Mitochondria Dynamically Transplant into Cells in Vitro and in Mice and Rescue Aerobic Respiration of Mitochondrial DNA-Depleted Motor Neuron NSC-34

It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare for mitochondrial transplantation study in human neurodegenerative diseases, we select human fibroblasts as mitochondrial donor because that fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). We isolate human primary fibroblasts and develop a mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34 ρ° cells). Fibroblast and NSC-34 cell’s mitochondria are co-cultured with NSC-34 ρ° cells. Mitochondrial transplantation is observed by fluorescent microscopy. Gene expression is determined by polymerase chain reaction (PCR) and real time PCR (qPCR). Also, mitochondria are injected to mice bearing mammary adenocarcinoma 4T1 cells. We find results as following: 1) There are abundant mitochondria in fibroblasts (337 ± 80 mitochondria per fibroblast). 42.4% of viable mitochondria are obtained by using differential centrifugation. The isolated mitochondria actively transplant into NSC-34 ρ° cells after co-culture. 2) Fibroblasts transfer mitochondria to human mammary adenocarcinoma MCF-7 cells. 3) There is no expression of HLA-I antigen in fibroblast’s mitochondria indicating they can be used for allogeneic mitochondrial transplantation without HLA antigen match. 4) PCR and qPCR show that NSC-34 ρ° cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate expression of glycolysis-associated genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA). 5) Transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates gene expression of HK2, SLC2A1 and LDHA. 6) Normal mammary epithelial mitochondria successfully enter to 4T1 cells in mice. Subcutaneous injection of mitochondria is safe for mice. In summary, mitochondrial transplantation replenishes mtDNA and rescues aerobic respiration of diseased cells with mitochondrial dysfunction. Human primary fibroblasts are potential mitochondrial donor for mitochondrial transplantation study in human neurodegenerative diseases.

Genetic modification of miR-34a enhances efficacy of transplanted human dental pulp stem cells after ischemic stroke

Human dental pulp stem cells(hDPSCs) promote recovery after ischemic stro ke;however,the therapeutic efficacy is limited by the poor survival of transplanted cells.For in vitro expe riments in the present study,we used oxygen-glucose deprivation/reoxygenation in hDPSCs to mimic cell damage induced by ischemia/reperfusion.We found that miRNA-34a-5p(miR-34a) was elevated under oxygen-glucose deprivation/reoxygenation conditions in hDPSCs.Inhibition of miR-34a facilitated the prolife ration and antioxidant capacity and reduced the apoptosis of hDPSCs.Moreove r,dual-luciferase reporter gene assay showed WNT1and SIRT1 as the targets of miR-34a.In miR-34a knockdown cell lines,WNT1 suppression reduced cell prolife ration,and SIRT1 suppression decreased the antioxidant capacity.Togethe r,these results indicated that miR-34a regulates cell prolife ration and antioxidant stress via targeting WNT1 and SIRT1,respectively.For in vivo expe riments,we injected genetically modified hDPSCs(anti34a-hDPSCs) into the brains of mice.We found that anti34a-hDPSCs significantly inhibited apoptosis,reduced cerebral edema and cerebral infarct volume,and improved motor function in mice.This study provides new insights into the molecular mechanism of the cell prolife ration and antioxidant capacity of hDPSCs,and suggests a potential gene that can be targeted to improve the survival rate and efficacy of transplanted hDPSCs in brain after ischemic stroke.

Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis

BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.

Repetitive administration of cultured human CD34+cells improve adenine-induced kidney injury in mice

