Report of the first live birth after uterus transplantation in People’s Republic of China
https://www.fertstert.org/article/S0015-0282(20)30543-4/fulltext
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考虑到各国出生率的差别，投胎到条件比5☆越南还差的地方应该是更大的可能……
The germ-cell lineage arises from primordial germ cells (PGCs) that go through a multi-step process to generate spermatozoa or oocytes. Methods for in vitro gametogenesis from pluripotent stem cells (PSCs) would provide a powerful tool to explore the mechanism of germ-cell development and its anomalies (1). Mouse PSCs have been induced into PGC-like cells (mPGCLCs), which contribute to spermatogenesis upon transplantation into testes (2, 3) and to oogenesis upon aggregation with embryonic ovarian somatic cells (reconstituted ovaries) followed by transplantation under ovarian bursa (4) or an appropriate culture (5). Resultant gametes from these procedures generate fertile offspring (2-5). Furthermore, human PSCs (hPSCs) have been induced into hPGCLCs bearing a gene-expression property of hPGCs just after their specification, opening the possibility for human in vitro gametogenesis (6, 7). However, further differentiation of hPGCLCs has not been successful, and whether hPGCLCs can develop as mature germ cells remains unknown.
At week 2 (Wk2) of development, hPGCs express key transcription factors (TFs) such as SOX17, TFAP2C, and BLIMP1 (also known as PRDM1). Then at Wk5 they migrate to and colonize the embryonic gonads to initiate differentiation into oogonia or gonocytes in embryonic ovaries or testes (8,9) that express RNA regulators such as DAZL and DDX4 (also known as human VASA homolog: hVH) (10-12). Oogonia and gonocytes are very similar in morphology, gene expression, and epigenetic properties until they start sexual differentiation at WklO into oocytes via meiotic prophase or fetal spermatogonia (8-12). A characteristic event in germ-cell development is epigenetic reprogramming, which occurs by WklO and leads to genome-wide DNA demethylation, imprint erasure, and X reactivation (10-12).
We explored whether hPGCLCs can undergo further development in vitro in xenogeneic reconstituted ovaries (xrOvaries) with mouse embryonic ovarian somatic cells (Fig. SI A). First, we induced male human induced PSCs (hiPSCs) with the BLIMPl-tdTomato; TFAP2C-EGFP alleles [585B1 BTAG (XY)] into incipient mesoderm-like cells (iMeLCs) and then into hPGCLCs (7,13). BLIMP1 and TFAP2C are expressed in oogonia/gonocytes at least until WklO (10-12). We isolated BTAG-positive (BT+AG+) hPGCLCs at day 6 (d6) of induction by fluorescence activated cell sorting (FACS), and generated xrOvaries. In agreement with a previous report (5), mPGCLCs differentiated efficiently into primary oocytes and formed secondary follicles after a 21-day culture in rOvaries (Fig. SIB). At culture day 7 (ag7), the xrOvaries exhibited a round and flattened shape, and the BT+AG+ cells were distributed uniformly within the xrOvaries (Fig. SIC). Subsequently, the xrOvaries expanded laterally with the formation of cyst-like structures (Fig. 1A). From ag21 to ag77, the xrOvaries exhibited auto-fluorescence under fluorescence microscopy (Fig. SID). There were about 2000 BT+AG+ cells per xrOvary at ag21
and -500 at ag77 (Fig. S1E), indicating that only a fraction of the initial hPGCLCs (-5,000) survived in xrOvaries.
At ag77, the AG+ cells in xrOvaries existed as clusters, were positive for a human mitochondrial antigen, bore faint DAPI staining, and were delineated by FOXL2+ mouse granulosa cells and their basement membrane (Fig. S2A-C). The intensity of the DAPI staining in AG+ cells appeared to decline progressively during the culture (Fig. S2D). The AG+ cells expressed key TFs for early germ cells (TFAP2C, SOX17, POU5F1), and some were mitotically active (Ki67+) or in apoptosis (cleaved CASPASE3+) (Fig. S2A). Importantly, at ag77, many AG+ cells up-regulated DAZL and DDX4 (Fig. IB, Fig. S2E), suggesting that in xrOvaries, hPGCLCs not only survive as germ cells, but also differentiate into oogonia/gonocytes. Accordingly, at ag77, electron microscopy revealed the presence of large cells, highly similar to oogonia/gonocytes, with clear cytoplasms with sparsely located mitochondria and with round nuclei with loosely packed chromatin and prominent nucleoli (Fig. S3) (8, 9).
