Mammalian target of rapamycin signaling is involved in the vasculogenic mimicry of glioma via hypoxia-inducible factor-1α.

The mammalian target of rapamycin (mTOR) is a crucial regulator in malignant gliomas. Vasculogenic mimicry (VM) describes functional channels established by highly malignant tumor cells that is different from endothelium-lined blood vessels. Our previous studies confirmed the existence and clinical significance of VM in medulloblastoma and glioblastoma. In the present study, by immunohistochemical and CD34/PAS histochemical double-staining, 34 cases (26.8%) with VM structures were identified among a total of 127 glioma cases, and these VM structures were associated with mTOR expression in the glioma specimens. In vitro, U87 malignant glioblastoma cells formed tube structures similar to HUVECs on Matrigel in 3D culture, and mTOR-specific inhibitor rapamycin inhibited VM formation in the U87 malignant glioblastoma cells under both normoxia and hypoxia. In addition, rapamycin and mTOR siRNA inhibited molecules in the signaling cascade of VM formation, particularly HIF-1α. Taken together, our results demonstrated that mTOR signaling is involved in VM formation, and may be a potential therapeutic target for gliomas.

Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells.

Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.

Nitric oxide-mediated immunosuppressive effect of human amniotic membrane-derived mesenchymal stem cells on the viability and migration of microglia.

Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered a novel and promising source of stem cells for cell replacement-based therapy. Current research is mostly limited to investigating the cellular differentiation potential of AMSCs, while few have focused on their immunosuppressive properties. This study is aimed at exploring and evaluating the immunosuppressive effect of human AMSCs on the viability and migratory properties of microglia. We found, from results of cell viability assays, that AMSCs can reduce the activity of inflammatory cells by secreting nitric oxide (NO). Also, based on results from wound healing and transwell migration assays, we show that AMSCs can inhibit the migration of human microglia as well as the mouse microglial cell line BV2, suggesting that they have the ability to inhibit the recruitment of certain immune cells to injury sites. Furthermore, we found that NO contributes significantly to this inhibitory effect. Our study provides evidence that human AMSCs can have detrimental effects on the viability and migration of microglia, through secretion of NO. This mechanism may contribute to anti-inflammatory processes in the central nervous system.

Blood-brain barrier disruption induced by hemoglobin in vivo: Involvement of up-regulation of nitric oxide synthase and peroxynitrite formation.

Accumulating evidence has demonstrated that up-regulation of nitric oxide synthase (NOS) and subsequent peroxynitrite (ONOO(-)) formation exert a devastating effect on the damage of BBB in multiple diseases. However, considerably less attention has been focused on the role of NOS/ONOO(-) in BBB disruption after intracerebral hemorrhage (ICH). Using an experimental stroke model by injecting hemoglobin (Hb) into the caudate nucleus of male Sprague Dawley rats, we explored the role of NOS/ONOO(-) in BBB disruption after ICH. Brain edema content, behavioral changes, alterations of TJ proteins (claudin-5 and ZO-1), expression of neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), formation of 3-nitrotyrosine (3-NT), as well as NO production were investigated. Hb in the rat brain led to a significant brain edema production and neurological deficits. Overexpressed NOS was concomitant with large quantities of 3-NT formation. Moreover, sites of enhanced nNOS, iNOS, eNOS and 3-NT immunoreactivity were colocalized with diminished or discontinuous ZO-1 and/or claudin-5 staining as evidenced by Western blot and immunofluorescence, indicating the involvement of NOS and ONOO(-) in the BBB disruption. Meaningfully, levels of 3-NT in serum, which had a similar tendency with that of in brain tissues (r=0.934, P<0.001), had a marked correlation with brain edema content (r=0.782, P<0.001) and neurological deficits (r=0.851, P<0.001). We concluded that ONOO(-) formation by the upregulation of NOS may play a central role in promoting the BBB damage following ICH. Moreover, ONOO(-) may be a promising biomarker for the judgment or prediction of brain injury and clinical prognosis after ICH.