BACKGROUND There is no established treatment to impede the progression or restore kidney function in human chronic kidney disease(CKD).AIM To examine the efficacy of cultured human CD34+cells with enhanced proliferating potential in kidney injury in mice.METHODS Human umbilical cord blood(UCB)-derived CD34+cells were incubated for one week in vasculogenic conditioning medium.Vasculogenic culture significantly increased the number of CD34+cells and their ability to form endothelial progenitor cell colony-forming units.Adenineinduced tubulointerstitial injury of the kidney was induced in immunodeficient non-obese diabetic/severe combined immunodeficiency mice,and cultured human UCB-CD34+cells were administered at a dose of 1×106/mouse on days 7,14,and 21 after the start of adenine diet.RESULTS Repetitive administration of cultured UCB-CD34+cells significantly improved the time-course of kidney dysfunction in the cell therapy group compared with that in the control group.Both interstitial fibrosis and tubular damage were significantly reduced in the cell therapy group compared with those in the control group(P<0.01).Microvasculature integrity was significantly preserved(P<0.01)and macrophage infiltration into kidney tissue was dramatically decreased in the cell therapy group compared with those in the control group(P<0.001).CONCLUSION Early intervention using human cultured CD34+cells significantly improved the progression of tubulointerstitial kidney injury.Repetitive administration of cultured human UCB-CD34+cells significantly improved tubulointerstitial damage in adenine-induced kidney injury in mice via vasculoprotective and anti-inflammatory effects.

Exploring hematopoietic stem cell population in human milk and its benefits for infants:A scoping review

Objective:To comprehensively explore hematopoietic stem cells(HSCs)in human milk,understanding their molecular markers,isolation methods,benefits for infants,and potential medical applications.Methods:We conducted a scoping literature review following the PRISMA-ScR guidelines.This review included studies investigating HSCs in human milk,utilizing molecular markers such as CD34^(+),CD113^(+),and CD117^(+)for characterization.Both in vitro and in vivo studies exploring the morphology,function,and clinical implications of these cells were considered.The diverse range of papers reviewed were indexed in PubMed,Science Direct,Scopus,Sage Journals,and Google Scholar,published between 2010 and 2023.Results:This scoping review explored 577 articles and selected 13 studies based on our inclusion criteria,focusing on HSCs in human milk.Most studies dilute samples prior to HSC isolation,followed by detection using markers such as CD34^(+),CD113^(+),and CD117^(+),with flow cytometry serving as the primary analysis tool,focusing on their isolation and detection methods.While no definitive benefits have been conclusively established,there is a strong belief in the potential of HSCs to positively impact infant immunity,growth,and tissue repair.Conclusions:This review presents significant evidence supporting the presence of HSCs in human milk,identified by markers such as CD34^(+),CD113^(+),and CD117^(+).These cells show considerable potential in enhancing infant health,including immunity,tissue repair,cognitive development,and gastrointestinal health.Despite methodological variations in isolation and detection techniques,the collective findings underscore the potential clinical relevance of HSCs in human milk.Moreover,this review highlights the noninvasive accessibility of human milk as a source of HSCs and emphasizes the need for further research to unlock their therapeutic potential.

IL-34介导的NF-кB信号通路对根尖牙乳头干细胞成牙成骨定向分化的调节作用

目的:探讨白细胞介素-34(IL-34)介导的核因子кB(NF-кB)对根尖牙乳头干细胞(SCAPs)成牙/成骨定向分化调节作用。方法:使用酶消化法从小鼠根尖牙乳头组织传代培养SCAPs;流式细胞术鉴定SCAPs中CD44、CD90、CD45及集落刺激因子1受体(CSF-1R)的表达;实验分为4组:空白对照组、IL-34组(100 ng/mL IL-34)、CSF-1R组(100 ng/mL IL-34+25 ng/mL anti-CSF-1R)、NF-кB组(100 ng/mL IL-34+1umol/L BMS-345541)。通过蛋白质印迹法(Western blot)分析各组30、60、120 min时SCAPs胞浆中NF-кB关键蛋白p-IкBα、IкBα、p-P65及P65蛋白的表达情况;矿化诱导4组SCAPs 3、7、14 d时,茜素红染色和半定量分析比较各组细胞的矿化结节形成能力;通过RT-qPCR检测各组SCAPs中成骨相关基因Runt相关转录因子-2(RUNX2)、牙本质涎磷蛋白(DSPP)、骨钙素(OCN)mRNA水平。结果:流式细胞术显示,SCAPs中CD44(90.4%)、CD90(93.5%)阳性表达,造血干细胞CD45(3.1%)阴性表达,SCAPs表达CSF-1R(23.2%)阳性。与对照组比较,IL-34组SCAPs胞浆蛋白内p-IкBα、p-P65表达水平上调,矿化诱导3、7、14 d矿化结节形成量增多,细胞中RUNX2、DSPP、OCN mRNA相对表达量升高,差异均有统计学意义(P<0.05)。与IL-34组比较,CSF-1R组和NF-кB组SCAPs胞浆蛋白内p-IкBα、p-P65表达水平下调(P<0.05),矿化诱导3、7、14 d矿化结节形成量减少,细胞中RUNX2、DSPP、OCN mRNA相对表达量降低,差异均有统计学意义(P<0.05)。结论:IL-34可通过IL-34/CSF-1R调节SCAPs中NF-кB信号通路,并通过IL-34/CSF-1R/NF-кB信号调节SCAPs的成牙/成骨定向分化能力。

