Hypoxia-preconditioned bone marrow-derived mesenchymal stem cells protect neurons from cardiac arrest-induced pyroptosis

Cardiac arrest can lead to severe neurological impairment as a result of inflammation,mitochondrial dysfunction,and post-cardiopulmonary resuscitation neurological damage.Hypoxic preconditioning has been shown to improve migration and survival of bone marrow–derived mesenchymal stem cells and reduce pyroptosis after cardiac arrest,but the specific mechanisms by which hypoxia-preconditioned bone marrow–derived mesenchymal stem cells protect against brain injury after cardiac arrest are unknown.To this end,we established an in vitro co-culture model of bone marrow–derived mesenchymal stem cells and oxygen–glucose deprived primary neurons and found that hypoxic preconditioning enhanced the protective effect of bone marrow stromal stem cells against neuronal pyroptosis,possibly through inhibition of the MAPK and nuclear factor κB pathways.Subsequently,we transplanted hypoxia-preconditioned bone marrow–derived mesenchymal stem cells into the lateral ventricle after the return of spontaneous circulation in an 8-minute cardiac arrest rat model induced by asphyxia.The results showed that hypoxia-preconditioned bone marrow–derived mesenchymal stem cells significantly reduced cardiac arrest–induced neuronal pyroptosis,oxidative stress,and mitochondrial damage,whereas knockdown of the liver isoform of phosphofructokinase in bone marrow–derived mesenchymal stem cells inhibited these effects.To conclude,hypoxia-preconditioned bone marrow–derived mesenchymal stem cells offer a promising therapeutic approach for neuronal injury following cardiac arrest,and their beneficial effects are potentially associated with increased expression of the liver isoform of phosphofructokinase following hypoxic preconditioning.

Bone marrow-derived mesenchymal stem cell-derived exosomeloaded miR-129-5p targets high-mobility group box 1 attenuates neurological-impairment after diabetic cerebral hemorrhage

BACKGROUND Diabetic intracerebral hemorrhage(ICH)is a serious complication of diabetes.The role and mechanism of bone marrow mesenchymal stem cell(BMSC)-derived exosomes(BMSC-exo)in neuroinflammation post-ICH in patients with diabetes are unknown.In this study,we investigated the regulation of BMSC-exo on hyperglycemia-induced neuroinflammation.AIM To study the mechanism of BMSC-exo on nerve function damage after diabetes complicated with cerebral hemorrhage.METHODS BMSC-exo were isolated from mouse BMSC media.This was followed by transfection with microRNA-129-5p(miR-129-5p).BMSC-exo or miR-129-5poverexpressing BMSC-exo were intravitreally injected into a diabetes mouse model with ICH for in vivo analyses and were cocultured with high glucoseaffected BV2 cells for in vitro analyses.The dual luciferase test and RNA immunoprecipitation test verified the targeted binding relationship between miR-129-5p and high-mobility group box 1(HMGB1).Quantitative polymerase chain reaction,western blotting,and enzyme-linked immunosorbent assay were conducted to assess the levels of some inflammation factors,such as HMGB1,interleukin 6,interleukin 1β,toll-like receptor 4,and tumor necrosis factorα.Brain water content,neural function deficit score,and Evans blue were used to measure the neural function of mice.RESULTS Our findings indicated that BMSC-exo can promote neuroinflammation and functional recovery.MicroRNA chip analysis of BMSC-exo identified miR-129-5p as the specific microRNA with a protective role in neuroinflammation.Overexpression of miR-129-5p in BMSC-exo reduced the inflammatory response and neurological impairment in comorbid diabetes and ICH cases.Furthermore,we found that miR-129-5p had a targeted binding relationship with HMGB1 mRNA.CONCLUSION We demonstrated that BMSC-exo can reduce the inflammatory response after ICH with diabetes,thereby improving the neurological function of the brain.

Overexpression of lentivirus-mediated glial cell line-derived neurotrophic factor in bone marrow stromal cells and its neuroprotection for the PC12 cells damaged by lactacystin

Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.

