Periodic formation of epithelial somites from human pluripotent stem cells.

During embryonic development, epithelial cell blocks called somites are periodically formed according to the segmentation clock, becoming the foundation for the segmental pattern of the vertebral column. The process of somitogenesis has recently been recapitulated with murine and human pluripotent stem cells. However, an in vitro model for human somitogenesis coupled with the segmentation clock and epithelialization is still missing. Here, we report the generation of human somitoids, organoids that periodically form pairs of epithelial somite-like structures. Somitoids display clear oscillations of the segmentation clock that coincide with the segmentation of the presomitic mesoderm. The resulting somites show anterior-posterior and apical-basal polarities. Matrigel is essential for epithelialization but dispensable for the differentiation into somite cells. The size of somites is rather constant, irrespective of the initial cell number. The amount of WNT signaling instructs the proportion of mesodermal lineages in somitoids. Somitoids provide a novel platform to study human somitogenesis.

ZGLP1 is a determinant for the oogenic fate in mice.

Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine.

Evolutionarily Distinctive Transcriptional and Signaling Programs Drive Human Germ Cell Lineage Specification from Pluripotent Stem Cells.

Germline specification underlies human reproduction and evolution, but it has proven difficult to study in humans since it occurs shortly after blastocyst implantation. This process can be modeled with human induced pluripotent stem cells (hiPSCs) by differentiating them into primordial germ cell-like cells (hPGCLCs) through an incipient mesoderm-like cell (iMeLC) state. Here, we elucidate the key transcription factors and their interactions with important signaling pathways in driving hPGCLC differentiation from iPSCs. Germline competence of iMeLCs is dictated by the duration and dosage of WNT signaling, which induces expression of EOMES to activate SOX17, a key driver of hPGCLC specification. Upon hPGCLC induction, BMP signaling activates TFAP2C in a SOX17-independent manner. SOX17 and TFAP2C then cooperatively instate an hPGCLC transcriptional program, including BLIMP1 expression. This specification program diverges from its mouse counterpart regarding key transcription factors and their hierarchies, and it provides a foundation for further study of human germ cell development.

Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells in vitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression in vivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.

PRDM14: a unique regulator for pluripotency and epigenetic reprogramming.

PRDM14 belongs to the PR domain-containing (PRDM) transcriptional regulators. Among the PRDM family members, PRDM14 shows specific expression in preimplantation embryos, primordial germ cells (PGCs), and embryonic stem cells (ESCs) in vitro, and accordingly plays a key role in the regulation of their pluripotency and epigenetic reprogramming, most notably, genome-wide DNA demethylation. The function of PRDM14 appears to be conserved between mice and humans, but it shows several characteristic differences between the two species. A precise understanding of the function of PRDM14 in mice and humans would shed new light on the regulation of pluripotency and the epigenome in these two species, providing a foundation for better control of stem cell fates in a broader context.

Induction of mouse germ-cell fate by transcription factors in vitro.

The germ-cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have previously demonstrated that, by using cytokines, embryonic stem cells and induced pluripotent stem cells can be induced into epiblast-like cells (EpiLCs) and then into primordial germ cell (PGC)-like cells with the capacity for both spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ-cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous overexpression of three transcription factors, Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not embryonic stem cells, swiftly and efficiently into a PGC state. Notably, Prdm14 alone, but not Blimp1 or Tfap2c, suffices for the induction of the PGC state in EpiLCs. The transcription-factor-induced PGC state, irrespective of the transcription factors used, reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC or PGC-like-cell specification by cytokines including bone morphogenetic protein 4. Notably, the transcription-factor-induced PGC-like cells contribute to spermatogenesis and fertile offspring. Our findings provide a new insight into the transcriptional logic for PGC specification, and create a foundation for the transcription-factor-based reconstitution and regulation of mammalian gametogenesis.

PD-1 and LAG-3 inhibitory co-receptors act synergistically to prevent autoimmunity in mice.

Stimulatory and inhibitory co-receptors play fundamental roles in the regulation of the immune system. We describe a new mouse model of spontaneous autoimmune disease. Activation-induced cytidine deaminase-linked autoimmunity (aida) mice harbor a loss-of-function mutation in the gene encoding lymphocyte activation gene 3 (LAG-3), an inhibitory co-receptor. Although LAG-3 deficiency alone did not induce autoimmunity in nonautoimmune-prone mouse strains, it induced lethal myocarditis in BALB/c mice deficient for the gene encoding the inhibitory co-receptor programmed cell death 1 (PD-1). In addition, LAG-3 deficiency alone accelerated type 1 diabetes mellitus in nonobese diabetic mice. These results demonstrate that LAG-3 acts synergistically with PD-1 and/or other immunoregulatory genes to prevent autoimmunity in mice.