CD19^(+)CD24^(+)CD27^(+)调节性B细胞及IL-10、IL-34与系统性红斑狼疮中医证型的关系

目的:探讨CD19^(+)CD24^(+)CD27^(+)调节性B细胞(Bregs细胞)及白细胞介素(IL)-10、IL-34与系统性红斑狼疮(SLE)中医证型的关系。方法:选取2022年4月~2024年4月在本院接受治疗的117例SLE患者为研究组,并纳入同期进行体检的86名健康体检者为对照组,检测对比两组的CD19^(+)CD24^(+)CD27^(+)Bregs细胞中CD24^(high) CD27^(+)比例、CD24^(mid) CD27^(+)比例以及血清IL-10、IL-34水平。另将研究组根据中医辨证分型分为热毒炽盛证、肝肾阴虚证、脾肾阳虚证,检测对比不同中医证型患者间CD24^(high) CD27^(+)比例、CD24^(mid) CD27^(+)比例、IL-10、IL-34、肾功能及系统性红斑狼疮疾病活动指数(SLEDAI)的差异。结果:研究组的CD24^(high) CD27^(+)比例、CD24^(mid) CD27^(+)比例、IL-10、IL-34水平均高于对照组,差异有统计学意义(P<0.05)。117例SLE患者中热毒炽盛证有49例、肝肾阴虚证33例、脾肾阳虚证35例。CD24^(high) CD27^(+)比例、CD24^(mid) CD27^(+)比例、IL-10、IL-34水平及SLEDAI评分均在肝肾阴虚证、脾肾阳虚证、热毒炽盛证中依次上升,差异有统计学意义(P<0.05);脾肾阳虚证患者的血清肌酐、尿素氮、24h尿蛋白定量均高于热毒炽盛证及肝肾阴虚证患者(P<0.05)。结论:SLE患者血清CD19^(+)CD24^(+)CD27^(+)Bregs细胞、IL-10、IL-34水平均与中医证型有关,可作为中医辨证论治的辅助指标。

THE EFFECT OF STEM CELL FACTOR, INTERLEUKIN-6 AND ERYTHROPOIETIN ON EXPANSION OF CD34^+ CELLS FROM HUMAN UMBILICAL CORD BLOOD

CD34+ cells from human umbilical cord blood were purified by Dynal beads M-450 CD34 immunoselection system and cultured in the presence of various cytokines alone or in combination, including stem cell factor (SCF), interleukin-6 (IL-6) and erythropoietin (EPO). The results revealed that: (D In methylcellulose culture, the plating efficiencies of purified cord blood CD34+ cells were much different when stimulated by various cytokines. IL-6 alone had the lowest colo-ny yield, while the combination of SCF, IL-6 and EPO had the highest yield. ② In the suspension culture, IL-6 alone or IL-6 + EPO had little expanding effect on cord blood CD34+ celis, the other cytokine combinations could expand cord blood CD34+ celis at different Ievels. Among them, the combination of SCF, IL-6 and EPO had the maximal expanding effect on cord blood CD34+ celis, the number of progenitor celis peaked at day 21, about 29-fold increase and nucleated celis increased approximately 3676-fold at day 28. The expanding effect of