Glial cell-derived neurotrophic factor mRNA expression in a rat model of spinal cord injury following bone marrow stromal cell transplantation

BACKGROUND： Several animal experiments utilizing bone marrow stromal cell （BMSC） transplantation for the treatment of spinal cord injury have proposed a hypothesis that BMSC transplantation effects are associated with increased glial cell-derived neurotrophic factor （GDNF） expression. OBJECTIVE： To confirm the effects of BMSC transplantation on GDNF mRNA expression in rats with spinal cord injury by reverse transcription-polymerase chain reaction （RT-PCR）. DESIGN, TIME AND SETTING： The present molecular, cell biology experiment was performed at the Key Laboratory of Children＇s Congenital Malformation, Ministry of Health of China ＆ Department of Developmental Biology, Basic Medical College, China Medical University between March 2006 and May 2007. MATERIALS： Sixty healthy Wistar rats aged 2-4-months and of either gender were included in this study. Spinal cord injury was induced in all rats by hemisection of T9 on the left side. RT-PCR kits were purchased from TaKaRa Company, China. Type 9600 RCR amplifier was provided by Perkin Elmer Company, USA. METHODS： Three rats were selected for BMSC culture and subsequent transplantation （after three passages）. Of the remaining 57 rats, nine were selected for sham-operation （sham-operated group）, where only the T9 spinal cord was exposed without hemisection. A total of 48 rats were randomly and evenly divided into BMSC transplantation and model groups. In the BMSC transplantation group, following spinal cord injury induction, each rat was administered a BMSC suspension tbrougb two injection sites selected on the gray and white matter boundary caudally and cephalically, seperately and near to injury site in the spinal cord. The model group received an equal volume of PBS through the identical injection sites. MAIN OUTCOME MEASURES： At 24 and 72 hours, as well as at 7 days, following spinal cord injury, the spinal cord at the T9 segment was removed. Eight rats were allocated to each time point in the BMSC transplantation and model groups, with three rats allocated to the sham-operated group. GDNF mRNA expression was semiquantitatively analyzed by RT-PCR. RESULTS： The sham-operated group exhibited extremely low GDNF mRNA expression. GDNF mRNA expression significantly increased at 24 hours after spinal cord injury, reached a peak level at 72 hours, and slowly decreased thereafter. However, it remained higher than normal levels at 7 days （P 〈 0.05）. At all time points following spinal cord injury, GDNF mRNA expression was significantly greater in the BMSC transplantation group than in the model group （P 〈 0.05）. CONCLUSION： Transplantation of BMSCs into the injured spinal cord up-regulated GDNF mRNA expression, thereby promoting repair of the injured spinal cord.

Stromal cell derived factor-1 enhances bone marrow mononuclear cell migration in mice with acute liver failure

AIM： To evaluate the number of bone marrow mononuclear cells （BMMC） that are migrated to the liver following transplantation of murine BMMC into mice with acute liver injury.METHODS： BMMC were isolated from the bone marrow of mice in a lymphocyte separation medium and then labeled with PKH26. The labeled cells were subsequently infused into the caudal veins of BALB/c mice with hepatic injury induced by carbon tetrachloride and 2-acetylaminofluorene. Mice in experimental group were treated with stromal cell-derived factor-1 （SDF-1） which was injected intraperitoneally after trans- plantation of BMMC. Mice in control group were injected intraperitoneally with 0.1 mL of saline （0.9% NaCl） after transplantation of BMMC. After 2 wk, migration of the cells in experimental group was studied by fluorescence microscopy. The expression of proliferating cell nuclear antigen and albumin was quantified with manual methods in both groups. The serum transaminase levels at different time points were compared between the two groups.RESULTS： The labeled ＂cells＂ were found in the portal region and central veins of hepatic Iobules. The PKH26labeled cells appeared at an average frequency of 108 ± 8/high power field in the experiment group and 65 ± 8/high power field in the control group （P 〈 0.05）. The total number of positive cells was 29 ± 7/high power field in the experimental group and 13 ± 2/high power field in the control group. The albumin expression level was also higher in the experimental group than in the control group （29 ± 7 vs 13 ± 2, P 〈 0.05）. The total number of crossing points was 156 ± 5/high power field in the experimental group and 53 ± 5/high power field in the control group （P 〈 0.05）. The serum alanine aminotransferase levels in experimental and control groups were measured at different time points （120 ± 40 vs 118.50 ± 1.75, P 〉 0.05; 80.60 ± 6.50 vs 101.08 ± 5.67, P 〈 0.05; 50.74 ± 5.38 vs 80.47 ± 4.62, P 〈 0.05; 30.54 ± 2.70 vs 60.72 ± 4.37, P 〈 0.05; 30.77 ± 5.36 vs 40.47 ± 6.50, P 〈 0.05）. At the same time, the serum aspartate aminotransferase levels were measured in experimental and control groups at different time points （122.55 ± 1.46 vs 120.70 ± 4.22, P 〉 0.05; 54.26 ± 6.50 vs 98.70 ± 8.20, P 〈 0.05; 39.47 ± 5.39 vs 78.34 ± 4.50, P 〈 0.05; 28.94 ±2.70 vs 56.44 ± 4.28, P 〈 0.05; 30.77 ± 5.45 vs 42.50 ± 6.28, P 〈 0.05）.CONCLUSION： SDF-1 can promote the migration of BMMC to the liver of mice with acute liver failure.

