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.

Is mandible derived mesenchymal stromal cells superior in proliferation and regeneration to long bone-derived mesenchymal stromal cells?

Mesenchymal stromal cells(MSCs)are cells with the characteristic ability of self-renewal along with the ability to exhibit multilineage differentiation.Bone marrow(BM)is the first tissue in which MSCs were identified and BM-MSCs are most commonly used among various MSCs in clinical settings.MSCs can stimulate and promote osseous regeneration.Due to the difference in the development of long bones and craniofacial bones,the mandibular-derived MSCs(M-MSCs)have distinct differentiation characteristics as compared to that of long bones.Both mandibular and long bone-derived MSCs are positive for MSC-associated markers such as CD-73,-105,and-106,stage-specific embryonic antigen 4 and Octamer-4,and negative for hematopoietic markers such as CD-14.

Commitment of human mesenchymal stromal cells to skeletal lineages is independent of their morphogenetic capacity

BACKGROUND Mesenchymal stromal cells(MSCs)are multipotent cell populations obtained from fetal and adult tissues.They share some characteristics with limb bud mesodermal cells such as differentiation potential into osteogenic,chondrogenic,and tenogenic lineages and an embryonic mesodermal origin.Although MSCs differentiate into skeletal-related lineages in vitro,they have not been shown to selforganize into complex skeletal structures or connective tissues,as in the limb.In this work,we demonstrate that the expression of molecular markers to commit MSCs to skeletal lineages is not sufficient to generate skeletal elements in vivo.AIM To evaluate the potential of MSCs to differentiate into skeletal lineages and generate complex skeletal structures using the recombinant limb(RL)system.METHODS We used the experimental system of RLs from dissociated-reaggregated human placenta(PL)and umbilical cord blood(UCB)MSCs.After being harvested and reaggregated in a pellet,cultured cells were introduced into an ectodermal cover obtained from an early chicken limb bud.Next,this filled ectoderm was grafted into the back of a donor chick embryo.Under these conditions,the cells received and responded to the ectoderm’s embryonic signals in a spatiotemporal manner to differentiate and pattern into skeletal elements.Their response to differentiation and morphogenetic signals was evaluated by quantitative poly-merase chain reaction,histology,immunofluorescence,scanning electron microscopy,and in situ hybridization.RESULTS We found that human PL-MSCs and UCB-MSCs constituting the RLs expressed chondrogenic,osteogenic,and tenogenic molecular markers while differentially committing into limb lineages but could not generate complex structures in vivo.MSCs-RL from PL or UCB were committed early to chondrogenic lineage.Nevertheless,the UCB-RL osteogenic commitment was favored,although preferentially to a tenogenic cell fate.These findings suggest that the commitment of MSCs to differentiate into skeletal lineages differs according to the source and is independent of their capacity to generate skeletal elements or connective tissue in vivo.Our results suggest that the failure to form skeletal structures may be due to the intrinsic characteristics of MSCs.Thus,it is necessary to thoroughly evaluate the biological aspects of MSCs and how they respond to morphogenetic signals in an in vivo context.CONCLUSION PL-MSCs and UCB-MSCs express molecular markers of differentiation into skeletal lineages,but they are not sufficient to generate complex skeletal structures in vivo.

Senescent mesenchymal stem/stromal cells in pre-metastatic bone marrow of untreated advanced breast cancer patients

Breast cancer is the predominant form of carcinoma among women worldwide,with 70%of advanced patients developing bone metastases,with a high mortality rate.In this sense,the bone marrow(BM)mesenchymal stem/stromal cells(MSCs)are critical for BM/bone homeostasis,and failures in their functionality,transform the BM into a premetastatic niche(PMN).We previously found that BM-MSCs from advanced breast cancer patients(BCPs,infiltrative ductal carcinoma,stage III-B)have an abnormal profile.This work aims to study some of the metabolic and molecular mechanisms underlying MSCs shift from a normal to an abnormal profile in this group of patients.A comparative analysis was undertaken,which included self-renewal capacity,morphology,proliferation capacity,cell cycle,reactive oxygen species(ROS)levels,and senescence-associatedβ‑galactosidase(SA‑β‑gal)staining of BMderived MSCs isolated from 14 BCPs and 9 healthy volunteers(HVs).Additionally,the expression and activity of the telomerase subunit TERT,as well as telomere length,were measured.Expression levels of pluripotency,osteogenic,and osteoclastogenic genes(OCT-4,SOX-2,M-CAM,RUNX-2,BMP-2,CCL-2,M-CSF,and IL-6)were also determined.The results showed that MSCs from BCPs had reduced,self-renewal and proliferation capacity.These cells also exhibited inhibited cell cycle progression and phenotypic changes,such as an enlarged and flattened appearance.Additionally,there was an increase in ROS and senescence levels and a decrease in the functional capacity of TERT to preserve telomere length.We also found an increase in pro-inflammatory/pro-osteoclastogenic gene expression and a decrease in pluripotency gene expression.We conclude that these changes could be responsible for the abnormal functional profile that MSCs show in this group of patients.

Harnessing and honing mesenchymal stem/stromal cells for the amelioration of graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation is a deterministic curative procedure for various hematologic disorders and congenital immunodeficiency.Despite its increased use,the mortality rate for patients undergoing this procedure remains high,mainly due to the perceived risk of exacerbating graft-versushost disease(GVHD).However,even with immunosuppressive agents,some patients still develop GVHD.Advanced mesenchymal stem/stromal cell(MSC)strategies have been proposed to achieve better therapeutic outcomes,given their immunosuppressive potential.However,the efficacy and trial designs have varied among the studies,and some research findings appear contradictory due to the challenges in characterizing the in vivo effects of MSCs.This review aims to provide real insights into this clinical entity,emphasizing diagnostic,and therapeutic considerations and generating pathophysiology hypotheses to identify research avenues.The indications and timing for the clinical application of MSCs are still subject to debate.

