A developmental landscape of 3D-cultured human pre-gastrulation embryos.

Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms1,2 that do not recapitulate the in vivo conditions3-5. Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior-posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.

Transcriptional dynamics of transposable elements when converting fibroblast cells of Macaca mulatta to neuroepithelial stem cells.

BACKGROUND: Transposable elements (TE) account for more than 50% of human genome. It has been reported that some types of TEs are dynamically regulated in the reprogramming of human cell lines. However, it is largely unknown whether some TEs in Macaca mulatta are also regulated during the reprogramming of cell lines of monkey. RESULTS: Here, we systematically examined the transcriptional activities of TEs during the conversion of Macaca mulatta fibroblast cells to neuroepithelial stem cells (NESCs). Hundreds of TEs were dynamically regulated during the reprogramming of Macaca mulatta fibroblast cells. Furthermore, 48 Long Terminal Repeats (LTRs), as well as some integrase elements, of Macaca endogenous retrovirus 3 (MacERV3) were transiently activated during the early stages of the conversion process, some of which were further confirmed with PCR experiments. These LTRs were potentially bound by critical transcription factors for reprogramming, such as KLF4 and ETV5. CONCLUSION: These results suggest that the transcription of TEs are delicately regulated during the reprogramming of Macaca mulatta fibroblast cells. Although the family of ERVs activated during the reprogramming of fibroblast cells in Macaca mulatta is different from those in the reprogramming of human fibroblast cells, our results suggest that the activation of some ERVs is a conserved mechanism in primates for converting fibroblast cells to stem cells.

CpG site DNA methylation of the CCAAT/enhancer-binding protein, alpha promoter in chicken lines divergently selected for fatness.

CCAAT/enhancer-binding protein (C/EBP), alpha (CEBPA) is a master regulator of adipogenesis and, together with peroxisome proliferator-activated receptor gamma (PPARG), plays a critical role in adipocyte differentiation. Previous studies have demonstrated that CEBPA is regulated by DNA methylation and involved in the osteogenesis and adipogenesis of mouse C3H10T1/2 and bone marrow stromal cells. However, it is unclear whether CEBPA is regulated by DNA methylation in adipose tissues. Thus, the objectives of the present study were to investigate CpG site methylation in a 357-bp CEBPA promoter region and to assess the correlation between promoter CpG site methylation and CEBPA gene expression in the abdominal adipose tissues of Northeast Agricultural University broiler lines divergently selected for abdominal fat content. The results showed that the methylation percentage of the analyzed CEBPA promoter region was significantly higher in lean broilers than in fat broilers at 2 weeks (80.3% vs. 43.4%, P < 0.0001), 3 weeks (95.4% vs. 74.0%, P < 0.0001) and 7 weeks of age (82.6% vs. 57.2%, P < 0.0001). Real-time quantitative RT-PCR analysis showed that CEBPA expression was significantly higher in the fat vs. the lean line at 2 weeks of age (P = 0.0013) but not at 3 or 7 weeks of age. The correlation analysis showed that only at 2 weeks of age was the methylation percentage negatively correlated with CEBPA expression (Pearson's r = -0.8312, P = 0.0029). Of all seven tested CpGs, only two, the CpGs at -1494 and -1478 bp, displayed a significantly negative correlation with CEBPA mRNA expression. These results suggest that the CEBPA is methylated in adipose tissue and may regulate chicken early adipose development.

