Interplay between mesenchymal stromal cells and the immune system after transplantation: implications for advanced cell therapy in the retina

Advanced mesenchymal stromal cell-based therapies for neurodegenerative diseases are widely investigated in preclinical models.Mesenchymal stromal cells are well positioned as therapeutics because they address the underlying mechanisms of neurodegeneration,namely trophic factor deprivation and neuroinflammation.Most studies have focused on the beneficial effects of mesenchymal stromal cell transplantation on neuronal survival or functional improvement.However,little attention has been paid to the interaction between mesenchymal stromal cells and the host immune system due to the immunomodulatory properties of mesenchymal stromal cells and the long-held belief of the immunoprivileged status of the central nervous system.Here,we review the crosstalk between mesenchymal stromal cells and the immune system in general and in the context of the central nervous system,focusing on recent work in the retina and the importance of the type of transplantation.

Exosomes from umbilical cord mesenchymal stromal cells promote the collagen production of fibroblasts from pelvic organ prolapse

BACKGROUND Pelvic organ prolapse(POP)involves pelvic organ herniation into the vagina due to pelvic floor tissue laxity,and vaginal structure is an essential factor.In POP,the vaginal walls exhibit abnormal collagen distribution and decreased fibroblast levels and functions.The intricate etiology of POP and the prohibition of trans-vaginal meshes in pelvic reconstruction surgery present challenges in targeted therapy development.Human umbilical cord mesenchymal stromal cells(hucMSCs)present limitations,but their exosomes(hucMSC-Exo)are promising therapeutic tools for promoting fibroblast proliferation and extracellular matrix remodeling.suppressed inflammation in POP group fibroblasts,stimulated primary fibroblast growth,and elevated collagen I(Col1)production in vitro.High-throughput RNA-seq of fibroblasts treated with hucMSC-Exo and miRNA sequencing of hucMSC-Exo revealed that abundant exosomal miRNAs downregulated matrix metalloproteinase 11(MMP11)expression.CONCLUSION HucMSC-Exo normalized the growth and function of primary fibroblasts from patients with POP by promoting cell growth and Col1 expression in vitro.Abundant miRNAs in hucMSC-Exo targeted and downregulated MMP11 expression.HucMSC-Exo-based therapy may be ideal for safely and effectively treating POP.

Mesenchymal stromal cells modulate unfolded protein response and preserve β-cell mass in type 1 diabetes

Introduction:Transplantation of mesenchymal stromal cells(MSCs)is a promising therapy for type 1 diabetes(T1D).However,whether the infused MSCs affect the endoplasmic reticulum stress or subsequent unfolded protein response inβcells remains unclear.Methods:To investigate this,we induced early-onset T1D in non-obese diabetic mice using streptozotocin.Subsequently,T1D mice were randomly assigned to receive either MSCs or phosphate-buffered saline.We observed the in vivo homing of MSCs and assessed their effectiveness by analyzing blood glucose levels,body weight,histopathology,pancreatic protein expression,and serum levels of cytokines,proinsulin,and C-peptide.Results:Infused MSCs were found in the lungs,liver,spleen,and pancreas of T1D mice.They exhibited various effects,including reducing blood glucose levels,regulating immunity,inhibiting inflammation,increasingβ-cell areas,and reducing the expression of key proteins in the unfolded protein response pathway.Fasting serum proinsulin and C-peptide levels were significantly higher in the MSCs treatment group than in the T1D model group.However,there was no significant difference in the biomarker ofβ-cell endoplasmic reticulum stress,the ratio of fasting serum proinsulin to C-peptide,between the two groups.Conclusion:Ourfindings reveal that MSCs infusion does not alleviate endoplasmic reticulum stress inβcells directly but modulates the unfolded protein response pathway to preserveβ-cell mass and function in T1D mice.