Adipose-derived stromal cells resemble bone marrow stromal cells in hepatocyte differentiation potential in vitro and in vivo

AIM To investigate whether mesenchymal stem cells(MSCs) from adipose-derived stromal cells(ADSCs) and bone marrow stromal cells(BMSCs) have similar hepatic differentiation potential.METHODS Mouse ADSCs and BMSCs were isolated and cultured. Their morphological and phenotypic characteristics, as well as their multiple differentiation capacity were compared. A new culture system was established to induce ADSCs and BMSCs into functional hepatocytes. Reverse transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to identify the induced hepatocytelike cells. CM-Dil-labeled ADSCs and BMSCs were then transplanted into a mouse model of CCl4-induced acute liver failure. fluorescence microscopy was used to track the transplanted MSCs. Liver function was tested by an automatic biochemistry analyzer, and liver tissue histology was observed by hematoxylin and eosin(HE) staining.RESULTS ADSCs and BMSCs shared a similar morphology and multiple differentiation capacity, as well as a similar phenotype(with expression of CD29 and CD90 and no expression of CD11 b or CD45). Morphologically, ADSCs and BMSCs became round and epithelioid following hepatic induction. These two cell types differentiated into hepatocyte-like cells with similar expression of albumin, cytokeratin 18, cytokeratin 19, alpha fetoprotein, and cytochrome P450. fluorescence microscopy revealed that both ADSCs and BMSCs were observed in the mouse liver at different time points. Compared to the control group, both the function of the injured livers and HE staining showed significant improvement in the ADSC-and BMSC-transplanted mice. There was no significant difference between the two MSC groups.CONCLUSION ADSCs share a similar hepatic differentiation capacity and therapeutic effect with BMSCs in an acute liver failure model. ADSCs may represent an ideal seed cell type for cell transplantation or a bio-artificial liver support system.

In vitro differentiation of adipose-derived stem cells and bone marrow-derived stromal stem cells into neuronal-like cells

Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analysis by western blot and quantitative real-time PCR showed that protein levels of Nanog, Oct4, and Sox2, and mRNA levels of miR/125a/3p were decreased, while expression of insulin-like growth factor-2 and neuron specific enolase was increased. In comparison the generation of neuron specific enolase-positive cells was most successful when adipose-derived stem cells were co-cultured with Aβ1-40-treated PC12 cells. Our results demonstrate that adipose-derived stem cells and bone marrow-derived stromal stem cells exhibit trends of neuronal-like cell differentiation after co-culture with Aβ1-40-treated PC12 cells. This process may relate to a downregulation of miR-125a-3p mRNA expression and increased levels of insulin-like growth factor-2 expression.

Improvement of learning and memory abilities and motor function in rats with cerebral infarction by intracerebral transplantation of neuron-like cells derived from bone marrow stromal cells