人TDP-43-M337V对NSC-34细胞增殖的影响

目的探讨人TDP-43-M337V对NSC-34细胞增殖的影响。方法实验分为转染空质粒pEGFP组、转染TDP-43-WT的pEGFP-TDP-43-WT组和转染TDP-43-M337V的pEGFP-TDP-43-M337V组3组,利用免疫组化方法观察转染细胞的形态变化;通过检测细胞内MDA含量来观察细胞氧化损伤的程度;MTT检测转染基因对NSC-34细胞的影响。结果转染各组细胞中,转染人TDP-43-M337V组细胞,胞体变圆,突起减少,且轴突变短;经过MDA检测发现,突变的细胞内MDA的含量明显高于其它两组细胞(P<0.05);突变的细胞内,其MTT水平明显低于其它两组细胞。结论人TDP-43-M337V削弱了NSC-34细胞活性,降低了NSC-34细胞的抗氧化损伤能力。

Autologous mobilized peripheral blood CD34^+ cell infusion in non-viral decompensated liver cirrhosis

AIM: To study the effect of mobilized peripheral blood autologous CD34 positive(CD34+) cell infusion in patients with non-viral decompensated cirrhosis.METHODS: Cirrhotic patients of non-viral etiology were divided into 2 groups based on their willingness to be listed for deceased donor liver transplant(DDLT)(control, n = 23) or to receive autologous CD34+ cell infusion through the hepatic artery(study group, n= 22). Patients in the study group were admitted to hospital and received granulocyte colony stimulating factor injections 520 μg/d for 3 consecutive days to mobilize CD34+ cells from the bone marrow. On day 4,leukapheresis was done and CD34+ cells were isolated using CliniMAC magnetic cell sorter. The isolated CD34+ cells were infused into the hepatic artery under radiological guidance. The patients were discharged within 48 h. The control group received standard of care treatment for liver cirrhosis and were worked up for DDLT as per protocol of the institute. Both groups were followed up every week for 4 wk and then every month for 3 mo.RESULTS: In the control and the study group, the cause of cirrhosis was cryptogenic in 18(78.2%) and16(72.72%) and alcohol related in 5(21.7%) and6(27.27%), respectively. The mean day 3 cell count(cells/μL) was 27.00 ± 20.43 with a viability of 81.84± 11.99%. and purity of 80%-90%. Primary end point analysis revealed that at 4 wk, the mean serum albumin in the study group increased significantly(2.83± 0.36 vs 2.43 ± 0.42, P = 0.001) when compared with controls. This improvement in albumin was,however, not sustained at 3 mo. However, at the end of3 mo there was a statistically significant improvement in serum creatinine in the study group(0.96 ± 0.33 vs 1.42 ± 0.70, P = 0.01) which translated into a significant improvement in the Model for End-Stage Liver Disease score(15.75 ± 5.13 vs 19.94 ± 6.68,P = 0.04). On statistical analysis of secondary end points, the transplant free survival at the end of 1 mo and 3 mo did not show any significant difference(P =0.60) when compared to the control group. There was no improvement in aspartate transaminase, alanine transaminase, and bilirubin at any point in the study population. There was no mortality benefit in the study group. The procedure was safe with no procedural or treatment related complications.CONCLUSION: Autologous CD 34+ cell infusion is safe and effectively improves liver function in the short term and may serve as a bridge to liver transplantation.

Autologous CD34^+ and CD133^+ stem cells transplantation in patients with end stage liver disease

AIM:To assess the utility of an autologous CD34 + and CD133 + stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases.METHODS:One hundred and forty patients with endstage liver diseases were randomized into two groups.Group 1,comprising 90 patients,received granulocyte colony stimulating factor for five days followed by autologous CD34 + and CD133 + stem cell infusion in the portal vein.Group 2,comprising 50 patients,received regular liver treatment only and served as a control group.RESULTS:Near normalization of liver enzymes and improvement in synthetic function were observed in 54.5% of the group 1 patients;13.6% of the patients showed stable states in the infused group.None of the patients in the control group showed improvement.No adverse effects were noted.CONCLUSION:Our data showed that a CD34 + and CD133 + stem cells infusion can be used as supportive treatment for end-stage liver disease with satisfactory tolerability.

