Photobiomodulation:a novel approach to promote trans-differentiation of adipose-derived stem cells into neuronal-like cells

Photobiomodulation,originally used red and near-infrared lasers,can alter cellular metabolism.It has been demonstrated that the visible spectrum at 451-540 nm does not necessarily increase cell proliferation,near-infrared light promotes adipose stem cell proliferation and affects adipose stem cell migration,which is necessary for the cells homing to the site of injury.In this in vitro study,we explored the potential of adipose-derived stem cells to differentiate into neurons for future translational regenerative treatments in neurodegenerative disorders and brain injuries.We investigated the effects of various biological and chemical inducers on trans-differentiation and evaluated the impact of photobiomodulation using 825 nm near-infrared and 525 nm green laser light at 5 J/cm2.As adipose-derived stem cells can be used in autologous grafting and photobiomodulation has been shown to have biostimulatory effects.Our findings reveal that adipose-derived stem cells can indeed trans-differentiate into neuronal cells when exposed to inducers,with pre-induced cells exhibiting higher rates of proliferation and trans-differentiation compared with the control group.Interestingly,green laser light stimulation led to notable morphological changes indicative of enhanced trans-differentiation,while near-infrared photobiomodulation notably increased the expression of neuronal markers.Through biochemical analysis and enzyme-linked immunosorbent assays,we observed marked improvements in viability,proliferation,membrane permeability,and mitochondrial membrane potential,as well as increased protein levels of neuron-specific enolase and ciliary neurotrophic factor.Overall,our results demonstrate the efficacy of photobiomodulation in enhancing the trans-differentiation ability of adipose-derived stem cells,offering promising prospects for their use in regenerative medicine for neurodegenerative disorders and brain injuries.

Conophylline Promotes the Proliferation of Immortalized Mesenchymal Stem Cells Derived from Fetal Porcine Pancreas (iPMSCs)

Conophylline, is a bis （indole） alkaloid consisting of two pentacyclic aspidosperma skeletons, isolated from Tabernaemontana divaricata, which has been found to induce β-cell differentiation in rat pancreatic acinar carcinoma cells and in cultured rat pancreatic tissue. However, the precise role of conophylline in the growth and survival of immortalized pancreatic mesenchymal stem cells （iPMSCs） derived from fetal porcine pancreas were not understood at present. To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effects of conophylline on iPMSCs. We found that conophylline can robustly stimulate iPMSCs proliferation, even promote their potential differentiation into islet-like clusters analyzed by cell counting, morphology, RT-PCR and real-time PCR, Western blotting, glucose-stimulated insulin release and insulin content analysis. The effects of conophylline were inhibited by LY294002, which is the inhibitor of the PI3K pathway. These results suggest that conophylline plays a key role in the regulation of cell mass proliferation, maintenance of the undifferentiated state of iPMSCs and also promotes iPMSCs differentiated into insulin-producing cells.

Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

Magnetic resonance image （MRI） systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields （SMF）. With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid （AL） cells, Chinese hamster ovary （CHO） cells, DNA double-strand break repair deficient mutant （XRS-5） cells, and human primary skin fibroblasts （AG1522） cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster ceils after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

Efficient generation of functional hepatocytelike cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells

BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Advancing Environmental Toxicology In Vitro:From Immortalized Cancer Cell Lines to 3D Models Derived from Stem Cells

In recent years,rapid industrial development has resulted in the production and exposure of a substantial number of compounds to the human body.This has created an urgent need in environmental toxicology for models that are efficient,accurate,and cost-effective in evaluating the health impacts of these compounds on humans.Over the past seven decades,various cancer cell lines and immortalized cell lines have made significant contributions to the advancement of research on organ toxicity.Pluripotent stem cell technology,especially toxicological models derived from pluripotent stem cells,presents modern environmental toxicologists with high-throughput,species-relevant,and predictive options.In this comprehensive review,we assess the characteristics of representative human cancer cell lines and immortalized cell lines in environmental toxicology,as well as introduce two distinct human pluripotent stem cell types and their innovative toxicological models.We explore their applications and prospects in the field of environmental toxicology,while also addressing the readiness of in vitro models to confront the emerging challenges of the future.

Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro

·AIM: To explore the feasibility that human amniotic epithelial cells (hAECs) have the potential to differentiate into corneal epithelial -like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells (S-ihCECs). ·METHODS: hAECs were isolated by enzyme digestion, and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA -DR. Recovered and cultured S -ihCECs, immunocytochemistry was used to detect the expression of CK3/12. The proliferation of S - ihCECs handled by different concentrations of mitomycin was detected by CCK -8. The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK -8. After filtered out the optimal conditions, we collected S-ihCECs culture media for 5 days, then prepared conditioned medium to incubate hAECs, inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs. Quantitative real -time reverse transcription -polymerase chain reaction (QRT - PCR) was carried out to evaluate the expression of Oct - 4, NANOG, PAX6, and CK12 in the differentiation period. Immunocytochemistry and western bloting were used to detect the expression of CK3/12. ·RESULTS: The culture media collected every 12h, from 20μg/mL mitomycin pretreatment S -ihCECs could significantly promote the proliferation of hAECs. In the period of differentiation, the morphology of differentiated hAECs was obviously different compared with the control group, and the distinctive CK3/12 for corneal epithelial cells was detected.·CONCLUSION: This study showed that hAECs can differentiate into corneal epithelial -like cells by replication of the corneal epithelial microenvironment, using the culture media collected from S -ihCECs, and it is possible that S -ihCECs culture media could be used in corneal tissue engineering. ·