Dual expression of hTERT and VEGF prolongs life span and enhances angiogenic ability of aged BMSCs.

Previous studies have confirmed the therapeutic effects of bone marrow stromal cells (BMSCs) transplantation on cerebral ischemia. However, the proliferative, differentiative, and homing capacity of BMSC from the elderly are significantly reduced, especially after several passages expansion in vitro. In this study, by introducing lentivirus-mediated hTERT and VEGF genes to modify human BMSCs from aged donors, we observed extended lifespan, promoted angiogenic capacity while less enhanced tumorigenicity of the genetically engineering BMSCs. These results therefore suggest that the modification of aged BMSCs by dual expression of hTERT and VEGF may be used for autologous cell replacement for ischemic cerebrovascular disease in elderly patients.

Hemoglobin-induced nitric oxide synthase overexpression and nitric oxide production contribute to blood-brain barrier disruption in the rat.

Hemoglobin (Hb) released from extravasated erythrocytes may have a critical role in the process of blood-brain barrier (BBB) disruption and subsequent edema formation after intracerebral hemorrhage (ICH). Excessive nitric oxide (NO) production synthesized by nitric oxide synthase (NOS) has been well documented to contribute to BBB disruption. However, considerably less attention has been focused on the role of NO in Hb-induced BBB disruption. This study was designed to examine the hypothesis that Hb-induced NOS overexpression and excessive NO production may contribute to the changes of tight junction (TJ) proteins and subsequent BBB dysfunction. Hemoglobin was infused with stereotactic guidance into the right caudate nucleus of male Sprague Dawley rats. Then, we investigated the effect of Hb on the BBB permeability, changes of TJ proteins (claudin-5, occludin, zonula occludens-1 (ZO-1), and junctional adhesion molecule-1 (JAM-1)), iron deposition, expression of inducible NOS (iNOS) and endothelial NOS (eNOS), as well as NO production. Hb injection caused a significant increase in BBB permeability. Significant reduction of claudin-5, ZO-1, and JAM-1 was observed after Hb injection as evidenced by PCR and immunofluorescence. After a decrease at early stage, occludin showed a fivefold increase in mRNA level at 7 days. Significant iron deposition was detectable from 48 h to 7 days in a time-dependent manner. The iNOS and eNOS levels dramatically increased after Hb injection concomitantly with large quantities of NO released. Furthermore, enhanced iNOS or eNOS immunoreactivity was co-localized with diffused or diminished claudin-5 staining. We concluded that overexpressed NOS and excessive NO production induced by Hb may contribute to BBB disruption, which may provide an important potential therapeutic target in the treatment of ICH.

PKH26 can transfer to host cells in vitro and vivo.

The fluorescent dye, PKH26, which mainly binds to the cell membrane, has been used as the cell tracer to locate the transplanted cells in host for a long time. However, there was no detailed report that whether the PKH26 dye was specific to the transplanted cells. Therefore, the aim of this article is to explore the effect of cells debris as the cracking cells from the PKH26-labeled adipose-derived stem cells (ADSCs) on the cells in vitro and the host in vivo. After we tested the proliferation and toxicity of PKH26 to the ADSCs by the Cell Count-8 kit and alamar blue assay, we constructed 2 models, coculturing lots of PKH26-labeled cell debris with the unlabeled ADSCs in vitro and injecting via the tail vein in rat, to evaluate the specificity of the PKH26 dye. The result indicated that the PKH26 didn't inhibit the proliferation and had no toxicity to the ADSCs compared with the unlabeled ADSCs, but the cell debris cracking from PKH26-labeled transplanted cells can cause the unlabeled cells to emit red fluorescence in vitro and also lead the tissues displaying red fluorescence in vivo. We can conclude that the PKH26 dye, used as a cell tracer for a long time, was not an ideal cell tracer.

Adult rat hippocampus soluble factors: a novel transplantation model mimicking intracranial microenvironment for tracing the induction and differentiation of adipose-derived stromal cells in vitro.