BACKGROUND： Transplantation of fetal cell suspension or blocks of fetal tissue can ameliorate the nerve function after the injury or disease in the central nervous system, and it has been used to treat neurodegenerative disorders induced by Parkinson disease. OBJECTIVE： To observe the effects of the transplantation of neuron-like cells derived from bone marrow stromal cells （rMSCs） into the brain in restoring the dysfunctions of muscle strength and balance as well as learning and memory in rat models of cerebral infarction. DESIGN ： A randomized controlled experiment.SETTING ： Department of Pathophysiology, Zhongshan Medical College of Sun Yat-sen University.MATERIALS ： Twenty-four male SD rats （3-4 weeks of age, weighing 200-220 g） were used in this study （Certification number：2001A027）. METHODS： The experiments were carried out in Zhongshan Medical College of Sun Yat-sen University between December 2003 and December 2004. ① Twenty-four male SD rats randomized into three groups with 8 rats in each： experimental group, control group and sham-operated group. Rats in the experiment al group and control group were induced into models of middle cerebral artery occlusion （MCAO）. After in vitro cultured, purified and identified with digestion, the Fischer344 rMSCs were induced to differentiate by tanshinone IIA, which was locally injected into the striate cortex （18 area） of rats in the experimental group, and the rats in the control group were injected by L-DMEM basic culture media （without serum） of the same volume to the corresponding brain area. In the sham-operated group, only muscle and vessel of neck were separated. ② At 2 and 8 weeks after the transplantation, the rats were given the screen test, prehensile-traction test, balance beam test and Morris water-maze test. ③ The survival and distribution of the induced cells in corresponding brain area were observed with Nissl stained with toluidine blue and hematoxylin and eosin （HE） staining in the groups.MAIN OUTCOME MEASURES ： ① Results of the behavioral tests （time of the Morris water-maze test screen test, prehensile-traction test, balance beam test）; ② Survival and distribution of the induced cells.RESULTS： All the 24 rats were involved in the analysis of results. ① Two weeks after transplantation, rats with neuron-like cells grafts in the experimental group had significant improvement on their general muscle strength than those in the control group [screen test： （9.4±1.7）, （4.7±1.0） s, P 〈 0.01]; forelimb muscle strength [prehensile-traction test： （7.6±1.4）, （5.2±1.2） s, P 〈 0.01], ability to keep balance [balance beam test： （7.9±0.74）, （6.1±0.91） s, P 〈 0.01] and abilities of learning and memory [latency to find the platform： （35.8±5.9）, （117.5±11.6） s, P 〈 0.01; distance： （623.1±43.4）, （1 902.3±98.6） cm, P 〈 0.01] as compared with those in the control group. The functional performances in the experimental group at 8 weeks were better than those at two weeks, which were still obviously different from those in the sham-operated group （P 〈 0.05）. ② The HE and Nissl stained brain tissue section showed that there was nerve cell proliferation at the infarcted cortex in the experiment group, the density was higher than that in the control group, plenty of aggregative or scattered cells could be observed at the site where needle was inserted for transplantation, the cells migrated directively towards the area around them, the cerebral vascular walls were wrapped by plenty of cells; In the control group, most of the cortices were destroyed, karyopyknosis and necrosis of neurons were observed, normal nervous tissue structure disappeared induced by edema, only some nerve fibers and glial cells remained.CONCLUSION： The rMSCs transplantation can obviously enhance the motor function and the abilities of learning and memory in rat models of cerebral infarction.

Neuron-like differentiation of adult rat bone marrow stromal cells induced by transforming growth factor-beta and brain-derived neurotrophic factor

BACKGROUND： It has been demonstrated that transforming growth factor-β （TGF-β） and brain- derived neurotrophic factor （BDNF） can induce stem cell differentiation into neuron-like cells. OBJECTIVE： To investigate the efficacy of TGF-β and BDNF at inducing the differentiation of adult rat bone marrow stromal cells （BMSCs） into neuron-like cells, both in combination or alone. DESIGN, TIME AND SETTING： A comparative observation experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Liaoning Medical University between October 2007 and January 2008. MATERIALS： TGF-~ and BDNF were purchased from Sigma, USA; mouse anti-rat neuron specific enolase, neurofilament and glial fibrillary acidic protein were purchased from Beijing HMHL Biochem Ltd., China. METHODS： BMSCs were isolated from rats aged 4 weeks and incubated with TGF-β（1μ g/L） and/or BDNF （50 μ g/mL）. MAIN OUTCOME MEASURES： Expression of neuron-specific enolase, neurofilament and glial fibrillary acidic protein were determined by immunocytochemistry. RESULTS： BMSCs differentiated into neuron-like cells following induction of TGF-β and BDNF, and expressed both neuron-specific enolase and neurofilament. The percent of positive cells was significantly greater in the combination group than those induced with TGF-β or BDNF alone （P 〈 0.01）. CONCLUSION： Treatment of BMSCs with a combination of TGF-β and BDNF induced differentiation into neuron-like cells, with the induction being significantly greater than with TGF-β or BDNF alone.