MiR-34a overexpression enhances the inhibitory effect of doxorubicin on HepG2 cells

BACKGROUND Hepatocellular carcinoma(HCC) is the third leading cause of death from malignant tumors worldwide. More than 50% of HCC cases occur in China. The prognosis remains poor and overall efficacy is still unsatisfactory. Chemotherapy resistance is the most important reason for the poor outcome. Much progress has been made in the study of chemotherapy resistance of HCC;however, the specific mechanisms of progression of HCC have still only been partially established.Therefore, the mechanism of chemotherapy resistance in HCC requires more research.AIM To investigate the effect of miR-34 a expression on the growth inhibition of HepG2 cells by doxorubicin.METHODS A recombinant lentiviral vector containing miR-34 a was constructed and transfected into HepG2 cells. The expression of miR-34 a was detected by reverse transcription-polymerase chain reaction(commonly known as RT-PCR) before and after transfection. Cells were exposed to 2 μM doxorubicin or phosphatebuffered saline before and after transfection. Cell viability in each group was detected by MTT assay, and cell cycle and apoptosis were detected by flow cytometry. Changes in expression levels of phospho(p)-p53, sirtuin(SIRT) 1,cyclin D1, cyclin-dependent kinase(CDK) 4, CDK6, BCL-2, multidrug resistance protein(MDR) 1/P glycoprotein(P-gp), and AXL were detected by Western blotting.RESULTS Recombinant lentiviral vector LV-hsa-mir-34 a was successfully constructed by restriction endonuclease digestion and sequencing. RT-PCR showed that expression of miR-34 a in HepG2 cells was significantly upregulated after transfection(P < 0.01). MTT assay showed that growth of HepG2 cells was inhibited after upregulation of miR-34 a, and viability was significantly decreased after combined treatment with doxorubicin(P < 0.01). Flow cytometry showed that the number of HepG2 cells in G1 phase increased, and G1 phase arrest was more obvious after intervention with doxorubicin(P < 0.01). The apoptosis rate of HepG2 cells was increased after upregulation of miR-34 a, and became more obvious after intervention with doxorubicin(P < 0.01). Western blotting showed that upregulation of miR-34 a combined with treatment with doxorubicin caused significant changes in the expression levels of p-p53, SIRT1, cyclin D1, CDK4,CDK6, BCL-2, MDR1/P-gp and AXL proteins(P < 0.01).CONCLUSION MiR-34 a may enhance the inhibitory effect of doxorubicin by downregulating MDR1/P-gp and AXL, which may be related to p53 expression.

MicroRNA-34a promoting apoptosis of human lens epithelial cells through down-regulation of B-cell lymphoma-2 and silent information regulator

AIM： To investigate the role of micro RNA-34a（mi R-34a） in the induction of apoptosis of human lens epithelial（HLE-B3） cells. METHODS： The apoptosis of HLE-B3 cells was detected by Annexin V-PE apoptosis detection kit after the treatment with 200 μmol/L H2O2 for 24h and lentiviral mi R-34 a vector transfection. The expression of mi R-34 a in the cells was quantified by quantitative real time polymerase chain reaction（q RT-PCR） in response to H2O2 exposure and the vector transfection. The effects of overexpression of mi R-34 a on the expression of B-cell lymphoma-2（Bcl-2） and silent information regulator 1（SIRT1） was determined by q RT-PCR and Western blot. RESULTS： The expression of mi R-34 a was up-regulated by the treatment of H2O2 in HLE-B3 cells. The increased expression of mi R-34 a is accompanied with the cell apoptosis. Consistence with the H2O2 exposure,ectopic overexpression of mi R-34 a in HLE-B3 cells promoted cells apoptosis. Importantly the anti-apoptosis factors Bcl-2 and SIRT1 were reduced significantly by up-regulation of mi R-34 a in HLE-B3 cells.CONCLUSION： Mi R-34 a promotes the apoptosis of HLE-B3 cells by down-regulating Bcl-2 and SIRT1,suggesting that mi R-34 a may involve in the pathogenesis of cataract formation and targeting mi R-34 a may be a potentially therapeutic approach for treatment of cataract.

