CRISPR loss of function screening to identify genes involved in human primordial germ cell-like cell development.

Despite our increasing knowledge of molecular mechanisms guiding various aspects of human reproduction, those underlying human primordial germ cell (PGC) development remain largely unknown. Here, we conducted custom CRISPR screening in an in vitro system of human PGC-like cells (hPGCLCs) to identify genes required for acquisition and maintenance of PGC fate. Amongst our candidates, we identified TCL1A, an AKT coactivator. Functional assessment in our in vitro hPGCLCs system revealed that TCL1A played a critical role in later stages of hPGCLC development. Moreover, we found that TCL1A loss reduced AKT-mTOR signaling, downregulated expression of genes related to translational control, and subsequently led to a reduction in global protein synthesis and proliferation. Together, our study highlights the utility of CRISPR screening for human in vitro-derived germ cells and identifies novel translational regulators critical for hPGCLC development.

Generation of a familial hypercholesterolemia model in non-human primate.

Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder that is associated with a high plasma level of low-density lipoprotein (LDL) cholesterol, leading to an increased risk of cardiovascular diseases. To develop basic and translational research on FH, we here generated an FH model in a non-human primate (cynomolgus monkeys) by deleting the LDL receptor (LDLR) gene using the genome editing technique. Six LDLR knockout (KO) monkeys were produced, all of which were confirmed to have mutations in the LDLR gene by sequence analysis. The levels of plasma cholesterol and triglyceride were quite high in the monkeys, and were similar to those in FH patients with homozygous mutations in the LDLR gene. In addition, periocular xanthoma was observed only 1 year after birth. Lipoprotein profile analysis showed that the plasma very low-density lipoprotein and LDL were elevated, while the plasma high density lipoprotein was decreased in LDLR KO monkeys. The LDLR KO monkeys were also strongly resistant to medications for hypercholesterolemia. Taken together, we successfully generated a non-human primate model of hypercholesterolemia in which the phenotype is similar to that of homozygous FH patients.

Reconstitution of Human Prospermatogonial Development from Human-Induced Pluripotent Stem Cells.

There is a scarcity of information regarding the molecular mechanisms underlying human germ cell development due to limitations in obtaining the relevant materials. Reconstitution of human germ cell development from pluripotent stem cells in vitro would provide critical insight into the etiology of various reproductive conditions and disorders, including infertility.Recently, we reported the in vitro reconstitution of human prospermatogonial development from human-induced pluripotent stem cells through human primordial germ cell (PGC)-like cells (hPGCLCs) using long-term cultured xenogeneic reconstituted testes. Here, we describe a method to generate M-prospermatogonia-like cells (MLCs) and T1-prospermatogonia-like cells (T1LCs), which closely resemble M- and T1-prospermatogonia present in second-trimester human fetal testes in vivo.

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 induced pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus [cj]), allowing stable propagation 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.

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 recapitulate human fetal adrenal cortex specification processes through stepwise induction of human-induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenocortical 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 Δ5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN, and WNT signaling, and steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of Δ5 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.

A hominoid-specific endogenous retrovirus may have rewired the gene regulatory network shared between primordial germ cells and naïve pluripotent cells.

Mammalian germ cells stem from primordial germ cells (PGCs). Although the gene regulatory network controlling the development of germ cells such as PGCs is critical for ensuring gamete integrity, substantial differences exist in this network among mammalian species, suggesting that this network has been modified during mammalian evolution. Here, we show that a hominoid-specific group of endogenous retroviruses, LTR5_Hs, discloses enhancer-like signatures in human in vitro-induced PGCs, PGC-like cells (PGCLCs). Human PGCLCs exhibit a transcriptome signature similar to that of naïve-state pluripotent cells. LTR5_Hs are epigenetically activated in both PGCLCs and naïve pluripotent cells, and the expression of genes in the vicinity of LTR5_Hs is coordinately upregulated in these cell types, contributing to the establishment of the transcriptome similarity between these cell types. LTR5_Hs are preferentially bound by transcription factors that are highly expressed in both PGCLCs and naïve pluripotent cells (KLF4, TFAP2C, NANOG, and CBFA2T2), suggesting that these transcription factors contribute to the epigenetic activation of LTR5_Hs in these cells. Comparative transcriptome analysis between humans and macaques suggests that the expression of many genes in PGCLCs and naïve pluripotent cells is upregulated by LTR5_Hs insertions in the hominoid lineage. Together, this study suggests that LTR5_Hs insertions may have finetuned the gene regulatory network shared between PGCLCs and naïve pluripotent cells and coordinately altered the gene expression in these cells during hominoid evolution.