PD-1 deficiency results in the development of fatal myocarditis in MRL mice.

The deficiency of programmed cell death 1 (PD-1, Pdcd1), a negative immuno-receptor belonging to the CD28/cytotoxic T lymphocyte antigen 4 (CTLA-4) family, can support various tissue-specific autoimmune conditions. Here, we analyzed the effect of PD-1 deficiency in MRL mice that is genetically predisposed to systemic autoimmunity. MRL-Pdcd1(-)(/-) mice developed a fatal myocarditis, which is reminiscent of CTLA-4-deficient (Ctla4(-)(/-)) mice. Massive infiltration of CD4(+) and CD8(+) T cells and myeloid cells was found in hearts of MRL-Pdcd1(-)(/-) mice concomitant with the production of high-titer auto-antibodies against cardiac myosin. In contrast to Ctla4(-)(/-) mice in which most of the CD4(+) T cells are non-specifically activated and invade various organs, T cells in the heart but not in the spleen and lymph nodes are activated in MRL-Pdcd1(-)(/-) mice, suggesting that myocarditis is mediated by antigen-specific autoimmune response. Heart infiltrating myeloid cells strongly suppressed the allogenic response of T cells in vitro, suggesting that these Mac1(+)Gr1(+) myeloid cells are phenotypically similar to myeloid suppressor cells, which can be found in tumor-bearing hosts. These findings unravel the hidden heart-specific autoimmune predisposition of MRL mice and provide MRL-Pdcd1(-)(/-) mice as a useful animal model of lymphocytic myocarditis.

Identification of QTLs that modify peripheral neuropathy in NOD.H2b-Pdcd1-/- mice.

The non-obese diabetic (NOD) mouse strain is prone to developing various autoimmune syndromes including type I diabetes mellitus (T1DM), sialadenitis, thyroiditis and pancreatitis. Although the genetic basis of T1DM has been extensively analyzed, genetic factors that modify the other autoimmune phenotypes are largely unknown. We have recently reported that NOD mice with anti-diabetogenic MHC haplotype (H-2(b)) and programmed cell death 1 (PD-1) deficiency (NOD.H2(b)-Pdcd1(-/-) mice) are protected from T1DM but develop various tissue-specific autoimmune diseases including peripheral neuropathy due to autoimmune neuritis, sialadenitis and gastritis. In the present study, we generated [(C57BL/6 x NOD.H2(b))(F1) x NOD-H2(b)](BC1)-Pdcd1(-/-) mice to screen non-MHC quantitative trait loci (QTLs) that modify autoimmune phenotypes other than T1DM. We identified seven QTLs for peripheral neuropathy and neuritis, one QTL for insulitis, four QTLs for gastritis, two QTLs for sialadenitis and seven QTLs for vasculitis throughout the genome and designated them as Annp loci for autoimmunity due to polymorphisms of non-MHC genes in NOD mice and PD-1 deficiency. Annp1, 5, 6 and 7 overlapped with reported loci for T1DM (Idd3, 9, 15 and 2, respectively), suggesting that these loci modify not only T1DM but also other autoimmune phenotypes. NOD allele was promotive at 9 of 14 Annp loci, while NOD allele was protective at the other loci. Half of Annp loci associated with a single phenotype, while the other seven loci associated with more than two phenotypes. These results indicate that NOD genetic background harbors various QTLs that modify autoimmune phenotypes either by organ-specific or by organ-non-specific manner.

Establishment of NOD-Pdcd1-/- mice as an efficient animal model of type I diabetes.

Mice deficient in programmed cell death 1 (PD-1, Pdcd1), an immunoinhibitory receptor belonging to the CD28/cytotoxic T lymphocyte-associated antigen-4 family, spontaneously develop lupus-like autoimmune disease and autoimmune dilated cardiomyopathy on C57BL/6 and BALB/c backgrounds, respectively. However, how PD-1 deficiency induces different forms of autoimmune diseases on these two strains was unknown. Here, we report that PD-1 deficiency specifically accelerates the onset and frequency of type I diabetes in NOD (nonobese diabetic) mice, with strong T helper 1 polarization of T cells infiltrating into islets. These results suggest that PD-1 deficiency accelerates autoimmune predisposition of the background strain, leading to the induction of different forms of autoimmune diseases depending on the genetic background of the strain. Using NOD-Pdcd1-/- mice as an efficient animal model of type I diabetes, we screened diabetes-susceptible loci by genetic linkage analysis. The diabetic incidence of NOD-Pdcd1-/- mice was controlled by five genetic loci, including three known recessive loci [Idd (insulin-dependent diabetes) 1, Idd17, and Idd20] and two previously unidentified dominant loci [Iddp (Idd under PD-1 deficiency) 1 and Iddp2].