⼈的滋养层⼲细胞是如何建⽴，并且稳定培养
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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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 esntial 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。
Mou trophoblast stem (TS) cells have been derived and ud as the best in vitro model for molecular and functional analysis of mou trophoblast lineages, but attempts to derive human TS c
ells 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 deacetyla (HDAC), and Rho-associated protein kina (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells.
胖人穿什么好看
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 ri to the three major trophoblast lineages, which show transcriptomes similar to tho 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。
olor【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 esntial 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（EVT）和合胞体滋养层（ST）。
CT cells are an undifferentiated and proliferative population that can give ri 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 bad 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。
virtueTho 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。
Tho that invade and remodel the spiral arteries are known as endovascular EVTs.
爱神丘比特的故事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 becau EVT cells have also been found in uterine glands, veins, and lymphatics (Mor 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。
霍顿与无名氏电影ST cells are directly in contact with maternal blood and mediate the exchange of gas 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。
sculleyAll of the trophoblast lineages ari from the trophectoderm (TE) cells of the blastocyst, and their coordinated proliferation and differentiation is esntial 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。
Mou 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 mou 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 prence of fibroblast growth factor 4 (FGF4) and transforming growth factor b1 (TGF-b1)/Activin, mou TS cells lf-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 esntial for maintaining the undifferentiated state of mou 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 the 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】
1. 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 quencing (RNA-q) (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-q。【CT：ITGA6； EVT：HLA-G；ST：SDC1】
We identified 377, 228, and 289 genes that were predominantly expresd in CT, EVT, and ST cells, respectively (fragments
per kiloba 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 ud 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 ConnsusPathDB (Herwig et al., 2
016; Figure 1A).
我们然后对基因列表进⾏功能注视，使⽤ConnsusPathDB。
Intriguingly, genes related to the Wingless/Integrated (Wnt) and epidermal growth factor (EGF) signal transduction pathways (“regulation of FZD by ubiquitination” and “EGFR1”) were overreprented 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]）
The data imply that CT cells might be maintained under conditions similar to tho 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。
2. Establishment of Proliferative Human CT Cells in Culture 建⽴体外培养增殖的⼈CT细胞
Bad 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 veral 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 veral 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。
wholeIn the prence of all of the 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 kina [ROCK] inhibitor) was found to be esntial for cell attachment and was added to all culture media in subquent 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 abnce 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。
流浪者英文
EGF, A83-01 and SB431542 (TGF-b inhibitors) and valproic acid (VPA) (a histone deacetyla [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).
什么叫三权分立最终，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).
传记英文
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 ction, 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。