Intracranial transplantation of ADSCs induces recovery of CNS diseases, but how they develop in host is poorly understood. The aim of this study is to observe induction and differentiation of ADSCs in the presence of hippocampus soluble factors (HiSF) extracted from the hippocampus of adult Wistar rats to mimic an intracranial microenvironment. To determine the optimal microenvironment, five conditions were tested: 0µg/ml (as control), 50µg/ml, 100µg/ml, 200µg/ml, and 400µg/ml of HiSF. The number of neurospheres was significantly higher in 200µg/ml group than in other groups on the sixth day. Immunofluorescence demonstrated that the neurospheres induced from ADSCs in 200µg/ml group expressed both nestin and CD133, which are more highly expressed in neurospheres than in ADSCs. This result was confirmed by Western blot analysis. Quantitative PCR revealed that the mRNA levels of nestin and CD133 in the neurospheres were 145- and 220-fold higher, respectively, than those in ADSCs. In the presence of 200µg/ml HiSF and 1% FBS, the neurospheres can further differentiate into Schwann-like cells which expressing characteristic markers GFAP, S100 and P75 NGFR. These data indicated that HiSF, mimicking a destination of ADSCs transplanted model in vitro, could effectively induce and differentiate neurospheres, representing a new method to obtain NSCs and Schwann-like cells from ADSCs.

Bone marrow-derived mesenchymal stem cells maintain the resting phenotype of microglia and inhibit microglial activation.

Many studies have shown that microglia in the activated state may be neurotoxic. It has been proven that uncontrolled or over-activated microglia play an important role in many neurodegenerative disorders. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown in many animal models to have a therapeutic effect on neural damage. Such a therapeutic effect is attributed to the fact that BMSCs have the ability to differentiate into neurons and to produce trophic factors, but there is little information available in the literature concerning whether BMSCs play a therapeutic role by affecting microglial activity. In this study, we triggered an inflammatory response situation in vitro by stimulating microglia with the bacterial endotoxin lipopolysaccharide (LPS), and then culturing these microglia with BMSC-conditioned medium (BMSC-CM). We found that BMSC-CM significantly inhibited proliferation and secretion of pro-inflammatory factors by activated microglia. Furthermore, we found that the phagocytic capacity of microglia was also inhibited by BMSC-CM. Finally, we investigated whether the induction of apoptosis and the production of nitric oxide (NO) were involved in the inhibition of microglial activation. We found that BMSC-CM significantly induced apoptosis of microglia, while no apoptosis was apparent in the LPS-stimulated microglia. Our study also provides evidence that NO participates in the inhibitory effect of BMSCs. Our experimental results provide evidence that BMSCs have the ability to maintain the resting phenotype of microglia or to control microglial activation through their production of several factors, indicating that BMSCs could be a promising therapeutic tool for treatment of diseases associated with microglial activation.

[Construction of a prokaryotic expression vector of human tau multi-epitope peptide and immunogenicity of the expressed product].

OBJECTIVE: To construct a prokaryotic expression vector of human tau multiepitope peptide for examining the immunogenicity of a TauP1/P2 DNA vaccine in mice using the expressed product. METHODS: The coding sequence of Tau multiepitope peptide gene was amplified from the plasmid pVAX1-Tau by PCR and inserted into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant plasmid pGEX-4T-2-TauP1/P2. The positive recombinants were transformed into E.coli BL21 cells, and the expression of fusion protein GST-TauP1/P2 was induced by IPTG and identified by SDS-PAGE. Mice was immunized with TauP1/P2 DNA vaccine and the production of the specific antibodies was detected by Dot-blot analysis using the purified fusion protein. RESULTS: A gene fragment 300 bp in length was amplified. Enzyme digestion and DNA sequencing verified correct construction of the prokaryotic expression plasmid pGEX-4T-2-TauP1/P2. The expression of target fusion protein GST-TauP1/P2 was detected by SDS-PAGE. Specific antibodies against TauP1/P2 were detected in the serum of mice immunized with the DNA vaccine using GST-TauP1/P2 fusion protein. CONCLUSION: The constructed prokaryotic expression plasmid of human Tau multiepitope peptide is capable of expressing the target fusion protein, which specifically recognizes the specific antibodies against TauP1/P2 in mice immunized with TauP1/P2 DNA vaccine.