Human dental pulp stem/stromal cells in clinical practice

Dental pulp stem/stromal cells(DPSCs)are fibroblast-like,neural crest-derived,and multipotent cells that can differentiate into several lineages.They are relatively easy to isolate from healthy and inflamed pulps,with little ethical concerns and can be successfully cryopreserved and thawed.The therapeutic effects of DPSCs derived from animal or human sources have been extensively studied through in-vitro and in-vivo animal experiments and the findings indicated that DPSCs are effective not only for dental diseases but also for systemic diseases.Understanding that translational research is a critical step through which the fundamental scientific discoveries could be translated into applicable diagnostics and therapeutics that directly benefit humans,several clinical studies were carried out to generate evidence for the efficacy and safety of autogenous or allogeneic human DPSCs(hDPSCs)as a treatment modality for use in cell-based therapy,regenerative medicine/dentistry and tissue engineering.In clinical medicine,hDPSCs were effective for treating acute ischemic stroke and human exfoliated deciduous teeth-conditioned medium(SHED-CM)repaired vascular damage of the corpus cavernous,which is the main cause of erectile dysfunction.Whereas in clinical dentistry,autologous SHED was able to rege-nerate necrotic dental pulp after implantation into injured teeth,and micrografts enriched with autologous hDPSCs and collagen sponge were considered a treatment option for human intrabony defects.In contrast,hDPSCs did not add a significant regenerative effect when they were used for the treatment of post-extraction sockets.Large-scale clinical studies across diverse populations are still lacking to provide robust evidence on the safety and efficacy of hDPSCs as a new treatment option for various human diseases including dental-related problems.

O-linkedβ-N-acetylglucosaminylation may be a key regulatory factor in promoting osteogenic differentiation of bone marrow mesenchymal stromal cells

Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.

Mesenchymal Stromal Cells and Their Uses in Bio-Regenerative Therapies for Bone and Cartilage: A Review

Mesenchymal stromal cells (MSCs) are a top candidate for new clinical treatments in the repair of bone and cartilage. In several clinical trials, they have shown reliable, effective, and safe management of inflammation, pain, and the regenerative capabilities of resident tissues. MSCs are likely derived from pericytes. They modulate the environment they are placed in by secreting immunomodulatory and signaling molecules to reduce inflammation and direct resident cells to create new tissues. They are easily isolated from several different adult tissues, and inexpensive to grow in a lab. However, a mistake made in the initial classification of MSCs as stem cells has created deeply engrained misconceptions that are still evident today. MSCs are not stem cells, despite a large fraction of research and therapies using the name “mesenchymal stem cells”. This mistake creates false narratives attributing the observed positive outcomes of MSC treatments to stem cell characteristics, which has led to distrust in MSC research. Despite inconsistencies in their classification, MSCs demonstrate consistent positive effects in numerous animal studies and human clinical trials for non-unions and osteoarthritis. With an aging population, regenerative techniques are very promising for novel therapies. To produce trusted and safe new treatments using MSCs, it is essential for the International Society for Cellular Therapies to re-establish common ground in the identity, mechanism of action, and isolation techniques of these cells.

Overexpression of lentivirus-mediated glial cell line-derived neurotrophic factor in bone marrow stromal cells and its neuroprotection for the PC12 cells damaged by lactacystin

Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.

Adipose-derived stromal cells resemble bone marrow stromal cells in hepatocyte differentiation potential in vitro and in vivo

AIM To investigate whether mesenchymal stem cells(MSCs) from adipose-derived stromal cells(ADSCs) and bone marrow stromal cells(BMSCs) have similar hepatic differentiation potential.METHODS Mouse ADSCs and BMSCs were isolated and cultured. Their morphological and phenotypic characteristics, as well as their multiple differentiation capacity were compared. A new culture system was established to induce ADSCs and BMSCs into functional hepatocytes. Reverse transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to identify the induced hepatocytelike cells. CM-Dil-labeled ADSCs and BMSCs were then transplanted into a mouse model of CCl4-induced acute liver failure. fluorescence microscopy was used to track the transplanted MSCs. Liver function was tested by an automatic biochemistry analyzer, and liver tissue histology was observed by hematoxylin and eosin(HE) staining.RESULTS ADSCs and BMSCs shared a similar morphology and multiple differentiation capacity, as well as a similar phenotype(with expression of CD29 and CD90 and no expression of CD11 b or CD45). Morphologically, ADSCs and BMSCs became round and epithelioid following hepatic induction. These two cell types differentiated into hepatocyte-like cells with similar expression of albumin, cytokeratin 18, cytokeratin 19, alpha fetoprotein, and cytochrome P450. fluorescence microscopy revealed that both ADSCs and BMSCs were observed in the mouse liver at different time points. Compared to the control group, both the function of the injured livers and HE staining showed significant improvement in the ADSC-and BMSC-transplanted mice. There was no significant difference between the two MSC groups.CONCLUSION ADSCs share a similar hepatic differentiation capacity and therapeutic effect with BMSCs in an acute liver failure model. ADSCs may represent an ideal seed cell type for cell transplantation or a bio-artificial liver support system.

