最近讀了一個文章劝篷，并做了一個匯報在學校的Journal club，如下：
【原文：http://www.cell.com/cell-stem-cell/fulltext/S1934-5909(17)30456-3】
簡單的翻譯了一下民宿。用于自己理解娇妓，肯定疏漏百出，還望見諒活鹰。
【Abstract】
Trophoblast cells play an essential role in the interactions between the fetus and mother.
滋養(yǎng)層細胞對于胎兒和母親之間的相互作用起到了關(guān)鍵作用哈恰。
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.
老鼠的滋養(yǎng)層干細胞已經(jīng)可以獲得只估，而且視作最佳的體外模型用來從分子和功能上分析老鼠滋養(yǎng)層的系譜。
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.
這里我們展示了如果激活 Wnt 和EGF信號通路着绷，同時抑制TGF-β蛔钙，HDAC和ROCH通路就有可能長期培養(yǎng)人的絨毛滋養(yǎng)層細胞。
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.
結(jié)果得到的細胞系具有分化成為三種主要滋養(yǎng)層細胞系譜的能力荠医，而且與對應原細胞有相似的轉(zhuǎn)錄組水平夸楣。
【什么是Primary cells：原代細胞，是從活的組織里提取然后用于體外培養(yǎng)的細胞子漩，這些細胞經(jīng)歷很少的分裂豫喧，所以能很大程度上代表原組織的功能〈逼茫】
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.
重要的是紧显，相同的細胞系也可以從人囊胚獲得。他們的數(shù)據(jù)支持絨毛滋養(yǎng)層細胞（CT）和囊胚導出的細胞系是人滋養(yǎng)層干細胞缕棵。這將提供一個非常有力的工具來研究人的滋養(yǎng)層細胞發(fā)育和功能孵班。
【Introduction】
The placenta is a multifunctional organ essential for fetal development and survival.
胎盤是具有多重功能的器官，它對胎兒的生長生存至關(guān)重要招驴。
Trophoblast cells are specialized cells in the placenta that mediate the interactions between the fetus and mother at the fetomaternal interface.
滋養(yǎng)層細胞是在胎盤中的母嬰結(jié)合處進行調(diào)節(jié)母嬰相互影響的專門細胞篙程。
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).
在人的胚胎中有三種滋養(yǎng)層亞型：細胞滋養(yǎng)層（CT），絨毛外的細胞滋養(yǎng)層（EVT）和合胞體滋養(yǎng)層（ST）别厘。
CT cells are an undifferentiated and proliferative population that can give rise to EVT and ST cells.
CT細胞是沒有分化的而且可以增殖后能夠產(chǎn)生EVT和ST細胞虱饿。
CT cells aggregate into cell columns at the tips of villi, where they differentiate into EVT cells.
CT細胞聚集成一列在絨毛端，在那里他們分化稱為EVT細胞触趴。
EVT cells can be subdivided based on their anatomical locations (Cierna et al., 2016).
EVT細胞可以根據(jù)解剖學上的位置而再細分氮发。
Those that invade the decidualized endometrium are called interstitial EVT cells.
侵襲到子宮內(nèi)膜的叫做間質(zhì)EVT細胞。
Those that invade and remodel the spiral arteries are known as endovascular EVTs.
那些侵襲并且改變動脈空間結(jié)構(gòu)的叫做血管內(nèi)EVT細胞冗懦。
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).
其他亞型也有存在爽冕，因為EVT也在子宮腺，靜脈以及淋巴中被發(fā)現(xiàn)披蕉。
Multinucleated ST cells are formed by fusion of CT cells and produce large quantities of placental hormones and other factors to maintain pregnancy.
多細胞核的ST細胞是由很多CT細胞融合產(chǎn)生颈畸，它們能夠產(chǎn)生大量的孕激素和其他因子，來維持妊娠没讲。
ST cells are directly in contact with maternal blood and mediate the exchange of gases and nutrients.