Constitutive aryl hydrocarbon receptor facilitates the regenerative potential of mouse bone marrow mesenchymal stromal cells

BACKGROUND Bone marrow mesenchymal stromal cells(BMSCs)are the commonly used seed cells in tissue engineering.Aryl hydrocarbon receptor(AhR)is a transcription factor involved in various cellular processes.However,the function of constitutive AhR in BMSCs remains unclear.AIM To investigate the role of AhR in the osteogenic and macrophage-modulating potential of mouse BMSCs(mBMSCs)and the underlying mechanism.METHODS Immunochemistry and immunofluorescent staining were used to observe the expression of AhR in mouse bone marrow tissue and mBMSCs.The overexpression or knockdown of AhR was achieved by lentivirus-mediated plasmid.The osteogenic potential was observed by alkaline phosphatase and alizarin red staining.The mRNA and protein levels of osteogenic markers were detected by quantitative polymerase chain reaction(qPCR)and western blot.After coculture with different mBMSCs,the cluster of differentiation(CD)86 and CD206 expressions levels in RAW 264.7 cells were analyzed by flow cytometry.To explore the underlying molecular mechanism,the interaction of AhR with signal transducer and activator of transcription 3(STAT3)was observed by co-immunoprecipitation and phosphorylation of STAT3 was detected by western blot.RESULTS AhR expressions in mouse bone marrow tissue and isolated mBMSCs were detected.AhR overexpression enhanced the osteogenic potential of mBMSCs while AhR knockdown suppressed it.The ratio of CD86+RAW 264.7 cells cocultured with AhR-overexpressed mBMSCs was reduced and that of CD206+cells was increased.AhR directly interacted with STAT3.AhR overexpression increased the phosphorylation of STAT3.After inhibition of STAT3 via stattic,the promotive effects of AhR overexpression on the osteogenic differentiation and macrophage-modulating were partially counteracted.CONCLUSION AhR plays a beneficial role in the regenerative potential of mBMSCs partially by increasing phosphorylation of STAT3.

Comparative effectiveness of adipose-derived mesenchymal stromal cells in the management of knee osteoarthritis:A meta-analysis

BACKGROUND Osteoarthritis(OA)is the most common joint disorder,is associated with an increasing socioeconomic impact owing to the ageing population.AIM To analyze and compare the efficacy and safety of bone-marrow-derived mesenchymal stromal cells(BM-MSCs)and adipose tissue-derived MSCs(AD-MSCs)in knee OA management from published randomized controlled trials(RCTs).METHODS Independent and duplicate electronic database searches were performed,including PubMed,EMBASE,Web of Science,and Cochrane Library,until August 2021 for RCTs that analyzed the efficacy and safety of AD-MSCs and BM-MSCs in the management of knee OA.The visual analog scale(VAS)score for pain,Western Ontario McMaster Universities Osteoarthritis Index(WOMAC),Lysholm score,Tegner score,magnetic resonance observation of cartilage repair tissue score,knee osteoarthritis outcome score(KOOS),and adverse events were analyzed.Analysis was performed on the R-platform using OpenMeta(Analyst)software.Twenty-one studies,involving 936 patients,were included.Only one study compared the two MSC sources without patient randomization;hence,the results of all included studies from both sources were pooled,and a comparative critical analysis was performed.RESULTS At six months,both AD-MSCs and BM-MSCs showed significant VAS improvement(P=0.015,P=0.012);this was inconsistent at 1 year for BM-MSCs(P<0.001,P=0.539),and AD-MSCs outperformed BM-MSCs compared to controls in measures such as WOMAC(P<0.001,P=0.541),Lysholm scores(P=0.006;P=0.933),and KOOS(P=0.002;P=0.012).BM-MSC-related procedures caused significant adverse events(P=0.003)compared to AD-MSCs(P=0.673).CONCLUSION Adipose tissue is superior to bone marrow because of its safety and consistent efficacy in improving pain and functional outcomes.Future trials are urgently warranted to validate our findings and reach a consensus on the ideal source of MSCs for managing knee OA.

Banking of perinatal mesenchymal stem/stromal cells for stem cellbased personalized medicine over lifetime:Matters arising

Mesenchymal stromal/stem cells(MSCs)are currently applied in regenerative medicine and tissue engineering.Numerous clinical studies have indicated that MSCs from different tissue sources can provide therapeutic benefits for patients.MSCs derived from either human adult or perinatal tissues have their own unique advantages in their medical practices.Usually,clinical studies are conducted by using of cultured MSCs after thawing or short-term cryopreserved-then-thawed MSCs prior to administration for the treatment of a wide range of diseases and medical disorders.Currently,cryogenically banking perinatal MSCs for potential personalized medicine for later use in lifetime has raised growing interest in China as well as in many other countries.Meanwhile,this has led to questions regarding the availability,stability,consistency,multipotency,and therapeutic efficiency of the potential perinatal MSC-derived therapeutic products after longterm cryostorage.This opinion review does not minimize any therapeutic benefit of perinatal MSCs in many diseases after short-term cryopreservation.This article mainly describes what is known about banking perinatal MSCs in China and,importantly,it is to recognize the limitation and uncertainty of the perinatal MSCs stored in cryobanks for stem cell medical treatments in whole life.This article also provides several recommendations for banking of perinatal MSCs for potentially future personalized medicine,albeit it is impossible to anticipate whether the donor will benefit from banked MSCs during her/his lifetime.

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.