The MORC2 p.S87L mutation reduces proliferation of pluripotent stem cells derived from a patient with the spinal muscular atrophy-like phenotype by inhibiting proliferation-related signaling pathways

Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.

Use of induced pluripotent stem cell-derived brain cells,organoids,assembloids,and blood-brain barrier models in understanding alcohol and anesthetic-induced brain injuries:an emerging perspective

Neurological disorders,including developmental disorders,Alzheimer’s disease(AD),and psychiatric conditions,have significant social and economic impacts globally.Despite extensive research into the underlying mechanisms of these disorders,effective treatments remain elusive,partly due to the complexity of the brain,the limited availability of human brain tissue,and the blood-brain barrier(BBB)’s impermeability to certain drugs.This perspective article discusses the potential of human induced pluripotent stem cell(iPSC)-based models of brain cells,organoids,assembloids,and BBB to advance our understanding of the etiology,progression,and mechanisms of brain injuries induced by alcohol consumption and general anesthesia.These models could also be used to develop protective and therapeutic approaches.

A perspective on age-related changes in cell environment and risk of neurodegenerative diseases

“Age-related neurodegenerative disease”underscores human age as the primary risk factor for disease development:Alzheimer’s,Parkinson’s,and Huntington’s disease(HD)as examples.Reasons for the age-dependent delay in disease manifestation,in particular for autosomal dominant forms of the disease,and the underlying cause(s)of specific neuron dysfunction and death to manifest as memory loss,anxiety,depression,and agitation in disease subjects remain unclear.We are interested in understanding age-related changes in cell environment that can modulate the structure,function,aggregation.

Pathologically successful conversion hepatectomy for advanced giant hepatocellular carcinoma after multidisciplinary therapy:A case report and review of literature

BACKGROUND Hepatocellular carcinoma(HCC)is one of the leading causes of death due to its complexity,heterogeneity,rapid metastasis and easy recurrence after surgical resection.We demonstrated that combination therapy with transcatheter arterial chemoembolization(TACE),hepatic arterial infusion chemotherapy(HAIC),Epclusa,Lenvatinib and Sintilimab is useful for patients with advanced HCC.CASE SUMMARY A 69-year-old man who was infected with hepatitis C virus(HCV)30 years previously was admitted to the hospital with abdominal pain.Enhanced computed tomography(CT)revealed a low-density mass in the right lobe of the liver,with a volume of 12.9 cm×9.4 cm×15 cm,and the mass exhibited a“fast-in/fast-out”pattern,with extensive filling defect areas in the right branch of the portal vein and an alpha-fetoprotein level as high as 657 ng/mL.Therefore,he was judged to have advanced HCC.During treatment,the patient received three months of Epclusa,three TACE treatments,two HAIC treatments,three courses of sintilimab,and twenty-one months of lenvatinib.In the third month of treatment,the patient developed severe side effects and had to stop immunotherapy,and the Lenvatinib dose had to be halved.Postoperative pathological diagnosis indicated a complete response.The patient recovered well after the operation,and no tumor recurrence was found.CONCLUSION Multidisciplinary conversion therapy for advanced enormous HCC caused by HCV infection has a significant effect.Individualized drug adjustments should be made during any treatment according to the patient's tolerance to treatment.

Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization

BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.

Sorl1 knockout inhibits expression of brain-derived neurotrophic factor:involvement in the development of late-onset Alzheimer's disease

Sortilin-related receptor 1(SORL1)is a critical gene associated with late-onset Alzheimer’s disease.SORL1 contributes to the development and progression of this neurodegenerative condition by affecting the transport and metabolism of intracellularβ-amyloid precursor protein.To better understand the underlying mechanisms of SORL1 in the pathogenesis of late-onset Alzheimer s disease,in this study,we established a mouse model of SorI1 gene knockout using cluste red regularly inters paced short palindro mic repeats-associated protein 9 technology.We found that Sorl1-knocko ut mice displayed deficits in learning and memory.Furthermore,the expression of brain-derived neurotrophic factor was significantly downregulated in the hippocampus and co rtex,and amyloidβ-protein deposits were observed in the brains of 5orl1-knockout mice.In vitro,hippocampal neuronal cell synapses from homozygous Sorl1-knockout mice were impaired.The expression of synaptic proteins,including Drebrin and NR2B,was significantly reduced,and also their colocalization.Additionally,by knocking out the Sorl1 gene in N2a cells,we found that expression of the N-methyl-D-aspartate receptor,NR2B,and cyclic adenosine monophosphate-response element binding protein was also inhibited.These findings suggest that SORL1 participates in the pathogenesis of late-onset Alzheimer s disease by regulating the N-methyl-D-aspartate receptor NR2B/cyclic adenosine monophosphate-response element binding protein signaling axis.