miR-124 and miR-128 differential expression in bone marrow stromal cells and spinal cord-derived neural stem cells

BACKGROUND： MicroRNA （miRNA） expression in stem cells provides important clues for the molecular mechanisms of stem cell proliferation and differentiation. Bone marrow stromal cells and spinal cord-derived neural stem cells exhibit potential for neural regeneration. However, miRNA expression in these cells has been rarely reported. OBJECTIVE： To explore differential expression of two nervous system-specific miRNAs, miR-124 and miR-128, in bone marrow stromal cells and spinal cord-derived neural stem cells. DESIGN, TIME AND SETTING： An In vitro, cell biology experiment was performed at the Department of Biotechnology, Shanxi Medical University from June 2008 to June 2009. MATERIALS： TaqMan miRNA assays were purchased from Applied Biosystems. METHODS： Rat bone marrow stromal cells were isolated and cultured using the whole-bone marrow method, and rat spinal cord-derived neural stem cells were obtained through neurosphere formation. TaqMan miRNA assays were used to measure miR-124 and miR-128 expression in bone marrow stromal cells and spinal cord-derived neural stem cells. MAIN OUTCOME MEASURES： Morphology of bone marrow stromal cells and spinal cord-derived neural stem cells were observed by inverted microscopy. Expression of the neural stem cell-specific marker, nestin, the bone marrow stromal cell surface marker, CD71, and expression of miR-124 and miR-128, were detected by real-time polymerase chain reaction. RESULTS： Cultured bone marrow stromal cells displayed a short fusiform shape. Flow cytometry revealed a large number of CD71-positive cells （〉 95%）. Cultured spinal cord-derived neural stem cells formed nestin-positive neurospheres, and quantitative detection of miRNA demonstrated that less miR-124 and miR-128 was expressed in bone marrow stromal cells compared to spinal cord-derived neural stem cells （P 〈 0.05）. CONCLUSION： Bone marrow stromal cells and spinal cord-derived neural stem cells exhibited differential expression of miR-124 and miR-128, which suggested different characteristics in miRNA expression.

Optimization of adipose tissue-derived mesenchymal stromal cells transplantation for bone marrow repopulation following irradiation

BACKGROUND Bone marrow(BM)suppression is one of the most common side effects of radiotherapy and the primary cause of death following exposure to irradiation.Despite concerted efforts,there is no definitive treatment method available.Recent studies have reported using mesenchymal stromal cells(MSCs),but their therapeutic effects are contested.AIM We administered and examined the effects of various amounts of adipose-derived MSCs(ADSCs)in mice with radiation-induced BM suppression.METHODS Mice were divided into three groups:Normal control group,irradiated(RT)group,and stem cell-treated group following whole-body irradiation(WBI).Mouse ADSCs(mADSCs)were transplanted into the peritoneal cavity either once or three times at 5×10^(5) cells/200μL.The white blood cell count and the levels of,plasma cytokines,BM mRNA,and BM surface markers were compared between the three groups.Human BM-derived CD34+hematopoietic progenitor cells were co-cultured with human ADSCs(hADSCs)or incubated in the presence of hADSCs conditioned media to investigate the effect on human cells in vitro.RESULTS The survival rate of mice that received one transplant of mADSCs was higher than that of mice that received three transplants.Multiple transplantations of ADSCs delayed the repopulation of BM hematopoietic stem cells.Anti-inflammatory effects and M2 polarization by intraperitoneal ADSCs might suppress erythropoiesis and induce myelopoiesis in sub-lethally RT mice.CONCLUSION The results suggested that an optimal amount of MSCs could improve survival rates post-WBI.

Effects of corneal stromal cell-and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

AIM： To explore the effects of conditioned media on the proliferation of corneal endothelial cells （CECa） and to compare the efficiency of different conditioned media （CM）. METHODS： Rat CECs, corneal stromal cells （CSCs）, bone marrow -derived endothelial progenitor cells （BEPCs）, and bone marrow-derived mesenchymal stem cells （BMSCs） were isolated and cultured in vitra CM was collected from CSCs, BEPCs, and BMSCSo CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed.~ RESULTS： After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone- like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na＋/K＋-ATP, aquaporin 1 （AQP1）, and zonula occludens 1 （ZO-1）. Based on quantitative polymerase chain reaction （qPCR） analysis, Na ＋/K ＋-ATP expression in CSC-CM was notably upregulated by 1.3-fold （＋0.036） （P〈0.05, n=3）. The expression levels of ZO-1, neuron specific enolase （NSE）, Vimentin, paired homebox 6 （PAX6）, and procollagen type VII （COL8A1） were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation.~ CONCLUSION： CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. KEYWORDS： conditioned medium; corneal endothelial cell; corneal stromal cell; bone marrow-derived endothelial progenitor cell; proliferation

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.

