OBJECTIVES: To reprogram human placental fibroblasts (HPFs) into chemically induced epithelioid-like cells (ciEP-Ls) using a combination of small-molecule compounds. METHODS: HPFs cultured under normoxic conditions were identified using immunofluorescence assay, PCR and chromosomal karyotyping. Under hypoxic conditions (37 ℃, 5% O₂), HPFs were cultured in a medium containing small-molecule compounds C6TSEDRVAJZ (CHIR99021, 616452, TTNPB, SAG, EPZ5676, DZNep, Ruxolitinib, VTP50469, Afuresertib, JNK-IN-8, and EZM0414), and the cell morphology was observed daily. The expression levels of epithelial cell markers in the induced cells were detected by immunofluorescence, Western blotting and PCR. Chromosomal karyotyping of the induced cells was performed and the induction efficiency was calculated. RESULTS: Before induction, HPFs showed positive expressions of fibroblast surface markers CD34 and vimentin and were negative for epithelial surface markers. PCR results showed high expressions of fibroblast-specific genes S100A4 and COL1A1 in HPFs with a normal human diploid karyotype. After one day of induction, the HPFs underwent morphological changes from a multinodular spindle shape to a round or polygonal shape, which was morphologically characteristic of ciEP-Ls. On day 4 of induction, the cells exhibited high expressions of the epithelial cell markers E-cadherin and Lin28A. RT-qPCR results also showed that the cells expressed the epithelial markers Smad3, GLi3, PAX8, WT1, KRT19, and KRT18 with significantly down-regulated expressions of all the fibroblast surface markers and a normal human diploid karyotype. The reprogramming efficiency of HPFs into ciEP-Ls ranged from (64.53±2.8)% to (68.10±3.6)%. CONCLUSIONS The small-molecule compound combination C6TSEDRVAJZ is capable of inducing HPFs into ciEP-Ls under hypoxic conditions with a high induction efficiency.
Humans ; Fibroblasts/drug effects* ; Pregnancy ; Female ; Cell Differentiation/drug effects* ; Pyrimidines/pharmacology* ; Placenta/cytology* ; Cells, Cultured ; Pyridines/pharmacology* ; Pyrazoles/pharmacology* ; Epithelial Cells/cytology*

Objective To propose a transfer learning-based method for breath sound feature recognition and autonomous determination of sputum suction timing.Methods An electronic stethoscope was used to collect breath sounds from the main airways of clinically ventilated patients before and after sputum suction,with pre-suction breath sounds labeled as requiring suction.The collected data underwent high-pass filtering and wavelet soft-threshold denoising,followed by the extraction of log-Mel spectrograms.A VGGish model pretrained on the Audio Set dataset was then employed to extract feature vectors from these spectrograms,which were subsequently classified using a support vector machine to determine whether suction was required.Results The precision,recall and F1 score for recognition of breath sounds requiring sputum suction were 86.73％,93.06％and 89.78％,respectively.Conclusions The proposed breath sound recognition method based on transfer learning effectively determines the timing of sputum suction and shows a significant clinical potential.

Objective To propose a transfer learning-based method for breath sound feature recognition and autonomous determination of sputum suction timing.Methods An electronic stethoscope was used to collect breath sounds from the main airways of clinically ventilated patients before and after sputum suction,with pre-suction breath sounds labeled as requiring suction.The collected data underwent high-pass filtering and wavelet soft-threshold denoising,followed by the extraction of log-Mel spectrograms.A VGGish model pretrained on the Audio Set dataset was then employed to extract feature vectors from these spectrograms,which were subsequently classified using a support vector machine to determine whether suction was required.Results The precision,recall and F1 score for recognition of breath sounds requiring sputum suction were 86.73％,93.06％and 89.78％,respectively.Conclusions The proposed breath sound recognition method based on transfer learning effectively determines the timing of sputum suction and shows a significant clinical potential.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

