VIM were poorly expressed (Figure 2G).
ST的marker蛋白CGB和SDC1在这些合胞体细胞里面高度表达。然而，ITGA6，CDH1，HLA-G和VIM的表达量很低。
The ST-like syncytia were designated ST(2D)-TSCT cells.
ST-like合胞体细胞被命名为ST（2D）-TSCT细胞。
It has also been reported that 3D culture enhances differentiation of choriocarcinoma cells into ST-like cells (McConkey et al., 2016).
也曾被报道过，3D培养会促进绒毛癌细胞分化称为ST-like细胞。
Therefore, we cultured the proliferative CT cells in low adhesion plates (Figure 2A).
因此我们在低附着培养皿中培养增殖的CT细胞。
These cells formed cyst-like structures (Figure 2F), expressed CGB and SDC1 (Figure 2H), and secreted a large amount of human chorionic gonadotropin (hCG) (Figure 2I).
这些细胞形成了囊状的结构，而且分泌大量的绒毛促性腺激素。
Forskolin and EGF synergistically enhanced the formation of the cyst-like structures (Figure S2F).
Forskolin和EGF同时增进这种囊状结构的形成。
These cyst-like structures were designated ST(3D)-TSCT cells.
这种囊状结构的细胞被命名为ST（3D）-TSCT细胞。
Expression of ST markers was higher in ST(3D)-TSCT cells than in ST(2D)-TSCT cells (Figure S2G).
ST的marker蛋白在3D分化的细胞中要比2D分化的细胞表达量要高。
TSCT cells maintained their ability to differentiate into EVT- and ST-like cells after 50 passages (Figures S2H and S2I).
TSCT细胞仍然保持分化称为EVT-like和ST-like细胞在50此传代之后。
We also cultured single TSCT cells (n = 50) and isolated 10 clonal lines (Figure 2J).
我们也培养了单一的TSCT细胞（n=50）并且分离了10个单克隆系。
We randomly selected three clonal lines and confirmed that they could differentiate into both EVT- and ST-like cells (Figures 2K and 2L), suggesting that individual TSCT cells were bipotent.
我们随机抽选了三个单克隆细胞系并确认了它们都能够分化为EVT-和ST-like细胞。
Establishment of Human TS Cells from Blastocysts
从胚囊建立人的TS细胞
We next investigated whether cells similar to TSCT cells could be derived directly from human blastocysts.
我们接下来，调查是否从人陪囊中也可以诱导出类似于从细胞滋养层细胞中得到一样的滋养层干细胞。
Sixteen blastocysts were cultured under the same conditions (Figure 3A), and eight cell lines were established.
16个陪囊在相同的条件下进行培养。8个细胞系被建立。
These cell lines, designated blastocyst-derived TS cells (TS blast cells), were morphologically similar to TSCT cells (Figure 3B).
这些细胞系被命名为陪囊诱导滋养层干细胞（TS-blast细胞），从外形上看和TS-ct很类似。
A normal karyotype was confirmed in all six TSblast cell lines examined (Figure S3A; Table S3).
染色体型在这六个细胞系中都被检测了。
TSblast cells continued to proliferate for at least 5 months (Figure 3B).
TS-blast细胞能够保持增殖至少5个月。
As in the case of TSCT cells, TSblast cells expressed KRT7, TP63, GATA3, and TEAD4 (Figures 3C and 3D), and HLA-ABC expression was very low (Figure S3B).
和TSct细胞一样，TS-blast细胞表达KRT7, TP63, GATA3, 和 TEAD4。而且HLA-ABC表达很低。
Furthermore, TSblast cells had the ability to differentiate into EVT-TSblast (Figures 3E and 3F), ST(2D)-TSblast (Figures 3G–3I), and ST(3D)-TSblast cells (Figures 3J and 3K) just as TSCT cells did (Figure 2A), although two TSblast lines (4 and 7) differentiated into EVT-like cells less efficiently than the other TSblast and TSCT lines (Figures S3C and S3D).
更进一步，TS-blast细胞也有分化为EVT-TSblast，ST（2D）-TSblast和ST（3D）-TSblast细胞的能力，就和TSCT细胞一样。但是有两个细胞系比起其他的效率上要低。
The differentiation ability of TSblast cells was maintained after 55 passages (Figures S3E and S3F).