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 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.

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. 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. We assembled PacBio long reads, and the resultant contigs were scaffolded with Hi-C data, which were further refined based on Hi-C contact maps and alternate de novo assemblies. The assemblies achieved scaffold N50 lengths of 149 Mb and 137 Mb for cynomolgus macaque and common marmoset, respectively. The high fidelity of our assembly is also ascertained by BAC-end concordance in common marmoset. 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.

The embryonic ontogeny of the gonadal somatic cells in mice and monkeys.

In the early fetal stage, the gonads are bipotent and only later become the ovary or testis, depending on the genetic sex. Despite many studies examining how sex determination occurs from biopotential gonads, the spatial and temporal organization of bipotential gonads and their progenitors is poorly understood. Here, using lineage tracing in mice, we find that the gonads originate from a T+ primitive streak through WT1+ posterior intermediate mesoderm and appear to share origins anteriorly with the adrenal glands and posteriorly with the metanephric mesenchyme. Comparative single-cell transcriptomic analyses in mouse and cynomolgus monkey embryos reveal the convergence of the lineage trajectory and genetic programs accompanying the specification of biopotential gonadal progenitor cells. This process involves sustained expression of epithelial genes and upregulation of mesenchymal genes, thereby conferring an epithelial-mesenchymal hybrid state. Our study provides key resources for understanding early gonadogenesis in mice and primates.

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 establish a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells differentiated from human induced pluripotent stem cells are further induced into M-prospermatogonia-like cells and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing is used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibit gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enables 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.

Establishment of macaque trophoblast stem cell lines derived from cynomolgus monkey blastocysts.

The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and exchange of gases and wastes. Establishing an in vitro culture model of human and non-human primate trophoblast stem/progenitor cells is important for investigating the process of early placental development and trophoblast differentiation. In this study, we have established five trophoblast stem cell (TSC) lines from cynomolgus monkey blastocysts, named macTSC #1-5. Fibroblast growth factor 4 (FGF4) enhanced proliferation of macTSCs, while other exogenous factors were not required to maintain their undifferentiated state. macTSCs showed a trophoblastic gene expression profile and trophoblast-like DNA methylation status and also exhibited differentiation capacity towards invasive trophoblast cells and multinucleated syncytia. In a xenogeneic chimera assay, these stem cells contributed to trophectoderm (TE) development in the chimeric blastocysts. macTSC are the first primate trophoblast cell lines whose proliferation is promoted by FGF4. These cell lines provide a valuable in vitro culture model to analyze the similarities and differences in placental development between human and non-human primates.

Generation of Transgenic Cynomolgus Monkeys Overexpressing the Gene for Amyloid-ß Precursor Protein.

Alzheimer's disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-ß precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models. Here we report the generation of two transgenic cynomolgus monkeys overexpressing the human gene for APP with Swedish, Artic, and Iberian mutations, and demonstrated expression of gene tagged green fluorescent protein marker in the placenta, amnion, hair follicles, and peripheral blood. We believe that these nonhuman primate models will be very useful to study the pathogenesis of dementia and AD. However, generated Tg monkeys still have some limitations. We employed the CAG promoter, which will promote gene expression in a non-tissue specific manner. Moreover, we used transgenic models but not knock-in models. Thus, the inserted transgene destroys endogenous gene(s) and may affect the phenotype(s). Nevertheless, it will be of great interest to determine whether these Tg monkeys will develop tauopathy and neurodegeneration similar to human AD.

Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments.

Poor second ovarian stimulation in cynomolgus monkeys (Macaca fascicularis) is associated with the production of antibodies against human follicle-stimulating hormone.

Cynomolgus monkeys (Macaca fascicularis) are a valuable model organism for human disease modeling because human physiology and pathology are closer to those of cynomolgus monkeys than rodents. It has been widely reported that mature oocytes can be recovered from cynomolgus monkeys through ovarian stimulation by human follicle-stimulating hormone (hFSH). However, it is unknown whether mature oocytes can be effectively obtained through a second ovarian stimulation by hFSH. Here, we report that some ovaries (eight ovaries from 14 female monkeys) were stimulated effectively by hFSH even after the first ovum pick up, whereas the others were stimulated poorly by hFSH. Furthermore, we found antibodies against hFSH only in the serum of female monkeys with poorly stimulated ovaries. Collectively, these data suggest that anti-hFSH antibodies in serum may cause a poor ovarian response to hFSH stimulation. Finally, detection of such antibodies as well as observation of the ovary over the course of hFSH administration might be useful to predict favorable second ovarian stimulation by hFSH.

Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†.

Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.

The Germ Cell Fate of Cynomolgus Monkeys Is Specified in the Nascent Amnion.

The germ cell lineage ensures reproduction and heredity. The mechanism for germ cell specification in primates, including humans, has remained unknown. In primates, upon implantation the pluripotent epiblast segregates the amnion, an extra-embryonic membrane eventually ensheathing an embryo, and thereafter initiates gastrulation to generate three germ layers. Here, we show that in cynomolgus monkeys, the SOX17/TFAP2C/BLIMP1-positive primordial germ cells (cyPGCs) originate from the dorsal amnion at embryonic day 11 (E11) prior to gastrulation. cyPGCs appear to migrate down the amnion and, through proliferation and recruitment from the posterior amnion, expand in number around the posterior yolk sac by E17. Remarkably, the amnion itself expresses BMP4 and WNT3A, cytokines potentially critical for cyPGC specification, and responds primarily to them. Moreover, human PGC-like cells in vitro exhibit a transcriptome similar to cyPGCs just after specification. Our study identifies the origin of PGCs and a unique function of the nascent amnion in primates.

A developmental coordinate of pluripotency among mice, monkeys and humans.

The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of pluripotent stem cells in vitro. To explore the ontogeny of human and primate pluripotency, here we perform comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys (Macaca fascicularis). We show that after specification in the blastocysts, EPI from cynomolgus monkeys (cyEPI) undergoes major transcriptome changes on implantation. Thereafter, while generating gastrulating cells, cyEPI stably maintains its transcriptome over a week, retains a unique set of pluripotency genes and acquires properties for 'neuron differentiation'. Human and monkey pluripotent stem cells show the highest similarity to post-implantation late cyEPI, which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI and epiblast-like cells in vitro. These findings not only reveal the divergence and coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro.

Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body.

Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson's disease and Alzheimer's disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediate-early enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys. We also found that injection into mature oocytes before fertilization achieved homogenous expression of GFP in each tissue, including the amnion, and fibroblasts, whereas injection into fertilized oocytes generated a transgenic cynomolgus monkey with mosaic GFP expression. Thus, the injection timing was important to create transgenic cynomolgus monkeys that expressed GFP homogenously in each of the various tissues. The strategy established in this work will be useful for the generation of transgenic cynomolgus monkeys for transplantation studies as well as biomedical research.

Molecular characteristics of horse phospholipase C zeta (PLCζ).

A sperm-specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca(2+) ]i ) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT-PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long-lasting [Ca(2+) ]i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca(2+) ]i oscillation-inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.

Antibody-directed lentiviral gene transduction for live-cell monitoring and selection of human iPS and hES cells.

The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular, in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers, a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells, but that specific genes, including positive and negative selection markers, regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies.