RESUMO
With the present paper, the Working Group on Cells, Tissues and Organs and other experts of the Superior Health Council of Belgium aimed to provide stakeholders in material of human origin with advice on critical aspects of serological and nucleic acid test (NAT) testing, to improve virological safety of cell- and tissue and organ donation. The current paper focusses on a number of preanalytical variables which can be critical for any medical biology examination: (1) sampling related variables (type of samples, collection of the samples, volume of the sample, choice of specific tubes, identification of tubes), (2) variables related to transport, storage and processing of blood samples (transport, centrifugation and haemolysis, storage before and after centrifugation, use of serum versus plasma), (3) variables related to dilution (haemodilution, pooling of samples), and (4) test dependent variables (available tests and validation). Depending on the type of donor (deceased donor (heart-beating or non-heart beating) versus living donor (allogeneic, related, autologous), and the type of donated human material (cells, tissue or organs) additional factors can play a role: pre- and post-mortem sampling, conditions of sampling (e.g. morgue), haemodilution, possibility of retesting.
RESUMO
BACKGROUND: Chronic lymphocytic leukemia is a neoplastic disorder that arises largely as a result of defective apoptosis leading to chemoresistance. Stromal cell-derived factor-1 and its receptor, CXCR4, have been shown to play an important role in chronic lymphocytic leukemia cell trafficking and survival. DESIGN AND METHODS: Since histone acetylation is involved in the modulation of gene expression, we evaluated the effects of suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, on chronic lymphocytic leukemia cells and in particular on cell survival, CXCR4 expression, migration, and drug sensitization. RESULTS: Here, we showed that treatment with suberoylanilide hydroxamic acid (20 microM) for 48 hours induced a decrease in chronic lymphocytic leukemia cell viability via apoptosis (n=20, P=0.0032). Using specific caspase inhibitors, we demonstrated the participation of caspases-3, -6 and -8, suggesting an activation of the extrinsic pathway. Additionally, suberoylanilide hydroxamic acid significantly decreased CXCR4 mRNA (n=10, P=0.0010) and protein expression (n=40, P<0.0001). As a result, chronic lymphocytic leukemia cell migration in response to stromal cell-derived factor-1 (n=23, P<0.0001) or through bone marrow stromal cells was dramatically impaired. Consequently, suberoylanilide hydroxamic acid reduced the protective effect of the microenvironment and thus sensitized chronic lymphocytic leukemia cells to chemotherapy such as fludarabine. CONCLUSIONS: In conclusion, suberoylanilide hydroxamic acid induces apoptosis in chronic lymphocytic leukemia cells via the extrinsic pathway and down-regulates CXCR4 expression leading to decreased cell migration. Suberoylanilide hydroxamic acid in combination with other drugs represents a promising therapeutic approach to inhibiting migration, chronic lymphocytic leukemia cell survival and potentially overcoming drug resistance.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Antineoplásicos , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Receptores CXCR4/antagonistas & inibidores , Células Tumorais Cultivadas , VorinostatAssuntos
Ritmo Circadiano/fisiologia , Sangue Fetal/fisiologia , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Estações do Ano , Adulto , Feminino , Sangue Fetal/citologia , Seguimentos , Idade Gestacional , Células-Tronco Hematopoéticas/citologia , Humanos , Recém-Nascido , Masculino , Gravidez , PrognósticoRESUMO
BACKGROUND AIMS: The transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production. METHODS: The migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel in the presence and absence of stromal cell-derived (SDF)-1alpha, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels. RESULTS: We have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli. CONCLUSIONS: Our observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.
Assuntos
Movimento Celular/efeitos dos fármacos , Citocinas/farmacologia , Mediadores da Inflamação/farmacologia , Metaloproteinases da Matriz/biossíntese , Células-Tronco Mesenquimais/citologia , Células Estromais/enzimologia , Inibidores Teciduais de Metaloproteinases/biossíntese , Células da Medula Óssea/citologia , Proliferação de Células/efeitos dos fármacos , Quimiocinas/farmacologia , Colágeno/metabolismo , Combinação de Medicamentos , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/patologia , Laminina/metabolismo , Metaloproteinases da Matriz/genética , Fenótipo , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Quimiocinas/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/genéticaRESUMO
BACKGROUND: Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC) could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. METHODS: BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin). By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. RESULTS: Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin). Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin). Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3), NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate receptor channel (AMPA3) in the presence of two agonist glutamate, AMPA or CNQX antagonist. CONCLUSION: Our results demonstrate that BM-MSC have the potential to differentiate in neuronal cells with specific gene expression and functional properties. BM-MSC are thus promising candidates for cell-based therapy of neurodegenerative diseases.