Combination immunotherapy of glioblastoma with dendritic cell cancer vaccines,anti-PD-1 and poly I:C

Glioblastoma(GBM)is a lethal cancer with limited therapeutic options.Dendritic cell(DC)-based cancer vaccines provide a promising approach for GBM treatment.Clinical studies suggest that other immunotherapeutic agents may be combined with DC vaccines to further enhance antitumor activity.Here,we report a GBM case with combination immunotherapy consisting of DC vaccines,anti-programmed death-1(anti-PD-1)and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy,and the patient remained disease-free for 69 months.The patient received DC vaccines loaded with multiple forms of tumor antigens,including mRNA-tumor associated antigens(TAA),mRNA-neoantigens,and hypochlorous acid(HOCl)-oxidized tumor lysates.Furthermore,mRNA-TAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histocompatibility complex(MHC)class I and II antigen presentation.The treatment consisted of 42 DC cancer vaccine infusions,26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions.The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells.No immunotherapy-related adverse events were observed during the treatment.Robust antitumor CD4t and CD8t T-cell responses were detected.The patient remains free of disease progression.This is the first case report on the combination of the above three agents to treat glioblastoma patients.Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient.A large-scale trial to validate these findings is warranted.

Loss of Notch signaling in skeletal stem cells enhances bone formation with aging

Skeletal stem and progenitor cells(SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

Sequential extraction of RNA,DNA and protein from cultured cells of the same group

BACKGROUND Efficient extraction of nucleic acids and proteins(ENAP)from cells is a prerequisite for precise annotation of gene function,and has become laboratory routine for revealing the mysteries of life.However,cell samples are often from different culture dishes,resulting in inevitable experimental errors and sometimes poor repeatability.AIM To explore a method to improve the efficiency of ENAP,minimizing errors in ENAP processes,enhancing the reliability and repeatability of subsequent experimental results.METHODS A protocol for the sequential isolation of RNA,DNA,and proteins from the same cultured HepG2 cells using RNAzol reagent is presented here.The first step involves culturing HepG2 cells to the exponential phase,followed by the sequential isolation of RNA,DNA,and proteins from the same cultured cells in the second step.The yield of nucleic acids and proteins is detected in the third step,and their purity and integrity are verified in the last step.RESULTS The procedure takes as few as 3-4 d from the start to quality verification and is highly efficient.In contrast to the existing kits and reagents,which are primarily based on independent isolation,this RNAzol reagent-based method is characterized by the sequential isolation of RNA,DNA,and proteins from the same cells,and therefore saves time,and has low cost and high efficiency.CONCLUSION The RNA,DNA,and proteins isolated using this method can be used for reverse transcription-polymerase chain reaction,polymerase chain reaction,and western blotting,respectively.

Recent progress in hair follicle stem cell markers and their regulatory roles

Hair follicle stem cells(HFSCs)in the bulge are a multipotent adult stem cell population.They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing.An increasing number of biomarkers have been used to isolate,label,and trace HFSCs in recent years.Considering more detailed data from single-cell transcriptomics technology,we mainly focus on the important HFSC molecular markers and their regulatory roles in this review.

New insights into the role of the endoplasmic reticulum in microglia

Microglial cells are the only resident immune cells in the central nervous system and constitute its frontline guardian.They are extremely reactive against infections,trauma,or toxins,but are also responsible for mediating inflammation,taking part in the pathogenic course of many neuropathologies(Sierra et al.,2019).Cell-specific staining,ultrastructural analysis by transmission electron microscopy(TEM),or two-photonmicroscopy imaging have been relevant for the characterization of microglia as well as their cell-cell interactions。

Intermittent fasting boosts antitumor immunity by restricting CD11b^(+)Ly6C^(low)Ly6G^(low) cell viability through glucose metabolism in murine breast tumor model