ST細胞是直接與母體血液連接的眯娱，而且能夠調(diào)節(jié)氣體和營養(yǎng)的交換。
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.
所有的滋養(yǎng)層族系的細胞都是由胚囊里滋養(yǎng)外胚層的細胞長成的食零。它們協(xié)調(diào)的增殖和分化對于成功妊娠起到至關(guān)重要作用困乒。
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).
滋養(yǎng)層發(fā)育和功能受到損害認為會導致不同的妊娠并發(fā)癥，包括流產(chǎn)贰谣，子癇前期以及宮內(nèi)生長抑制娜搂。
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.
老鼠的滋養(yǎng)層干細胞迁霎，是最先由胚囊和著床后的外胚層誘導而來，是目前最佳的體外模型用來研究老鼠滋養(yǎng)層細胞分子和功能分析百宇。
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存在的情況下考廉，老鼠的TS細胞就能持續(xù)自我增值而不失去分化稱為其他滋養(yǎng)層細胞族系的功能。
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).
一些轉(zhuǎn)錄因子携御，包括Cdx2昌粤，Eomes，Elf5啄刹，和Gata3都被證明了具有對老鼠TS細胞保持不分化起到關(guān)鍵作用涮坐。
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).
盡管之前被推斷，人的囊胚里的滋養(yǎng)胚層以及早期胎盤的CT細胞都包含了干細胞群誓军，企圖從這細胞中引出人TS細胞袱讹，但是還未成功。
In this study, we analyzed the transcriptomes of primary trophoblast cells to infer how CT cells are maintained in their undifferentiated state in vivo.
在這個研究里昵时，我們分析了早期滋養(yǎng)層細胞的轉(zhuǎn)錄組來推斷CT細胞是如何可以維持在體內(nèi)不分化捷雕。
Using this knowledge, we optimized the culture conditions and derived human TS cells from CT cells and blastocysts.
通過知道這些，我們優(yōu)化培養(yǎng)的條件壹甥，并且從CT細胞中已經(jīng)胚囊中引導出了人的TS細胞救巷。
Our culture system will provide a powerful tool to study human trophoblast development and function.
我們的培養(yǎng)系統(tǒng)會提高強有力的工具用于研究人的培養(yǎng)層細胞發(fā)育和功能。
【Result】
- Transcriptome Analysis of Primary Trophoblast Cells
轉(zhuǎn)錄組分析原代滋養(yǎng)層細胞
We isolated CT, EVT, and ST cells from first-trimester placentas (Figures S1A–S1C) and performed RNA sequencing (RNA-seq) (Table S1).
我們分離第一階段的胚胎得到了CT句柠，EVT和ST細胞浦译，然后執(zhí)行了RNA-seq《碚迹【CT：ITGA6管怠； EVT：HLA-G淆衷；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).
我們鑒定出來在CT缸榄，EVT和ST細胞中顯著表達的基因數(shù)量分別是，377祝拯，228和289個甚带。【該細胞型里FPKM>10高表達的基因佳头，倍數(shù)變化>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.
我們確認了廣泛運用的marker基因ITGA6和TP63（CT）鹰贵，ITGA5和HLA-G（EVT），和CGB和CSH1（ST）都在各自高表達的基因列表里康嘉。
We then conducted functional annotation of the gene lists using ConsensusPathDB (Herwig et al., 2016; Figure 1A).
我們?nèi)缓髮蛄斜磉M行功能注視碉输，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.
有趣的是亭珍，和wnt信號通路以及EGF信號通路有關(guān)的基因在CT里面高表達敷钾。
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信號通路在一些表皮干細胞的增殖起關(guān)鍵作用枝哄。包括皮膚干細胞，腸干細胞阻荒。
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]).
一致地挠锥，CT高表達的基因富集的通路中，排在最前面的是“毛囊發(fā)育”侨赡，這個通路里包括一些對于維持毛囊干細胞關(guān)鍵的基因（(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.