Synergistic effects of brain-derived neurotrophic factor and retinoic acid on inducing the differentiation of bone marrow stromal cells into neuron-like cells in adult rats in vitro

BACKGROUND： Under induction of retinoic acid （RA）, bone marrow stromal cells （BMSCs） can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an application. Other neurotrophic factors can also differentiate into neuronal cells through inducing BMSCs; especially, brain-derived neurotrophic factor （BDNF） can delay natural death of neurons and play a key role in survival and growth of neurons. The combination of them is beneficial for differentiation of BMSCs. OBJECTIVE： To investigate the effects of BDNF combining with RA on inducing differentiation of BMSCs to nerve cells of adult rats and compare the results between common medium group and single BDNF group. DESIGN： Randomized controlled animal study SETTING： Department of Neurology, Affiliated Hospital of Xuzhou Medical College MATERIALS： The experiment was carried out in the Clinical Neurological Laboratory of Xuzhou Medical College from September 2003 to April 2005. A total of 24 SD rats, of either gender, 2 months old, weighing 130-150 g, were provided by Experimental Animal Center of Xuzhou Medical College [certification： SYXK （su） 2002-0038]. Materials and reagents： low-glucose DMEM medium, bovine serum, BDNF, RA, trypsin, separating medium of lymphocyte, monoclonal antibody of mouse-anti-nestin, neuro-specific enolase, glial fibrillary acidic protein （GFAP） antibody, SABC kit, and diaminobenzidine （DAB） color agent. All these mentioned above were mainly provided by SIGMA Company, GIBCO Company and Boshide Company. METHODS： Bone marrow of SD rats was selected for density gradient centrifugation. BMSCs were undertaken primary culture and subculture; and then, those cells were induced respectively in various mediums in total of 3 groups, including control group （primary culture）, BDNF group （20 μg/L BDNF） and BDNF＋RA group （20 μg/L BDNF plus 20 μg/L RA）. On the 3^rd and the 7^th days after induction, BMSCs were stained immunocytochemically with nestin （sign of nerve stem cells）, neuron-specific enolase （NSE, sign of diagnosing neurons） and GFAP （diagnosing astrocyte）, and evaluated cellular property. MAIN OUTCOME MEASURES ： Induction and differentiation in vitro of BMSCs in 3 groups RESULTS： （1） Induction and differentiation of BMSCs： Seven days after induction, cells having 2 or more apophyses were observed. Soma shaped like angle or erose form, which were similar to neurons and glial cells having strong refraction. （2） Results of immunocytochemical detection： Three days after induction, rate of positive cells in BDNF＋RA group was higher than that in BDNF group and control group [（86.15±4.58）%, （65.43±4.23）%, （4.18±1.09）%, P 〈 0.01]. Seven days after induction, rate of positive cells was lower in BDNF group and BDNF＋RA group than that in both groups at 3 days after induction [（31.12±3.18）%, （29.35±2.69）%, P 〈 0.01]; however, amounts of positive cells of NSE and GFAP were higher than those at 3 days after induction （P 〈 0.01）; meanwhile, the amount in BDNF＋RA group was remarkably higher than that in BDNF group （P 〈 0.01）. CONCLUSION： Combination of BDNF and RA can cooperate differentiation of BMSCs into neurons and astrocyte, and the effect is superior to single usage of BDNF.

EXPRESSION OF rhBMP-7 GENE IN TRANSDUCED BONE MARROW DERIVED STROMAL CELLS

OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. RESULTS: The exogenous gene could be expressed efficiently in transduced BMSCs. CONCLUSION: The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.