TSblast的分化能力可以持续到第55代。
We also cultured single TSblast cells (n = 50) and isolated 8 clonal lines.
我们也培养了但细胞TS-blast（n=50），并且分离了8个单克隆细胞系。
We randomly selected three clonal lines and confirmed that they could differentiate into both EVT- and ST-like cells (Figures 3L and 3M).
我们随机挑选了三个单克隆细胞系，并且确认了它们可以分化成为EVT-和ST-like的细胞。
Transcriptome Profiling of Human TS Cells
人滋养层干细胞转录组谱
To determine whether TSCT and TSblast cells had gene expression patterns similar to primary trophoblast cells, we performed RNA-seq of TSCT and TSblast cells and their derivatives (Figure 4A).
为了确认是否TSCT和TSblast细胞拥有和原代滋养层细胞一样的基因表达水平，我们对TSCT，TSblast以及它们的派生细胞进行RNA-seq。
A preliminary investigation suggested that ST(2D) and ST(3D) cells had very similar transcriptome profiles, but ST(3D) cells were a little more similar to primary ST cells (Figure S4A).
最初调查显示了ST（2D）和ST（3D）细胞有很相似的转录组，但是ST（3D）相比，跟接近原代ST细胞。
Therefore, we chose ST(3D) cells as the model of ST cells.
因此我们，选择ST（3D）细胞作为ST细胞模型。
We compared the RNA-seq data with those of primary trophoblast cells and placenta-derived stromal cells.
我们比较原代滋养层细胞和胚胎诱导的基质细胞。
Hierarchical clustering revealed that TSCT and TSblast cells had very similar gene expression patterns to each other, both before and after their differentiation (R > 0.98) (Figure 4A).
聚类分析揭示TSCT和TSblast细胞有分寸类似的基因表达情况，同样也包括它们分化后。
Importantly, the gene expression profiles of CT cells were closest to those of TSCT and TSblast cells (Figure 4A).
关键的是，CT细胞的基因表达水平和TSCT与TSblast细胞的很接近。
The profiles of TSCT- and TSblast-derived EVT- and ST-like cells were closely related to those of primary EVT and ST cells, respectively (Figure 4A).
TSCT-和TSblast分化诱导得到的EVT-和ST-like细胞也与对应的EVT和ST细胞很类似。
Furthermore, most of the genes predominantly expressed in CT, EVT, or ST cells (the genes shown in Figure 1A) showed similar expression patterns in TSCT and TSblast cells and their derivatives (Figure 4B).
更进一步，绝大多数在CT，EVT或者ST细胞里面显著表达的基因在TSCT和TSBlast细胞的诱导细胞里也有表达。
We then focused on some representative lineage markers.
我们接下来专注于一些有代表性的组系marker。
All CT markers we examined exhibited the expected expression patterns, although some genes, such as LRP5, TP63, and ELF5, showed lower expression in TSCT and TSblast cells (Figure 4C).
全部的CT marker我们都发现了它们的表达，尽管一些基因如LRP5，TP63和ELF5在TSCT和TSblast细胞里面表达很低。
Most EVT and ST markers also showed comparable expression patterns and levels in the primary and cultured cells, with a few exceptions (e.g., CD9 and CSH1) (Figures 4D and 4E).
绝大多数EVT和ST的marker在原代和培养的细胞里面也呈现出类似的表达水平（如， CD9 和CSH1）。
Although TSCT and TSblast cells had gene expression profiles similar to those of primary cells, they were not exactly the same, presumably reflecting the artificial in vitro conditions.
尽管TSCT和TSblast细胞在基因表达水平上很相似于原代的TS细胞，但是它们不会是完全一样的，因为它们是在大致类似的体外培养的条件下。
Gene set enrichment analysis (GSEA) revealed that genes associated with various gene ontology (GO) terms were differentially expressed between the primary and cultured cells.
GSEA分析显示不同的GO term在原代和培养的细胞中富集。
Notably, genes related to ribosome biogenesis were especially enriched in TS cells (Figure S4B), which might contribute to TS cell proliferation because ribosomes drive cell proliferation and growth.
显著地，和核糖体合成又关系的基因在TS细胞里面富集，这些基因或许能够促进TS细胞的增殖，因为核糖体驱动细胞的增殖与生长。
We also found that DNA replication-related genes were significantly depleted in TS-derived EVT-like cells (Figure S4C), consistent with our observation that TS cells differentiating into EVT-like cells gradually lost their proliferative capacity.