這些數(shù)據(jù)意味著CT細胞或許保持增殖的條件與其他的表皮干細胞類似蓖租。
- Establishment of Proliferative Human CT Cells in Culture 建立體外培養(yǎng)增殖的人CT細胞
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.
根據(jù)之前的結(jié)論，我們嘗試在培養(yǎng)基里培養(yǎng)CT細胞羊壹。培養(yǎng)基包含CHIR99021（是Wnt激活物）和EGF蓖宦，但是這些細胞并不貼壁在培養(yǎng)皿上生長，只能存活幾天時間油猫。
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).
我們接下來測試了一些抑制因子和生長因子球昨。它們是已知的體外增殖培養(yǎng)表皮干細胞的因子。
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).
當所有的這些抑制因子和生長因子都存在時候眨攘，從CT細胞里面高度增殖的一個細胞系被誘導出來主慰。（條件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.
在這些抑制因子與生長因子里面Y27632（ROCK抑制因子）被發(fā)現(xiàn)它對細胞能夠貼壁增殖起到至關(guān)重要作用。所以在后續(xù)的實驗中鲫售，ROCK抑制因子都被持續(xù)使用共螺。
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是細胞增殖不可或缺的因子，在缺少它的時候情竹，會導致CT細胞分化稱為HLA-G顯陽性的EVT類似細胞藐不。
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抑制劑）和丙戊酸（VPA秦效，是組蛋白去乙醭化抑制劑）也明顯的起到了增強CT細胞增殖的作用。
Eventually, we found that CHIR99021, EGF, TGF-b inhibitors, VPA, and Y27632 together were sufficient for long-term culture of CT cells (Figure 1C).
最終阱州，我們發(fā)現(xiàn)CHIR99021, EGF, TGF-b inhibitors, VPA和Y27632一起能夠有效的讓CT細胞在體外培養(yǎng)挑秉。
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).
我們能夠從1000個CT細胞誘導增殖的CT細胞（做了5次同樣的實驗，得到了五個細胞系）苔货，但是沒能得到TS從單獨CT細胞里面（n=200）犀概。
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這些因子對于CT細胞長期保持增殖很關(guān)鍵。
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）或者異羥肟酸（SAHA）來代替夜惭。
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).
盡管A83-01 或者SB431542可以支持誘導和增殖CT細胞姻灶，我們保留了它們兩個抑制劑，考慮到了它們的一些特異性诈茧。
The culture conditions tested in this study are summarized in Table S2.
培養(yǎng)的條件在Table S2里列出來了产喉。
We successfully derived proliferative CT cell lines from all first- trimester placental samples tested (n = 8) (Table S3).
我們從八個不同個體的第一階段胚胎組織中都成功的引導出了可以增殖的CT細胞系。
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，使用同樣條件曾沈，我們不能在后面的胚胎（n=5尘颓，剖腹產(chǎn)得到的胚胎）中獲取CT細胞系。
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細胞有正常的染色體型晦譬，而且能持續(xù)增殖達到5個月疤苹。
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).
這些細胞有一個凡滋養(yǎng)層marker，KRT7敛腌。但是卧土，HLA-ABC表達很低。
They also expressed TP63 and TEAD4 (CT markers) and GATA3 (a mononuclear trophoblast marker) (Figure 1G).
它們也表達TP63和TEAD4（CT的marker）和GATA3（是單核滋養(yǎng)層細胞的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細胞被指定為CT-誘導的滋養(yǎng)層干細胞像樊，因為它們有能力分化稱為EVT和ST樣子的細胞尤莺，如下所述。
Directed Differentiation of TSCT Cells into EVT- and ST-like Cells
TSCT定向分化為EVT和ST類似的細胞
To analyze the differentiation potential of TSCT cells, we first cultured TS cells in a basal medium containing only Y27632.
