New insights into the biological roles of immune cells in neural stem cells in post-traumatic injury of the central nervous system

Traumatic injuries in the central nervous system,such as traumatic brain injury and spinal cord injury,are associated with tissue inflammation and the infiltration of immune cells,which simultaneously affect the self-renewal and differentiation of neural stem cells.Howeve r,the tissue repair process instigated by endogenous neural stem cells is incapable of restoring central nervous system injuries without external intervention.Recently,resident/peripheral immune cells have been demonstrated to exert significant effects on neural stem cells.Thus,the resto ration of traumatic injuries in the central nervous system by the immune intervention in neural stem cells represents a potential therapeutic method.In this review,we discuss the roles and possible mechanisms of immune cells on the selfrenewal and differentiation of neural stem cells along with the prognosis of central nervous system injuries based on immune intervention.Finally,we discuss remaining research challenges that need to be considered in the future.Further elucidation of these challenges will fa cilitate the successful application of neural stem cells in central nervous system injuries.

Intranasal administration of stem cell-derived exosomes for central nervous system diseases

Exosomes,lipid bilayer-enclosed small cellular vesicles,are actively secreted by various cells and play crucial roles in intercellular communication.These nanosized vesicles transport internalized proteins,mRNA,miRNA,and other bioactive molecules.Recent findings have provided compelling evidence that exosomes derived from stem cells hold great promise as a therapeutic modality for central nervous system disorders.These exosomes exhibit multifaceted properties including antiapoptotic,anti-inflammatory,neurogenic,and vasculogenic effects.Furthermore,exosomes offer several advantages over stem cell therapy,such as high preservation capacity,low immunogenicity,the ability to traverse the blood-brain barrier,and the potential for drug encapsulation.Consequently,researchers have turned their attention to exosomes as a novel therapeutic avenue.Nonetheless,akin to the limitations of stem cell treatment,the limited accumulation of exosomes in the injured brain poses a challenge to their clinical application.To overcome this hurdle,intranasal administration has emerged as a non-invasive and efficacious route for delivering drugs to the central nervous system.By exploiting the olfactory and trigeminal nerve axons,this approach enables the direct transport of therapeutics to the brain while bypassing the blood-brain barrier.Notably,exosomes,owing to their small size,can readily access the nerve pathways using this method.As a result,intranasal administration has gained increasing recognition as an optimal therapeutic strategy for exosomebased treatments.In this comprehensive review,we aim to provide an overview of both basic and clinical research studies investigating the intranasal administration of exosomes for the treatment of central nervous system diseases.Furthermore,we elucidate the underlying therapeutic mechanisms and offer insights into the prospect of this approach.

Culture and identification of neonatal rat brain-derived neural stem cells

BACKGROUND Timing of passaging,passage number,passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells(NSCs)culture.How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.AIM To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.METHODS First,curved tip operating scissors were used to dissect brain tissues from new born rats(2 to 3 d)and the brain tissues were cut into approximately 1 mm^(3)sections.Filter the single cell suspension through a nylon mesh(200-mesh)and culture the sections in suspensions.Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques.Second,identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation.BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells.Different NSCs specific antibodies(anti-nestin,NF200,NSE and GFAP antibodies)were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.RESULTS Brain derived cells from newborn rats(2 to 3 d)proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging.When BrdU was incorporated into the 5th generation of passaged cells,positive BrdU cells and nestin cells were observed by immunofluorescence staining.After induction of dissociation using 5%fetal bovine serum,positive NF200,NSE and GFAP cells were observed by immunofluorescence staining.CONCLUSION This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.

High mobility group box 1 in the central nervous system:regeneration hidden beneath inflammation

High-mobility group box 1 was first discovered in the calf thymus as a DNA-binding nuclear protein and has been widely studied in diverse fields,including neurology and neuroscience.High-mobility group box 1 in the extracellular space functions as a pro-inflammatory damage-associated molecular pattern,which has been proven to play an important role in a wide variety of central nervous system disorders such as ischemic stroke,Alzheimer’s disease,frontotemporal dementia,Parkinson’s disease,multiple sclerosis,epilepsy,and traumatic brain injury.Several drugs that inhibit high-mobility group box 1 as a damage-associated molecular pattern,such as glycyrrhizin,ethyl pyruvate,and neutralizing anti-high-mobility group box 1 antibodies,are commonly used to target high-mobility group box 1 activity in central nervous system disorders.Although it is commonly known for its detrimental inflammatory effect,high-mobility group box 1 has also been shown to have beneficial pro-regenerative roles in central nervous system disorders.In this narrative review,we provide a brief summary of the history of high-mobility group box 1 research and its characterization as a damage-associated molecular pattern,its downstream receptors,and intracellular signaling pathways,how high-mobility group box 1 exerts the repair-favoring roles in general and in the central nervous system,and clues on how to differentiate the pro-regenerative from the pro-inflammatory role.Research targeting high-mobility group box 1 in the central nervous system may benefit from differentiating between the two functions rather than overall suppression of high-mobility group box 1.