A familiar stranger:CD34 expression and putative functions in SVF cells of adipose tissue

Human adipose tissue obtained by liposuction is easily accessible and an abundant potential source of autologous cells for regenerative medicine applications. After digestion of the tissue and removal of differentiated adipocytes, the so-called stromal vascular fraction (SVF) of adipose, a mix of various cell types, is obtained. SVF contains mesenchymal fibroblastic cells, able to adhere to culture plastic and to generate large colonies in vitro , that closely resemble bone marrow-derived colony forming units-fibroblastic, and whose expanded progeny, adipose mesenchymal stem/stromal cells (ASC), show strong similarities with bone marrow mesenchymal stem cells. The sialomucin CD34, which is well known as a hematopoietic stem cell marker, is also expressed by ASC in native adipose tissue but its expression is gradually lost upon standard ASC expansion in vitro . Surprisingly little is known about the functional role of CD34 in the biology and tissue forming capacity of SVF cells and ASC. The present editorial provides a short introduction to the CD34 family of sialomucins and reviews the data from the literature concerning ex- pression and function of these proteins in SVF cells and their in vitro expanded progeny.

Expression of Caspase－3 in Cord Blood CD34^＋ Cells during Culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34^+ cells from cord blood (CB) during culture in vitro with different growth factors. Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34^+ CB cells during culture in vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34^+ cells. The expression of caspase-3 mRNA and protein was upregulated when these cellswere first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34^+ cells as well as during the first 3 days expansion with cytokines. With longer culture time in vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34^+ cells during expansion in vitro.

Effect and mechanism of miR-34a on proliferation, apoptosis and invasion of laryngeal carcinoma cells

Objective: To discuss the effect and mechanism of miR-34 a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells. Methods: The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34 a mimics and miR-34 a NC. The MTT, colony-forming assay, Hoechst staining and Annexin V-PI double staining flow cytometry were employed to detect the effect of miR-34 a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34 a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RTPCR assay to defect the effect of miR-34 a mimics on the expression of survivin and Ki-67 m RNA in laryngeal squamous carcinoma Hep2 cells. Results: Compared with miR-34 a NC group, the cell viability in miR-34 mimics group was significantly decreased(P<0.01), the cell apoptosis rate was significantly increased(P<0.01), the abilities of cell migration and invasion were significantly reduced(P<0.01) and the expression of survivin and Ki-67 m RNA was significantly decreased(P<0.01). Conclusions: The increased expression of miR-34 a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.

Demethylation of miR-34a upregulates expression of membrane palmitoylated proteins and promotes the apoptosis of liver cancer cells

BACKGROUND Liver cancer is a common cancer and the main cause of cancer-related deaths worldwide.Liver cancer is the sixth most common cancer in the world.Although miR-34a and palmitoyl membrane palmitoylated protein(MPP2)are reportedly involved in various cell processes,their precise roles in liver cancer are still unclear.AIM To investigate the expression of micro RNA 34a(miR-34a),methylation of the miR-34a promoter and the expression of MPP2 in liver cancer cells and their related mechanisms.METHODS Together,78 cases of liver cancer tissues and 78 cases of adjacent tissues were collected.The methylation degree of miR-34a promoter in liver cancer/paracancerous tissue and liver cancer cells/normal liver cells,and the expression levels of miR-34a and MPP2 in the above samples were detected.Demethylation of liver cancer cells or transfection of liver cancer cells with miR-34a mimetic was performed.The MPP2 overexpression vector was used to transfect liver cancer cells,and the changes in proliferation,invasion,apoptosis,migration,and other biological functions of liver cancer cells after the above interventions were observed.Double luciferase reporter genes were used to detect the targeting relationship between miR-34a and MPP2.RESULTS Clinical samples showed that the expression levels of miR-34a and MPP2 in liver cancer tissues were lower than those in the normal tissues.The methylation degree of miR-34a promoter region in liver cancer cells was higher than that in normal liver cells.After miR-34a demethylation/mimetic transfection/MPP2 overexpression,the apoptosis of liver cancer cells was increased;the proliferation,invasion and migration capabilities were decreased;the expression levels of caspase 3,caspase 9,E-cadherin,and B-cell lymphoma 2(Bcl-2)-associated X protein were increased;and the expression levels of Bcl-2,N-cadherin,andβ-catenin were decreased.Double luciferase reporter genes confirmed that MPP2 is targeted by miR-34a.Rescue experiments showed that small interfering MPP2 could counteract the promoting effect of miR-34a demethylation on apoptosis and the inhibitory effect on cell proliferation,invasion,and migration.CONCLUSION miR-34a demethylation upregulates the expression level of MPP2 in liver cancer cells and promotes the apoptosis of liver cancer cells.miR-34a demethylation is a potential method for liver cancer treatment.