為了分析TSCT細胞具有分化的潛能生棍，我們首先培養(yǎng)TS細胞颤霎，在只含有Y27632的培養(yǎng)基里。
Most of the cells differentiated into multinucleated ST-like cells, but some cells remained mononucleated (Figure S2A).
絕大多數(shù)細胞分化成了多細胞核的ST-like 細胞涂滴，但是一些細胞仍然是單核細胞
The culture conditions did not support the survival of the differentiated cells, and most of them died within 5 days.
培養(yǎng)條件不能夠支持分化細胞的存活友酱，絕大多數(shù)的細胞在5天內(nèi)死亡。
Therefore, additional factor(s) may be required for the efficient and directed differentiation of TSCT cells.
因此柔纵，附近的因子或許需要TSCT細胞的定向分化缔杉。
Matrigel is widely used to induce outgrowth of EVT cells from placental explants (Miller et al., 2005).
人工基質(zhì)膠體被廣泛用于誘導胚胎移植得到EVT細胞衍生物。
A recent study also revealed that decidua-derived NRG1 promotes EVT formation in placental explant cultures (Fock et al., 2015).
最近的研究也顯示經(jīng)期脫膜得到的NRG1能夠推動EVT形成在胚胎移植培養(yǎng)中搁料。
In addition, CT cells preferentially differentiated into EVT-like cells under condition 2 shown in Figure 1B (see also Figure S1E).
另外或详，CT細胞有潛力分化為EVT-like細胞在條件二中。
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).
在條件二里的抑制劑和生長因子中TGF-β抑制劑被發(fā)現(xiàn)能夠促進CT細胞的分化稱為EVT-like細胞郭计。而且A83-01比SB431542更有效霸琴。
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昭伸，和人工基質(zhì)膠的培養(yǎng)系統(tǒng)中暂雹，TSCT細胞經(jīng)歷了EMT轉(zhuǎn)化形成了EVT-like 細胞格仲。而且這些細胞高表達HLA-G蛋白幔欧。
The resulting cells were named EVT-TSCT cells.
結(jié)果得到的細胞被稱為EVT-TSCT細胞梨撞。
We confirmed that NRG1, A83-01, and Matrigel were all important for the induction of EVT-TSCT cells (Figure S2D).
我們確認了NRG1哩陕，A83-01岗宣，和人工基質(zhì)膠對于誘導EVT-TSCT細胞都很關(guān)鍵褐荷。
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 細胞的marker）计济，SDC1（ST 細胞的marker）罩引，VIM（基質(zhì)marker各吨？）在EVT-TSCT細胞里面表達很低或者沒有檢測到。
CGB (an ST marker) is expressed at low levels in EVT cells (Pr?ll et al., 2000) and, consistently, was detectable in EVT-TSCT cells (Figure 2G).
已知CGB（ST細胞的marker）在EVT細胞里面表達很低，EVT-TSCT同樣也沒有檢測到CGB的表達揭蜒。
Previous studies on choriocarcinoma cell lines revealed that cyclic AMP (cAMP) enhances ST formation (Strauss et al., 1992).
以往的研究絨毛癌細胞系揭示了cAMP能夠增強ST形成横浑。
Thus, we treated TSCT cells with forskolin, a cAMP agonist (Figure 2A).
因此，我們用forskolin細胞去處理TSCT屉更。forskolin是cAMP的增筋劑徙融。
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存在的情況下這些細胞開始聚集并且高效的成為合胞體。
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在這些合胞體細胞里面高度表達瑰谜。然而欺冀，ITGA6，CDH1萨脑，HLA-G和VIM的表達量很低隐轩。
The ST-like syncytia were designated ST(2D)-TSCT cells.
ST-like合胞體細胞被命名為ST（2D）-TSCT細胞。
It has also been reported that 3D culture enhances differentiation of choriocarcinoma cells into ST-like cells (McConkey et al., 2016).