Objective To investigate the effects of long noncoding RNA(lncRNA)-NRON on apoptosis following myocardial infarc-tion(MI)in mice.Methods The C57BL/6 mice were randomly divided into four groups:sham operation(Sham)group,MI group,MI combined with lncRNA-NRON interference lentivirus(MI+shNRON)group,and MI combined with the negative control(NC)lentivirus(MI+NC)group.The expression of lncRNA-NRON was detected using real-time PCR.In addition,the pathology of the myocardial tissue injury was analyzed using HE staining,the myocardial infarction size was examined using TTC staining,and the extent of apoptosis was assessed using the TUNEL assay,respectively.The RPISeq database was used to predict the probability of interaction between lncR-NA-NRON and the voltage-dependent anionic channel protein(VDAC).The effect of lncRNA-NRON on the expression of VDAC protein was detected using Western blotting.Results The lncRNA-NRON expression was significantly increased in the MI group,and the tar-geted knockdown of lncRNA-NRON resulted in alleviation of the pathological myocardial tissue injury,reduction in the myocardial infarc-tion area,and inhibition of apoptosis.The probability of interaction between lncRNA-NRON and VDAC reached 0.9,indicating a high probability of their association.Additionally,lncRNA-NRON could regulate the protein expression of VDAC.Conclusion Knockdown of lncRNA-NRON could reduce the occurrence of myocardial injury following myocardial infarction.This effect may be attributable to a spe-cific mechanism wherein lncRNA-NRON affects the process of apoptosis by binding to VDAC,consequently suppressing its expression.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

A triple-transgenic (tyrosine hydroxylase/dopamine decarboxylase/GTP cyclohydrolase 1, TH/DDC/GCH1) bone marrow mesenchymal stem cell line (BMSCs) capable of stably synthesizing dopamine (DA) transmitters were established to provide experimental evidence for the clinical treatment of Parkinson's disease (PD) by using this cell line. The DA-BMSCs cell line that could stably synthesize and secrete DA transmitters was established by using the triple transgenic recombinant lentivirus. The triple transgenes (TH/DDC/GCH1) expression in DA-BMSCs was detected using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Moreover, the secretion of DA was tested by enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). Chromosome G-banding analysis was used to detect the genetic stability of DA-BMSCs. Subsequently, the DA-BMSCs were stereotactically transplanted into the right medial forebrain bundle (MFB) of Parkinson's rat models to detect their survival and differentiation in the intracerebral microenvironment of PD rats. Apomorphine (APO)-induced rotation test was used to detect the improvement of motor dysfunction in PD rat models with cell transplantation. The TH, DDC and GCH1 were expressed stably and efficiently in the DA-BMSCs cell line, but not expressed in the normal rat BMSCs. The concentration of DA in the cell culture supernatant of the triple transgenic group (DA-BMSCs) and the LV-TH group was extremely significantly higher than that of the standard BMSCs control group (P < 0.000 1). After passage, DA-BMSCs stably produced DA. Karyotype G-banding analysis showed that the vast majority of DA-BMSCs maintained normal diploid karyotypes (94.5%). Moreover, after 4 weeks of transplantation into the brain of PD rats, DA-BMSCs significantly improved the movement disorder of PD rat models, survived in a large amount in the brain microenvironment, differentiated into TH-positive and GFAP-positive cells, and upregulated the DA level in the injured area of the brain. The triple-transgenic DA-BMSCs cell line that stably produced DA, survived in large numbers, and differentiated in the rat brain was successfully established, laying a foundation for the treatment of PD using engineered culture and transplantation of DA-BMSCs.
Rats ; Animals ; Dopamine ; Parkinson Disease/metabolism* ; Mesenchymal Stem Cells/metabolism* ; Cell Line ; Brain/metabolism* ; Cell Differentiation ; Mesenchymal Stem Cell Transplantation

