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.

Identification of potential M2 macrophage-associated diagnostic biomarkers in coronary artery disease.

BACKGROUND: M2 macrophages have been reported to be important in the progression of coronary artery disease (CAD). Thus, the present study aims at exploring the diagnostic value of M2 macrophage-associated genes in CAD. METHODS: Transcriptome profile of CAD and control samples were downloaded from Gene Expression Omnibus database. The proportion of immune cells was analyzed using cell type identification by estimating relative subsets of RNA transcripts. Weighted Gene Co-expression Network Analysis (WGCNA) was carried out to screen the relevant module associated with M2 macrophages. Differential CAD and control samples of expressed genes (DEGs) were identified by the limma R package. Functional enrichment analysis by means of the clusterProfiler R package. Least absolute shrinkage and selection operator (LASSO) and random forest (RF) algorithms were carried out to select signature genes. Receiver operating curves (ROC) were plotted to evaluate the diagnostic value of selected signature genes. The expressions of potential diagnostic markers were validated by RT-qPCR. The ceRNA network of diagnostic biomarkers was constructed via miRwalk and Starbase database. CMap database was used to screen candidate drugs in the treatment of CAD by targeting diagnostic biomarkers. RESULTS: A total of 166 M2 macrophage-associated genes were identified by WGCNA. By intersecting those genes with 879 DEGs, 53 M2 macrophage-associated DEGs were obtained in the present study. By LASSO, RF, and ROC analyses, C1orf105, CCL22, CRYGB, FRK, GAP43, REG1P, CALB1, and PTPN21 were identified as potential diagnostic biomarkers. RT-qPCR showed the consistent expression patterns of diagnostic biomarkers between GEO dataset and clinical samples. Perhexiline, alimemazine and mecamylamine were found to be potential drugs in the treatment of CAD. CONCLUSION: We identified eight M2 macrophage-associated diagnostic biomarkers and candidate drugs for the CAD treatment.

Prevalence and risk factors for congenital heart defects among children in the Multi-Ethnic Yunnan Region of China.

Background: To determine the congenital heart defect (CHD) prevalence and identify the associated risk factors in children within the multi-ethnic Yunnan Region of China. Methods: This is a prospective matched case-control screening study. Screening for CHD in children residing within 28 county districts of Yunnan Province during the period of January 2001 to December 2016 was conducted. A total of 2,421 and CHD cohort and 24,210 control cohort were derived from a total population of 400,855 children (under 18 years of age). Results: A total of 2,421 children were diagnosed with CHD, yielding a CHD prevalence of 6.04 cases per 1,000 children. The prevalence of CHD by sex was 6.54 per 1,000 females versus 5.59 per 1,000 males. The ethnic groups displaying the highest CHD prevalence were the Lisu (15.51 per 1,000), Achang (13.18 per 1,000), Jingpo (12.32 per 1,000), Naxi (9.68 per 1,000), and Tibetan (8.57 per 1,000), respectively. The most common CHD was atrial septal defect, amounting to 1.94 instances per 1,000 children. We identified a number of child-associated parameters that significantly correlated with greater CHD risk, such as lower mass at birth, shorter duration of gestation, and younger age at the time of screening. We also identified a number of maternal and familial risk factors. Conclusions: This ultrasonic color Doppler imaging study revealed a relatively commonplace prevalence of CHD. Moreover, the prevalence of CHD in Yunnan Region significantly varied with sex and ethnic status. Certain child-associated, maternal, and familial risk factors may contribute to CHD risk.

SLC2A3 variants in familial and sporadic congenital heart diseases in a Chinese Yunnan population.