也曾被報道過渤早，3D培養(yǎng)會促進絨毛癌細胞分化稱為ST-like細胞职车。
Therefore, we cultured the proliferative CT cells in low adhesion plates (Figure 2A).
因此我們在低附著培養(yǎng)皿中培養(yǎng)增殖的CT細胞。
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).
這些細胞形成了囊狀的結(jié)構(gòu)鹊杖，而且分泌大量的絨毛促性腺激素悴灵。
Forskolin and EGF synergistically enhanced the formation of the cyst-like structures (Figure S2F).
Forskolin和EGF同時增進這種囊狀結(jié)構(gòu)的形成。
These cyst-like structures were designated ST(3D)-TSCT cells.
這種囊狀結(jié)構(gòu)的細胞被命名為ST（3D）-TSCT細胞骂蓖。
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分化的細胞中要比2D分化的細胞表達量要高称勋。
TSCT cells maintained their ability to differentiate into EVT- and ST-like cells after 50 passages (Figures S2H and S2I).
TSCT細胞仍然保持分化稱為EVT-like和ST-like細胞在50此傳代之后。
We also cultured single TSCT cells (n = 50) and isolated 10 clonal lines (Figure 2J).
我們也培養(yǎng)了單一的TSCT細胞（n=50）并且分離了10個單克隆系涯竟。
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.
我們隨機抽選了三個單克隆細胞系并確認了它們都能夠分化為EVT-和ST-like細胞赡鲜。
Establishment of Human TS Cells from Blastocysts
從胚囊建立人的TS細胞
We next investigated whether cells similar to TSCT cells could be derived directly from human blastocysts.
我們接下來，調(diào)查是否從人陪囊中也可以誘導出類似于從細胞滋養(yǎng)層細胞中得到一樣的滋養(yǎng)層干細胞庐船。
Sixteen blastocysts were cultured under the same conditions (Figure 3A), and eight cell lines were established.
16個陪囊在相同的條件下進行培養(yǎng)银酬。8個細胞系被建立。
These cell lines, designated blastocyst-derived TS cells (TS blast cells), were morphologically similar to TSCT cells (Figure 3B).
這些細胞系被命名為陪囊誘導滋養(yǎng)層干細胞（TS-blast細胞）筐钟，從外形上看和TS-ct很類似揩瞪。
A normal karyotype was confirmed in all six TSblast cell lines examined (Figure S3A; Table S3).
染色體型在這六個細胞系中都被檢測了。
TSblast cells continued to proliferate for at least 5 months (Figure 3B).
TS-blast細胞能夠保持增殖至少5個月篓冲。
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細胞一樣李破，TS-blast細胞表達KRT7, TP63, GATA3, 和 TEAD4。而且HLA-ABC表達很低壹将。
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).
更進一步嗤攻，TS-blast細胞也有分化為EVT-TSblast，ST（2D）-TSblast和ST（3D）-TSblast細胞的能力诽俯，就和TSCT細胞一樣妇菱。但是有兩個細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的分化能力可以持續(xù)到第55代。
We also cultured single TSblast cells (n = 50) and isolated 8 clonal lines.
我們也培養(yǎng)了但細胞TS-blast（n=50）闯团，并且分離了8個單克隆細胞系辛臊。
We randomly selected three clonal lines and confirmed that they could differentiate into both EVT- and ST-like cells (Figures 3L and 3M).
我們隨機挑選了三個單克隆細胞系，并且確認了它們可以分化成為EVT-和ST-like的細胞房交。
Transcriptome Profiling of Human TS Cells
人滋養(yǎng)層干細胞轉(zhuǎn)錄組譜
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).
為了確認是否TSCT和TSblast細胞擁有和原代滋養(yǎng)層細胞一樣的基因表達水平彻舰，我們對TSCT，TSblast以及它們的派生細胞進行RNA-seq候味。
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).
