MHC-DRB alleles with amino acids Val11, Phe13, and the shared epitopes promote collagen-induced arthritis and a rapid IgG1 response in Filipino cynomolgus macaques.

Macaques are useful animal models for studying the pathogenesis of rheumatoid arthritis (RA) and the development of anti-rheumatic drugs. The purpose of this study was to identify the major histocompatibility complex (MHC) polymorphisms associated with the pathology of collagen-induced arthritis (CIA) and anti-collagen IgG induction in a cynomolgus macaque model, as MHC polymorphisms affect the onset of CIA in other animal models. Nine female Filipino cynomolgus macaques were immunized with bovine type II collagen (b-CII) to induce CIA, which was diagnosed clinically by scoring the symptoms of joint swelling over 9 weeks. MHC polymorphisms and anti-b-CII antibody titers were compared between symptomatic and asymptomatic macaques. Four of 9 (44%) macaques were defined as the CIA-affected group. Anti-b-CII IgG in the affected group increased in titer approximately 3 weeks earlier compared with the asymptomatic group. The mean plasma IgG1 titer in the CIA-affected group was significantly higher (p < 0.05) than that of the asymptomatic group. Furthermore, the cynomolgus macaque MHC (Mafa)-DRB1*10:05 or Mafa-DRB1*10:07 alleles, which contain the well-documented RA-susceptibility five amino acid sequence known as the shared epitope (SE) in positions 70 to 74, with valine at position 11 (Val11, V11) and phenylalanine at position 13 (Phe13, F13), were detected in the affected group. In contrast, no MHC polymorphisms specific to the asymptomatic group were identified. In conclusion, the presence of V11 and F13 along with SE in the MHC-DRB1 alleles seems essential for the production of IgG1 and the rapid induction of severe CIA in female Filipino cynomolgus macaques.

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.

Impact of administering umbilical cord-derived mesenchymal stem cells to cynomolgus monkeys with endometriosis.

Purpose: This study aimed to explore whether umbilical cord-derived mesenchymal stem cells (UC-MSCs) could be used as a therapeutic resource for endometriosis. Methods: Of seven cynomolgus monkeys with endometriosis, five were administered UC-MSCs (intervention group) and two were administered saline (control group). First, intravenous US-MSC treatment was administered for three months. Second, weekly intravenous US-MSC administration combined with monthly intraperitoneal US-MSC administration was conducted for 3 months. Finally, weekly intraperitoneal US-MSC administration was conducted for 3 months. The dose of UC-MSCs was set to 2 × 106 cells/kg for all administration routes. Laparoscopic findings and serum cancer antigen 125 (CA125) levels were also evaluated. The Revised American Society for Reproductive Medicine classification was used for laparoscopic evaluation. Results: Laparoscopic findings showed exacerbation of endometriosis after intraperitoneal UC-MSC administration, although no changes were observed in the control group. Intravenous UC-MSC administration decreased the level of CA125 in all monkeys; however, the difference was not significant. Intraperitoneal UC-MSC administration significantly exacerbated endometriosis compared with intravenous administration (p = 0.02). Conclusions: This study revealed that intraperitoneal UC-MSC administration exacerbates endometriosis in a nonhuman primate model of the disease.

Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys.

Human in vitro oogenesis provides a framework for clarifying the mechanism of human oogenesis. To create its benchmark, it is vital to promote in vitro oogenesis using a model physiologically close to humans. Here, we establish a foundation for in vitro oogenesis in cynomolgus (cy) monkeys (Macaca fascicularis): cy female embryonic stem cells harboring one active and one inactive X chromosome (Xa and Xi, respectively) differentiate robustly into primordial germ cell-like cells, which in xenogeneic reconstituted ovaries develop efficiently into oogonia and, remarkably, further into meiotic oocytes at the zygotene stage. This differentiation entails comprehensive epigenetic reprogramming, including Xi reprogramming, yet Xa and Xi remain epigenetically asymmetric with, as partly observed in vivo, incomplete Xi reactivation. In humans and monkeys, the Xi epigenome in pluripotent stem cells functions as an Xi-reprogramming determinant. We further show that developmental pathway over-activations with suboptimal up-regulation of relevant meiotic genes impede in vitro meiotic progression. Cy in vitro oogenesis exhibits critical homology with the human system, including with respect to bottlenecks, providing a salient model for advancing human in vitro oogenesis.

Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys.

In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced into primordial germ cell-like cells and into oogonia with epigenetic reprogramming, yet further reconstitutions remain a challenge. Here, we demonstrate ex vivo reconstitution of fetal oocyte development in both humans and cynomolgus monkeys (Macaca fascicularis). With an optimized culture of fetal ovary reaggregates over three months, human and monkey oogonia enter and complete the first meiotic prophase to differentiate into diplotene oocytes that form primordial follicles, the source for oogenesis in adults. The cytological and transcriptomic progressions of fetal oocyte development in vitro closely recapitulate those in vivo. A comparison of single-cell transcriptomes among humans, monkeys, and mice unravels primate-specific and conserved programs driving fetal oocyte development, the former including a distinct transcriptomic transformation upon oogonia-to-oocyte transition and the latter including two active X chromosomes with little X-chromosome upregulation. Our study provides a critical step forward for realizing human in vitro oogenesis and uncovers salient characteristics of fetal oocyte development in primates.

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.

The X chromosome dosage compensation program during the development of cynomolgus monkeys.

X chromosome dosage compensation ensures balanced gene dosage between the X chromosome and autosomes and between the sexes, involving divergent mechanisms among mammals. We elucidated a distinct mechanism for X chromosome inactivation (XCI) in cynomolgus monkeys, a model for human development. The trophectoderm and cytotrophoblast acquire XCI around implantation through an active intermediate bearing repressive modifications and compacted structure, whereas the amnion, epiblast, and hypoblast maintain such an intermediate protractedly, attaining XCI by a week after implantation. Males achieve X chromosome up-regulation (XCU) progressively, whereas females show XCU coincidentally with XCI, both establishing the X:autosome dosage compensation by 1 week after implantation. Conversely, primordial germ cells undergo X chromosome reactivation by reversing the XCI pathway early during their development. Our findings establish a foundation for clarifying the dosage compensation mechanisms in primates, including humans.

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.

Capturing human trophoblast development with naive pluripotent stem cells in vitro.

Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program.

The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17, TFAP2C, and BLIMP1, which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2, immediate BMP effectors, combined with SOX17 and TFAP2C, generated hPGCLCs. GATA3/GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3/GATA2 expression remained unaffected in SOX17, TFAP2C, or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3, SOX17, and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis.