Daiji Okamura

Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently, differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately, differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether, our results provide a new platform for the study of kidney diseases and lineage commitment, and open new avenues for the future application of regenerative strategies in the clinic.

An alternative pluripotent state confers interspecies chimaeric competency

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

Selective Elimination of Mitochondrial Mutations in the Germline by Genome Editing

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Lebers hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.

Cadherin-mediated cell interaction regulates germ cell determination in mice

The germ cell lineage segregates from the somatic cell lineages in early embryos. Germ cell determination in mice is not regulated by maternally inherited germplasm, but is initiated within the embryo during gastrulation. However, the mechanisms of germ cell specification in mice remain unknown. We located precursors to primordial germ cells (PGCs) within early embryos, and show here that cell-cell interaction among these precursors is required for germ cell specification. We found that the expression of a calcium-dependent cell adhesion molecule, E-cadherin, is restricted to the proximal region of extra-embryonic mesoderm that contains PGC precursors, and that blocking the functions of E-cadherin with an antibody inhibits PGC formation in vitro. These results showed that E-cadherin-mediated cell-cell interaction among cells containing PGC precursors is essential to directing such cells to the germ cell fate.

Requirement of Oct3/4 function for germ cell specification

In mammalian embryos, PGCs (primordial germ cells) are specified from a pluripotent epiblast cell population after implantation. In this study, we demonstrated an essential role for the germline-specific transcription factor Oct3/4 in PGC specification. We generated chimeric embryos with ZHBTc4 ES cells lacking both alleles of the Oct3/4 gene (pou5f1). Pluripotency was maintained by an Oct3/4 transgene, and its expression was suppressed by doxycycline (Dox). Transcription of the Oct3/4 transgene in the ES-derived cells unexpectedly suffered constitutive suppression in chimeric embryos without Dox, and the ES-derived cells contributed to PGC precursor-like cells, but failed to form PGCs. We then attempted to rescue Oct3/4 expression in the ES-derived cells in the chimeric embryos by introducing an additional Oct3/4 transgene. The ES cell-derived cells indeed recovered Oct3/4 transcription in these chimeric embryos, and were successfully specified to PGCs. We further confirmed the requirement of Oct3/4 by using another derivative of ZHBTc4 ES cells in which a Dex (dexamethasone)-dependent Oct3/4 transgene was introduced. In the presence of Dox, Oct3/4 protein was absent in the nuclei of the ES-derived cells, which failed to form PGCs. In contrast, the ES-derived cells could be specified to PGCs after activation of Oct3/4 function in the presence of Dex.

Discordant developmental waves of angioblasts and hemangioblasts in the early gastrulating mouse embryo

Vasculogenesis and hematopoiesis are thought to arise in hemangioblasts, the common progenitors of cells in vessels and in blood. This scheme was challenged by kinetic analysis of vascular endothelial and hematopoietic progenitors in early gastrulating mouse embryos. The OP-9 co-culture system with a combination of cytokines permitted the detection of endothelial progenitors, as well as stroma-dependent hematopoietic progenitors. Endothelial progenitors were detected as early as embryonic day (E) 5.50, after which time their numbers increased. Stroma-dependent hematopoietic progenitors were detected at E6.75, the time point when hemangioblasts reportedly emerge. Colony-forming units in culture (CFU-c), most likely generated from stroma-dependent hematopoietic progenitors via contact with the microenvironment, were detected at E7.50, concomitant with the onset of primitive hematopoiesis in the yolk sac. The presence of nucleated erythrocytes and the expression of an embryonic-type globin in erythroid colonies derived from stroma-dependent hematopoietic progenitors and from CFU-c support the notion that these progenitors coordinately establish primitive hematopoiesis. Using Oct3/4 promoter-driven GFP transgenic mice, early endothelial progenitors, stroma-dependent hematopoietic progenitors, and CFU-c were all shown to express the Oct3/4 transcription factor. Among Oct3/4-positive cells, both endothelial and hematopoietic progenitors were present in the CD31-positive fraction, leaving a subset of endothelial progenitors in the CD31-negative fraction. These data imply that Oct3/4-positive mesoderm gives rise to CD31-negative angioblasts, CD31-positive angiboblasts and CD31-positive hemangioblasts. We propose a distinct developmental pathway in which the angioblast lineage directly diverges from mesoderm prior to and independent of hemangioblast development.

Max is a repressor of germ cell-related gene expression in mouse embryonic stem cells

Embryonic stem cells and primordial germ cells (PGCs) express many pluripotency-associated genes, but embryonic stem cells do not normally undergo conversion into primordial germ cells. Thus, we predicted that there is a mechanism that represses primordial germ cell-related gene expression in embryonic stem cells. Here we identify genes involved in this putative mechanism, by using an embryonic stem cell line with a Vasa reporter in an RNA interference screen of transcription factor genes expressed in embryonic stem cells. We identify five genes that result in the expression of Vasa when silenced. Of these, Max is the most striking. Transcriptome analysis reveals that Max knockdown in embryonic stem cells results in selective, global derepression of germ cell-specific genes. Max interacts with histone H3K9 methyltransferases and associates with the germ cell-specific genes in embryonic stem cells. In addition, Max knockdown results in a decrease in histone H3K9 dimethylation at their promoter regions. We propose that Max is part of protein complex that acts as a repressor of germ cell-related genes in embryonic stem cells.

Loss of MAX results in meiotic entry in mouse embryonic and germline stem cells

Meiosis is a unique process that allows the generation of reproductive cells. It remains largely unknown how meiosis is initiated in germ cells and why non-germline cells do not undergo meiosis. We previously demonstrated that knockdown of Max expression, a gene encoding a partner of MYC family proteins, strongly activates expression of germ cell-related genes in ESCs. Here we find that complete ablation of Max expression in ESCs results in profound cytological changes reminiscent of cells undergoing meiotic cell division. Furthermore, our analyses uncovers that Max expression is transiently attenuated in germ cells undergoing meiosis in vivo and its forced reduction induces meiosis-like cytological changes in cultured germline stem cells. Mechanistically, Max depletion alterations are, in part, due to impairment of the function of an atypical PRC1 complex (PRC1.6), in which MAX is one of the components. Our data highlight MAX as a new regulator of meiotic onset.

On the fate of primordial germ cells injected into early mouse embryos.

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells – pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs.

Cell cycle gene-specific control of transcription has a critical role in proliferation of primordial germ cells

Transcription elongation is stimulated by positive transcription elongation factor b (P-TEFb), for which activity is repressed in the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. We show here a critical role of 7SK snRNP in growth control of primordial germ cells (PGCs). The expression of p15(INK4b), a cyclin-dependent kinase inhibitor (CDKI) gene, in PGCs is selectively activated by P-TEFb and its recruiting molecule, Brd4, when the amount of active P-TEFb is increased due to reduction of the 7SK snRNP, and PGCs consequently undergo growth arrest. These results indicate that CDKI gene-specific control of transcription by 7SK snRNP plays a pivotal role in the maintenance of PGC proliferation.