Assuntos
Células da Medula Óssea/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Doenças Neurodegenerativas/terapia , Neurônios/metabolismo , Transplante de Células-Tronco/métodos , Células Estromais/metabolismo , Diferenciação Celular/fisiologia , Humanos , Neurônios/citologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
OBJECTIVE: Chronic lymphocytic leukemia (CLL) cells develop chemoresistance over time associated with defects in apoptosis pathway. Novel treatment strategies are required to overcome resistance of cells to commonly used agents. The effects of valproic acid (VPA), an antiepileptic drug with histone deacetylase inhibitory activity, on mononuclear cells isolated from 40 CLL patients were evaluated. METHODS: CLL cells were treated with increasing doses of VPA (0.5, 1, 2, and 5 mM). The mode of cytotoxic drug action was determined by annexin binding, DNA fragmentation, and caspase activation. RESULTS: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in the 40 CLL patients. VPA treatment induced apoptotic changes in CLL cells including phosphatidylserine externalization and DNA fragmentation. The mean apoptotic rates were similar between IgV(H) mutated and unmutated patients, the latter presenting a more aggressive clinical course. VPA induced apoptosis via the extrinsic pathway involving engagement of the caspase-8-dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA significantly increased sensitivity of leukemic cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and led to downregulation of c-FLIP (L) expression. VPA caused no potentialization of TRAIL-induced apoptosis on normal B cells. In addition, VPA overcame the prosurvival effects of bone marrow stromal cells. CONCLUSION: These findings point out that the combination of TRAIL and VPA, at clinically relevant concentration, may be valuable in the treatment of CLL.
Assuntos
Anticonvulsivantes/farmacologia , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ácido Valproico/farmacologia , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Leucêmica da Expressão Gênica/genética , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Fosfatidilserinas/metabolismo , Transdução de Sinais/genética , Células Tumorais Cultivadas , Ácido Valproico/uso terapêuticoRESUMO
OBJECTIVE: We evaluated the cytotoxic effect of ultrasonic irradiation at low energy on the viability of normal and leukemic cells and the potential mechanisms of action inducing this cytotoxicity. MATERIALS AND METHODS: Human leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic cells, and normal mononuclear cells are treated by ultrasound at a frequency of 1.8 MHz during various exposure times (acoustical power of 7 mW/mL) and immediately tested for cell viability by the trypan blue exclusion assay. Apoptosis is evaluated by cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax ratio are tested by flow cytometry. Cloning efficiency is evaluated by assays in methylcellulose. RESULTS: The technique we describe here, using minute amounts of energy and in the absence of any chemical synergy, specifically triggers apoptosis in leukemic cells while necrosis is significantly reduced. Ultrasonic treatment of 20 seconds' duration induces a series of successive phases showing the characteristic features of apoptosis: mitochondrial transmembrane potential disturbances, loss of phosphatidylserine asymmetry, morphological changes, and, finally, DNA fragmentation. In contrast to K562 cells, for which a significant reduction of cloning efficiency is observed, the growth of hematopoietic progenitors is totally unaffected. Ultrasound treatment is also associated with depletion of cellular glutathione content, suggesting a link with the oxidative stress. Moreover, the fact that active oxygen scavengers reduce ultrasonic-induced apoptosis suggests a sonochemical mechanism. CONCLUSION: The cell damage observed after exposure of leukemic cells to ultrasound is associated with the apoptotic process and may be a promising tool for a smooth, specific, and effective ex vivo purging of leukemic cells.