Hematopoietic stem cells in research and clinical applications:The“CD34 issue”

In this paper,experimental findings concerning the kinetics of hematopoietic reconstitution are compared to corresponding clinical data.Although not clearly apparent,the transplantation practice seems to confirm the basic proposals of experimental hematology concerning hematopoietic reconstitution resulting from successive waves of repopulation stemming from different subpopulations of progenitor and stem cells.One of the "f irst rate" parameters in clinical transplantations in hematology;i.e.the CD34+ positive cell dose,has been discussed with respect to the functional heterogeneity and variability of cell populations endowed by expression of CD34.This parameter is useful only if the relative proportion of stem and progenitor cells in the CD34+ cell population is more or less maintained in a series of patients or donors.This proportion could vary with respect to the source,pathology,treatment,processing procedure,the graft ex vivo treatment and so on.Therefore,a universal dose of CD34+ cells cannot be def ined.In addition,to avoid further confusion,the CD34+ cells should not be named "stem cells" or "progenitor cells" since these denominations only concern functionally characterized cell entities.

The effects of microRNA-34a regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide-induced human umbilical vein endothelial cells

BACKGROUND: Notch-1/NF-κB signaling plays a key role in the cecal ligation and puncture(CLP)-induced sepsis. This study aims to investigate the intervention effects of microRNA-34a(miR-34a) lentivirus regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide(LPS)-induced human umbilical vein endothelial cells(HUVEC).METHODS: HUVEC were divided into four groups as the following: they were infected with negative control lentivirus(NC group) or miR-34a lentivirus(OE group); LPS(1 g/mL) was added on the third day on the basis of NC group and OE group for 24 hours(NC+LPS group or OE+LPS group). The levels of TNF-α, IL-1β, IL-6, and IL-10 in the cell supernatants, and the mRNA and protein expression of Notch-1 and NF-κB in the HUVEC were evaluated.RESULTS: After 24 hours, the levels of TNF-α, IL-1β, IL-6 in the cell supernatants and the protein expression of NF-κB from NC+LPS group were significantly higher than those of NC group, but IL-10 level and the protein expression of Notch-1 in NC+LPS group were the opposite. After intervention of miR-34a lentivirus, the cell supernatants TNF-α and the protein expression of NF-κB in OE+LPS group after 24 hours markedly decreased compared to NC+LPS group. While the cell supernatants IL-1β and IL-6 and the mRNA expression of NF-κB slightly decreased in OE+LPS group, IL-10 and the mRNA and protein expression of Notch-1 were the opposite.CONCLUSION: miR-34a regulating Notch-1/NF-κB signaling pathway can reduce the HUVEC damage caused by LPS stimulation.

HUMAN PRIMITIVE HEMATOPOIETIC PROGENITOR CELLS ARE MORE ENRICHED IN CD34^+CD38^- POPULATION THAN IN CD34^+CD38^+ POPULATION

To clarify the hematopoietic potential of various sub-classes of human hematopoietic progenitor cells, we used a multicolor staining protocol in conjunction with anti-CD34 and -CD38 McAb. We characterized two cell fractions in CD34+cells with or without CD38 expression. A clonogenic assay showed that most CFC were present in CD34+CD38+ population. Morphologic analysis showed that blast-like cells were more enriched in the CD34+CD38 fraction. To clarify the biologic differences between both fractions, we examined the more primitive progenitor cell function by assessing long-term culture-initiating cells (LTC-IC) on the stromal cells. At the first two weeks, more CF.C harvested from the culture in the fractions initiated with both populations. However, more LTC-IC were present during weeks 4 to 12 in the CD34+CD38- population. These results indicate the primitive progenitors are more enriched in CD34+CD38 population than in CD34+CD38+ cells.