The extracellular matrix plays a key role in stem cell maintenance, expansion, and differentiation. Laminin, a basement membrane protein, is a widely used substrate for cell culture including the growth of human induced pluripotent stem cells (hiPSCs). Here, we show that different isoforms of laminin lead to the selective differentiation of hiPSCs into different eye-like tissues. Specifically, the 211 isoform of the E8 fragment of laminin (LN211E8) promotes differentiation into neural crest cells via Wnt activation, whereas LN332E8 promotes differentiation into corneal epithelial cells. The immunohistochemical distributions of these laminin isoforms in the developing mouse eye mirrors the hiPSC type that was induced in vitro. Moreover, LN511E8 enables generation of dense hiPSC colonies due to actomyosin contraction, which in turn led to cell density-dependent YAP inactivation and subsequent retinal differentiation in colony centers. Thus, distinct laminin isoforms determine the fate of expanded hiPSCs into eye-like tissues.

Chronic heart failure (CHF) with preserved left ventricular (LV) ejection fraction (HFpEF) is observed in half of all patients with CHF and carries the same poor prognosis as CHF with reduced LV ejection fraction (HFrEF). In contrast to HFrEF, there is no established therapy for HFpEF. Chronic inflammation contributes to cardiac fibrosis, a crucial factor in HFpEF; however, inflammatory mechanisms and mediators involved in the development of HFpEF remain unclear. Therefore, we sought to identify novel inflammatory mediators involved in this process.

Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein. Previously, we demonstrated that Tinagl1 expression was restricted to extraembryonic regions during the postimplantation period and detected marked expression in mouse Reichert's membranes. In uteri, Tinagl1 is markedly expressed in the decidual endometrium during the postimplantation period, suggesting that it plays a physical and physiological role in embryo development and/or decidualization of the uterine endometrium during pregnancy. In the present study, in order to determine the role of Tinagl1 during embryonic development and pregnancy, we generated Tinagl1-deficient mice. Although Tinagl1(-/-) embryos were not lethal during development to term, homologous matings of Tinagl1(-/-) females and Tinagl1(-/-) males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. To examine ovarian function, ovulation was induced with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); the number of ovulated oocytes did not differ between Tinagl1(-/-) and Tinagl1(flox/flox). In vitro fertilization followed by embryo culture also demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. Our results demonstrate that Tinagl1 deficiency affects female mice and results in subfertility phenotypes, and they suggest that although the potential of Tinagl1(-/-) oocytes is normal, Tinagl1 is related to fertility in adult females but is not essential for either fertilization or preimplantation development in vitro.

Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized, the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here, using a short fragment of laminin 411 (LM411-E8), an ECM predominantly expressed in the vascular endothelial basement membrane, we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (>95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate.

Neural tube defects (NTDs) are the second most common structural birth defect. Senescence, a state of permanent cell cycle arrest, occurs only after neural tube closure. Maternal diabetes-induced NTDs are severe diabetic complications that lead to infant mortality or lifelong morbidity and may be linked to premature senescence. Here, we report that premature senescence occurs in the mouse neuroepithelium and disrupts neurulation, leading to NTDs in diabetic pregnancy. Premature senescence and NTDs were abolished by knockout of the transcription factor Foxo3a, the miR-200c gene, and the cell cycle inhibitors p21 and p27; transgenic expression of the dominant-negative FoxO3a mutant; or the senomorphic rapamycin. Double transgenic expression of p21 and p27 mimicked maternal diabetes in inducing premature neuroepithelium senescence and NTDs. These findings integrate transcription- and epigenome-regulated miRNAs and cell cycle regulators in premature neuroepithelium senescence and provide a mechanistic basis for targeting premature senescence and NTDs using senomorphics.

During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon.

A treatment for intractable diseases is expected to be the replacement of damaged tissues with products from human induced pluripotent stem cells (hiPSCs). Target cell purification is a critical step for realizing hiPSC-based therapy. Here, we found that hiPSC-derived ocular cell types exhibited unique adhesion specificities and growth characteristics on distinct E8 fragments of laminin isoforms (LNE8s): hiPSC-derived corneal epithelial cells (iCECs) and other non-CECs rapidly adhered preferentially to LN332/411/511E8 and LN211E8, respectively, through differential expression of laminin-binding integrins. Furthermore, LN332E8 promoted epithelial cell proliferation but not that of the other eye-related cells, leading to non-CEC elimination by cell competition. Combining these features with magnetic sorting, highly pure iCEC sheets were fabricated. Thus, we established a simple method for isolating iCECs from various hiPSC-derived cells without using fluorescence-activated cell sorting. This study will facilitate efficient manufacture of iCEC sheets for corneal disease treatment and provide insights into target cell-specific scaffold selection.

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.

X-chromosome inactivation (XCI) is an essential epigenetic process in female mammalian development. Although cell-based studies suggest the potential importance of the Ftx long non-protein-coding RNA (lncRNA) in XCI, its physiological roles in vivo remain unclear. Here we show that targeted deletion of X-linked mouse Ftx lncRNA causes eye abnormalities resembling human microphthalmia in a subset of females but rarely in males. This inheritance pattern cannot be explained by X-linked dominant or recessive inheritance, where males typically show a more severe phenotype than females. In Ftx-deficient mice, some X-linked genes remain active on the inactive X, suggesting that defects in random XCI in somatic cells cause a substantially female-specific phenotype. The expression level of Xist, a master regulator of XCI, is diminished in females homozygous or heterozygous for Ftx deficiency. We propose that loss-of-Ftx lncRNA abolishes gene silencing on the inactive X chromosome, leading to a female microphthalmia-like phenotype.

Laminin-integrin interactions regulate various adhesion-dependent cellular processes. γ1C-Glu, the Glu residue in the laminin γ1 chain C-terminal tail, is crucial for the binding of γ1-laminins to several integrin isoforms. Here, we investigated the impact of γ1C Glu to Gln mutation on γ1-laminin binding to all possible integrin partners in vitro, and found that the mutation specifically ablated binding to α3, α6, and α7 integrins. To examine the physiological significance of γ1C-Glu, we generated a knock-in allele, Lamc1 EQ , in which the γ1C Glu to Gln mutation was introduced. Although Lamc1 EQ/EQ homozygotes developed into blastocysts and deposited laminins in their basement membranes, they died just after implantation because of disordered extraembryonic development. Given the impact of the Lamc1 EQ allele on embryonic development, we developed a knock-in mouse strain enabling on-demand introduction of the γ1C Glu to Gln mutation by the Cre-loxP system. The present study has revealed a crucial role of γ1C-Glu-mediated integrin binding in postimplantation development and provides useful animal models for investigating the physiological roles of laminin-integrin interactions in vivo.