Chizuru Iwatani

Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent Stem Cells.

Mechanisms underlying human germ cell development are unclear, partly due to difficulties in studying human embryos and lack of suitable experimental systems. Here, we show that human induced pluripotent stem cells (hiPSCs) differentiate into incipient mesoderm-like cells (iMeLCs), which robustly generate human primordial germ cell-like cells (hPGCLCs) that can be purified using the surface markers EpCAM and INTEGRINα6. The transcriptomes of hPGCLCs and primordial germ cells (PGCs) isolated from non-human primates are similar, and although specification of hPGCLCs and mouse PGCs rely on similar signaling pathways, hPGCLC specification transcriptionally activates germline fate without transiently inducing eminent somatic programs. This includes genes important for naive pluripotency and repression of key epigenetic modifiers, concomitant with epigenetic reprogramming. Accordingly, BLIMP1, which represses somatic programs in mice, activates and stabilizes a germline transcriptional circuit and represses a default neuronal differentiation program. Together, these findings provide a foundation for understanding and reconstituting human germ cell development in vitro.

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.

Vitrification and transfer of cynomolgus monkey (Macaca fascicularis) embryos fertilized by intracytoplasmic sperm injection

Protocols for cryopreservation of monkey embryos are not well established. The objective of the current study was to determine the efficacy of the polypropylene strip method for cryopreserving cynomolgus monkey embryos. Cynomolgus monkey embryos, 63 and 56 at the 4- to 8-cell and 56 blastocyst stages, respectively, were produced by intracytoplasmic sperm injection and in vitro culture, and vitrified using a polypropylene strip. For these two stages, 95 and 86% survived after thawing and pregnancy rates were 29.2% (7 pregnant females/24 recipients, with three live births) and 0% (n = 16 recipients). These were apparently the first live births obtained from embryos fertilized by ICSI. In conclusion, 4- to 8-cell preimplantation cynomolgus monkey embryos were successfully cryopreserved using a polypropylene strip.

Single-cell transcriptome of early embryos and cultured embryonic stem cells of cynomolgus monkeys

In mammals, the development of pluripotency and specification of primordial germ cells (PGCs) have been studied predominantly using mice as a model organism. However, divergences among mammalian species for such processes have begun to be recognized. Between humans and mice, pre-implantation development appears relatively similar, but the manner and morphology of post-implantation development are significantly different. Nevertheless, the embryogenesis just after implantation in primates, including the specification of PGCs, has been unexplored due to the difficulties in analyzing the embryos at relevant developmental stages. Here, we present a comprehensive single-cell transcriptome dataset of pre- and early post-implantation embryo cells, PGCs and embryonic stem cells (ESCs) of cynomolgus monkeys as a model of higher primates. The identities of each transcriptome were also validated rigorously by other way such as immunofluorescent analysis. The information reported here will serve as a foundation for our understanding of a wide range of processes in the developmental biology of primates, including humans.

155 LAPAROSCOPIC EVALUATION OF OVARIAN REACTION TO HORMONE STIMULATION IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS)

Oocyte collection is a key step to development of a system for modeling human regenerative medicine and assisted reproductive technology in cynomolgus monkeys (Macaca fascicularis), but collecting oocytes at a suitable developmental stage (metaphase II) is difficult. Metaphase II (MII) oocytes can be collected from cynomolgus ovaries stimulated by hormone injection. In this study, we developed a useful method for collecting a large number of MII oocytes by monitoring the morphology and size of ovaries with laparoscopic observation. Controlled ovarian stimulation and oocyte recovery in mature cynomolgus monkeys have been previously described by Torii et al. (2000 Biochemistry 39, 3197–3205). Beginning at menses, levels of estrogens were down-regulated by the subcutaneous injection of a GnRH antagonist (Leuplin, 0.9 mg animal-1; Takeda Chemical Industries, Ltd., Osaka, Japan). Two weeks later, human follicle stimulating hormone (FSH, Fertinorm, 25 IU kg-1; Serono, Canton Zug, Switzerland) was administered for 9 days. On the day after that of the last FSH administration, human chorionic gonadotropin (hCG, Puberogen, 400 IU kg-1; Sankyo Co., Ltd., Tokyo, Japan) was intramuscularly injected. Follicular aspiration was performed at 404-41 hours post-hCG injection. Oocyte collection was monitored using a 3-mm laparoscope attached to a video system. Oocytes were aspirated from the follicles using a 20-guage needle. Follicle development of the ovary was rated morphologically as A (small follicles), B (many small follicles), or C (many large follicles), and size relative to the uterus was rated as 1 (no response), 2 (smaller), 3 (equal), or 4 (larger) at oocyte collection. Regarding morphology, the highest ratio of MII/total oocytes was obtained from B–C ovaries (rating of 2 ovaries, one each left or right with a rating of B or C, n = 8, 26.9 ± 22.4, 70.7%), followed by B–B (n = 21, 19.5 ± 14.3, 55.9%), and C–C (n = 19, 10.6 ± 5.8, 51.9%). No MII oocytes were collected from A–A (n = 1, 0%) ovaries. Ovaries appeared to be over-stimulated in the ovaries rated C–C but under-stimulated in B–B. Regarding ovary size, the highest ratio of MII/total oocytes was obtained from 4–4 (both ovaries with rating of 4, n = 10, 21.9 ± 11.2, 68.8%), followed by 3–3 (n = 21, 19.1 ± 14.3, 58.2%), and 2–2 (n = 9, 11.6 ± 6.5, 53.3%). No MII oocytes were collected from 1–1 ovaries (n = 1, 0%). The number of MII oocytes collected was directly related to ovary size: more MII oocytes were collected from larger ovaries. These data demonstrate that the number of oocytes collected is directly related to ovary size. Our results suggest that the ratio of MII oocytes can be predicted by the morphology and the size of ovaries. In addition, we found that ovarian development can be controlled by adjusting FSH dosage. Therefore, laparoscopic observation of ovaries during FSH treatment and adjusting FSH dosage are necessary to collect MII oocytes efficiently.