Transforming growth factor-ß is required for vasculogenic mimicry formation in glioma cell line U251MG.

Both vasculogenic mimicry (VM) and transforming growth factor-ß (TGFß) are positively correlated with malignancy in glioma. Accordingly, we supposed that TGFß might be related with VM, and aimed to detect whether TGFß could influence VM formation in two glioma cell lines U251MG and SHG44, which were different in malignancy. We found that the VM-positive U251MG had a significantly higher TGFß expression than the VM-negative SHG44. Downregulating TGFß in U251MG by RNAi technology resulted in a significantly impaired VM formation, which could be rescued by rhTGFß. However, adding rhTGFß could not induce VM in SHG44. To investigate the possible mechanism, we detected the changes of some VM-related genes including EphA2, VE-cadherin, MMP-2, MMP-9, MT1-MMP and LAMC2 by RT-PCR and found that MT1-MMP transcript was affected by TGFß expression. Gelatin zymography showed a declined MMP-2 activity in the TGFß-inhibited cells. Further studies showed that MT1-MMP inhibition impaired VM formation in U251MG. Moreover, TGFß induced MT1-MMP expression and VM formation in a dose-dependent manner. These findings indicated us that TGFß was required for VM formation in U251MG. MT1-MMP was correlated with TGFß-induced VM formation. Thus, TGFß might be a potential target for VM inhibition in glioma.

Passive immunization with LINGO-1 polyclonal antiserum afforded neuroprotection and promoted functional recovery in a rat model of spinal cord injury.

LINGO-1 (leucine-rich repeat and Ig domain-containing, Nogo receptor-interacting protein) is an important component of the NgR receptor complex involved in RhoA activation and axon regeneration. The authors report on passive immunization with LINGO-1 polyclonal antiserum, a therapeutic approach to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). The intrathecally administered high-titer rabbit-derived antiserum can be detected around the injury site within a wide time window; it blocks LINGO-1 in vivo with high molecular specificity. In this animal model, passive immunization with LINGO-1 antiserum significantly decreased RhoA activation and increased neuronal survival. Adult rats immunized in this manner show recovery of certain hindlimb motor functions after dorsal hemisection of the spinal cord. Thus, passive immunotherapy with LINGO-1 polyclonal antiserum may represent a promising repair strategy following acute SCI.

Therapeutic DNA vaccination as a repair strategy following spinal cord injury.

Myelin-derived proteins, such as tenascin-R (TN-R), myelin associate glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), and Nogo-A, inhibit the central nervous system regeneration. In this study, the DNA vaccine encoding for oligodendrocyte and myelin-related antigens was employed to attenuate the axonal growth inhibitory properties of myelin in the setting of spinal cord injury. Using a rat spinal cord dorsal hemisection model, the vaccine directed against the inhibitory epitopes of Nogo-A, MAG, OMgp, and TN-R was administered intramuscularly once a week following spinal cord injury, supplemented with local application of specific anti-sera against the four antigens. Anterograde labeling of dorsal column fibers showed active axonal regeneration through the lesion site at the eighth week following the treatment in experimental group but not in control groups. Light microscopic and ultrastructural analysis revealed that vaccination with these myelin-related antigens did not lead to demyelinating disease. OMgp and TN-R levels were down-regulated at the lesion site together with a parallel increase in growth-associated protein 43 levels in the treatment groups. This study reveals the effective approach of a DNA vaccine strategy by attaining the special antibody to direct neutralization of the myelin inhibitors during spinal cord injury.

[Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody].

OBJECTIVE: To express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb). METHODS: The 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting. RESULTS: The prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity. CONCLUSION: The fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

[Transfection of pEGFP-C2 in brain mediated by targeting liposome P-MMA-DOSPER].