Intermittent fasting can benefit breast cancer patients undergoing chemotherapy or immunotherapy.However,it is still uncertain how to select immunotherapy drugs to combine with intermittent fasting.Herein we observed that two cycles of fasting treatment significantly inhibited breast tumor growth and lung tissue metastasis,as well as prolonged overall survival in mice bearing 4T1 and 4T07 breast cancer.During this process,both the immunosuppressive monocytic-(M-)and granulocytic-(G-)myeloid-derived suppressor cell(MDSC)decreased,accompanied by an increase in interleukin(IL)7R^(+)and granzyme B^(+)T cells in the tumor microenvironment.Interestingly,we observed that Ly6G^(low)G-MDSC sharply decreased after fasting treatment,and the cell surface markers and protein mass spectrometry data showed potential therapeutic targets.Mechanistic investigation revealed that glucose metabolism restriction suppressed the splenic granulocytemonocyte progenitor and the generation of colony-stimulating factors and IL-6,which both contributed to the accumulation of G-MDSC.On the other hand,glucose metabolism restriction can directly induce the apoptosis of Ly6G^(low)G-MDSC,but not Ly6G^(high)subsets.In summary,these results suggest that glucose metabolism restriction induced by fasting treatment attenuates the immune-suppressive milieu and enhances the activation of CD3^(+)T cells,providing potential solutions for enhancing immune-based cancer interventions.

Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells

人的导致的 pluripotent 茎(iPS ) 房间类似于胚胎的茎(ES ) 房间，和罐头强烈地增殖并且区分进许多房间类型。然而，人的 iPS 房间的肝的区别还没被报导了。在这份报告，人的 iPS 房间被导致由一个逐步的协议区分进肝的房间。肝房间标记的表达式和人的 iPS 的肝相关的函数导出房间的房间被监视并且与区分的人的 ES 房间和主要人的 hepatocytes 的相比。在白天 7 点的约 60% 区分的人的 iPS 房间表示了肝的标记 alpha fetoprotein 和白长袍的。在白天 21 点的区分的房间包括白朊 Asecretion，肝糖合成，脲生产和可诱导的细胞色素 P450 活动展出了肝房间功能。肝的标记的表示和 iPS 的肝相关的功能导出房间的肝的房间比得上人的 ES 导出房间的肝的房间的。这些结果证明人的 iPS 房间，类似于人的 ES 房间，能高效地被导致区分房间进象 hepatocyte 一样。

Gene expression alteration during redox-dependent enhancement of arsenic cytotoxicity by emodin in HeLa cells

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) could enhance the sensitivity of tumor cells to arsenic trioxide(As2O3)–induced apoptosis via generation of ROS, but the molecular mechanism has not been elucidated. Here, wecarried out cDNA microarray-based global transcription profiling of HeLa cells in response to As2O3/emodin cotreatment,comparing with As2O3–only treatment. The results showed that the expression of a number of genes was substantiallyaltered at two time points. These genes are involved in different aspects of cell function. In addition to redox regulationand apoptosis, ROS affect genes encoding proteins associated with cell signaling, organelle functions, cell cycle,cytoskeleton, etc. These data suggest that based on the cytotoxicity of As2O3, emodin mobilize every genomic resourcethrough which the As2O3–induced apoptosis is facilitated.

Roles of liver innate immune cells in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease in the United States and other developed countries and is expected to increase in the next few years. Emerging data suggest that some patients with NAFLD may progress to nonalcoholic steatohepatitis (NASH), cirrhosis and even hepatocellular carcinoma. NAFLD can also promote the development and progression of disease in other organ systems, such as the cardiovascular and endocrine (i.e. diabetes) systems. Thus, understanding the pathogenesis of NAFLD is of great clinical importance and is critical for the prevention and treatment of the disease. Although the "two-hit hypothesis" is generally accepted, the exact pathogenesis of NAFLD has not been clearly established. The liver is an important innate immune organ with large numbers of innate immune cells, including Kupffer cells (KCs), natural killer T (NKT) cells and natural killer (NK) cells. Recent data show that an imbalance in liver cytokines may be implicated in the development of fatty liver disease. For example, Th1 cytokine excess may be a common pathogenic mechanism for hepatic insulin resistance and NASH. Innate immune cells in the liver play important roles in the excessive production of hepatic Th1 cytokines in NAFLD. In addition, liver innate immune cells participate in the pathogenesis of NAFLD in other ways. For example, activated KCs can generate reactive oxygen species, which induce liver injury. This review will focus primarily on the possible effect and mechanism of KCs, NKT cells and NK cells in the development of NAFLD.

Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model

人的间充质的干细胞(hMSCs ) 装家到肿瘤地点并且禁止肿瘤细胞的生长。很少对内在的分子的机制被知道连接 hMSCs 到肿瘤房间的指向的抑制。在这研究，我们从 hMSCs 用一个动物移植模型，一个合作文化系统和调节媒介在二根人的 hepatoma 房间线(H7402 和 HepG2 ) 上调查了 hMSCs 的效果。当 SCID 老鼠与 H7402 细胞和 Z3 hMSCs 的一个相等的数字被注射时，动物移植研究证明肿瘤形成的潜伏的时间被延长并且肿瘤尺寸更小。当 co 有教养时与 Z3 房间， H7402 细胞增殖减少了，增加的 apoptosis，和 Bcl-2 的表示， c-Myc，原子抗原(PCNA ) 和 survivin 低是的增殖的房间调整了。在有调节媒介的处理源于 Z3 hMSC 文化以后， H4702 房间出现了减少的形成殖民地的能力和减少的增长。Immunoblot 分析证明 beta-catenin， Bcl-2， c-Myc， PCNA 和 survivin 表示是在 H7402 和 HepG2 房间调整的 down。总起来说，我们的调查结果证明 hMSCs 禁止 H7402 和 HepG2 人的肝癌症房间线的恶意的显型，它包括增长，形成殖民地的能力和 oncogene 表示试管内和体内。而且，我们的研究提供表明小径的 Wnt 可以在调停 hMSC 的指向和肿瘤房间抑制有一个角色的证据。

The therapeutic effects of recombinant adenovirus RA538 on human gastric carcinoma cells in vitro and in vivo

AIM To evaluate the potential of RA-538 genetherapy for gastric carcinoma.METHODS Human gastric carcinoma cell lineSGC7901 treated with Ad-RA538 or Ad-LacZ wereanalysed by X-gal stain,MTT,DNA ladder,Tunel,flow cytometric analysis,PCR,andWestern Blot in vitro.The tumorigenicity andexperimental therapy in nude mice model wereassessed in vivo.RESULTS Ad-LacZ could efficiently transferthe LacZ gene into SGC7901 cells.X-gal-positivecells at MOI 25,50,100,and 200 were 90%,100%,100%,and 100% respectively.Ad-RA538could strongly inhibit cell growth and inducedapoptosis in SGC7901 cells.The proliferation ofthe Ad-RA538-infected SGC7901 cells wasreduced by 76.3%.The mechanism of killing ofgastric carcinoma cells by Ad-RA538 was foundto be apoptosis by DNA ladder,Tunel and flowcytometric analysis.The tumorigenicity in nudemice using Ad-RA538 showed that all three micefailed to form tumor from 7 to 30 days comparedwith Ad-LacZ and parent SGC7901 cells.Experimental therapy on the nude mice modelbearing subcutaneous tumor of SGC790| cells showed that intratumor instillation of Ad-RA538inhibited the growth of the tumors.Ad-RA538-treated tumors were inhibited by 60.66 %,compared with that of the tumor injected withAd-LacZ and mock.CONCLUSION The expression of Ad-RA538 can inhibit growth and induce apoptosis of gastric cancer cell in vitro and in vivo. Ad-RA538 can be used potentially in gene therapy for gastric carcinoma.

In vitro derivation of functional insulin-producing cells from human embryonic stem cells

为人的胚胎的茎(ES ) 的自强和区别的能力细胞为对待类型 Idiabetes mellitus 为胰腺的贝它细胞的产生使他们成为潜在的来源。这里，我们报导一最新发展了并且有效方法，在aserum免费的系统执行了，区分进生产胰岛素的 cells.Activin A 的导致的人的 ES 房间它在起始的阶段被使用从人的 EScells 导致权威的内胚叶区别，是由权威的内胚叶标记 Sox17 和 Brachyury.Further 的表示检测了， all-trans retinoic 酸( RA )被用来支持胰腺的区别，由早胰腺的抄写因素 pdx1 和 hlxb9 的表示显示了。在成熟 inDMEM/F12 以后有 bFGF 和菸碱的没有浆液的媒介，区分的房间表示了小岛特定的标记象 C 肽，胰岛素，胰高血糖素和 glut2 那样。百分比 ofC-peptide-positive 房间超过了 15% 。由这些房间的胰岛素和 C 肽的分泌物在葡萄糖层次对应于变化。当移植了进肾的囊时， ofStreptozotocin (STZ ) 对待裸体老鼠，这些区分的人的 ES 房间熬过并且维持贝它房间标记基因的表示包括 C 肽， pdx1， glucokinase， nkx6.1， IAPP， pax6and Tcf1。百分之三十只移植裸体老鼠展出了 stableeuglycemia 的明显的恢复；并且改正的显型被支撑超过六个星期。我们的新方法为学习人的胰开发的机制提供一个有希望的试管内区别模特儿并且说明为类型 Idiabetes mellitus 的处理使用人的 ES 房间的潜力。