最初調(diào)查顯示了ST（2D）和ST（3D）細胞有很相似的轉(zhuǎn)錄組刃唤，但是ST（3D）相比，跟接近原代ST細胞负溪。
Therefore, we chose ST(3D) cells as the model of ST cells.
因此我們透揣，選擇ST（3D）細胞作為ST細胞模型。
We compared the RNA-seq data with those of primary trophoblast cells and placenta-derived stromal cells.
我們比較原代滋養(yǎng)層細胞和胚胎誘導的基質(zhì)細胞川抡。
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).
聚類分析揭示TSCT和TSblast細胞有分寸類似的基因表達情況辐真，同樣也包括它們分化后。
Importantly, the gene expression profiles of CT cells were closest to those of TSCT and TSblast cells (Figure 4A).
關(guān)鍵的是崖堤，CT細胞的基因表達水平和TSCT與TSblast細胞的很接近侍咱。
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分化誘導得到的EVT-和ST-like細胞也與對應的EVT和ST細胞很類似。
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).
更進一步密幔，絕大多數(shù)在CT楔脯，EVT或者ST細胞里面顯著表達的基因在TSCT和TSBlast細胞的誘導細胞里也有表達。
We then focused on some representative lineage markers.
我們接下來專注于一些有代表性的組系marker胯甩。
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我們都發(fā)現(xiàn)了它們的表達昧廷，盡管一些基因如LRP5，TP63和ELF5在TSCT和TSblast細胞里面表達很低偎箫。
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).
絕大多數(shù)EVT和ST的marker在原代和培養(yǎng)的細胞里面也呈現(xiàn)出類似的表達水平（如木柬， 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.
盡管TSCT和TSblast細胞在基因表達水平上很相似于原代的TS細胞淹办，但是它們不會是完全一樣的眉枕，因為它們是在大致類似的體外培養(yǎng)的條件下。
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分析顯示不同的GO term在原代和培養(yǎng)的細胞中富集怜森。
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.
顯著地速挑，和核糖體合成又關(guān)系的基因在TS細胞里面富集，這些基因或許能夠促進TS細胞的增殖副硅，因為核糖體驅(qū)動細胞的增殖與生長姥宝。
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.
我們同樣發(fā)現(xiàn)了在TS-誘導的EVT-like細胞中DNA復制相關(guān)的基因也顯著的減少，這個和我觀察到的TS分化為EVT-like細胞后就嚴重失去了增殖的能力的現(xiàn)象一致想许。
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被報道過具有胚胎器官特異性啟動子伶授。
We found that these placenta-specific promoters were active in ST(3D)-TSCT and ST(3D)-TSblast cells (Figure S4D).
我們發(fā)現(xiàn)這些胚胎器官特異性啟動子在ST（3D）-TSCT和ST（3D）-TSblast細胞中也是活躍的断序。
As shown in Figure 4C, FGFR2 was predominantly expressed in CT cells and undifferentiated TSCT and TSblast cells.
FGFR2在CT細胞以及TSCT和TSblast細胞里都顯著表達流纹。
We found that, of the two major isoforms of FGFR2 (FGFR2b and FGFR2c), FGFR2b was expressed almost exclusively (Figure S4E).
我們發(fā)現(xiàn)糜烹，F(xiàn)GFR2的兩種亞型（FGFR2b和FGFR2c），其中FGFR2b幾乎不表達漱凝。
This is intriguing because the essential role of Fgfr2c was reported in mouse trophoblast cells (Arman et al., 1998).
這個很有趣疮蹦，因為Fgfr2c在老鼠的滋養(yǎng)層細胞里被報道過。
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).
更進一步茸炒，CDX2, EOMES, ESRRB, 和 SOX2這些轉(zhuǎn)譯成為轉(zhuǎn)錄因子的基因很少表達（在CT愕乎，TSCT，和TSblast細胞）壁公。這些轉(zhuǎn)錄因子是在老鼠TS細胞自我更新所必需的感论。
DNA Methylome Profiling of Human TS Cells
人滋養(yǎng)層干細胞DNA甲基化組水平
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).
