Modeling human pregastrulation development by 3D culture of blastoids generated from primed-to-naïve transitioning intermediates

Human pluripotent stem cells provide an inexhaustible model to study human embryogenesis in vitro. Recent studies have provided diverse models to generate human blastoids by self-organization of different pluripotent stem cells or somatic reprogramming intermediates. However, whether blastoids can be generated from other cell types or whether they can recapitulate postimplantation development in vitro is unknown. Here, we develop a strategy to generate human blastoids from heterogeneous intermediates with epiblast, trophectoderm, and primitive endoderm signatures of the primed-to-naïve conversion process, which resemble natural blastocysts in morphological architecture, composition of cell lineages, transcriptome, and lineage differentiation potential. In addition, these blastoids reflect many features of human peri-implantation and pregastrulation development when further cultured in an in vitro 3D culture system. In summary, our study provides an alternative strategy to generate human blastoids and offers insights into human early embryogenesis by modeling peri- and postimplantation development in vitro.

Bilineage embryo-like structure from EPS cells can produce live mice with tetraploid trophectoderm

Self-organized blastoids from extended pluripotent stem (EPS) cells possess enormous potential for investigating postimplantation embryo development and related diseases. However, the limited ability of postimplantation development of EPS-blastoids hinders its further application. In this study, single-cell transcriptomic analysis indicated that the "trophectoderm (TE)-like structure" of EPS-blastoids was primarily composed of primitive endoderm (PrE)-related cells instead of TE-related cells. We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure. Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation. Furthermore, we demonstrated that blastocyst-like structures reconstituted by combining the EPS-derived bilineage embryo-like structure (BLES) with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses. In summary, our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.

Safety and prognostic analysis of partial nephrectomy for cT 1N 0M 0 non-clear renal cell carcinoma with high grade malignancy / 中华泌尿外科杂志

Objective:To investigate the safety and prognosis of partial nephrectomy (PN) in the treatment of highly malignant non-clear renal cell carcinoma (nccRCC).Methods:Clinical data of 47 patients with cT 1N 0M 0 high malignant nccRCC treated in Changhai Hospital from March 2016 to March 2022 were retrospectively analyzed. All patients received PN. There were 34(72.3%) males and 13(27.7%) females. The mean age was (53.5±15.0) years, and average BMI, was(23.7±3.4)kg/m 2.The maximum tumor diameter was (29.8±12.6) mm, and R. E.N.A.L. score was 7(5-9), with 37(78.7%) cases of T 1a and 10(21.3%) cases of T 1b. The mean estimated glomerular filtration rate (eGFR) before surgery was (96.3±25.5) ml/ (min·1.73m 2). All patients underwent PN, including 1 patient (2.1%) undergoing open surgery, 29 patients (61.7%) undergoing laparoscopic surgery, and 17 patients (36.2%) undergoing robotic surgery. There were a total of 22(46.8%) cases of papillary cell carcinoma(pRCC)type Ⅱ, 4(8.5%) cases of collecting duct carcinoma (cdRCC), 9(19.1%) cases of MiT family translocated renal cell carcinoma (tRCC), 5(10.6%) cases of mucoid tubular and spindle cell carcinoma (mtSCC)and 7(14.9%) cases of unclassified renal cell carcinoma (uRCC). The surgical conversion rate, positive margin rate, operative time, intraoperative blood loss, complications, and postoperative hospital stay were analyzed. Preoperative and postoperative eGFR were analyzed, and overall survival (OS) and cancer specific survival (CSS) were calculated. Results:All the operations were successfully completed. No radical operation or open operation was performed, with operation time of(100±60) min and intraoperative blood loss of(100±59) ml. There were no intraoperative complication and 1 case (2.1%) suffered from postoperative complication. Postoperative hospital stay were 5 (4-6) days. The mean eGFR after surgery was (86.5±27.1) ml/(min·1.73m 2), and the difference was statistically significant ( P=0.041). In this study, the mean follow-up time was (45.7±20.9)months, and no adjuvant therapy was used after surgery. During the follow-up period, 2 patients died, who all of them were kidney cancer-related death, and both OS and CSS were 95.7% (45/47). Conclusions:PN is safe, feasible and has a good prognosis in the treatment of high malignant T 1 nccRCC. For tumors with clear imaging boundaries and complete envelope, complete tumor resection is more likely, postoperative follow-up should be strict, and no remedial radical or systemic treatment was required.

