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1.
Stem Cells ; 37(10): 1357-1368, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31184411

RESUMO

Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34+ cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34+ cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. Stem Cells 2019;37:1357-1368.

2.
Br J Haematol ; 187(1): 93-104, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31172513

RESUMO

Patients with low-risk myelodysplastic syndromes (MDS) usually develop iron overload. This leads to a high level of oxidative stress in the bone marrow (BM) and increases haematopoietic cell dysfunction. Our objective was to analyse whether chelation with deferasirox (DFX) alleviates the consequences of oxidative stress and improves BM cell functionality. We analysed 13 iron-overloaded MDS patients' samples before and 4-10 months after treatment with DFX. Using multiparametric flow cytometry analysis, we measured intracellular reactive oxygen species (ROS), DNA oxidation and double strand breaks. Haematopoietic differentiation capacity was analysed by colony-forming unit (CFU) assays. Compared to healthy donors, MDS showed a higher level of intracellular ROS and DNA oxidative damage in BM cells. DNA oxidative damage decreased following DFX treatment. Furthermore, the clonogenic assays carried out before treatment suggest an impaired haematopoietic differentiation. DFX seems to improve this capacity, as illustrated by a decreased cluster/CFU ratio, which reached values similar to controls. We conclude that BM cells from MDS are subject to higher oxidative stress conditions and show an impaired haematopoietic differentiation. These adverse features seem to be partially rectified after DFX treatment.

3.
Stem Cell Res Ther ; 10(1): 63, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30795797

RESUMO

BACKGROUND: Posterolateral spinal fusion with autologous bone graft is considered the "gold standard" for lumbar degenerative disc disease (DDD) when surgical treatment is indicated. The potential role of mesenchymal stromal cells (MSCs) to replace the bone graft in this setting has not been fully addressed. OBJECTIVE: To analyze the safety, feasibility and potential clinical efficacy of the implantation of autologous MSCs embedded with tricalcium phosphate as a therapeutic alternative to bone graft in patients with DDD during posterolateral spine fusion. STUDY DESIGN: Phase I/II single-arm prospective clinical trial. METHODS: Eleven patients with monosegmental DDD at L4-L5 or L5-S1 level were included. Autologous bone marrow-derived MSC were expanded in our Good Manufacturing Practice (GMP) Facility and implanted during spinal surgery embedded in a tricalcium phosphate carrier. Monitoring of patients included a postoperative period of 12 months with four visits (after the 1st, 3rd, 6th, and 12th month), with clinical and radiological assessment that included the visual analog scale (VAS), the Oswestry disability index (ODI), the Short-Form Health Survey (SF-36), the vertebral fusion grade observed through a simple Rx, and the evaluation of possible complications or adverse reactions. In addition, all patients were further followed up to 5 years for outcome. RESULTS: Median age of patients included was 44 years (range 30-58 years), and male/female ratio was (6/5) L4-L5 and L5-S1 DDD was present five and six patients, respectively. Autologous MSCs were expanded in all cases. There were no adverse effects related to cell implantation. Regarding efficacy, both VAS and ODI scores improved after surgery. Radiologically, 80% of patients achieved lumbar fusion at the end of the follow-up. No adverse effects related to the procedure were recorded. CONCLUSIONS: The use of autologous MSCs for spine fusion in patients with monosegmental degenerative disc disease is feasible, safe, and potentially effective. TRIAL REGISTRATION: no. EudraCT: 2010-018335-17 ; code Identifier: NCT01513694 ( clinicaltrials.gov ).

4.
Stem Cells Int ; 2018: 7089484, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30123292

RESUMO

Significant research efforts have been undertaken during the last decades to treat musculoskeletal disorders and improve patient's mobility and quality of life. The goal is the return of function as quickly and completely as possible. Cellular therapy has been increasingly employed in this setting. The design of this study was focused on cell-based alternatives. The present study aimed at investigating the bone regeneration capacity of xenogeneic human bone marrow-derived mesenchymal stromal cell (hMSC) implantation with tricalcium phosphate (TCP) granules in an immunocompetent rabbit model of critical-size bone defects at the femoral condyles. Two experimental groups, TCP and hMSC + TCP, were compared. Combination of TCP and hMSC did not affect cell viability or osteogenic differentiation. We also observed significantly higher bone regeneration in vivo in the hMSC + TCP group, which also displayed better TCP osteointegration. Also, evidence of hMSC contribution to a better TCP osteointegration was noticed. Finally, no inflammatory reaction was detected, besides the xenotransplantation of human cells into an immunocompetent recipient. In summary, hMSC combined with TCP granules is a potential combination for bone regeneration purposes that provides better preclinical results compared to TCP alone.