BACKGROUND: Solute carrier family 2 member 3 (SLC2A3), is a member of a superfamily of transport protein genes. SLC2A3 played an important role in embryonic development. Previous research reported SLC2A3 duplication was reportedly associated with congenital syndromic heart defects. However, it is not clear whether the gene is associated with non-syndromic congenital heart disease. Our study aimed to elucidate the relationship between its variation and congenital heart disease. METHODS: Genomic DNA extracted from the peripheral blood leukocytes of two families with CHD were sequenced with whole-exome sequencing to identify variations, used Sanger sequencing to investigate SLC2A3 variants in 494 Chinese patients with CHD and 576 healthy unrelated individuals. RESULTS: In members from the two families, three with CHD had the SLC2A3 (rs3931701) C > T variant. Of the 494 patients with CHD, 394 had gene variants (86 had the TT type and 308 had the CT type). Of the 576 healthy controls, 272 participants had gene variants (36 had the TT type and 236 had the CT type). The TT type [p < 0.0001, odds ratio (OR) =7.262, 95% confidence interval (CI) =4.631-11.388] and CT type (p < 0.0001, OR =3.967, 95% CI =2.991-5.263) of SLC2A3 (rs3931701) significantly increased the risk of sporadic ASD in a Chinese Yunnan population. CONCLUSIONS: Single nucleotide variations of SLC2A3, particularly, the SLC2A3 (rs3931701) C > T variant increased the risk of CHD among the studied population.

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.

Nkx2-5 Regulates the Proliferation and Migration of H9c2 Cells.

BACKGROUND The protein NKX2-5 affects mammalian heart development. In mice, the disruption of Nkx2-5 has been associated with arrhythmias, abnormal myocardial contraction, abnormal cardiac morphogenesis, and death. However, the details of the mechanisms are unclear. This study was designed to investigate them. MATERIAL AND METHODS Rat cardiomyocytes from the H9c2 cell line were used in our study. First, we knocked down Nkx2-5 in the H9c2 cells and then validated consequent changes in cell proliferation and migration. We then used RNA sequencing to determine the changes in transcripts. Finally, we validated these results by quantitative reverse transcription-polymerase chain reaction. RESULTS We confirmed that Nkx2-5 regulates the proliferation and migration of H9c2 cells. In our experiments, Nkx2-5 regulated the expression of genes related to proliferation, migration, heart development, and disease. Based on bioinformatics analysis, knockdown of Nkx2-5 caused differential expression of genes involved in cardiac development, calcium ion-related biological activity, the transforming growth factor (TGF)-ß signaling pathway, pathways related to heart diseases, the MAPK signaling pathway, and other biological processes and signaling pathways. CONCLUSIONS Nkx2-5 may regulate proliferation and migration of the H9c2 cells through the genes Tgfb-2, Bmp10, Id2, Wt1, Hey1, and Cacna1g; rno-miR-1-3p; the TGF­ß signaling pathway; the MAPK signaling pathway; as well as other genes and pathways.

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.

Endothelial retinoblastoma protein reduces abdominal aortic aneurysm development via promoting DHFR/NO pathway-mediated vasoprotection.

Cardiovascular disease (CVD) is a major cause of global mortality. The proper functioning of the endothelial layer of arteries is crucial to cardiovascular health. Retinoblastoma protein (Rb), encoded by the Rb1 gene, has been shown to offer vasoprotective effects. Herein, we investigated endothelial Rb's effects on arterial function using an endothelial-specific conditional Rb1 knockout (Rb cKO) mouse model. We found that Rb deficiency reduced dihydrofolate reductase (DHFR) activity and downstream NO production in mouse aortic endothelial cells and blocked arterial vasodilation in an endothelial DHFR-dependent manner. Rb deficiency also increased phenylephrine-triggered arterial vasoconstriction, BP levels, and pathological aortic remodeling without significantly affecting prostanoid synthesis. Employing an angiotensin II (AngII)-stimulated apolipoprotein E knockout (apoE -/-) mice fed a standard, non-atherogenic diet, Rb deficiency increased aortic diameter, stimulated abdominal aortic aneurysm (AAA) development, and reduced survival. These pathological responses to Rb deficiency in AngII-stimulated apoE-/- mice were rescued by DHFR overexpression. Cumulatively, our findings reveal that endothelial Rb positively impacts arterial function by supporting vasoprotective endothelial DHFR/NO pathway activity, leading to reduced AAA development.