Relevance of iPSC-derived human PGC-like cells at the surface of embryoid bodies to prechemotaxis migrating PGCs.
Relevance of iPSC-derived human PGC-like cells at the surface of embryoid bodies to prechemotaxis migrating PGCs.
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DOI：
10.1073/pnas.1707779114
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发表时间：
2017-11-14
影响因子：
11.1
通讯作者：
Shioda T
中科院分区：
文献类型：
--
作者：
Mitsunaga S;Odajima J;Yawata S;Shioda K;Owa C;Isselbacher KJ;Hanna JH;Shioda T
人原始生殖细胞样细胞（human primordial germ cell like cells，hPGCLC）是由多能干细胞体外培养而成的，在人类生殖细胞研究中具有广泛的应用前景。我们表明，使用几种不同的方案产生的hPGCLC在转录组上是相当的，并且引发的多能性人iPSC在仅72小时的重编程后就获得了产生hPGCLC的能力，该重编程朝向ERK独立的幼稚状态多能性。hPGCLC定位于胚状体的最外表面层，并强烈表达CXCR 4。活细胞成像显示hPGCLC具有活跃的迁移活性，并且它们暴露于CXCR 4配体CXCL 12/SDF-1诱导了促迁移基因和抗凋亡基因的富集表达。这些结果支持了hPGCLC与趋化前人胚胎原始生殖细胞在胚胎中线区域迁移的相似性。多能干细胞来源的人原始生殖细胞样细胞（human primordial germ cell-like cells，hPGCLC）为研究原始生殖细胞（primordial germ cells，PGCs）提供了重要的机会。我们在四种化学抑制剂（4 i）-幼稚重编程培养基中短暂孵育72小时后，从致敏状态诱导多能干细胞（iPSC）中稳健地产生了CD 38 + hPGCLC [约43%的FACS分选的胚状体（EB）细胞]，并显示了我们的hPGCLC与先前研究中使用各种不同方案产生的hPGCLC的转录一致性。CD 38 + hPGCLC和CD 38 − EB细胞均显著表达PRDM 1和TFAP 2C，尽管CD 38 −细胞中的PRDM 1 mRNA缺乏3′-UTR，该UTR含有调节mRNA稳定性的miRNA结合位点。hPGCLC中上调的基因富集细胞迁移基因，其启动子富集TFAP 2（在T、NANOS 3和SOX 17的启动子中鉴定）和RREB-1细胞粘附调节因子的结合基序。在EB中，hPGCLC仅在最外表面单层中被鉴定为具有POU 5 F1/OCT 4蛋白的强和特异性表达的分散细胞或细胞聚集体。延时活细胞成像显示hPGCLC在基质胶上的主动迁移。而所有hPGCLC强烈表达CXCR 4趋化受体，其配体CXCL 12/SDF 1在整个EB中不显着表达。CXCL 12/SDF 1诱导hPGCLC细胞迁移基因和抗凋亡基因的表达。因此，我们的研究表明，使用各种方法，在hiPSC的多能性从致敏状态转变后，可以容易地从hiPSC产生转录一致的hPGCLC，并且hPGCLC类似于在CXCL 12/SDF 1引导的趋化性开始之前在人胚胎中线区域随机迁移的早期PGCs。
Human primordial germ cell-like cells (hPGCLCs) generated from pluripotent stem cells in vitro hold promise, with broad applications for studies of human germline cells. We show that hPGCLCs generated using several distinct protocols are transcriptomally comparable and that primed pluripotency human iPSCs gain competence to generate hPGCLCs after only 72 hours of reprogramming toward ERK-independent state-naïve pluripotency. hPGCLCs were localized in the outermost surface layer of embryoid bodies and strongly expressed CXCR4. Live cell imaging showed active migratory activity of hPGCLCs, and their exposure to the CXCR4 ligand CXCL12/SDF-1 induced enriched expression of promigratory genes and antiapoptotic genes. These results support the resemblance of hPGCLCs to prechemotaxis human embryonic primordial germ cells migrating in the midline region of embryos. Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38+ hPGCLCs [∼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38+ hPGCLCs and CD38− EB cells significantly expressed PRDM1 and TFAP2C, although PRDM1 mRNA in CD38− cells lacked the 3′-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of T, NANOS3, and SOX17) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs on Matrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis.
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DOI：
10.1126/science.1229277
发表时间：
2013-01-25
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Hackett JA;Sengupta R;Zylicz JJ;Murakami K;Lee C;Down TA;Surani MA
通讯作者：
Surani MA
影响因子：
11.4
作者：
Ma J;Nie K;Redmond D;Liu Y;Elemento O;Knowles DM;Tam W
通讯作者：
Tam W
影响因子：
64.8
作者：
Kobayashi T;Zhang H;Tang WWC;Irie N;Withey S;Klisch D;Sybirna A;Dietmann S;Contreras DA;Webb R;Allegrucci C;Alberio R;Surani MA
通讯作者：
Surani MA
人类原始生殖细胞的转录组和 DNA 甲基化组景观
影响因子：
64.5
作者：
Guo, Fan;Yan, Liying;Qiao, Jie
通讯作者：
Qiao, Jie
影响因子：
--
作者：
Brickman, Joshua M.;Serup, Palle
通讯作者：
Serup, Palle