EXPRESSION OF rhBMP-7 GENE IN TRANSDUCED BONE MARROW DERIVED STROMAL CELLS

OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. RESULTS: The exogenous gene could be expressed efficiently in transduced BMSCs. CONCLUSION: The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.

Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro.However,because of the short survival time of the differentiated cells,clinical applications for this technique are limited.As such,we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy.The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended.Taken together,these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death.However,the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

Tumor formation of prostate cancer cells influenced by stromal cells from the transitional or peripheral zones of the normal prostate

This study was designed to investigate the different involvements of prostatic stromal cells from the normal transitional zone(TZ)or peripheral zone(PZ)in the carcinogenesis of prostate cancer(PCa)epithelial cells(PC-3)in vitro and in vivo co-culture models.Ultra-structures and gene expression profiles of primary cultures of human prostatic stromal cells from the normal TZ or PZ were analyzed by electron microscopy and microarray analysis.In vitro and in vivo co-culture models composed of normal TZ or PZ stromal cells and human PCa PC-3 cells were established.We assessed tumor growth and weight in the in vivo nude mice model.There are morphological and ultra-structural differences in stromal cells from TZ and PZ of the normal prostate.In all,514 differentially expressed genes were selected by microarray analysis;483 genes were more highly expressed in stromal cells from TZ and 31 were more highly expressed in those from PZ.Co-culture with PZ stromal cells and transforming growth factor-β1(TGF-β1)increased the tumor growth of PC-3 cells in vitro and in vivo,as well as Bcl-2 expression.On the other hand,stromal cells of TZ suppressed PC-3 cell tumor growth in the mouse model.We conclude that ultra-structures and gene expression differ between the stromal cells from TZ or PZ of the normal prostate,and stroma-epithelium interactions from TZ or PZ might be responsible for the distinct zonal localization of prostate tumor formation.

Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells

β-mercaptoethanol induces in vitro adult adipose-derived stromal cells （ADSCs） to differentiate into neurons. However, the ultrastructural features of the differentiated neuronal-like cells remain unknown. In the present study, inverted phase contrast microscopy was utilized to observe β-mercaptoethanol-induced differentiation of neuronal-like cells from human ADSCs, and immunocytochemistry and real-time polymerase chain reaction were employed to detect expression of a neural stem cells marker （nestin）, a neuronal marker （neuron-specific enolase）, and a glial marker （glial fibrillary acidic protein）. In addition, ultrastructure of neuronal-like cells was observed by transmission election microscopy. Results revealed highest expression rate of nestin and neuron-specific enolase at 3 and 5 hours following induced differentiation; cells in the 5-hour induction group exhibited a neuronal-specific structure, i.e., Nissl bodies. However, when induction solution was replaced by complete culture medium after 8-hour induction, the differentiated cells reverted to the fibroblast-like morphology from day 1. These results demonstrate that β-mercaptoethanol-induced ADSCs induced differentiation into neural stem cells, followed by morphology of neuronal-like cells. However, this differentiation state was not stable.