Developments in cell culture systems for human pluripotent stem cells

Human pluripotent stem cells(hPSCs)are important resources for cell-based therapies and pharmaceutical applications.In order to realize the potential of hPSCs,it is critical to develop suitable technologies required for specific applications.Most hPSC technologies depend on cell culture,and are critically influenced by culture medium composition,extracellular matrices,handling methods,and culture platforms.This review summarizes the major technological advances in hPSC culture,and highlights the opportunities and challenges in future therapeutic applications.

Osteogenic Differentiation of Bone Mesenchymal Stem Cells Regulated by Osteoblasts under EMF Exposure in a Co-culture System

This study examined the osteogenic effect of electromagnetic fields （EMF） under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells （BMSCs） and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit （CCK-8）. For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase （ALP） staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure （2 h/day） could obviously promote prolifera- tion of BMSCs and osteoblasts, while long-term EMF （8 h/day） could promote osteogenic differen- tiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when ceils were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differ- entiation of BMSCs.

Mesenchymal stem cell-derived exosomes regulate microglia phenotypes:a promising treatment for acute central nervous system injury

There is growing evidence that long-term central nervous system(CNS)inflammation exacerbates secondary deterioration of brain structures and functions and is one of the major determinants of disease outcome and progression.In acute CNS injury,brain microglia are among the first cells to respond and play a critical role in neural repair and regeneration.However,microglial activation can also impede CNS repair and amplify tissue damage,and phenotypic transformation may be responsible for this dual role.Mesenchymal stem cell(MSC)-derived exosomes(Exos)are promising therapeutic agents for the treatment of acute CNS injuries due to their immunomodulatory and regenerative properties.MSC-Exos are nanoscale membrane vesicles that are actively released by cells and are used clinically as circulating biomarkers for disease diagnosis and prognosis.MSC-Exos can be neuroprotective in several acute CNS models,including for stroke and traumatic brain injury,showing great clinical potential.This review summarized the classification of acute CNS injury disorders and discussed the prominent role of microglial activation in acute CNS inflammation and the specific role of MSC-Exos in regulating pro-inflammatory microglia in neuroinflammatory repair following acute CNS injury.Finally,this review explored the potential mechanisms and factors associated with MSCExos in modulating the phenotypic balance of microglia,focusing on the interplay between CNS inflammation,the brain,and injury aspects,with an emphasis on potential strategies and therapeutic interventions for improving functional recovery from early CNS inflammation caused by acute CNS injury.

Research progress of the relationship between microvesicles derived from stem cells and nervous system diseases

Microvesicles, also called microparticles, are membranous vesicles released from the cell membrane surface or by exocytose. Almost any type of cells can secrete vesicles, especially stem cells. Recent years, stem cells are becoming a research hotspot of cytotherapy for their capacity of self-renewing, expansion and proliferation in vitro and the microvesicles derived from the conditioned medium of stem cells have been widely used to regenerative medicine because they are safer, easily obtained, measurable and cause no obvious immune rejection. Stem cells derived microvesicles have been confirmed to be closely related to the progress and treatment of atherosclerosis, diabetes, inflammation and tumor. This review focuses on the new progress of stem cells derived microvesicles treating various nervous system diseases and its application in biological therapy and the behind molecule mechanisms.

Different priming strategies improve distinct therapeutic capabilities of mesenchymal stromal/stem cells:Potential implications for their clinical use

Mesenchymal stromal/stem cells(MSCs)have shown significant therapeutic potential,and have therefore been extensively investigated in preclinical studies of regenerative medicine.However,while MSCs have been shown to be safe as a cellular treatment,they have usually been therapeutically ineffective in human diseases.In fact,in many clinical trials it has been shown that MSCs have moderate or poor efficacy.This inefficacy appears to be ascribable primarily to the heterogeneity of MSCs.Recently,specific priming strategies have been used to improve the therapeutic properties of MSCs.In this review,we explore the literature on the principal priming approaches used to enhance the preclinical inefficacy of MSCs.We found that different priming strategies have been used to direct the therapeutic effects of MSCs toward specific pathological processes.Particularly,while hypoxic priming can be used primarily for the treatment of acute diseases,inflammatory cytokines can be used mainly to prime MSCs in order to treat chronic immune-related disorders.The shift in approach from regeneration to inflammation implies,in MSCs,a shift in the production of functional factors that stimulate regenerative or anti-inflammatory pathways.The opportunity to fine-tune the therapeutic properties of MSCs through different priming strategies could conceivably pave the way for optimizing their therapeutic potential.