Immortalized Sertoli cell lines sk11 and sk9 and binding of spermatids in vitro

Aim： To determine the effectiveness of the ski 1, sk9 and ski 1 TNUA5 Sertoli cell lines in binding germ cells in vitro. Methods： The immortalized Sertoli cell lines sk9, ski 1 and ski 1 TNUA5 were used in co-culture experiments with germ cells in media with or without reproductive hormones and incubated for 44 h at 32℃. The number of germ cells bound to Sertoli cells was then determined and statistically analyzed. Western blot analysis and reverse transcriptasepolymerase chain reaction （RT-PCR） studies were employed to investigate the presence of cell adhesion proteins and follicle stimulating hormone （FSH） receptor, respectively. Results： No statistical difference between the number of bound step-8 spermatids and bound pre-step 8 spermatids on Sertoli cells from any of the cell lines existed. After the addition of germ cells, Sertoli ceils showed more lipid accumulation in their cytoplasm, indicating active phagocytosis. Western blot analysis in the ski I TNUA5 line indicated the expression of N-cadherin. FSH-only and testosterone-only treatments increased N-cadherin expression, regardless of germ cell addition. The addition of germ cells to the ski l TNUA5 Sertoli cells increased the expression of espin, as did the addition of FSH with germ cells. RT-PCR studies of the ski I TNUA5 cells indicated that the mRNA for FSH receptor decreased with successive passages. Conclusion： In vitro binding between isolated germ cells and sk9, skll or skll TNUA5 Sertoli cells is not feasible, and therefore these cell lines are not useful for the in vitro investigation of Sertoli-germ cell interactions and primary Sertoli cell isolates must still be used.

Differential Transcriptional Responses in Corneal and Lung Epithelial Cells to Seasonal Influenza With Potential Function of LGALS9

Seasonal flu,primarily caused by influenza A H1N1 and H3N2 subtype viruses or influenza B viruses,is the most prevalent respiratory tract infection globally and leads to substantial morbidity andmortality annually.Despite the influenza virus being initially recognized as a respiratory pathogenwithwell-characterized transmission through respiratory droplets,its impact on the ocular epithelium and associated gene expression remains relatively unexplored.In this study,we investigated the transcriptional profiles of immortalized human corneal epithelial cells(HCE-S)and A549 human lung epithelial cells infected with H1N1 and H3N2 influenza virus.In comparison with A549 cells,a reduced number of differentially expressed geneswas observed in HCE-S upon influenza virus infection.Specifically,there was a significant upregulation of the genes IFI44L and OAS1,along with lower release of the CCL5/RANTES protein.Notably,our findings revealed uniquely upregulated LGALS9(encoding galectin-9)in HCE-S following infection with the 2009 pandemic H1N1 virus.Furthermore,targeted knockdown of LGALS9 in these cells resulted in a measurable decrease in viral infection,highlighting its role in the cellular responses to influenza virus and suggesting a novel avenue for antiviral therapy.Overall,our findings provide insight into the distinct mechanisms of influenza virus interactions with different epithelial cells and underscore the importance of studying the ocular surface in understanding influenza pathogenesis.

Mechanisms of cell immortalization mediated by EB viral activation of telomerase in nasopharyngeal carcinoma

Nasopharyngeal carcinoma （NPC） is a common cancer in Southern China and Southeast Asia. The disease is a poorly differentiated carcinoma without effective cure, and the mechanism underlying its development remains largely unknown. Of several factors identified in NPC aetiology in recent years, Epstein-Barr virus （EBV） infection has emerged to be most important. In almost all NPC cells, EBV uses several intracellular mechanisms to cause oncogenic evolution of the infected cells. One such mechanism by which EBV infection induces cellular immortalization is believed to be through the activation of telomerase, an enzyme that is normally repressed but becomes activated during cancer development. Studies show that greater than 85% of primary NPC display high telomerase activity by mechanisms involving EBV infection, consistent with the notion that EBV is commonly involved in inducing cell immortalization. More recently, different EBV proteins have been shown to activate or inhibit the human telomerase reverse transcriptase gene, by modulating intracellular signalling pathways. These findings suggest a new model with a number of challenges towards our understanding, molecular targeting and therapeutic intervention in NPC.