Assuntos
Apoptose , Leucemia/patologia , Células-Tronco Neoplásicas/diagnóstico por imagem , Terapia por Ultrassom , Caspase 3 , Caspases/análise , Sobrevivência Celular , Fragmentação do DNA , Sequestradores de Radicais Livres/farmacologia , Glutationa/análise , Células HL-60/química , Células HL-60/diagnóstico por imagem , Células HL-60/patologia , Histidina/farmacologia , Humanos , Radical Hidroxila , Membranas Intracelulares/diagnóstico por imagem , Células K562/química , Células K562/diagnóstico por imagem , Células K562/patologia , Manitol/farmacologia , Lipídeos de Membrana/análise , Mitocôndrias/diagnóstico por imagem , Proteínas de Neoplasias/análise , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/patologia , Estresse Oxidativo , Fosfatidilserinas/análise , Poli(ADP-Ribose) Polimerases/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Oxigênio Singlete , Ensaio Tumoral de Célula-Tronco , Ultrassonografia , Proteína X Associada a bcl-2RESUMO
We tested the effects of hydroxychloroquine (HCQ), an anti-rheumatic drug, on the viability of chronic lymphocytic leukemia (CLL) cells. HCQ induced a decrease in cell viability in a dose- and time-dependent manner. The mean LC50 calculated for the cells of 20 patients was 32 +/- 7 microg/ml (range, 10-75 microg/ml). We observed a large increase in apoptotic cell number after 24 h of incubation with 50 microg/ml HCQ (55 +/- 6 vs. 23 +/- 3% in medium alone, p < 0.001). Indeed, HCQ in leukemic cells induced the features of apoptosis (cell shrinkage, decrease in mitochondrial transmembrane potential, phosphatidylserine externalization, chromatin condensation and DNA fragmentation). HCQ had marked selective cytotoxicity when compared with normal blood mononuclear cells, in which the LC50 was >100 microg/ml at 24 h. HCQ induced the proteolytic cleavage of poly(ADP(adenosine 5'-diphosphate)ribose) polymerase (PARP) and increased the activity of caspase-3. The expression of bcl-2 and bax proteins was significantly modified after incubation with the drug and HCQ activity against CLL cells occurred independently of the presence of IL-4, sCD40L and bone marrow stromal cells.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Hidroxicloroquina/farmacologia , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas/análise , Caspase 3 , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína X Associada a bcl-2RESUMO
The co-infusion of mesenchymal stromal cells (MSCs) with hematopoietic stem cells could improve the hematopoietic engraftment after cord blood transplant. Adult bone marrow is the major source of MSCs for cell therapy. However, bone marrow aspiration involves an invasive procedure and, in the case of a cord blood transplant, requires the use of a third party. The umbilical cord matrix, called Wharton's jelly (WJ), was previously shown to be a valuable source of MSCs. However, the process of cell separation is not standardized and needs to be optimized. In this study, we focused on the efficiency of the isolation procedure and expansion of cells from WJ MSCs isolated from human full-term umbilical cords. MSCs were isolated from the WJ without enzyme digestion or dissection. The procedure was based only on the plastic adhesion capacities of MSCs. Briefly, umbilical cord segments of 5-10 cm were cut longitudinally and plated with the WJ onto a plastic surface for 5 days in an appropriate culture medium. After removing the cord segment, the culture was pursued until subconfluency. The number of cells and their phenotypes, clonogenic capacities, differentiation capacities, immunomodulation, and hematopoietic supportive functions were evaluated. Using this method, we were able to isolate MSCs from all human umbilical cords analyzed (n = 50). We obtained a mean of 1.4 × 10(8) cells at the second passage and >7 × 10(9) cells at the third. The expanded cells expressed characteristic markers and presented typical functional properties of MSCs such as differentiation capacities, immunologic properties, and hematopoietic supportive functions. In conclusion, we have established a simple, rapid, and reproducible protocol to isolate abundant MSCs from short segments of umbilical cords.
Assuntos
Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Antígenos de Diferenciação/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fenótipo , Reprodutibilidade dos Testes , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Mesenchymal stromal cells (MSCs) are important in the support of hematopoiesis. In this pilot study, we evaluated the safety and efficiency of donor-expanded MSC infusion after allogeneic hematopoietic stem cell transplantation (HSCT) in six patients with poor hematopoietic recovery. MSCs were infused without HSC and without conditioning at a dose of 1 x 10(6)/kg weight. Two patients displayed rapid hematopoietic recovery (days 12 and 21), and four patients showed no response. The two patients who showed hematopoietic recovery were in first complete remission (CR1) compared to the other heavily pretreated patients. There were no toxic side effects linked to MSC infusion. One patient developed cytomegalovirus (CMV) reactivation 12 days following the MSC infusion and died from CMV disease. We found that infusion of MSCs without HSC co-infusion can restore medullary function in some patients with poor hematopoietic recovery. Our data suggest that patients with a less damaged stroma could benefit from this approach.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células Estromais/transplante , Condicionamento Pré-Transplante/métodos , Adulto , Infecções por Citomegalovirus/etiologia , Rejeição de Enxerto/prevenção & controle , Hematopoese , Humanos , Leucemia/sangue , Leucemia/terapia , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/terapia , Projetos Piloto , Aspergilose Pulmonar/etiologia , Recuperação de Função Fisiológica , Indução de Remissão , Células Estromais/citologia , Fatores de Tempo , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
The expansion of mesenchymal stem cells (MSCs) strongly depends on the culture conditions and requires medium supplemented with 10-20% fetal calf serum (FCS) to generate relevant numbers of cells. However, the presence of FCS is a major obstacle for their clinical use. Therefore, we have evaluated the capacity of expansion of MSC in a commercial serum-free medium (UC) supplemented with a serum substitute (ULTROSER) in comparison with a classical medium alpha-MEM containing 15% FBS. Bone marrow-mononuclear cells collected from 12 volunteer healthy donors were expanded in two different culture media. MSCs isolated in the both media were morphologically similar and expressed identical phenotypic markers. After the primoculture (P0) and one passage, we obtained significantly more MSC and CFU-F progenitors in UC medium than in alphaMEM. Their multipotentiality was preserved during culture, as well as their capacity to support haematopoiesis. In conclusion, our observations strongly suggest that UC is an optimal medium for ex vivo expansion of MSC: it allows a better cell expansion, preserves cell multipotentiality, reduces the culture period and contains low concentration of serum substitute. This medium seems suitable for clinical scale expansion of MSC.