5.
J Tissue Eng Regen Med ; 12(3): e1450-e1460, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28865091

RESUMO

Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein-based ones, such as elastin-like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix-like hydrogels through either physical or chemical cross-linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase-expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR-based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL-1ß, IL-4, IL-6, and IL-10 concentrations were measured by enzyme-linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine-related applications.


Assuntos
Materiais Biocompatíveis/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Elastina/farmacologia , Hidrogéis/farmacologia , Proteínas Recombinantes/farmacologia , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Animais , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Rastreamento de Células , Citocinas/sangue , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/patologia , Injeções Subcutâneas , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos
6.
Biol Blood Marrow Transplant ; 24(3): 443-451, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29155314

RESUMO

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.


Assuntos
Adipogenia/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Raios gama , Hematopoese/efeitos da radiação , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos da radiação , Adulto , Idoso , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade
7.
PLoS One ; 12(8): e0182470, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28796790

RESUMO

There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HD-MSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cells.


Assuntos
Neoplasias Hematológicas/patologia , Janus Quinase 2/metabolismo , Células-Tronco Mesenquimais/fisiologia , Adulto , Idoso , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Expressão Gênica , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/enzimologia , Hematopoese , Humanos , Masculino , Pessoa de Meia-Idade
8.
J Mater Sci Mater Med ; 28(8): 115, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28647792

RESUMO

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.


Assuntos
Materiais Biocompatíveis/química , Elastina/química , Cartilagem Hialina/crescimento & desenvolvimento , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Regeneração , Animais , Fenômenos Biomecânicos , Células da Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Cartilagem Articular/patologia , Humanos , Imagem Tridimensional , Imagem por Ressonância Magnética , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Coelhos , Reprodutibilidade dos Testes , Engenharia Tecidual/métodos , Tomografia Computadorizada por Raios X , Transplante Heterólogo
9.
Oncotarget ; 8(17): 28187-28202, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28390197

RESUMO

Histone deacetylases (HDACs) are involved in epigenetic modulation and their aberrant expression has been demonstrated in myeloproliferative neoplasms (MPN). HDAC8 inhibition has been shown to inhibit JAK2/STAT5 signaling in hematopoietic cells from MPN. Nevertheless, the role of HDAC8 expression in bone marrow-mesenchymal stromal cells (BM-MSC) has not been assessed. In the current work we describe that HDAC8 is significantly over-expressed in MSC from in JAK-2 positive MPN compared to those from healthy-donors (HD-MSC). Using a selective HDAC8 inhibitor (PCI34051), we verified that the subsequent decrease in the protein and mRNA expression of HDAC8 is linked with an increased apoptosis of malignant MSC whereas it has no effects on normal MSC. In addition, HDAC8 inhibition in MPN-MSC also decreased their capacity to maintain neoplastic hematopoiesis, by increasing the apoptosis, cell-cycle arrest and colony formation of JAK2+-hematopoietic cells. Mechanistic studies using different MPN cell lines revealed that PCI34051 induced their apoptosis, which is enhanced when were co-cultured with JAK2V617F-MSC, decreased their colony formation and the phosphorylation of STAT3 and STAT5. In summary, we show for the first time that the inhibition of HDAC8 in MSC from JAK2+ MPN patients selectively decreases their hematopoietic-supporting ability, suggesting that HDAC8 may be a potential therapeutic target in this setting by acting not only on hematopoietic cells but also on the malignant microenvironment.


Assuntos
Histona Desacetilases/genética , Janus Quinase 2/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transtornos Mieloproliferativos/genética , Proteínas Repressoras/genética , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Hematopoese/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Janus Quinase 2/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Terapia de Alvo Molecular , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo
10.
Eur J Haematol ; 97(6): 528-537, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27118602

RESUMO

INTRODUCTION: The ability of mesenchymal stromal cells (MSC) to suppress T-cell function has prompted their therapeutic use for graft-versus-host disease (GVHD) control. However, as MSC also modulate the activity of NK cells, which play an important role in graft-versus-leukemia (GVL) reaction, their administration could hamper this beneficial effect of allogeneic hematopoietic stem cell transplantation. MSC can be expanded from several sources, especially bone marrow and fat, but it is not well established if the cell source makes a difference in their immunoregulatory capacity. OBJECTIVE: The aim of this study was to compare the immunomodulatory effect of MSC derived from bone marrow (BM-CSM) or adipose tissue (AT-MSC) on NK cells, to determine whether the use of MSC from one or the other origin could be more favorable to preserve NK cell activity and, therefore, GVL. METHODS: Human NK cells were stimulated with IL-15 in the presence of BM-MSC or AT-MSC. The effect of both MSC populations on NK cell proliferation, cell cycle progression, and CD56 expression was analyzed by flow cytometry. Cytokine secretion was measured by ELISA, and cytotoxic activity was assessed by calcein release assays. RESULTS: Although both BM-MSC and AT-MSC induced a similar inhibition of NK cell proliferation, only BM-MSC decreased significantly NK cell cytotoxic activity and showed a trend for a higher reduction of IFN-γ secretion. CONCLUSION: These results suggest that, in the context of GVHD inhibition, the use of AT-MSC rather than BM-MSC could further preserve NK cell activity and, thus, favor GVL.