Clinical features of familial adenomas polyps in Chinese and establishment of its immortal lymphocyte cell lines

AIM： To reserve the rare Chinese familial adenomas polyp （FAP） family resource and to investigate the clinical features of FAP in Chinese for its diagnosis. METHODS： Clinical features of patients with FAP were investigated. If there is any question, their medical records were verified. Blood sample was taken and lymphocyte immortal cell lines were established with modified EB-transformation methods. Congenital hypertrophy of retinal pigment epithelium （CHRPE） was checked by an experienced ophthalmologist. RESULTS： Twenty seven families including 21 classical FAP （CFAP） families, 3 attenuated FAP （AFAP） families, and 3 suspected AFAP families were investigated. A total of 116 lymphocyte immortal cell lines were established from 26 families. In all the FAP families, colorectal cancer occurred at the mean age of 42.84 years. Of the 16 families checked, 15 （93.75%） had CHRPE. The mean number of patients suffering from colorectal neoplasm was 3.14 in CFAP families and 2.0 in AFAP families （P 〈 0.01）. The mean oldest age at diagnosis of FAP was 41.75 years in CFAP families, and 58.67 years in AFAP families, respectively （P 〈 0.01）. Mean age of development of colorectal cancer was 42.23 in CFAP and 57.33 years old in AFAP （P 〈 0.01）. Mean of the earliest age at diagnosis of FAP was 29.95 years in the FAP families with a positive family history and 46.80 years in the FAP families with a negative family history （P 〈 0.01）. The ratio of extra-intestinal tumors to colorectal neoplasms was different in the two kinds of families with positive and negative family history （P 〈 0.01）. CONCLUSION： Additional use of ciclosporin will effectively improve to establish lymphocyte immortal cell lines with modified EB- transformation methods. In Chinese FAP, there was a high frequency of CHRPE, and a later age at diagnosis and a later age of development of colorectal cancer in AFAR And earlier age at diagnosis in FAP with positive family history was also found that will help to diagnose various kinds of FAP in Chinese.

Bone morphogenetic protein-9 effectively induces osteogenic differentiation of reversibly immortalized calvarial mesenchymal progenitor cells

Critical-sized craniofacial defect repair represents a significant challenge to reconstructive surgeons.Many strategies have been employed in an effort to achieve both a functionally and cosmetically acceptable outcome.Bone morphogenetic proteins(BMPs)provide a robust osteoinductive cue to stimulate bony growth and remodeling.Previous studies have suggested that the BMP-9 isoform is particularly effective in promoting osteogenic differentiation of mesenchymal progenitor cells.The aim of this study is to characterize the osteogenic capacity of BMP-9 on calvarial mesenchymal progenitor cell differentiation.Reversibly immortalized murine calvarial progenitor cells(iCALs)were infected with adenoviral vectors encoding BMP-9 or GFP and assessed for early and late stages of osteogenic differentiation in vitro and for osteogenic differentiation via in vivo stem cell implantation studies.Significant elevations in alkaline phosphatase(ALP)activity,osteocalcin(OCN)mRNA transcription,osteopontin(OPN)protein expression,and matrix mineralization were detected in BMP-treated cells compared to control.Specifically,ALP activity was elevated on days 3,7,9,11,and 13 post-infection and OCN mRNA expression was elevated on days 8,10,and 14 in treated cells.Additionally,treatment groups demonstrated increased OPN protein expression on day 10 and matrix mineralization on day 14 post-infection relative to control groups.BMP-9 also facilitated the formation of new bone in vivo as detailed by gross,microcomputed tomography,and histological analyses.Therefore,we concluded that BMP-9 significantly stimulates osteogenic differentiation in iCALs,and should be considered an effective agent for calvarial tissue regeneration.

Comprehensive Characterization and Global Transcriptome Analysis of Human Fetal Liver Terminal Erythropoiesis

The fetal liver(FL)is the key erythropoietic organ during fetal development,but knowledge on human FL erythropoiesis is very limited.In this study,we sorted primary erythroblasts from FL cells and performed RNA sequencing(RNA-seq)analyses.We found that temporal gene expression patterns reflected changes in function during primary human FL terminal erythropoiesis.Notably,the expression of genes enriched in proteolysis and autophagy was up-regulated in orthochromatic erythroblasts(OrthoEs),suggesting the involvement of these pathways in enucleation.We also performed RNA-seq of in vitro cultured erythroblasts derived from FL CD34+cells.Comparison of transcriptomes between the primary and cultured erythroblasts revealed significant differences,indicating impacts of the culture system on gene expression.Notably,the expression of lipid metabolism-related genes was increased in cultured erythroblasts.We further immortalized erythroid cell lines from FL and cord blood(CB)CD34+cells(FL-iEry and CB-iEry,respectively).FL-iEry and CB-iEry were immortalized at the proerythroblast stage and can be induced to differentiate into OrthoEs,but their enucleation ability was very low.Comparison of the transcriptomes between OrthoEs with and without enucleation capability revealed the down-regulation of pathways involved in chromatin organization and mitophagy in OrthoEs without enucleation capacity,indicating that defects in chromatin organization and mitophagy contribute to the inability of OrthoEs to enucleate.Additionally,the expression of HBE1,HBZ,and HBG2 was up-regulated in FL-iEry compared with CB-iEry,and such up-regulation was accompanied by down-regulated expression of BCL11A and up-regulated expression of LIN28B and IGF2BP1.Our study provides new insights into human FL erythropoiesis and rich resources for future studies.