Identification and characterization of transcript variants of chicken peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma regulates adipogenesis. The genomic structure of the chicken peroxisome proliferator-activated receptor gamma (cPPARγ) gene has not been fully characterized, and only one cPPARγ gene mRNA sequence has been reported in genetic databases. Using 5' rapid amplification of cDNA ends, we identified five different cPPARγ mRNAs that are transcribed from three transcription initiation sites. The open reading frame analysis showed that these five cPPARγ transcript variants (cPPARγ1 to 5) could encode two cPPARγ protein isoforms (cPPARγ1 and cPPARγ2), which differ only in their N-terminal region. Quantitative real-time RT-PCR analysis showed that, of these five cPPARγ transcript variants, cPPARγ1 was ubiquitously highly expressed in various chicken tissues, including adipose tissue, liver, kidney, spleen and duodenal; cPPARγ2 was exclusively highly expressed in adipose tissue; cPPARγ3 was highly expressed in adipose tissue, kidney, spleen and liver; cPPARγ4 and cPPARγ5 were ubiquitously weakly expressed in all the tested tissues, and comparatively, cPPARγ5 was highly expressed in adipose tissue, heart, liver and kidney. The comparison of the expression of the five cPPARγ transcript variants showed that adipose tissue cPPARγ1 expression was significantly higher in the fat line than in the lean line from 2 to 7 wk of age (P < 0.05 or P < 0.01). Adipose tissue cPPARγ3 expression was significantly higher in the fat line than in the lean line at 3, 5 and 6 wk of age (P < 0.01, P < 0.05), but lower at 4 wk of age (P < 0.05). Adipose tissue cPPARγ5 expression was significantly higher in the fat line than in the lean line at 3, 4, and 6 wk of age (P < 0.01) and at 2 and 7 wk of age (P < 0.05). This is the first report of transcript variants and protein isoforms of cPPARγ gene. Our findings provided a foundation for future investigations of the function and regulation of cPPARγ gene in adipose tissue development.

Flexible Capacitive Sensing and Ultrasound Calibration for Skeletal Muscle Deformations.

Skeletal muscles are critical to human-limb motion dynamics and energetics, where their mechanical states are seldom explored in vitro due to practical limitations of sensing technologies. This article aims to capture mechanical deformations of muscle contraction using wearable flexible sensors, which is justified with model calibration and experimental validation. The capacitive sensor is designed with the composite of conductive fabric electrodes and the porous dielectric layer to increase the pressure sensitivity and prevent lateral expansions. In this way, the compressive displacement of muscle deformation is captured in the muscle-sensor coupling model in terms of sensor deformation and parameters of pretension, material, and shape properties. The sensing model is calibrated in a linear form using ultrasound medical imaging. The sensor is capable of measuring muscle strain of 70% with an error of <3.6% and temperature disturbance of <5.6%. After 10K cycles of compression, the drift is only 3.3%. Immediate application of the proposed method is illustrated by gait pattern identification, where the K-nearest neighbor prediction accuracy of squats, level walking, stair ascent/descent, and ramp ascent is over 97% with a standard deviation below 2.6% compared to that of 94.61 ± 4.24% for ramp descent, and the response time is 14.37 ± 0.52 ms. The wearable sensing method is valid for muscle deformation monitoring and gait pattern identification, and it provides an alternative approach to capture mechanical motions of muscles, which is anticipated to contribute to understand locomotion biomechanics in terms of muscle forces and metabolic landscapes.

Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids.

Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage development as well as underlying mechanisms remains obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling pathways synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast development and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights into human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis.

Meta-Analysis of the Relationship between the Prognosis of Acute Cerebral Infarction Intravenous Lysis and Cerebral Microbleeds Based on Intelligent Medical Care.

The accelerated pace of life leads to people's unhealthy living habits such as irregular diet, irregular work and rest, and fatigued work. The incidence of acute cerebral infarction (ACI) is increasing year by year. Intravenous thrombolysis is the best solution for clinical treatment of ACI, but intravenous thrombolysis increases the risk of cerebral microbleeds and even seriously damages the brain of patients. It is crucial to analyze the relationship between intravenous thrombolysis of ACI and cerebral microbleeds. Using intelligent medical methods such as BP neural network (BPNN), meta-analysis was carried out on the prognosis of ACI intravenous thrombolysis and cerebral microbleeds, and the basic data indicators, living habits indicators, and ACI intravenous thrombolysis indicators of ACI patients were analyzed. The experimental results showed that the odds ratios (OR) of systolic blood pressure before ACI intravenous thrombolysis, blood glucose concentration after ACI intravenous thrombolysis, diastolic blood pressure before ACI intravenous thrombolysis, and diastolic blood pressure after ACI intravenous thrombolysis on cerebral microbleeds after ACI intravenous thrombolysis were 0.97, 0.44, 0.13, and 0.07, respectively. Long-term intravenous thrombolysis with ACI, high systolic blood pressure after NIHSS thrombolysis with high scores, and blood glucose concentration before thrombolysis had OR >1, which were risk factors for cerebral microbleeds after intravenous thrombolysis with ACI. Therefore, paying attention to the risk factors of cerebral microbleeds during ACI intravenous thrombolysis can effectively reduce cerebral microbleeds after ACI intravenous thrombolysis and improve the treatment efficiency of ACI patients.