Objective:To investigate the safety and the efficacy of percutaneous and surgical approach in femoro-femoral veno-arterial extracorporeal membrane oxygenation (VA-ECMO) cannulation.Methods:All consecutive patients implanted with femoro-femoral VA-ECMO between January 2018 and December 2020 in Beijing Anzhen Hospital, Capital Medical University. Propensity score matching was used to compare outcomes of percutaneous and surgical groups while controlling for confounders.Results:Among the 276 patients who received femoro-femoral VA-ECMO (62 surgical and 214 percutaneous), propensity-score matching selected 52 pairs of patients with similar characteristics with mean age of(59.6±13.0)years old, in which 26 patients were female. There were a lower ECMO cannulation-associated complication (28.8% vs. 48.1%, P=0.044) and a lower hospital mortality (42.3% vs. 67.3%, P=0.010) in the percutaneous group. The circuit blood flow after ECMO initiation was similar in both groups[(3.3±0.8)L·min -1·kg -1 in percutaneous group vs. (3.2±0.7)L·min -1·kg -1 in surgical group, P=0.738]. The serum lactate was declined in both group after ECMO initiation[(5.4±5.8)mmol/L vs. (9.2±6.9)mmol/L, P<0.001 in percutaneous group; (6.3±6.2)mmol/L vs. (10.5±7.0)mmol/L, P=0.003 in surgical group]. Conclusion:Percutaneous approach is a safe and efficient technique in emoro-femoral VA-ECMO cannulation. Compared with surgical cannulation, percutaneous approach is associated with lower ECMO cannulation-associated complication and lower hospital mortality.

Objective:To research the clinical effect of early comprehensive intervention for physical and intelligence development of premature infant (test-tube baby).Methods:One hundred and eleven premature infant in Dalian Municipal Women and Children′s Medical Center from June 2016 to June 2018 were enrolled. Study group (57 cases) received comprehensive intervention including health education, exercise training, nutrition guidance and rehabilitation therapy, and control group (54 cases) received health education. The weight, height, head circumference and CDCC score of two groups were analyzed in 3, 6 and 12 month.Results:Physical development: the weight [3 month: (4.72 ± 0.19) kg vs. (4.34 ± 0.29) kg; 6 month: (6.49 ± 0.37) kg vs. (6.25 ± 0.41) kg; 12 month: (9.58 ± 1.15) kg vs. (8.76 ± 0.92) kg] and height [3 month: (59.63 ± 5.51) cm vs. (56.29 ± 5.86) cm; 6 month: (65.09 ± 5.94) cm vs. (62.36 ± 6.20) cm] in 3, 6 and 12 month, and head circumference in 3 and 6 month [3 month: (37.71 ± 1.77) cm vs. (35.90 ± 1.48) cm; 6 month: (43.18 ± 1.96) cm vs. (41.82 ± 2.61) cm] of study group were higher than those in control group, and all difference had statistical significant ( P<0.05). Intelligence development: the MDI [3 month: (84.49 ± 9.78) scores vs. (80.58 ± 10.40) scores; 6 month: (89.65 ± 13.21) scores vs. (83.24 ± 17.66) scores; 12 month: (96.03 ± 15.43) scores vs. (89.71 ± 17.85) scores] and PDI score of study group was higher than that in control group in 3, 6 and 12 month [3 month: (82.68 ± 5.35) scores vs. (79.43 ± 7.21) scores; 6 month: (86.34 ± 8.33) scores vs. (82.51 ± 9.67) scores; 12 month: (94.86 ± 10.27) scores vs. (90.32 ± 11.65) scores] ( P<0.05). Conclusions:Early comprehensive intervention has a good clinical efficacy for physical and intelligence development of premature infant (test-tube baby) and is worthy of popularizing and applying.

Objective To evaluate the effect of early postoperative delirium on outcomes in elderly patients undergoing joint replacement.Methods Elderly patients,aged ≥ 60 yr,of American Society of Anesthesiologists physical status Ⅰ-Ⅲ,scheduled for elective hip or knee joint replacement,were divided into delirium group and non-delirium group according to whether the patients developed postoperative delirium.The case-matching criteria was set up with difference in age less than 5 yr and difference in Charlson comorbidity index less than 2 and with a ratio of 1 ∶ 4 for match.The development of complications was recorded within 28 days after operation.Complications happened after delirium was recorded in delirium group.Results There were 53 cases in delirium group and 212 cases in non-delirium group.There was no significant difference in preoperative baseline data or intraoperative variables between two groups (P ＞0.05).Compared with non-delirium group,the total incidence of postoperative complications was significantly increased,the incidence of postoperative cognitive dysfunction,cardiovascular complications,pulmonary complications and deep venous thrombosis was increased,and the length of hospitalization was prolonged in delirium group (P＜0.05).Conclusion Early postoperative delirium is not helpful for outcomes in elderly patients undergoing joint replacement.