Cultured meat from muscle stem cells: A review of challenges and prospects

Growing muscle tissue in culture from animal stem cells to produce meat theoretically eliminates the need to sacrifice animals. So-called ＂cultured＂ or ＂synthetic＂ or ＂in vitro＂ meat could in theory be constructed with different characteristics and be produced faster and more efficiently than traditional meat. The technique to generate cultured muscle tissues from stem cells was described long ago, but has not yet been developed for the commercial production of cultured meat products. The technology is at an early stage and prerequisites of implementation include a reasonably high level of consumer acceptance, and the development of commercially-viable means of large scale production. Recent advancements in tissue culture techniques suggest that production may be economically feasible, provided it has physical properties in terms of colour, flavour, aroma, texture and palatability that are comparable to conventional meat. Although considerable progress has been made during recent years, important issues remain to be resolved, including the characterization of social and ethical constraints, the fine-tuning of culture conditions, and the development of culture media that are cost-effective and free of animal products. Consumer acceptance and confidence in in vitro produced cultured meat might be a significant impediment that hinders the marketing process.

Culture and Identification of Human Amniotic Mesenchymal Stem Cells

Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45. Conclusions This study establishes a potential method for isolation of hAMSCs from human amnion, in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.

Isolation and Identification of Cancer Stem Cells from Human Osteosarcom by Serum-free Three-dimensional Culture Combined with Anticancer Drugs

The cancer stem cells(CSCs)from human osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs were isolated and identified.The primary cells derived from human osteosarcoma were digested by trypsin to prepare a single-cell suspension,and mixed homogeneously into 1.2% alginate gel.Single-cell alginate gel was cultured with serum-free DMEM/F12 medium.Epirubicin(0.8μg/mL)was added to the medium to enrich CSCs.After cultured conventionally for 7 to 10 days,most of cells suspended in ...

In vitro Culture of Bone Marrow Mesenchymal Stem Cells in Rats and Differentiation into Retinal Neural-like Cells

In order to study the in vitro culture and expansion of bone marrow mesenchymal stem cells in rats （rMSCs） and the possibility of rMSCs differentiation into retinal neural cells, the bone marrow-derived cells in SD rats were isolated and cultured in vitro. The retinal neural cells in SD rats were cultured and the supernatants were collected to prepare conditioned medium. The cultured rMSCs were induced to differentiate by two steps. Immunofluorescence method and anti-nestin, anti-NeuN, anti-GFAP and anti-Thyl. 1 antibodies were used to identify the cells derived from the rMSCs. The results showed that the in vitro cultured rMSCs grew well and expanded quickly. After induction with two conditioned media, rMSCs was induced to differentiate into neural progenitor cells, then into retinal neural-like cells which were positive for nestin, NeuN, GFAP and Thyl. 1 detected by fluorescence method. The findings suggested that rMSCs could be culture and expanded in vitro, and induced to differentiate into retinal neural-like cells.

Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells

Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells （hdFs）. Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen （SSEA）-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction （RT-PCR） analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the ‘niche＇ that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research,the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems.Recently,researchers have been actively developing and evaluating three-dimensional(3D)cell culture-based platforms using microfluidic technologies,such as organ-on-a-chip and organoid-on-a-chip platforms,and they have achieved promising breakthroughs in stem cell engineering.In this review,we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery.In a subsequent section,we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research.In addition,some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.

Progress in in vitro culture and gene editing of porcine spermatogonial stem cells

Research on in vitro culture and gene editing of domestic spermatogonial stem cells (SSCs) is of considerable interest but remains a challenging issue in animal science. In recent years, some progress on the isolation, purification, and genetic manipulation of porcine SSCs has been reported. Here, we summarize the characteristics of porcine SSCs as well current advances in their in vitro culture, potential usage, and genetic manipulation. Furthermore, we discuss the current application of gene editing in pig cloning technology. Collectively, this commentary aims to summarize the progress made and obstacles encountered in porcine SSC research to better serve animal husbandry, improve livestock fecundity, and enhance potential clinical use.

Culture and Identification of Monoclonal Neural Stem Cells Derived from Cerebral Cortex

Summary： To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells （Nestin） of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

Background： The use of equine bone marrow mesenchymal stem cells （BMSC） is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine： 1） if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2） the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum （FBS） or autologous horse serum （HS）, and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 （BMP2）, dexamethasone （DEX）, or combination of the two. Alkaline phosphatase （ALP） activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-reloted tronscrJption foctor2 （Runx2）, osterix （Osx）, and T-box3 （Tbx3）. Data were analyzed by ANOVA. Results： Relative to control, FBS and HS increased cell number （133 ± 5 and 116 ± 5%, respectively; P 〈 0.001） and 5-bromo- 2＇-deoxyuridine （BrdU） incorporation （167 ± 6 and 120 ± 6%, respectively; P 〈 0.001）. Treatment with DEX increased ALP activity compared with control （1,638 ± 38%; P 〈 0.001）. In the absence and presence of Dex, BMP-2 did not alter ALP activity （P 〉 0.8）. Runt-reloted transcription foctor2 expression increased 3-fold （P 〈 0.001） by d 6 of culture. Osterix expression increased 94old （P 〈 0.05） by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 （P 〈 0.01）; however expression was reduced 4-fold at d 18 （P 〈 0.01）. Conclusions： Dexamethasone, but not BMP-2, is required for differentiation of equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation.

Co-culture of Mesenchymal Stem Cells with Umbilical Vein Endothelial Cells under Hypoxic Condition

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.