BRN2 as a key gene drives the early primate telencephalon development.

Evolutionary mutations in primate-specific genes drove primate cortex expansion. However, whether conserved genes with previously unidentified functions also play a key role in primate brain expansion remains unknown. Here, we focus on BRN2 (POU3F2), a gene encoding a neural transcription factor commonly expressed in both primates and mice. Compared to the limited effects on mouse brain development, BRN2 biallelic knockout in cynomolgus monkeys (Macaca fascicularis) is lethal before midgestation. Histology analysis and single-cell transcriptome show that BRN2 deficiency decreases RGC expansion, induces precocious differentiation, and alters the trajectory of neurogenesis in the telencephalon. BRN2, serving as an upstream factor, controls specification and differentiation of ganglionic eminences. In addition, we identified the conserved function of BRN2 in cynomolgus monkeys to human RGCs. BRN2 may function by directly regulating SOX2 and STAT3 and maintaining HOPX. Our findings reveal a previously unknown mechanism that BRN2, a conserved gene, drives early primate telencephalon development by gaining novel mechanistic functions.

The Long Terminal Repeats of ERV6 Are Activated in Pre-Implantation Embryos of Cynomolgus Monkey.

Precise gene regulation is critical during embryo development. Long terminal repeat elements (LTRs) of endogenous retroviruses (ERVs) are dynamically expressed in blastocysts of mammalian embryos. However, the expression pattern of LTRs in monkey blastocyst is still unknown. By single-cell RNA-sequencing (seq) data of cynomolgus monkeys, we found that LTRs of several ERV families, including MacERV6, MacERV3, MacERV2, MacERVK1, and MacERVK2, were highly expressed in pre-implantation embryo cells including epiblast (EPI), trophectoderm (TrB), and primitive endoderm (PrE), but were depleted in post-implantation. We knocked down MacERV6-LTR1a in cynomolgus monkeys with a short hairpin RNA (shRNA) strategy to examine the potential function of MacERV6-LTR1a in the early development of monkey embryos. The silence of MacERV6-LTR1a mainly postpones the differentiation of TrB, EPI, and PrE cells in embryos at day 7 compared to control. Moreover, we confirmed MacERV6-LTR1a could recruit Estrogen Related Receptor Beta (ESRRB), which plays an important role in the maintenance of self-renewal and pluripotency of embryonic and trophoblast stem cells through different signaling pathways including FGF and Wnt signaling pathways. In summary, these results suggest that MacERV6-LTR1a is involved in gene regulation of the pre-implantation embryo of the cynomolgus monkeys.

Generation of cortical neurons through large-scale expanding neuroepithelial stem cell from human pluripotent stem cells.

BACKGROUND: Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into cortical neurons for disease modeling and regenerative medicine. However, these procedures are hard to provide sufficient cells for their applications. Using a combination of small-molecules and growth factors, we previously identified one condition which can rapidly induce hPSCs into neuroepithelial stem cells (NESCs). Here, we developed a scalable suspension culture system, which largely yields high-quality NESC-spheres and subsequent cortical neurons. METHODS: The NESC medium was first optimized, and the suspension culture system was then enlarged from plates to stirred bioreactors for large-scale production of NESC-spheres by a stirring speed of 60 rpm. During the expansion, the quality of NESC-spheres was evaluated. The differentiation potential of NESC-spheres into cortical neurons was demonstrated by removing bFGF and two pathway inhibitors from the NESC medium. Cellular immunofluorescence staining, global transcriptome, and single-cell RNA sequencing analysis were used to identify the characteristics, identities, purities, or homogeneities of NESC-spheres or their differentiated cells, respectively. RESULTS: The optimized culture system is more conducive to large-scale suspension production of NESCs. These largely expanded NESC-spheres maintain unlimited self-renewal ability and NESC state by retaining their uniform sizes, high cell vitalities, and robust expansion abilities. After long-term expansion, NESC-spheres preserve high purity, homogeneity, and normal diploid karyotype. These expanded NESC-spheres on a large scale have strong differentiation potential and effectively produce mature cortical neurons. CONCLUSIONS: We developed a serum-free, defined, and low-cost culture system for large-scale expansion of NESCs in stirred suspension bioreactors. The stable and controllable 3D system supports long-term expansion of high-quality and homogeneous NESC-spheres. These NESC-spheres can be used to efficiently give rise to cortical neurons for cell therapy, disease modeling, and drug screening in future.