We next generated female hiPSCs bearing the AG; DDX4/hVH-tdTomato alleles [1390G3 AGVT (XX)] (Fig. S4) (13), created xrOvaries, and cultured them up to agl20. The female AG+ hPGCLCs in xrOvaries developed similarly to the male hPGCLCs (Fig. S5), and up-regulated VT at ag77 and the AG+VT+ cells appeared to differentiate into AG-negative (AG-) VT+ cells at agl20 (Fig. 1C, S5A). We examined the expression of key genes by quantitative (q)-PCR. Both male and female hPGCLC-derived cells expressed early germ-cell genes (BLIMP1, TFAP2C, S0X17, NAN0S3), core/naive pluripotency genes (P0U5F1, NANOG, TCL1B, TFCP2L1), and up-regulated genes for oogonia/gonocytes (DAZL, DDX4) from around ag35/49 onwards, and also genes for meiosis (SYCP3, REC8) (Fig. S6). The AG-VT+ cells at agl20 down-regulated early germ-cell and core/naive pluripotency genes, and up-regulated STRA8, a gene essential for meiosis initiation (Fig. S6), suggesting their developmentally advanced character. Consistently, the DDX4+ cells at agl20 expressed SYCP3, but not YH2AX, DMC1 and SYCP1 proteins, indicating that they have not yet initiated meiotic recombination (Fig. S7).
We analyzed the transcriptomes of these cell types (Fig. S8A, Table SI). Unsupervised hierarchical clustering (UHC) showed that hPGCLC-derived cells are distinct from hiP- SCs/iMeLCs, and can be sub-classified according to their culture period (Fig. S8B). Principle component analysis (PCA) gave a concordant result (Fig. S8C). We identified the genes with significantly positive or negative PC1/2 loadings (453 genes) (Fig. S8D, Table S2). UHC classified them into five major clusters (Fig. 2A, S8D, Table S2): Cluster 1 represents the genes up-regulated upon hPGCLC specification or early in xrOvaries and expressed essentially continuously thereafter. Clusters 2 and 5 signify the genes transiently expressed in early hPGCLC-derived cells or down-regulated early in xrOvaries, and cluster 4 represents the genes down-regulated upon hPGCLC specification (Fig. 2A, Table S2). Cluster 3, which shows a progressive and coordinated up-regulation from ag35 onwards, signifies genes for oogonia/gonocytes and is enriched in genes with gene ontology (GO) functional terms for male meiosis, fertilization, and piRNA metabolic process (Fig. 2A, Table S2). Using the 453 genes, we compared the gene-expression properties of hPGCLC-derived cells with those of oogonia/gonocytes (12). hPGCLC-derived cells from ag35 onward, particularly the ag77 BT+AG+/agl20 AG+ cells, exhibited a strong similarity to Wk7/9 oogonia/gonocytes (Fig. 2B, Fig. S10).
The agl20 AG_VT+ cells exhibited a developmentally advanced character (Fig. S6, S7, S8B, C). To clarify this point further, we examined the expression of genes that distinguish relevant human fetal germ cells [FGCs: mitotic (oogonia/gonocytes), retinoic acid (RA)-responsive (female), meiotic (female), oogenesis (female)] (14) in agl20 AG_VT+ cells, which, intriguingly, revealed their similarity to RA-responsive FGCs. They down-regulate genes for early germ cells, further up- regulate DAZL, DDX4, MAEL and KRB0X1, up-regulate RA- or bone morphogenetic protein (BMP)-responsive genes (STRA8, REC8, or IDI/2/3/4, MSX1/2), yet do not sufficiently up-regulate key meiosis genes (SYCP1, DMC1, SPO11, PRDM9) (Fig. 2C, Sil). Thus, hPGCLC development in xrOvaries reconstitutes human germ-cell development, albeit with protracted kinetics, leading to the generation of oogonia and RA- responsive FGCs, a state responding to the signal for the meiotic entry and in preparation for the meiotic recombination Q4).