滋養(yǎng)層細胞具有獨特的DNA甲基化特點，存在大量的PMD紊册，胚胎特異性啟動子去甲基化比肄，和胚胎特異性的生殖細胞分化的甲基化區(qū)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).
為了檢測是否這些獨特的甲基化狀態(tài)在TSCT和TSblast細胞中也依然保持囊陡，我們進行了全基因組酸性亞硫酸鹽測序（WGBS）在TSCT和TSblast細胞并于之前發(fā)表的CT細胞的甲基化水平進行比較芳绩。
TSCT and TSblast cells showed almost identical global DNA methylation patterns (R = 0.97).
TSCT和TSblast細胞的DNA甲基化水平機會一致。
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).
然而撞反，DNA甲基化平均水平上TSCT和TSblast要同時比CT細胞低妥色，但是甲基化區(qū)域都很類似。
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).
大多數(shù)的PMD區(qū)域在之前的研究中被認定遏片，會保持在CT細胞甲基化中間水平嘹害。但是在TSCT和TSblast細胞中沒有甲基化。
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.
活躍轉(zhuǎn)錄區(qū)域會有很高的甲基化水平比起其他區(qū)域（CT吮便，TSCT笔呀，和TSblast細胞），與之前的人胚胎細胞發(fā)現(xiàn)的一致线衫。因此凿可，大多數(shù)的胚胎特異性DNA甲基化在TSCT和TSblast細胞里面都保持，盡管PMD去甲基化的區(qū)域的原因和影響依然還不清楚授账。
We next analyzed the ELF5 promoter, which is hypomethylated in trophoblast cells but hypermethylated in many other cell types (Hemberger et al., 2010).
我們接下來分析了ELF5基因的啟動子枯跑，這個地方在滋養(yǎng)層細胞里是低甲基化的，但是在其他很多細胞型里是高甲基化的白热。
We found that the ELF5 promoter was hypomethylated in both TSCT and TSblast cells (Figure 5D).
我們發(fā)現(xiàn)ELF5的啟動子在TSCT和TSblast細胞里都是低甲基化的敛助。
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啟動子以為，我們還認定了55個啟動子和ELF5有類似的DNA甲基化水平屋确，包括了一些啟動子被認為是胚胎特異性的低甲基化的纳击。
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).
我們發(fā)現(xiàn)這些啟動子都保持很低的甲基化水平在TSCT和TSblast細胞里续扔。
We confirmed the hypomethylation of three selected promoters (DSCR4, ELF5, and ZNF750) in all TSCT and TSblast lines established in this study (Figure S5B).
我們確認三個基因的低甲基化水平在全部的TSCT和TSblast細胞系里。
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).
我們也找到5個啟動子是相反的甲基化樣子焕数，而且他們在TSCT和TSblast能夠有80%以上的甲基化水平纱昧。
A number of placenta-specific gDMRs, which maintain allele-specific DNA methylation in a placenta-specific manner, have been identified.
一些胚胎特異性的gDMRs，它們保持等位基因一致的特異性堡赔。
We focused on placenta-specific gDMRs associated with imprinted genes (n = 33) (Table S5).
我們專注于胚胎特異性的gDMR相關(guān)的印記基因 （n=33）识脆。
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).
絕大多數(shù)的gDMRs保持理想的中間甲基化水平。
We analyzed allele-specific DNA methylation in two TSCT cell lines using targeted bisulfite sequencing (see STAR Methods for details).
我們分析了等位基因特異性的DNA甲基化水平在TSCT細胞系里面善已，這里使用了定點重硫酸鹽測序（STAR）灼捂。
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個胚胎特異性的gDMR區(qū)域被成功獲得，其中9個保持了母親的等位基因特意甲基化水平换团。
We did not analyze the allelic DNA methylation patterns in TSblast cells because the maternal genotype was not available.