This research tried improving the specificity and efficiency of gene transfection in gene therapy and tried making the liposome a better gene transfer vector to brain by use of the monoclonal antibody (anti-Lex/SSEA-1)-mediated targeting of liposome. The derivatized monoclonal antibody was conjugated to the liposome DOSPER to form the targeting liposome P-MMA-DOSPER. Then, the pEGFP-C2 encapsulated in P-MMA-DOSPER or DOSPER was injected into the lateral ventricle of SD rats respectively, and the brains were taken for frosted slice 1, 3, 7 or 14 days later. The expression of GFP was observed under fluorescent microscope. There was a lot of expression of GFP around the lateral ventricle of rats in each group. But the indirect fluorescence antibody test showed the ratio of GFP+/nestin+ cells to nestin+ cells of every marking time point in the group of P-MMA-DOSPER was higher than the one in the group of DOSPER; the difference was found to be statistically significant (P<0.01). The results proved that the P-MMA-DOSPER can permeat the ependyma and can transfer gene into the nerve stem cells in vivo safely and effectively.

Beneficial effect of autologous transplantation of bone marrow stromal cells and endothelial progenitor cells on cerebral ischemia in rabbits.

We tested the therapeutic effect of autologous transplanted bone marrow stromal cells (BMSCs) and endothelial progenitor cells (EPCs) on cerebral ischemia in rabbits. Rabbit permanent middle cerebral artery occlusion (MCAO) models were intravenously injected with ex vivo expanded autologous BMSCs (n = 8), EPCs (n = 8), or phosphate-buffered saline (n = 6). 14 days after the transplantation, both infusion groups witnessed a functional improvement, a decrease in the number of apoptotic cells and an increase in the microvessel density in the ischemic boundary area, as compared to vehicle-treated control group. The EPCs treated group also exhibited a diminished infarct area in comparison with the control group. Moreover, immunohistochemistry revealed that few transplanted BMSCs expressed markers for astrocytes (GFAP+) and neurons (NeuN+), and most of EPCs were capable of binding to UEA-1 lectin and were incorporated into capillaries. Our data suggest that both BMSCs and EPCs, despite differences in their action mechanism, can be functional cytoreagents for treatment of cerebral ischemia in rabbits.

Bone marrow stromal cells can be delivered to the site of traumatic brain injury via intrathecal transplantation in rabbits.

Recent studies suggest that bone marrow stromal cells (BMSCs) are promising grafts for treatment of traumatic brain injury (TBI). Neural precursor cells (NPCs) have been detected in the site of cervical cord injury following intrathecal injection by lumbar puncture. So, this study is designed to determine whether BMSCs (after intrathecal administration by lumbar puncture) could also migrate to the TBI site. The cells were cultured in vitro and transfected with adenovirus green fluorescent protein (Ad-GFP), and then transplanted intrathecally or intravenously into an autologous rabbit model of TBI. The labeled, grafted cells were identified in the injured cerebral tissue using fluorescence microscopy. Results showed that the intrathecal protocol was more efficient than the intravenous one. And motor dysfunction was improved after autologous transplantation of BMSCs. This study suggests another attractive minimally invasive option for treating TBI.

Functional analysis of neuron-like cells differentiated from neural stem cells derived from bone marrow stroma cells in vitro.