Clinical study of high risk factors for poor prognosis in the neonatal respiratory distress syndrome / 中国综合临床

Objective To analyze high risk factors for poor prognosis in the refractory neonatal respiratory distress syndrome(NRDS).Methods A retrospective analysis was conducted at NICU in the Affiliated Hospital of Xuzhou Medical university.The newborns with NRDS,chest radiograph for grades 3 and 4,who required mechanical ventilation and pulmonary surfactant(PS)therapy were recruited from January 2014 to December 2015.According to treatment rusults,they were divided into death group(21 cases)and survival group(25 cases).Data regarding antenatal corticosteroid administration,maternal high risk factors,the basic situation of neonatal in the perinatal period,surfactant applications,the blood routine,albumin,lactate dehydrogenase(LDH),creatine kinase(CK),creatine kinase isoenzyme(CK-MB),blood coagulation function with the first 24 hours after birth were collected and analyzed.Results The frequency of using PS in the death group and the survival group was(2±0.9)times and(1.5±0.6)times,the difference between the two groups was statistically significant(t=2.131,P=0.039).Dangerous placenta previa in death group was 19％(4/21),in the survival group was 0％(0/25),the two groups had statistical differences(x2=5.215,P=0.022).CK in death group was(541.5±399.1)U/L,in the survival group was(345.4±173.3)U/L,the difference between of the two groups was statistically significant(t=2.224,P=0.031).Prothrombin time(PT)in the death group was(23.2±6.3)s,in the survival group was(18.5±3.6)s,there was a significant difference between the two groups(t=3.008,P=0.039).Maternal risk factors of premature rupture of the survival group(38.9％)was higher than that of death group(5.0％),the two groups was statistically significant(x2=4.29,P=0.038).The application of prenatal hormone NRDS newborns were more likely to survive,there was statistical difference between two groups(x2=5.197,P=0.023).Multivariate Logistic analysis showed that PT was an independent risk factor for poor prognosis of NRDS.Conclusion PT is an independent risk factor for poor prognosis of neonatal respiratory distress syndrome.

Effects of inhibiting NgR on retinal ganglion cells density and synaptophysin expression of diabetic rats / 眼科新进展

Objective To investigate the effects of inhibiting NgR on retinal ganglion cells density and synaptophysin expression of diabetic rat.Methods Thirty-two SD male rats were randomly divided into normal control group,diabetic group,siNgR group and scNgR group,8 rats in each group.Normal control group was given no any treatment.Diabetes model was induced by intraperitoneal injection of 50 mg · kg-1 streptozotocin in diabetic group,siNgR group and scNgR group,and the blood giucose more than 16.7 mmol · L-1 at 72 hours was set as the successfully model.The rats of siNgR group were intravitreally administrated with anti-NgR nucleotide and the rats of scNgR group intravitreally administrated with negative nucleotide.Eight weeks later,HE staining was conducted to detect density of retinal ganglion cell (RGC),immunofluorescence was used to evaluate the expression and distribution of synaptophysin (a marker of synaptic number).Relative expression of NgR and synaptophysin in retina were analyzed by Western blot.Results RGC density in normal control group,diabetes group,siNgR group and scNgR group were (624.33 ± 3.51) mm-2,(420.00 ± 2.65) mm-2,(621.67 ± 1.53) mm-2,(416.67 ± 2.52) mm-2,respectively.There was significant difference among four groups (F =5985.37,P ＜ 0.01).Compared with normal control group,RGC density in diabetes group and scNgR group were obviously decreased (all P ＜0.01),but siNgR group had no obviously change (P ＞ 0.05).The synaptophysin mainly expressed in the inner and outer network layer.Compared with normal control group,the positive expression of synaptophysin in diabetes group and scNgR group were decreased,but siNgR group had no obviously change.The relative expression of NgR in normal control group,diabetes group,siNgR group and scNgR group were (11.26 ±0.02) ％,(19.38 ± 0.10) ％,(11.17 ± 0.02) ％,(19.47 ± 0.31) ％,respectively.There was significant difference among four groups (F =2466.09,P ＜ 0.01).Compared with normal control group,the relative expression of NgR in diabetes group and scNgR group were obviously decreased (all P ＜ 0.01),but siNgR group had no obviously change (P ＞0.05).The relative expression of synaptophysin in normal control group,diabetes group,siNgR group and scNgR group were (35.76 ± 0.15) ％,(25.47 ± 0.36) ％,(35.28 ± 0.12) ％,(25.03 ± 0.75) ％,respectively.There was significant difference among four groups (F =583.70,P ＜ 0.01).Compared with the normal control group,the expression of synaptophysin in diabetic group and scNgR group were decreased increased (all P ＜ 0.01),while there was no significant difference in siNgR group (P ＞ 0.05).Conclusion Inhibiting the expression of NgR in the retina of diabetic rats can help to restore the number of synapses and protect the damaged RGC.

