Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1–13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4–7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.

The objective of this study was to evaluate the use of corpus luteum (CL) color doppler ultrasonography (CD) for early pregnancy diagnosis in Bos taurus beef replacement heifers. Beef heifers (n = 183) from two locations were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate CL morphometries and blood perfusion, respectively. Heifers were considered non-pregnant when CL area was < 20 mm2 or estimated luteal blood perfusion was ≤ 25%. Conventional ultrasonography on day 29 was utilized to determine pregnancy status and considered the gold standard method for pregnancy diagnosis. Pregnant heifers had greater CL diameter, CL area, and CL volume when compared to non-pregnant heifers on days 20 and 22 (P < 0.001). Additionally, percentage of central, peripheral, and total luteal blood perfusion, as well as the respective blood perfusion scores were greater (P < 0.001) in pregnant compared with non-pregnant heifers on both day 20 and 22. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and accuracy for CD on day 20 were 100, 70, 86, 100, and 90, respectively. Sensitivity, specificity, PPV, NPV, and accuracy for CD on day 22 were 100, 76, 90, 100, and 92, respectively. Pairwise comparison of receiver operating characteristics curve analysis indicated no differences (P = 0.47) between CD on days 20 and 22 (area under the curve = 0.82 and 0.84, respectively). In conclusion, CD successfully detected most non-pregnant replacement heifers on day 20 and 22, while false negative results were absent (NPV = 100%).

Background: The transcriptome of peripheral white blood cells (PWBCs) contains valuable physiological information, thus making them a prime biological sample for investigating mRNA-based biomarkers. However, prolonged storage of whole blood samples can alter gene transcript abundance in PWBCs, compromising the results of biomarker discovery. Here, we designed an experiment to interrogate the impacts of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 hours at 4°C would cause RNA degradation resulting in altered transcriptome profiles. Results: We produced RNA-sequencing data for 30 samples collected from five estrus synchronized heifers (Bos taurus). We quantified transcript abundance for 12,414 protein-coding genes in PWBCs. Analysis of parameters of RNA quality revealed no statistically significant differences (P>0.05) between samples collected from the jugular vein and coccygeal vein, as well as among samples processed after one, three, six, or eight hours. However, samples processed after 24 hours of storage had a lower RNA integrity number value (P=0.03) in comparison to those processed after one hour of storage. Next, we analyzed RNA-sequencing data between samples using those processed after one hour of storage as the baseline for comparison. Interestingly, evaluation of 3/5 prime bias revealed no differences between genes with lower transcript abundance in samples stored for 24 hours relative to one hour. In addition, sequencing coverage of transcripts was similar between samples from the 24-hour and one-hour groups. We identified four and 515 genes with differential transcript abundance in samples processed after storage for eight and 24 hours, respectively, relative to samples processed after one hour. Conclusions: The PWBCs respond to prolonged cold storage by increasing genes related to active chromatin compaction which in turn reduces gene transcription. This alteration in transcriptome profiles can impair the accuracy of mRNA-based biomarkers. Therefore, blood samples collected for mRNA-based biomarker discovery should be refrigerated immediately and processed within six hours post sampling.

Paternal chromatin undergoes extensive structural and epigenetic changes during mammalian spermatogenesis, producing sperm that contain an epigenome optimal for the transition to embryogenesis. Histone modifiers play an important role in this process by encoding specialized regulatory information in the sperm epigenome. Lysine demethylase 6a (KDM6A) promotes gene activation via demethylation of H3K27me3, a developmentally important repressive modification abundant throughout the epigenome of sperm and embryonic stem cells. Despite its developmental importance in pluripotent cells and germ cell progenitors, the function of KDM6A during spermatogenesis has not been described. Here, we show that Kdm6a is transiently expressed in the male germline in late spermatogonia and during the early stages of meiotic entry. Deletion of Kdm6a in the male mouse germline (Kdm6a cKO) yielded a modest increase in sperm head defects but did not affect fertility or the overall progression of spermatogenesis. However, hundreds of genes were deregulated upon loss of Kdm6a in spermatogenic cells and in an immortalized spermatogonia cell line (GC-1 spg) with a strong bias towards downregulation. Single cell RNA-seq revealed that most of these genes were deregulated in spermatogenic cells at the same stage when Kdm6a is expressed and encode epigenetic factors involved in chromatin organization and modification. A subset of these genes was persistently deregulated in the male germ line across two generations of offspring of Kdm6a cKO males. Our findings highlight KDM6A as a transcriptional activator in the mammalian male germline that is dispensable for spermatogenesis but important for safeguarding gene regulatory state intergenerationally.