Stem Cell Ophthalmology Treatment Study (SCOTS)： bone marrow-derived stem cells in the treatment of Leber＇s hereditary optic neuropathy

The Stem Cell Ophthalmology Treatment Study （SCOTS） is currently the largest-scale stem cell ophthal- mology trial registered at ClinicalTrials.gov （identifier： NCT01920867）. SCOTS utilizes autologous bone marrow-derived stem cells （BMSCs） to treat optic nerve and retinal diseases. Treatment approaches include a combination of retrobulbar, subtenon, intravitreal, intra-optic nerve, subretinal, and intravenous injection of autologous BMSCs according to the nature of the disease, the degree of visual loss, and any risk factors related to the treatments. Patients with Leber＇s hereditary optic neuropathy had visual acuity gains on the Early Treatment Diabetic Retinopathy Study （ETDRS） of up to 35 letters and Snellen acuity improvements from hand motion to 20/200 and from counting fingers to 20/100. Visual field improvements were noted. Macular and optic nerve head nerve fiber layer typically thickened. No serious complications were seen. The increases in visual acuity obtained in our study were encouraging and suggest that the use of autolo- gous BMSCs as provided in SCOTS for ophthalmologic mitochondrial diseases including Leber＇s hereditary optic neuropathy may be a viable treatment option.

Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in-vivo research reviews revealed more controversies in this issue. We expect the new researchers can have a quick understanding of the progress in this filed and design a more comprehensive research based on this review.

Human urokinase-type plasminogen activator gene-modifiedbone marrow-derived mesenchymal stem cells attenuateliver fibrosis in rats by down-regulating the Wnt signalingpathway

AIM: To evaluate the therapeutic effects of bone marrow-derived mesenchymal stem cells(BMSCs) with human urokinase-type plasminogen activator(u PA) on liver fibrosis, and to investigate the mechanism of gene therapy.METHODS: BMSCs transfected with adenovirusmediated human urokinase plasminogen activator(Adu PA) were transplanted into rats with CCl4-induced liver fibrosis. All rats were sacrificed after 8 wk, and their serum and liver tissue were collected for biochemical, histopathologic, and molecular analyzes. The degree of liver fibrosis was assessed by hematoxylin and eosin or Masson's staining. Western blot and quantitative reverse transcription-polymerase chain reaction were used to determine protein and m RNA expression levels.RESULTS: Serum levels of alanine aminotransferase, aminotransferase, total bilirubin, hyaluronic acid, laminin, and procollagen type Ⅲ were markedly decreased, whereas the levels of serum albumin were increased by u PA gene modified BMSCs treatment. Histopathology revealed that chronic CCl4-treatment resulted in significant fibrosis while u PA gene modified BMSCs treatment significantly reversed fibrosis. By quantitatively analysing the fibrosis area of liver tissue using Masson staining in different groups of animals, we found that model animals with CCl4-induced liver fibrosis had the largest fibrotic area(16.69% ± 1.30%), while fibrotic area was significantly decreased by BMSCs treatment(12.38% ± 2.27%) and was further reduced by u PA-BMSCs treatment(8.31% ± 1.21%). Both protein and m RNA expression of β-catenin, Wnt4 and Wnt5 a was down-regulated in liver tissues following u PA gene modified BMSCs treatment when compared with the model animals.CONCLUSION: Transplantation of u PA gene modified BMSCs suppressed liver fibrosis and ameliorated liver function and may be a new approach to treating liver fibrosis. Furthermore, treatment with u PA gene modified BMSCs also resulted in a decrease in expression of molecules of the Wnt signaling pathway.

Autologous bone marrow derived stem cell therapy in patients with type 2 diabetes mellitus-defining adequate administration methods