我們沒有分析等位基因DNA甲基化水平在TSblast細胞里悉稠，因為他們沒有辦法知道對應的母親基因型。
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個里面的9個gDMR保持里中間的甲基化水平（30-70%）在TSblast細胞艘包，暗示TSCT和TSblast細胞甲基化印記應該類似的猛。
Among the genes shown in Figure 5F, four genes had SNPs available for allelic expression analysis.
圖5F里的基因中，其中四個有SNP辑甜，在分析等位表達分析之后衰絮。
The four genes
showed similar allelic expression patterns in primary CT and TSCT cells (Figure S5D).
這四個基因有相似的等位基因狀態(tài)，在原代細胞和TSCT一致磷醋。
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.
我們也發(fā)現(xiàn)x-linked基因在男性女性的TS細胞里表達水平一致猫牡，XIST只在女性TS細胞里表達，說明其中一個X染色體可能被抑制活性在雌性TS細胞里面邓线。
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里面淌友，染色體19的miRNA DMR調(diào)節(jié)在胚胎里C19MC的表達。
C19MC miRNAs are almost exclusively expressed in the placenta (Bortolin-Cavaillé et al., 2009).
C19MC miRNA在胚胎里幾乎不表達骇陈。
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).
我們做了miRNA測序在CT震庭，TSCT和TSblast細胞里面，并且和熱ESC以及IMR90細胞比較你雌。
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).
我們發(fā)現(xiàn)C19MC miRNA在CT TSCT和TSblast細胞里面高表達器联，然而在人的胚胎干細胞以及IMR90里面表達量很低。
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).
另外婿崭，TSCT和TSblast細胞雨CT細胞在整體的miRNA表達水平很類似拨拓。
Engraftment of Human TS Cells into NOD-SCID Mice
移植人的滋養(yǎng)層胚胎干細胞到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.
為了知道TSCT和TSblast細胞在體內(nèi)試驗中可以增殖氓栈，我們用免疫缺陷小鼠實驗渣磷，注射了1X107cell。
The injected cells formed ～5-mm lesions by day 7 and were then gradually resorbed.
注射的細胞在7天時間內(nèi)形成了5mm 傷痕然后逐漸消失掉了授瘦。
Immunohistochemical staining of KRT7 revealed that the injected cells invaded the dermal and subcutaneous tissues (Figure 6A).
組織染色KRT7顯示注射的細胞侵襲到了真皮與皮下組織醋界。
The central area of the lesions was necrotic and surrounded by ITGA6-positive CT-like cells (Figure 6B).
傷痕中間處的細胞是壞死的竟宋，包圍其為ITGA6陽性的CT-like細胞。
We identified EVT-like cells migrating as single cells (Figure 6C), but they were few in number.
我們識別了EVT-like細胞以單細胞形式移動形纺，但是數(shù)量很少丘侠。
We analyzed 14 sections from four lesions, and only 4–18 HLA-G-positive cells were observed per section.
我們分析了14個損傷部位組織切片。
SDC1-positive ST-like cells were observed at the peripheral region of the lesions (Figure 6D).
SDC1陽性的 ST-like細胞在損傷部位末端被觀察到挡篓。
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).
有趣的是婉陷，ST-like細胞保護了血液帚称，與其聯(lián)系的是ST細胞在著床后侵襲母體子宮內(nèi)膜會產(chǎn)生血竇官研。
We also found that the host mouse serum contained a substantial amount of hCG (Figure 6E).
我們也發(fā)現(xiàn)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.
雖然絨毛形狀的結(jié)構(gòu)沒有觀察到闯睹，但是這個數(shù)據(jù)表明TS細胞注射到NOD老鼠戏羽，模擬了一些關(guān)鍵的特性類似滋養(yǎng)層細胞在著床后的侵襲。