Over-expression of VEGF in Marrow Stromal Cells Promotes Angiogenesis in Rats with Cerebral Infarction via the Synergistic Effects of VEGF and Ang-2

This study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction.MSCs were isolated by using a direct adherent method and cultured.Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection.The transfection efficiency was measured by the optical density method.The protein expression of VEGF gene before and after transfection was measured by Western blotting.SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion.Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction.ELISAs were used to measure the levels of VEGF in the rat cerebral tissues.The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically oserved.Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting.VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%.ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction,peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days.The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day.The immunohistochemical results showed that 10 days after the infarction,the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than the MSC group.A similar trend in Ang-2 protein expression was revealed by Western blotting.In the MCAO rat model transfected with modified MSCs over-expressing VEGF,compared to the MSC transplantation group,the concentration of VEGF was significantly increased in the brain tissue after cerebral infarction.In addition,the level of Ang-2 was up-regulated,with angiogenesis promoted,the blood supply to the areas surrounding the cerebral infarction increased,and neurological function improved.We are led to speculate that the synergistic effects of VEGF and Ang-2 may be responsible for the angiogenesis following cerebral infarction.

Proliferation and phenotypic changes of stromal cells in response to varying estrogen/androgen levels in castrated rats

It is known that human benign prostatic hyperplasia might arise from an estrogen/androgen （E/T） imbalance. We studied the response of castrated rat prostate to different ratios of circulating E/T. The castrated male Wistar rats were randomly injected with E/T at different ratios for 4 weeks. The prostates of E/T （1：100） group showed a distinct prostatic hyperplasia response by prostatic index, hematoxylin and eosin staining, and quantitative immunohistochemical analysis of a-smooth muscle actin （SMA）. In this group, cells positive for Vimentin, non-muscle myosin heavy chain （NMMHC） and proliferating cell nuclear antigen （PCNA） increased in the stroma and epithelium. Furthermore, the mRNA levels of smooth muscle myosin heavy chain （SMMHC） and NMMHC increased. So E/T at a ratio of 1：100 can induce a stromal hyperplastic response in the prostate of castrated rats. The main change observed was an increase of smooth muscle cells, whereas some epithelial changes were also seen in the rat prostates.

A novel ethanol-based method to induce differentiation of adipose-derived stromal cells into astrocytes

The quantity and survival time of astrocytes,which were differentiated from adult adipose-derived stromal cells after exposure to an inducer containing 3-isobutyl-1-methylxanthine,have thus far been unsatisfactory.The present study investigated the growth and differentiation characteristics of induced astrocytes by observing their growth curves.After induction for 48 hours with an inducer containing 0.5% ethanol,some adult adult adipose-derived stromal cells displayed typical astrocytic morphology.The cell quantity gradually decreased with prolonged induction time.Nestin,glial fibrillary acidic protein,and S-100 expression reached peak levels at 14 days,but neuron-specific enolase was not expressed.These results suggest that the induced astrocytes reached their peak at 14 days.Further optimization of the culture environment may yield mature astrocytes with normal functions,in greater quantity,and prolonged survival time.

Adult adipose-derived stromal cells differentiate into neurons with normal electrophysiological functions

β-mercaptoethanol was used to induce in vitro neuronal differentiation of adipose-derived stromal cells. Within an 8-hour period post-differentiation, the induced cells exhibited typical neuronal morphology, and expression of microtubule-associated protein 2 and neuron-specific enolase, which are markers of mature neurons, reached a peak at 5 hours. Specific organelle Nissl bodies of neurons were observed under transmission electron microscopy. Results of membrane potential showed that fluorescence intensity of cells was greater after 5 hours than adipose-derived stromal cells prior to induction. In addition, following stimulation with high-concentration potassium solution, fluorescence intensity increased. These experimental findings suggested that neurons differentiated from adipose-derived stromal cells and expressed mature K^＋ channels. In addition, following stimulation with high potassium solution, the membrane potential depolarized and fired an action potential, confirming that the induced cells possessed electrophysiological functions.