AIM To carry out randomized trial for evaluating effects of autologous bone marrow derived stem cell therapy(ABMSCT) through different routes.METHODS Bone marrow aspirate was taken from the iliac crest of patients. Bone marrow mononuclear cells were separatedand purified using centrifugation. These cells were then infused in a total of 21 patients comprising three groups of 7 patients each. Cells were infused into the superior pancreaticoduodenal artery(Group Ⅰ), splenic artery(Group Ⅱ) and through the peripheral intravenous route(Group Ⅲ). Another group of 7 patients acted as controls and a sham procedure was carried out on them(Group Ⅳ). The cells were labelled with the PET tracer F18-FDG to see their homing and in vivo distribution. Data for clinical outcome was expressed as mean ± SE. All other data was expressed as mean ± SD. Baseline and post treatment data was compared at the end of six months, using paired t-test. Cases and controls data were analyzed using independent t-test. A probability(P) value of < 0.05 was regarded as statistically significant. Measures of clinical outcome were taken as the change or improvement in the following parameters:(1) C-peptide assay;(2) HOMA-IR and HOMA-B;(3) reduction in Insulin dose; subjects who showed reduction of insulin requirement of more than 50% from baseline requirement were regarded as responders; and(4) reduction in HbA 1c. RESULTS All the patients, after being advised for healthy lifestyle changes, were evaluated at periodical intervals and at the end of 6 mo. The changes in body weight, body mass index, waist circumference and percentage of body fat in all groups were not significantly different at the end of this period. The results of intra-group comparison before and after ABMSCT at the end of six months duration was as follows:(1) the area under C-peptide response curve was increased at the end of 6 mo however the difference remained statistically non-significant(P values for fasting C-peptide were 0.973, 0.103, 0.263 and 0.287 respectively and the P values for stimulated C-peptide were 0.989, 0.395, 0.325 and 0.408 respectively for groups Ⅰ?to Ⅳ);(2) the Insulin sensitivity indices of HOMA IR and HOMA B also did not show any significant differences(P values for HOMA IR were 0.368, 0.223, 0.918 and 0.895 respectively and P values for HOMA B were 0.183, 0.664, 0.206 and 0.618 respectively for groups Ⅰto Ⅳ);(3) Group Ⅰshowed a significant reduction in Insulin dose requirement(P < 0.01). Group Ⅱ patients also achieved a significant reduction in Insulin dosages(P = 0.01). The Group Ⅰand Group Ⅱ patients together constituted the targeted group wherein the feeding arteries to pancreas were used for infusing stem cells. Group Ⅲ, which was the intravenous group, showed a non-significant reduction in Insulin dose requirement(P = 0.137). Group Ⅳ patients which comprised the control arm also showed a significant reduction in Insulin dosages at the end of six months(P < 0.05); and(4) there was a non-significant change in the Hb A1 c levels at the end of 6 mo across all groups(P = 0.355, P = 0.351, P = 0.999 and P = 0.408 respectively for groups Ⅰto Ⅳ). CONCLUSION Targeted route showed a significant reduction in Insulin requirement at the end of six months of study period whereas the intravenous group failed to show reduction.

Growth-associated protein 43 and neural cell adhesion molecule expression following bone marrow-derived mesenchymal stem cell transplantation in a rat model of ischemic brain injury

BACKGROUND： Transplantation of bone marrow-derived mesenchymal stem cells （BMSCs） improves motor functional recovery, but the mechanisms remain unclear. OBJECTIVE： To investigate expression of growth-associated protein 43 （GAP-43） and neural cell adhesion molecule following BMSC transplantation to the lateral ventricle in rats with acute focal cerebral ischemic brain damage. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment using immunohistochemistry was performed at the laboratories of Department of Neurology, Renmin Hospital of Wuhan University and Doctoral Scientific Research Work Station of C-BONS PHARMA, Hubei Province, China, from January 2007 to December 2008. MATERIALS： Monoclonal mouse anti-rat 5-bromo-2-deoxyuridine and neural cell adhesion molecule antibodies were purchased from Sigma, USA; monoclonal mouse anti-rat GAP-43 antibody was purchased from Wuhan Boster, China. METHODS： Rat models of right middle cerebral artery occlusion were established using the thread method. At 1 day after middle cerebral artery occlusion, 20μL culture solution, containing 5×10^5 BMSCs, was transplanted to the left lateral ventricle using micro-injection. MAIN OUTCOME MEASURES： Scores of neurological impairment were measured to assess neural function. Expression of GAP-43 and neural cell adhesion molecule at the lesion areas was examined by immunohistochemistry. RESULTS： GAP-43 and neural cell adhesion molecule expression was low in brain tissues of the sham-operated group, but expression increased at the ischemic boundary （P 〈 0.05）. Transplantation of BMSCs further enhanced expression of GAP-43 and neural cell adhesion molecule （P 〈 0.05） and remarkably improved neurological impairment of ischemic rats （P 〈 0.05）. CONCLUSION： BMSC transplantation promoted neurological recovery in rats by upregulating expression of GAP-43 and neural cell adhesion molecule.