The expression of intestinal trefoil factor (ITF) in colon of cytomegalovirus infected mouse and the involvement of ganciclovir / 重庆医学

Objective To observe the expression of ITF in colon of mice infected by mouse cytomegalovirus (MCMV) and the in-volvement of ganciclovir(GCV) .Methods Forty-eight BALB/c young mice were randomly divided into blank group ,virus group and GCV group ,each with 8 mice .Mice in virus group and GCV group received injection of 100 μL MCMV virus suspension (TCID50105 .31 /mL) ,and GCV group was given intraperitoneal injection of GCV once a day at the dose of 50 mg/kg from day 0 (24 hours after vaccination of virus ) ,for 14 days .At the same time the virus group and blank group were given the same dose of normal saline as controls .Murine cytomegalovirus loads in livers and colons were measured by PCR .The expression levels of ITF in mRNA in colon were detected by RT-PCR .Results After MCMV injection ,mice in virus group manifested aggressively apparent poor appetite ,less activity ,fur laxly ,unresponsiveness to stimuli ,growth retardation ,body weight not increased .All liver and colon tissue MCMV-DNA PCR electrophoresis of virus group had positive strip ,while the blank group did not .GCV group also showed less bright positive strip when compared with the virus group .Expression level of ITF mRNA was significant higher in GCV group than virus group ,there was statistically significant difference(P<0 .05) .Expression of ITF mRNA in virus group were higher than that in blank group ,there was statistical difference(P<0 .05) .Conclusion ITF is regarded as a fast reaction peptide in the course of mucosa impairments ,so ITF plays a protective role in delayed healing process after acute MCMV infection .

Expression of insulin-like growth factor Ⅱ mRNA-binding protein 3 in transitional cell carcinoma of bladder and its clinical significance / 中国综合临床

Objective To investigate the expression of insulin-like growth factor Ⅱ mRNA-binding protein 3( IMP3 ) in transitional cell carcinoma of bladder( TCCB ) and its clinical significance. Methods The IMP3 expressions in 6 normal bladder tissues and 48 TCCB tissues were determined by immunohistochemistry staining, and the relationships between IMP3 expression and gender, age, pathological grading and staging were analyzed statistically. Results The positive expression rate of IMP3 was 62.5% ( 30/48 ) in bladder cancer,which were significantly higher than that in normal control ( 0/6 ) ( P ＜ 0. 05 ). The positive rate of IMP3 expression were significantly lower in superficial TCCB ( 46. 15%, 12/22 ) or early TCCB [ Grade Ⅰ 12. 5%(1/8) ,Grade Ⅱ 60. 0% (12/20) ] than that in invasive TCCB [T2 or above,81.8% (18/22) ] or advanced TCCB [ Grade Ⅲ ,85% ( 17/20 ) ] ( Ps ＜ 0. 05 ). Conclusion IMP3 is a promising biomarker for eraly detection and assessment of the malignancy degree in TCCB.

Establishment of goat limbal stem cell strain expressing Venus fluorescent protein and construction of limbal epithelial sheets / 生物工程学报

The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Further, we cultivated the cells on denude amniotic membrane to construct tissue engineered fluorescent corneal epithelial sheets. Histology and HE staining showed that the constructed fluorescent corneal epithelial sheets consisted of 5-6 layers of epithelium. Only the lowest basal cells of fluorescent corneal epithelial sheets expressed P63 analyzed by immunofluorescence, but not superficial epithelial cells. These results showed that our constructed fluorescent corneal epithelial sheets were similar to the normal corneal epithelium in structure and morphology. This demonstrated that they could be transplanted for patents with corneal impair, also may provide a foundation for the study on the mechanisms of corneal epithelial cell regeneration after LSCT.