Biology subjects

Seita, Y.

Publications and source records attributed to Seita, Y..

8 recordsLinked to original sources

bioRxiv2024

Generation of spermatogonia from pluripotent stem cells in humans and non-human primates

Failures in germline development drive male infertility, but the lack of model systems that recapitulate human spermatogenesis hampers therapeutic development. Here, we have developed a system to differentiate human induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells that self-organize within xenogeneic reconstituted testes (xrTestes) generated from mouse fetal testicular cells. Subsequent transplant of xrTestes into immunodeficient mice resulted in efficient generation of undifferentiated and differentiated spermatogonia as well as preleptotene spermatocytes with striking similarities to their in vivo counterparts. As future clinical application will require testing in non-human primates, we utilized a similar strategy to differentiate rhesus iPSCs through all fetal germ cell stages into spermatogonia-like cells. Together, these models will serve as steppingstone to completion of human male in vitro gametogenesis.

developmental biology↗

bioRxiv2024

Defining the cellular origin of seminoma by transcriptional and epigenetic mapping to the normal human germline

Aberrant male germline development can lead to the formation of seminoma, a testicular germ cell tumor. Seminomas are biologically similar to primordial germ cells (PGCs) and many bear an isochromosome 12p [i(12p)] with two additional copies of the short arm of chromosome 12. By mapping seminoma transcriptomes and open chromatin landscape onto a normal human male germline trajectory, we find that seminoma resembles premigratory/migratory primordial germ cells, but exhibit enhanced germline and pluripotency programs, and upregulation of genes involved in apoptosis, angiogenesis, and MAPK/ERK pathways. Using pluripotent stem cell-derived PGCs from Pallister Killian syndrome patients mosaic for i(12p) to model seminoma, we identify gene dosage effects that may contribute to transformation. As murine seminoma models do not exist, our analyses provide critical insights into genetic, cellular and signaling programs driving seminoma transformation, and the newly developed in vitro platform permits evaluation of additional signals required for seminoma tumorigenesis.

developmental biology↗

bioRxiv2022

Reconstitution of human adrenocortical specification and steroidogenesis using induced pluripotent stem cells

The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we faithfully recapitulate human fetal adrenal cortex specification processes through stepwise induction of human induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenal progenitor-like states to ultimately generate fetal zone adrenal cortex-like cells (FZLCs), as evidenced by histomorphological, ultrastructural, and transcriptome features and adrenocorticotropic hormone (ACTH)-independent {Delta}5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN and WNT signaling, and that steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of {Delta}5 steroid synthesis enzymes. Finally, NR5A1 promotes FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro paving the way for cell-based therapies of adrenal insufficiency.

developmental biology↗

bioRxiv2022

Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells

Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for pluripotent stem cells (iPSCs) culture in the common marmoset (Callithrix jacchus, cj), which stably propagate in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes culminating in the establishment of a primate specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.

developmental biology↗

bioRxiv2022

The developmental origin and the specification of the adrenal cortex in humans and cynomolgus monkeys

Development of the adrenal cortex, a vital endocrine organ, originates in the adrenogonadal primordium, a common progenitor for both the adrenocortical and gonadal lineages in rodents. In contrast, we find that in humans and cynomolgus monkeys, the adrenocortical lineage originates in a temporally and spatially distinct fashion from the gonadal lineage, arising earlier and more anteriorly within the coelomic epithelium. The adrenal primordium arises from adrenogenic coelomic epithelium via an epithelial-to- mesenchymal-like transition, which then progresses into the steroidogenic fetal zone via both direct and indirect routes. Notably, we find that adrenocortical and gonadal lineages exhibit distinct HOX codes, suggesting distinct anterior-posterior regionalization. Together, our assessment of the early divergence of these lineages provides a molecular framework for understanding human adrenal and gonadal disorders. One Sentence SummarySpecification of the adrenal cortex occurs in adrenogenic coelomic epithelium independent of gonadogenesis in humans and cynomolgus monkeys

developmental biology↗

bioRxiv2021

Endogenous retrovirus rewired the gene regulatory network shared between primordial germ cells and naïve pluripotent cells in hominoids

Although the gene regulatory network controlling germ cell development is critical for gamete integrity, this network has been substantially diversified during mammalian evolution. Here, we show that several hundred loci of LTR5_Hs, a hominoid-specific endogenous retrovirus (ERV), function as enhancers in both human primordial germ cells (PGCs) and naive pluripotent cells. PGCs and naive pluripotent cells exhibit a similar transcriptome signature, and the enhancers derived from LTR5_Hs contribute to establishing such similarity. LTR5_Hs appears to be activated by transcription factors critical in both cell types (KLF4, TFAP2C, NANOG, and CBFA2T2). Comparative transcriptome analysis between humans and macaques suggested that the expression of many genes in PGCs and naive pluripotent cells has been upregulated by LTR5_Hs insertions in the hominoid lineage. Together, this study suggests that LTR5_Hs insertions have rewired and finetuned the gene regulatory network shared between PGCs and naive pluripotent cells during hominoid evolution. TeaserA hominoid-specific ERV has rewired the gene regulatory network shared between PGCs and naive pluripotent cells.

genetics↗

bioRxiv2020

Chromosomal-scale De novo Genome Assemblies of Cynomolgus Macaque and Common Marmoset

Cynomolgus macaque (Macaca fascicularis) and common marmoset (Callithrix jacchus) have been widely used in human biomedical research. Their genomes were sequenced and assembled initially using short-read sequences, with the advent of massively parallel sequencing. However, the resulting contig sequences tended to remain fragmentary, and long-standing primate genome assemblies used the human genome as a reference for ordering and orienting the assembled fragments into chromosomes. Here we performed de novo genome assembly of these two species without any human genome-based bias observed in the genome assemblies released earlier. Firstly we assembled PacBio long reads, and the resultant contigs were scaffolded with Hi-C data. The scaffolded sequences obtained were further refined based on assembly results of alternate de novo assemblies and Hi-C contact maps by resolving identified inconsistencies. The final assemblies achieved N50 lengths of 149 Mb and 137 Mb for cynomolgus macaque and common marmoset, respectively, and the numbers of scaffolds longer than 10Mb are equal to their chromosome numbers. The high fidelity of our assembly is ascertained by concordance to the BAC-end read pairs observed for common marmoset, as well as a high resemblance of their karyotypic organization. Our assembly of cynomolgus macaque outperformed all the available assemblies of this species in terms of contiguity. The chromosome-scale genome assemblies produced in this study are valuable resources for non-human primate models and provide an important baseline in human biomedical research.

genomics↗

bioRxiv2020

Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells

Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Here, we established a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells (hPGCLCs) differentiated from human induced pluripotent stem cells were further induced into M-prospermatogonia-like cells (MLCs) and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing was used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibited gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enabled us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. Together, our findings pave the way for understanding and reconstructing human male germline development in vitro.

developmental biology↗