The transversal differentiation of bone marrow stroma cell (BMSCs) into neural stem cells (NSCs) has attracted much attention in recent years because of their therapeutic potential. However, the problem in therapeutic application of NSCs was how to confirm whether neuron-like cells differentiated from bone marrow stroma cell-derived neural stem cells (BMSCs-D-NSCs) possess corresponding functions of neurochemistry and electrophysiology. In the present study, we tried to affirm the function of neuron-like cells differentiated from BMSCs-D-NSCs in vitro. The BMSCs were harvested by gradient centrifugation in Ficoll-Paque and cultured in "NSCs medium". Immunocytochemistry was used to detect positive expression of neuron-specific nuclear protein (NeuN) in neuron-like cells derived from the BMSCs-D-NSCs. High-pressure liquid chromatography (HPLC) was used to identify neuron-like cells by detecting excitable amino acids [aspartic acid (Asp), glutamic acid (Glu)], inhibited amino acids [glycine (Gly), gamma (gamma) -aminobutyric acid (GABA), alanine (Ala)] or monoamines [noradrenaline (NE), 5-hydroxytryptamine (5-HT), dopamine (DA)]. Electrophysiological properties of the neuron-like cells were also examined using patch clamp analysis to verify their neuron-like functions. It was found that the neuron-like cells differentiated from the BMSCs-D-NSCs could express positive NeuN, synthesize and excrete amino acids, and show some typical electrophysiological properties including the typical Na+ and K+ ion channel membrane current under the voltage patch clamp condition, the typical static electrical membrane potential under the current patch clamp condition, and the differential membrane capacitance and resistance values in series between undifferentiated BMSCs-D-NSCs and differentiated neuron-like cells under the whole-cell patch clamp condition. The neuron-like cells differentiated from BMSCs-D-NSCs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties.

Experimental study on trace marking and oncogenicity of neural stem cells derived from bone marrow.

It has been well accredited that the neural stem cells (NSCs) derived from bone marrow stroma cells (BMSCs) can be used as the therapeutic application. However, their efficacy and safety in therapeutic application are uncertain. In this experiment, the trace marking and oncogenicity of NSCs derived from BMSCs (BMSCs-D-NSCs) were studied. The BMSCs were harvested by gradient centrifugation and cultured in "NSCs medium" in vitro. The verified CD133/Nestin-positive BMSCs-D-NSCs were then transplanted into nude mice to detect the oncogenicity, into the right lateral cerebral ventricle or right caudae putamen and substantia nigra to examine, whether the symptoms were improved in Parkinson's Disease (PD) models after transplantation, by both SPECT image assay of dopamine transporter (DAT) in corpus striatum and its average standard uptake value (SUVave) in corpus striatum and thalamus. Tissue samples and surviving model animals were studied at 1, 3, and 6 months post-transplantation. Before transplantation, the cells were labeled with BrdU or rAAV-GFP for the pathological sections, and with Feridex for the in vivo trace by MRI assay. The concanavalin A (ConA) agglutination test, stop-dependence test with soft agar, karyotype analysis of chromosome G zone in BMSCs-D-NSCs, and the nude mouse neoplasia test were also performed. The BrdU, rAAV-GFP or Feridex can be used as trace markers of BMSCs-D-NSCs during transplantation. The transplanted BMSCs-D-NSCs displayed neither toxicity nor neoplasia up to 6 months in vivo, but could play an important role in improving the symptoms of the animals with degenerative diseases like PD.

Expression profile of cancer-related genes in human adult bone marrow-derived neural stemlike cells highlights the need for tumorigenicity study.

Human adult bone marrow-derived neural stemlike cells (MDNSCs) may serve as ideal seed cells for cell replacement therapy for human neurological disorders and injuries. However, the long-term safety of this cell population after transplantation must be thoroughly explored before clinical application, and tumorigenicity is a major concern. In this study, we generated MDNSCs capable of forming neurospherelike aggregates and with the potency to differentiate into neural lineage cells in vitro and investigated hundreds of cancer-related genes in MDNSCs in order to determine whether there were any characteristics that could help in the evaluation of their tumorigenic potential. According to the results of testing by PCR and DNA sequencing, there were no mutations at the frequent mutation sites of tumor-suppressor genes p53, p16, and Rb1. Of the 440 cancer-related genes covered by Oligo GEArray Human Cancer Microarray OHS-802, 63 were found to be significantly overexpressed compared with that in fresh normal human adult bone marrow depleted of red blood cells (RBCs). In particular, the overexpressed genes included those promoting cell proliferation and cell invasion and metastasis and members of several oncogenic signaling pathways. The overexpression of MYC, MMP2, Notch2, STC1, ITGA3, STAT5b, RhoC, and Wnt1 was also revealed by quantitative real-time RT-PCR. Because it has been shown that activation of some of these genes promote tumorigenesis, our findings highlight the need for further studies of long-term tumorigenicity in MDNSCs.