Millimeter-wave Exposure Promotes the Differentiation of Bone Marrow Stromal Cells into Cells with a Neural Phenotype

This study investigated the ability of millimeter-wave （MMW） to promote the differentiation of bone marrow stromal cells （BMSCs） into cells with a neural phenotype. The BMSCs were primarily cultured. At passage 3, the cells were induced by β-mercaptoethanol （BME） in combination with MMW or BME alone. The expressions of nucleostemin （NS） and neuron-specific enolase （NSE） were detected by immunofluorescent staining and Western blotting respectively to identify the differentiation. The untreated BMSCs predominately expressed NS. After induced by BME and MMW, the BMSCs exhibited a dramatic decrease in NS expression and increase in NSE expression. The differentiation rate of the cells treated with BME and MMW in combination was significantly higher than that of the cells treated with BME alone （P〈0.05）. It was concluded that MMW exposure enhanced the inducing effect of BME on the differentiation of BMSCs into cells with a neural phenotype.

Comparison of human amniotic fluid-derived and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells： Characterization and myocardial differentiation capacity

Objective To compare the characterization and myocardial differentiation capacity of arnniotic fluid-derived mesenchymal stromal cells （AF MSCs） and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells （WJ MSCs）. Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton＇s Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes （GATA-4, c-TnT, a-actin, and Cx43） were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities ofWJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility （MHC I） antigens （HLA-ABC）, and were negative, or mildly positive, for MHC Class II （HLA-DR） antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, a-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.

Differences in phenotype and gene expression of prostate stromal cells from patients of varying ages and their influence on tumour formation by prostate epithelial cells

Prostate cancer （PCa） is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and old tissue are still obscure. We established primary cultured stromal cells from normal prostatic peripheral zone （PZ） of donors of varying ages and found that cultured stromal cells from old donors （PZ-old） were more enlarged and polygonal than those from young donors （PZ-young）. Furthermore, based on immunocytochemical and ultrastructural analysis, the components of stromal cells changed from a majority of fibroblasts to a mixture of fibroblasts and myofibroblasts with increasing donor age. Using a three-dimensional in vitro culture system, we found that PZ-old stromal cells could enhance the proliferation, migration and invasion of cocultured benign BPH-1 and PC-3 cells. Using an in vivo tissue recombination system, we also found that PZ-old stromal cells are more effective than PZ-young cells in promoting tumour formation by BPH-1 cells of high passage（〉100） and PC-3 cells. To probe the possible mechanism of these effects, we performed cDNA microarray analysis and profiled 509 upregulated genes and 188 downregulated genes in PZ-old cells. Among the changed genes, we found genes coding for a subset of paracrine factors that are capable of influencing adjacent epithelial cells; these include hepatocyte growth factor （HGF）, fibroblast growth factor 5 （FGF5）, insulin-like growth factor 2 （IGF2）, insulin-like growth factor-binding protein 4 （IGFBP4）, IGFBP5 and matrix metallopeptidase 1 （MMP1）. Changes in the expression of these genes were further confirmed by quantitative real-time polymerase chain reaction （PCR）, Western blotting and enzyme-linked immunosorbent assays. Overall, our findings indicate that stromal cells from prostate PZ of old donors are more active than similar cells from young donors in promoting the malignant process of adjacent epithelial cells. This finding hints at a new potential strategy for the prevention of PCa.

Autophagy and apoptosis during adult adipose-derived stromal cells differentiation into neuron-like cells in vitro

β-mercaptoethanol can induce adult adipose-derived stromal cells to rapidly and efficiently differentiate into typical neuron-like cells in vitro. Immunohistochemistry showed that neuron specific enolase and neurofilament-200 expression gradually increased with the extension of induction time, and peaked at 5 hours. By contrast, glial fibrillary acidic protein was negatively expressed at all time points. Induced cells possessed a typical Nissl body, apoptosis showing condensed chromatin in the nucleus, autophagosomes with a bilayered membrane and autolysosomes in the cytoplasm at 5 hours. TUNEL assay and immunohistochemistry and immunofluorescence demonstrated that apoptosis and caspase-3 expression increased and peaked at 8 hours. Immunohistochemistry and immunofluorescence showed that microtubuleassociated protein light chain 3 gradually increased with induction and reached a peak at 5 hours These results indicate that autophagy played an important role in protecting cells during adult adipose-derived stromal cells differentiation into neuron-like cells in vitro.