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.

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.

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.

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.

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.

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.

Anti-fibrotic and anti-inflammatory effect of mesenchymal stromal cell-derived extracellular vesicles in chronic kidney disease

Renal fibrosis and inflammation are common pathological features of chronic kidney disease(CKD).Since currently available treatments can only delay the progression of CKD,the outcome of patients with CKD is still poor.One therapeutic option for the prevention of CKD-related complications could be the use of mesenchymal stromal cells(MSCs),which have shown beneficial effects in tissue fibrosis and regeneration after damage.However,safety issues,such as cellular rejection and carcinogenicity,limit their clinical application.Among the bioactive factors secreted by MSCs,extracellular vesicles(EVs)have shown the same beneficial effect of MSCs,without any notable side effects.This heterogeneous population of membranous nano-sized particles can deliver genetic material and functional proteins to injured cells,prompting tissue regeneration.Here we describe the anti-fibrotic and antiinflammatory properties of MSC-derived EVs in CKD preclinical models and summarize the potential molecular mechanisms involved in the regulation of renal fibrosis and inflammation.

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.

Amniotic membrane mesenchymal stromal cell-derived secretome in the treatment of acute ischemic stroke:A case report

BACKGROUND Stroke is the second and third leading cause of death and disability,respectively.To date,no definitive treatment can repair lost brain function.Recently,various preclinical studies have been reported on mesenchymal stromal cells(MSCs)and their derivatives and their potential as alternative therapies for stroke.CASE SUMMARY A 45-year-old female suffered an acute stroke,which led to paralysis in the left upper and lower limbs.The amniotic membrane MSC-derived secretome(MSCsecretome)was intravenously transplanted once a week for 4 wk.MSC-secretomeregulated regulatory T cells were investigated for the beneficial effects.The clinical improvement of this patient was accompanied by an increased frequency of regulatory T cells after transplantation.CONCLUSION Intravenous administration of MSC-secretome can potentially treat patients who suffer from acute ischemic stroke.

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.

Therapeutic Approach for Hair Growth and Regeneration Using Bioactive Formulation Containing Mesenchymal Stromal Cell-Derived Conditioned Medium

Background and Aims: Androgenetic alopecia (AGA) is a common form of hair loss in both men and women. Despite its high prevalence and associated patient morbidity, the approved therapeutic options are limited to finasteride and minoxidil. The present study is aimed at assessing the efficacy of hair serum formulation, Trichosera®containing Bone marrow-derived mesenchymal stromal cells conditioned media as an active ingredient, for hair fall control and hair regrowth in healthy Indian human volunteers. Methods: The product was made using a 20% concentration of 10X Conditioned Media along with excipients. The final product was tested for physicochemical parameters, biomarkers, total protein content and microbial limits as per our in-house specifications. Results: The primary irritation patch test showed that the product is non-irritant and dermatologically safe. A clinical study on 40 subjects was conducted to evaluate the effectiveness of the bioactive formulation in hair fall control and hair regrowth in healthy volunteers. Phototrichogram measurement showed hair density and hair growth rate increased significantly by 11.54% and 18.66% at week 24. Hair tensile strength also increased significantly by 41.10% at 12 weeks follow-up. Hair pull test, to see a reduction in pulled hair and comb’s test to show a decrease in hair fall significantly improved from week 4 onwards. There were no significant adverse events in response to the product application. Conclusion: It is concluded that the hair serum product is completely safe on direct application to the scalp and showed significant improvement in the hair growth rate, hair density, scalp condition and reduction in hair fall. .

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.

Exosomal microRNAs from mesenchymal stem/stromal cells: Biology and applications in neuroprotection

Mesenchymal stem/stromal cells(MSCs)are extensively studied as cell-therapy agents for neurological diseases.Recent studies consider exosomes secreted by MSCs as important mediators for MSCs’neuroprotective functions.Exosomes transfer functional molecules including proteins,lipids,metabolites,DNAs,and coding and non-coding RNAs from MSCs to their target cells.Emerging evidence shows that exosomal microRNAs(miRNAs)play a key role in the neuroprotective properties of these exosomes by targeting several genes and regulating various biological processes.Multiple exosomal miRNAs have been identified to have neuroprotective effects by promoting neurogenesis,neurite remodeling and survival,and neuroplasticity.Thus,exosomal miRNAs have significant therapeutic potential for neurological disorders such as stroke,traumatic brain injury,and neuroinflammatory or neurodegenerative diseases and disorders.This review discusses the neuroprotective effects of selected miRNAs(miR-21,miR-17-92,miR-133,miR-138,miR-124,miR-30,miR146a,and miR-29b)and explores their mechanisms of action and applications for the treatment of various neurological disease and disorders.It also provides an overview of state-of-the-art bioengineering approaches for isolating exosomes,optimizing their yield and manipulating the miRNA content of their cargo to improve their therapeutic potential.

Transcriptomic alterations underline aging of osteogenic bone marrow stromal cells

BACKGROUND Multipotent bone marrow stromal cells(BMSCs)are adult stem cells that form functional osteoblasts and play a critical role in bone remodeling.During aging,an increase in bone loss and reduction in structural integrity lead to osteoporosis and result in an increased risk of fracture.We examined age-dependent histological changes in murine vertebrae and uncovered that bone loss begins as early as the age of 1 mo.AIM To identify the functional alterations and transcriptomic dynamics of BMSCs during early bone loss.METHODS We collected BMSCs from mice at early to middle ages and compared their selfrenewal and differentiation potential.Subsequently,we obtained the transcriptomic profiles of BMSCs at 1 mo,3 mo,and 7 mo.RESULTS The colony-forming and osteogenic commitment capacity showed a comparable finding that decreased at the age of 1 mo.The transcriptomic analysis showed the enrichment of osteoblastic regulation genes at 1 mo and loss of osteogenic features at 3 mo.The BMSCs at 7 mo showed enrichment of adipogenic and DNA repair features.Moreover,we demonstrated that the WNT and MAPK signaling pathways were upregulated at 1 mo,followed by increased pro-inflammatory and apoptotic features.CONCLUSION Our study uncovered the cellular and molecular dynamics of bone aging in mice and demonstrated the contribution of BMSCs to the early stage of age-related bone loss.

Bone Marrow Stromal Cells Express Neural Phenotypes in vitro and Migrate in Brain After Transplantation in vivo

Objective To investigate the differentiation of bone marrow stromal cells （BMSC） into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC （hBMSC） to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilamentl （NF1）, nestin and neuron-specific enolase （NSE） in rat BMSC （rBMSC）. Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution. Results rBMSC expressed NSE, NFI and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S 100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated, rBMSC could migrate and adapted in the host brains after being transplanted. Conclusion Bone marrow stromal cells could express phenotypes of neurons, and Salvia milliorrhiza could induce hBMSC to differentiate into neuron-like cells, If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.

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.

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.