我们同样发现了在TS-诱导的EVT-like细胞中DNA复制相关的基因也显著的减少，这个和我观察到的TS分化为EVT-like细胞后就严重失去了增殖的能力的现象一致。
Several genes such as CYP19A1, EDNRB, IL2RB, and PTN are reported to have placenta-specific promoters (Cohen et al., 2011, Rawn and Cross, 2008).
一些基因如CYP19A1, EDNRB, IL2RB, 和 PTN被报道过具有胚胎器官特异性启动子。
We found that these placenta-specific promoters were active in ST(3D)-TSCT and ST(3D)-TSblast cells (Figure S4D).
我们发现这些胚胎器官特异性启动子在ST（3D）-TSCT和ST（3D）-TSblast细胞中也是活跃的。
As shown in Figure 4C, FGFR2 was predominantly expressed in CT cells and undifferentiated TSCT and TSblast cells.
FGFR2在CT细胞以及TSCT和TSblast细胞里都显著表达。
We found that, of the two major isoforms of FGFR2 (FGFR2b and FGFR2c), FGFR2b was expressed almost exclusively (Figure S4E).
我们发现，FGFR2的两种亚型（FGFR2b和FGFR2c），其中FGFR2b几乎不表达。
This is intriguing because the essential role of Fgfr2c was reported in mouse trophoblast cells (Arman et al., 1998).
这个很有趣，因为Fgfr2c在老鼠的滋养层细胞里被报道过。
Furthermore, CDX2, EOMES, ESRRB, and SOX2, which encode transcription factors required for mouse TS cell self-renewal (Latos and Hemberger, 2016), were poorly expressed (< 1 FPKM) in CT, TSCT, and TSblast cells (Table S1).
更进一步，CDX2, EOMES, ESRRB, 和 SOX2这些转译成为转录因子的基因很少表达（在CT，TSCT，和TSblast细胞）。这些转录因子是在老鼠TS细胞自我更新所必需的。
DNA Methylome Profiling of Human TS Cells
人滋养层干细胞DNA甲基化组水平
Trophoblast cells have unique DNA methylation patterns characterized by large partially methylated domains (PMDs) (Schroeder et al., 2013), placenta-specific promoter hypomethylation (Robinson and Price, 2015), and placenta-specific germline differentially methylated regions (gDMRs) (Court et al., 2014).
滋养层细胞具有独特的DNA甲基化特点，存在大量的PMD，胚胎特异性启动子去甲基化，和胚胎特异性的生殖细胞分化的甲基化区域。
To examine whether these unique methylation patterns were maintained in TSCT and TSblast cells, we performed whole-genome bisulfite sequencing (WGBS) of TSCT and TSblast cells and compared the data with those of CT cells (Hamada et al., 2016), human embryonic stem cells (ESCs) (Lister et al., 2011), and cord blood cells (Okae et al., 2014) (Figure 5A).
为了检测是否这些独特的甲基化状态在TSCT和TSblast细胞中也依然保持，我们进行了全基因组酸性亚硫酸盐测序（WGBS）在TSCT和TSblast细胞并于之前发表的CT细胞的甲基化水平进行比较。
TSCT and TSblast cells showed almost identical global DNA methylation patterns (R = 0.97).
TSCT和TSblast细胞的DNA甲基化水平机会一致。
Although the average methylation levels of TSCT (33.7%) and TSblast cells (33.6%) were substantially lower than that of CT cells (52.3%), their methylation patterns were similar to each other (R ≥ 0.80).
然而，DNA甲基化平均水平上TSCT和TSblast要同时比CT细胞低，但是甲基化区域都很类似。
Most of the PMDs defined in a previous study (Schroeder et al., 2013) maintained the intermediate methylation levels in CT cells but were hypomethylated in TSCT and TSblast cells (Figures 5B and 5C).
大多数的PMD区域在之前的研究中被认定，会保持在CT细胞甲基化中间水平。但是在TSCT和TSblast细胞中没有甲基化。
Actively transcribed regions showed higher methylation levels compared with other regions in CT, TSCT, and TSblast cells (Figure S5A), consistent with previous findings in the human placenta (Schroeder et al., 2013). Therefore, the placenta-specific DNA methylome was largely maintained in TSCT and TSblast cells, although the cause and significance of the PMD hypomethylation remain unclear.
