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 establish a methodological system for reprogramming rat embryonic fibroblasts(REF)into chemically induced neurons(ciNCs)via small molecule compounds to provide safe and effective donor cells for treatment of neurodegenerative diseases.Methods:Based on the method established by PEI Gang's research group to directly reprogram human fibroblasts into neurons,the induction medium and maturation medium was optimized by replacing the coating solution,mitigating oxidative stress injury,adding neurogenic protective factors,adjusting the concentration of trichothecenes,performing small-molecule removal experiments,and carrying out immunofluorescence and Western blotting on cells at different stages of induction to validate the effect of induction.Results:When the original protocol was used for induction,the cell survival rate was(34.24±2.77)%.After replacing the coating solution gelatin with matrigel,the cell survival rate increased to(45.41±4.27)%;after adding melatonin,the cell survival rate increased to(67.95±5.61)%and(23.43±1.42)%were transformed into neural-like cells;after adding the small molecule P7C3-A20,the cell survival rate was further increased to(76.27±1.41)%,and(39.72±4.75)%of the cells were transformed into neural-like cells.When the concentration of trichothecene was increased to 30 μmol/L,the proportion of neural-like cells reached(55.79±1.90)%;after the removal of SP600125,(86.96±2.15)%of the cells survived,and the rate of neural-like cell production increased to(63.43±1.60)%.With the optimized protocol,REF could be successfully induced into ciNC through the neural precursor cell stage,in which the neural precursor cells were able to highly express the neural precursor cell markers SRY-related HMG-box gene 2(Sox2)and paired box 6(Pax6)as well as neuron-specific marker tubulin 1(Tuj1),while the expression of fiber-associated protein vimentin was reduced.After two weeks of induction of neural precursor cells in a maturation medium,most cells displayed neuronal-like cell morphology.The induced ciNCs were able to highly express the mature neuronal surface markers Tuj1 and microtubule-associated protein 2(MAP2),while the expression of vimentin was reduced.Conclusion:The small molecule combinations optimized in this study can reprogram REF to ciNCs under normoxic conditions.

Objective:This study aimed to explore the status of radiological Kashin-Beck disease (KBD) among school-aged children in Chamdo City, Tibet, through a 3-year monitoring survey, providing epidemiological evidence for prevention and control strategies.Methods:The target areas for this study were Luolong, Bianba, and Basu counties in Chamdo City, Tibet Autonomous Region, identified as having the most severe historical cases of KBD. Children aged 7-12 years attending school were enrolled as study subjects. Anteroposterior X-ray films of the right-hand were taken, and radiological diagnoses were made based on the "Diagnosis of Kashin-Beck Disease" criteria (WS/T 207-2010). Two experienced researchers independently reviewed the X-rays, and intra- and inter-group consistency were assessed using weighted Kappa values and percentage agreement. Cross-sectional surveys were conducted in 2017 and 2020 to describe the X-ray detection rates of KBD, and logistic regression analysis was employed to construct a predictive model of risk factors for radiological KBD cases.Results:In 2017, a total of 5,711 children aged 7-12 years in Chamdo City, Tibet, participated in the baseline cross-sectional survey (average age 9.2 years, 48.0% female), with 28 cases of radiological KBD. The age- and gender-standardized prevalence rate was 0.527%. In 2020, 6,771 participants (average age 9.3 years, 49.5% female) underwent a second cross-sectional survey, with 9 cases of radiological KBD and a standardized prevalence rate of 0.134%. Logistic regression analysis indicated that older age [ OR=2.439, 95% CI(1.299, 4.580), P=0.006] and female gender [ OR=8.157, 95% CI(1.016, 65.528), P=0.048] were independent risk factors for radiological KBD cases. Conversely, higher residential altitude, under the premise of Tibet's high altitude, was a protective factor [ OR=0.995, 95% CI(0.990, 0.999), P=0.032). Conclusion:The radiographically positive detection rate of KBD among school-aged children in Chamdo City, Tibet Autonomous Region, is at an extremely low level and showing a declining trend, reaching the historical standard in 2020. Considering the absence of positive signs in affected children, it suggests that local KBD has been effectively eliminated.