Modulation of Wnt and Activin/Nodal supports efficient derivation, cloning and suspension expansion of human pluripotent stem cells.

Various culture systems have been used to derive and maintain human pluripotent stem cells (hPSCs), but they are inefficient in sustaining cloning and suspension expansion of hPSCs. Through systematically modulating Wnt and Activin/Nodal signaling, we developed a defined medium (termed AIC), which enables efficient cloning and long-term expansion of hPSCs (AIC-hPSCs) through single-cell passage on feeders, matrix or in suspension (25-fold expansion in 4 days) and maintains genomic stability of hPSCs over extensive expansion. Moreover, the AIC medium supports efficient derivation of hPSCs from blastocysts or somatic cells under feeder-free conditions. Compared to conventional hPSCs, AIC-hPSCs have similar gene expression profiles but down-regulated differentiation genes and display higher metabolic activity. Additionally, the AIC medium shows a good compatibility for different hPSC lines under various culture conditions. Our study provides a robust culture system for derivation, cloning and suspension expansion of high-quality hPSCs that benefits GMP production and processing of therapeutic hPSC products.

Improving Cell Survival in Injected Embryos Allows Primed Pluripotent Stem Cells to Generate Chimeric Cynomolgus Monkeys.

Monkeys are an optimal model species for developing stem cell therapies. We previously reported generating chimeric cynomolgus monkey fetuses using dome-shaped embryonic stem cells (dESCs). However, conventional primed pluripotent stem cells (pPSCs) lack chimera competency. Here, by altering the media in which injected morulae are cultured, we observed increased survival of cynomolgus monkey primed ESCs, induced PSCs, and somatic cell nuclear transfer-derived ESCs, thereby enabling chimeric contributions with 0.1%-4.5% chimerism into the embryonic and placental tissues, including germ cell progenitors in chimeric monkeys. Mechanically, dESCs and pPSCs belong to different cell types and similarly express epiblast ontogenic genes. The host embryonic microenvironment could reprogram injected PSCs to embryonic-like cells. However, the reprogramming level and chimerism were associated with the cell state of injected PSCs. Our findings provide a method to understand pluripotency and broaden the use of embryonic chimeras for basic developmental biology research and regenerative medicine.

Expression and induction of CYP2C P450 enzymes in primary cultures of human hepatocytes.

Although CYP2C8, CYP2C9, and CYP2C19 play an important role in drug biotransformation, factors influencing the expression and activity of these CYP2C P450s in human liver remain largely undefined. We used primary cultures of human hepatocytes from 15 subjects to assess the inducibility of CYP2C enzyme expression by prototypical inducer agents, including rifampicin, dexamethasone, and phenobarbital. After culture for 72 h in serum-free medium on collagen, Western blotting revealed that CYP2C9 was the only CYP2C enzyme expressed at appreciable levels in untreated hepatocytes. Subsequent treatment with 25 microMrifampicin for 48 h elicited marked increases in CYP2C8 (700 +/- 761%), CYP2C19 (854%), and CYP2C9 (209 +/- 176%) protein content versus a 550 +/- 170% enhancement of CYP3A4 enzyme levels. Parallel increases in CYP2C mRNAs, measured by Northern blotting and/or RNase protection, were found in rifampicin-treated hepatocytes, with CYP2C8, CYP2C9, and CYP2C19 transcripts exhibiting increases of 688 +/- 635, 207 +/- 49, and 230 +/- 60%, respectively, versus an 8.8-fold enhancement of CYP3A4 mRNA levels. Dexamethasone (10 microM) treatment enhanced CYP2C8 mRNA (360 +/- 100%) and protein (274%) content, although this steroid had less effect on CYP2C9 and CYP2C19 transcripts (23 +/- 21% and 21 +/- 36%, respectively) and enzyme levels (55 and 143%, respectively). Phenobarbital (100 microM) was a powerful inducer of CYP2C9 (850%) and CYP2C19 (735%) mRNA content, and also increased CYP2C8 (610%) and CYP3A4 (205%) transcripts. Our results show that CYP2C enzyme expression in human hepatocytes is highly inducible by rifampicin, dexamethasone, and phenobarbital. Because these xenobiotics are ligands and/or activators of the pregnane X receptor and/or constitutive androstane receptor, such orphan nuclear receptors and their response elements may partake in regulating CYP2C gene expression in humans.