生殖细胞谱系起源于原始生殖细胞 (PGC)，这些细胞经过多步过程产生精子或卵母细胞。多能干细胞 (PSC) 的体外配子发生方法将为探索生殖细胞发育及其异常的机制提供强有力的工具。小鼠 PSC 已被诱导成 PGC 样细胞 (mPGCLC)，其在移植到睾丸后有助于精子发生，并在与胚胎卵巢体细胞（重组卵巢）聚集后促进卵子发生，然后在卵巢囊或适当的培养物下移植。这些过程产生的配子产生可育后代。此外，人类 PSCs (hPSCs) 已被诱导成具有 hPGCs 基因表达特性的 hPGCLCs，这为人类体外配子发生开辟了可能性。然而，hPGCLCs 的进一步分化尚未成功，hPGCLCs 是否可以发育为成熟的生殖细胞仍然未知。
在发育的第 2 周 (Wk2)，hPGC 表达关键转录因子 (TF)，例如 SOX17、TFAP2C 和 BLIMP1（也称为 PRDM1）。然后在 Wk5 时，它们迁移到胚胎性腺并定植于胚胎性腺，开始分化为胚胎卵巢或睾丸中的卵原细胞或生殖母细胞，表达 RNA 调节因子，如 DAZL 和 DDX4（也称为人类 VASA 同源物：hVH）。卵原细胞和生殖母细胞在形态、基因表达和表观遗传特性方面非常相似，直到它们在 Wk10 开始通过减数分裂前期或胎儿精原细胞有性分化为卵母细胞。生殖细胞发育中的一个特征事件是表观遗传重编程，它发生在 Wk10 并导致全基因组 DNA 去甲基化、印记擦除和 X 染色体再激活。
我们探讨了 hPGCLCs 是否可以在小鼠胚胎卵巢体细胞的异种重组卵巢 (xrOvaries) 中进行体外进一步发育。 2首先，我们用【 BLIMP1-tdTomato; TFAP2C-EGFP alleles [585B1 BTAG (XY)]】 将
人类的诱导 PSCs (hiPSCs) 诱导成初始中胚层样细胞 (iMeLCs)，然后诱导成 hPGCLCs。*BLIMP1 和 TFAP2C 至少在 Wk10 之前在卵原细胞/生殖细胞中表达。我们在诱导的第 6 天 (d6) 通过荧光激活细胞分选 (FACS) 分离了 BTAG 阳性 (BT+AG+) hPGCLC，并产生了 xrOvaries。与先前的报道一致，mPGCLCs 在 rOvaries 中培养 21 天后有效地分化为初级卵母细胞并形成次级卵泡。在培养第7天（ag7），xrOvaries呈圆形扁平状，BT+AG+细胞在xrOvaries内均匀分布。随后，xrOvaries 横向扩张，形成囊状结构。从 ag21 到 ag77，xrOvaries 在荧光显微镜下表现出自发荧光。在 ag21 时，每个 xrOvary 大约有 2000 个 BT+AG+ 细胞在 ag77 处约为 500，表明只有一小部分初始 hPGCLC（约 5,000）在 xrOvaries 中存活。
在 ag77 时，xrOvaries 中的 AG+ 细胞以簇的形式存在，对人线粒体抗原呈阳性，具有微弱的 DAPI 染色，并被 FOXL2+ 小鼠颗粒细胞及其基底膜包围。 AG+ 细胞中 DAPI 染色的强度似乎在培养过程中逐渐下降。 AG+细胞表达早期生殖细胞的关键转录因子（TFAP2C、SOX17、POU5F1），一些细胞处于有丝分裂活性（Ki67+）或凋亡状态（断裂的 CASPASE3+）。重要的是，在 ag77，许多 AG+ 细胞上调 DAZL 和 DDX4，这表明在 xrOvaries 中，hPGCLCs 不仅作为生殖细胞存活，而且还分化为卵原细胞/生殖细胞。因此，在 ag77，电子显微镜显示存在大细胞，与卵原细胞/生殖细胞高度相似，细胞质清晰，线粒体分布稀疏，核圆形，染色质松散，核仁突出。