Assuntos
Células da Medula Óssea/fisiologia , Técnicas de Cultura de Células , Meios de Cultura Livres de Soro , Hematopoese/fisiologia , Células-Tronco Mesenquimais/fisiologia , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Compostos OrgânicosRESUMO
In this study, we used a common procedure to assess the potential of mobilized peripheral blood (MPB) and umbilical cord blood (UCB) as sources of mesenchymal stem cells (MSCs) in comparison with bone marrow (BM). We tested three methods: plastic adhesion supplemented with 5% of BM-MSC conditioned medium, unsupplemented plastic adhesion, and selection of CD133-positive cells. MSCs derived from MPB or UCB are identified by their positive expression of mesenchymal (SH2, SH3) and negative expression of hematopoietic markers (CD14, CD34, CD45, HLA-DR). We observed that the CD133-positive cell fraction contains more MSCs with high proliferative potential. Placed in appropriate conditions, these cells proved their capacity to differentiate into adipocytes, osteocytes, chondrocytes, and neuronal/glial cells. MPB- and UCB-MSCs express Oct4, a transcriptional binding factor present in undifferentiated cells with high proliferative capacity. The selection of CD133-positive cells enabled us to obtain a homogeneous population of MSCs from UCB and MPB. These sources may have a major clinical importance thanks to their easy accessibility.
Assuntos
Células Sanguíneas/citologia , Diferenciação Celular , Sangue Fetal/citologia , Células-Tronco Mesenquimais/citologia , Antígeno AC133 , Adipócitos/citologia , Antígenos CD/análise , Biomarcadores/análise , Células Sanguíneas/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem da Célula , Proliferação de Células , Separação Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Glicoproteínas/análise , Humanos , Células-Tronco Mesenquimais/metabolismo , Neurônios/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Peptídeos/análise , RNA Mensageiro/metabolismoRESUMO
Bone marrow mesenchymal stem cells (MSC) are multipotent cells. To explain their plasticity, we postulated that undifferentiated MSC may express proteins from other tissues such as neuronal tissues. MSC are obtained by two different approaches: plastic adhesion or negative depletion (RosetteSep and magnetic beads CD45/glycophorin A). MSC are evaluated through FACS analysis using a panel of antibodies (SH2, SH3, CD14, CD33, CD34, CD45, etc.). To confirm the multipotentiality in vitro, we have differentiated MSC into adipocytes, chondrocytes, osteocytes, and neuronal/glial cells using specific induction media. We have evaluated neuronal and glial proteins (Nestin, Tuj-I, betaIII Tubulin, tyrosine hydroxylase [TH], MAP-2, and GFAP) by using flow cytometry, Western blots, and RT-PCR. We found that MSC constituently express native immature neuronal proteins such as Nestin and Tuj-1. After only five passages, MSC can already express more mature neuronal or glial proteins, such as TH, MAP-2, and GFAP, without any specific induction. We noticed an increase in the expression of more mature neuronal/glial proteins (TH, MAP-2, and GFAP) after exposure to neural induction medium, thus confirming the differentiation of MSC into neurons and astrocytes. The constitutive expression of Nestin or Tuj-1 by MSC suggests that these cells are "multidifferentiated" cells and thus can retain the ability for neuronal differentiation, enhancing their potentiality to be employed in the treatment of neurological diseases.