活跃转录区域会有很高的甲基化水平比起其他区域（CT，TSCT，和TSblast细胞），与之前的人胚胎细胞发现的一致。因此，大多数的胚胎特异性DNA甲基化在TSCT和TSblast细胞里面都保持，尽管PMD去甲基化的区域的原因和影响依然还不清楚。
We next analyzed the ELF5 promoter, which is hypomethylated in trophoblast cells but hypermethylated in many other cell types (Hemberger et al., 2010).
我们接下来分析了ELF5基因的启动子，这个地方在滋养层细胞里是低甲基化的，但是在其他很多细胞型里是高甲基化的。
We found that the ELF5 promoter was hypomethylated in both TSCT and TSblast cells (Figure 5D).
我们发现ELF5的启动子在TSCT和TSblast细胞里都是低甲基化的。
In addition to the ELF5 promoter, we identified 55 promoters with methylation patterns similar to that of the ELF5 promoter (methylation level < 20% in CT cells and > 80% in ESCs and blood cells), which included some promoters that are known to be specifically hypomethylated in the placenta (e.g., the promoters of INSL4 and DSCR4) (Du et al., 2011, Macaulay et al., 2011).
除了ELF5启动子以为，我们还认定了55个启动子和ELF5有类似的DNA甲基化水平，包括了一些启动子被认为是胚胎特异性的低甲基化的。
We found that most of these promoters (48 of 55) maintained less than 20% methylation levels in TSCT and TSblast cells (Figure 5E; Table S4).
我们发现这些启动子都保持很低的甲基化水平在TSCT和TSblast细胞里。
We confirmed the hypomethylation of three selected promoters (DSCR4, ELF5, and ZNF750) in all TSCT and TSblast lines established in this study (Figure S5B).
我们确认三个基因的低甲基化水平在全部的TSCT和TSblast细胞系里。
We also identified 5 promoters with the opposite pattern, and all of them had more than 80% methylation levels in TSCT and TSblast cells (Figure 5E; Table S4).
我们也找到5个启动子是相反的甲基化样子，而且他们在TSCT和TSblast能够有80%以上的甲基化水平。
A number of placenta-specific gDMRs, which maintain allele-specific DNA methylation in a placenta-specific manner, have been identified.
一些胚胎特异性的gDMRs，它们保持等位基因一致的特异性。
We focused on placenta-specific gDMRs associated with imprinted genes (n = 33) (Table S5).
我们专注于胚胎特异性的gDMR相关的印记基因 （n=33）。
Most of the gDMRs maintained the expected intermediate methylation levels (30%–70%) in CT (33 of 33), TSCT (26 of 33), and TSblast cells (24 of 33) but not in ESCs (0 of 33) or blood cells (0 of 33).
绝大多数的gDMRs保持理想的中间甲基化水平。
We analyzed allele-specific DNA methylation in two TSCT cell lines using targeted bisulfite sequencing (see STAR Methods for details).
我们分析了等位基因特异性的DNA甲基化水平在TSCT细胞系里面，这里使用了定点重硫酸盐测序（STAR）。
The allelic methylation patterns were successfully obtained for ten placenta-specific gDMRs, and nine of them maintained maternal allele-specific DNA methylation in TSCT cells (Figure 5F).
等位基因甲基化形式在10个胚胎特异性的gDMR区域被成功获得，其中9个保持了母亲的等位基因特意甲基化水平。
We did not analyze the allelic DNA methylation patterns in TSblast cells because the maternal genotype was not available.
我们没有分析等位基因DNA甲基化水平在TSblast细胞里，因为他们没有办法知道对应的母亲基因型。
However, nine of the ten gDMRs maintained intermediate methylation levels (30%–70%) in TSblast cells (Figure S5C), implying that their imprinted methylation patterns might be similar in TSCT and TSblast cells.
然而10个里面的9个gDMR保持里中间的甲基化水平（30-70%）在TSblast细胞，暗示TSCT和TSblast细胞甲基化印记应该类似。
Among the genes shown in Figure 5F, four genes had SNPs available for allelic expression analysis.
图5F里的基因中，其中四个有SNP，在分析等位表达分析之后。
The four genes
showed similar allelic expression patterns in primary CT and TSCT cells (Figure S5D).