PBX3 promotes migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway

AIM:To investigate the role of pre-B-cell leukemia homeobox(PBX)3 in migration and invasion of colorectal cancer(CRC)cells.METHODS:We detected PBX3 expression in five cell lines and surgical specimens from 111 patients with CRC using real-time reverse transcription-polymerase chain reaction.We forced expression of PBX3 in low metastatic HT-29 and SW480 cells and knocked down expression of PBX3 in highly metastatic LOVO and HCT-8 cells.Wound healing and Boyden chamber assays were used to detect cell migration and invasionafter altered expression of PBX3.Western blot was performed to detect the change of signaling molecule ERK1/2 following PBX3 overexpression.RESULTS:High level of PBX3 expression was correlated with the invasive potential of CRC cells,and significantly associated with lymph node invasion(P=0.02),distant metastasis(P=0.04),advanced TNM stage(P=0.03)and poor overall survival of patients(P<0.05).Ectopic expression of PBX3 in low metastatic cells was shown to promote migration and invasion,while inhibited PBX3 expression in highly metastatic cells suppressed migration and invasion.Furthermore,upregulation of phosphorylated extracellular signal-regulated kinase(ERK)1/2 was found to be one of the targeted molecules responsible for PBX3-induced CRC cell migration and invasion.CONCLUSION:PBX3 induces invasion and metastasis of CRC cells partially through activation of the MAPK/ERK signaling pathway.

Migration of bone marrow progenitor cells in the adult brain of rats and rabbits

Neurogenesis takes place in the adult mammalian brain in three areas:Subgranular zone of the dentate gyrus(DG);subventricular zone of the lateral ventricle;olfactory bulb.Different molecular markers can be used to characterizethe cells involved in adult neurogenesis.It has been recently suggested that a population of bone marrow(BM)progenitor cells may migrate to the brain and differentiate into neuronal lineage.To explore this hypothesis,we injected recombinant SV40-derived vectors into the BM and followed the potential migration of the transduced cells.Long-term BM-directed gene transfer using recombinant SV40-derived vectors leads to expression of the genes delivered to the BM firstly in circulating cells,then after several months in mature neurons and microglial cells,and thus without central nervous system(CNS)lesion.Most of transgene-expressing cells expressed NeuN,a marker of mature neurons.Thus,BM-derived cells may function as progenitors of CNS cells in adult animals.The mechanism by which the cells from the BM come to be neurons remains to be determined.Although the observed gradual increase in transgene-expressing neurons over 16mo suggests that the pathway involved differentiation of BM-resident cells into neurons,cell fusion as the principal route cannot be totally ruled out.Additional studies using similar viral vectors showed that BM-derived progenitor cells migrating in the CNS express markers of neuronal precursors or immature neurons.Transgene-positive cells were found in the subgranular zone of the DG of the hippocampus 16 mo after intramarrow injection of the vector.In addition to cells expressing markers of mature neurons,transgene-positive cells were also positive for nestin and doublecortin,molecules expressed by developing neuronal cells.These cells were actively proliferating,as shown by short term BrdU incorporation studies.Inducing seizures by using kainic acid increased the number of BM progenitor cells transduced by SV40vectors migrating to the hippocampus,and these cells were seen at earlier time points in the DG.We show that the cell membrane chemokine receptor,CCR5,and its ligands,enhance CNS inflammation and seizure activity in a model of neuronal excitotoxicity.SV40-based gene delivery of RNAi targeting CCR5 to the BM results in downregulating CCR5 in circulating cells,suggesting that CCR5 plays an important role in regulating traffic of BM-derived cells into the CNS,both in the basal state and in response to injury.Furthermore,reduction in CCR5 expression incirculating cells provides profound neuroprotection from excitotoxic neuronal injury,reduces neuroinflammation,and increases neuronal regeneration following this type of insult.These results suggest that BM-derived,transgeneexpressing,cells can migrate to the brain and that they become neurons,at least in part,by differentiating into neuron precursors and subsequently developing into mature neurons.