这四个基因有相似的等位基因状态，在原代细胞和TSCT一致。
We also found that the expression levels of X-linked genes were comparable between male and female TS cells and that XIST was expressed only in female TS cells (Figure S5E), suggesting that one X chromosome may be inactivated in female TS cells.
我们也发现x-linked基因在男性女性的TS细胞里表达水平一致，XIST只在女性TS细胞里表达，说明其中一个X染色体可能被抑制活性在雌性TS细胞里面。
Among the gDMRs shown in Figure 5F, the chromosome 19 microRNA (miRNA) cluster (C19MC) differentially methylated region (DMR) regulates the imprinted expression of C19MC in the placenta (Noguer-Dance et al., 2010).
在gDMR里面，染色体19的miRNA DMR调节在胚胎里C19MC的表达。
C19MC miRNAs are almost exclusively expressed in the placenta (Bortolin-Cavaillé et al., 2009).
C19MC miRNA在胚胎里几乎不表达。
We performed miRNA sequencing in CT, TSCT, and TSblast cells and compared the data with those of human ESCs (GEO: GSM438362) and IMR90 cells (GEO: GSM438364).
我们做了miRNA测序在CT，TSCT和TSblast细胞里面，并且和热ESC以及IMR90细胞比较。
We found that C19MC miRNAs were highly expressed in CT, TSCT, and TSblast cells, weakly expressed in human ESCs, and almost absent in IMR90 cells (Figure 5G; Table S6).
我们发现C19MC miRNA在CT TSCT和TSblast细胞里面高表达，然而在人的胚胎干细胞以及IMR90里面表达量很低。
In addition, TSCT and TSblast cells had global miRNA expression patterns more similar to CT cells (R > 0.84) than to ESCs or IMR90 cells (R < 0.64) (Figure S5F).
另外，TSCT和TSblast细胞雨CT细胞在整体的miRNA表达水平很类似。
Engraftment of Human TS Cells into NOD-SCID Mice
移植人的滋养层胚胎干细胞到NOD-SCID mice。
To assess the in vivo potential of TSCT and TSblast cells, we subcutaneously injected them (1 × 107) into non-obese diabetic (NOD)-severe combined immunodeficiency (SCID) mice.
为了知道TSCT和TSblast细胞在体内试验中可以增殖，我们用免疫缺陷小鼠实验，注射了1X107cell。
The injected cells formed ∼5-mm lesions by day 7 and were then gradually resorbed.
注射的细胞在7天时间内形成了5mm 伤痕然后逐渐消失掉了。
Immunohistochemical staining of KRT7 revealed that the injected cells invaded the dermal and subcutaneous tissues (Figure 6A).
组织染色KRT7显示注射的细胞侵袭到了真皮与皮下组织。
The central area of the lesions was necrotic and surrounded by ITGA6-positive CT-like cells (Figure 6B).
伤痕中间处的细胞是坏死的，包围其为ITGA6阳性的CT-like细胞。
We identified EVT-like cells migrating as single cells (Figure 6C), but they were few in number.
我们识别了EVT-like细胞以单细胞形式移动，但是数量很少。
We analyzed 14 sections from four lesions, and only 4–18 HLA-G-positive cells were observed per section.
我们分析了14个损伤部位组织切片。
SDC1-positive ST-like cells were observed at the peripheral region of the lesions (Figure 6D).
SDC1阳性的 ST-like细胞在损伤部位末端被观察到。
Interestingly, some of the ST-like cells contained blood-filled lacunae, reminiscent of primitive ST cells that form during implantation, invade the maternal endometrium and erode maternal sinusoids (James et al., 2012).
有趣的是，ST-like细胞保护了血液，与其联系的是ST细胞在着床后侵袭母体子宫内膜会产生血窦。
We also found that the host mouse serum contained a substantial amount of hCG (Figure 6E).
我们也发现宿主老鼠血清里含有一定量的hCG？
Although a villous-like structure was not observed, these data suggest that TS cells injected into NOD-SCID mice mimic some key features of trophoblast invasion during implantation.
虽然绒毛形状的结构没有观察到，但是这个数据表明TS细胞注射到NOD老鼠，模拟了一些关键的特性类似滋养层细胞在着床后的侵袭。
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