Assuntos
Tecido Adiposo/citologia , Comunicação Celular/imunologia , Imunomodulação , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adulto , Idoso , Biomarcadores , Ciclo Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Humanos , Imunofenotipagem , Ativação Linfocitária , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Fenótipo
11.
PLoS One ; 11(2): e0146722, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26836120

RESUMO

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.


Assuntos
Comunicação Celular , Exossomos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Síndromes Mielodisplásicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34 , Sobrevivência Celular , Microambiente Celular , Feminino , Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética
12.
Cell Commun Signal ; 14: 2, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26754424

RESUMO

BACKGROUND: Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer. METHODS: Human MSC from bone marrow of healthy donors, mesenchymal cell lines (HS-5 and hTERT) and a leukemic cell line (K562 cells) were used to obtain EV for FCM characterization. EV released from the different cell lines were isolated by ultracentrifugation and were characterized, using a multi-parametric analysis, in a conventional flow cytometer. EV characterization by transmission electron microscopy (TEM), western blot (WB) and Nano-particle tracking analysis (NTA) was also performed. RESULTS: EV membranes are constituted by the combination of specific cell surface molecules depending on their cell of origin, together with specific proteins like tetraspanins (e.g. CD63). We have characterized by FCM the EV released from BM-hMSC, that were defined as particles less than 0.9 µm, positive for the hMSC markers (CD90, CD44 and CD73) and negative for CD34 and CD45 (hematopoietic markers). In addition, hMSC-derived EV were also positive for CD63 and CD81, the two characteristic markers of EV. To validate our characterization strategy, EV from mesenchymal cell lines (hTERT/HS-5) were also studied, using the leukemia cell line (K562) as a negative control. EV released from mesenchymal cell lines displayed the same immunophenotypic profile as the EV from primary BM-hMSC, while the EV derived from K562 cells did not show hMSC markers. We further validated the panel using EV from hMSC transduced with GFP. Finally, EV derived from the different sources (hMSC, hTERT/HS-5 and K562) were also characterized by WB, TEM and NTA, demonstrating the expression by WB of the exosomal markers CD63 and CD81, as well as CD73 in those from MSC origin. EV morphology and size/concentration was confirmed by TEM and NTA, respectively. CONCLUSION: We described a strategy that allows the identification and characterization by flow cytometry of hMSC-derived EV that can be routinely used in most laboratories with a standard flow cytometry facility.


Assuntos
5'-Nucleotidase/análise , Vesículas Extracelulares/química , Citometria de Fluxo/métodos , Receptores de Hialuronatos/análise , Células-Tronco Mesenquimais/citologia , Antígenos Thy-1/análise , Adulto , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/química , Pessoa de Meia-Idade , Adulto Jovem
13.
Leuk Res ; 39(8): 921-4, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26105049

RESUMO

The expression of BCR-ABL in hematopoietic stem cells is a well-defined primary event in chronic myeloid leukemia (CML). Some reports have described the presence of BCR-ABL on endothelial cells from CML patients, suggesting the origin of the disease in a primitive hemangioblastic cell. On the other hand, extracellular vesicles (EVs) released by CML leukemic cells are involved in the angiogenesis modulation process. In the current work we hypothesized that EVs released from BCR-ABL(+) cells may carry inside the oncogene that can be transferred to endothelial cells leading to the expression of both BCR-ABL transcript and the oncoprotein. EVs from K562 cells and plasma of newly diagnosed CML patients were isolated by ultracentrifugation. RT-PCR analysis detected the presence of BCR-ABL RNA in the EVs isolated from both K562 cells and plasma of CML patients. The incorporation of these EVs into endothelial cells was demonstrated by flow cytometry and fluorescence microscopy showed that after 24h of incubation most EVs were incorporated. BCR-ABL transcripts were detected in all experiments on endothelial cells incubated with EVs from both sources. The presence of BCR-ABL on endothelial cells incubated with Philadelphia(+) EVs was also confirmed by Western blot assays. In summary, endothelial cells acquire BCR-ABL RNA and the oncoprotein after incubation with EVs released from Ph(+) positive cells (either from K562 cells or from plasma of newly diagnosed CML patients). This results challenge the hypothesis that endothelial cells may be part of the Philadelphia(+) clone in CML.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/patologia , Vesículas Secretórias/fisiologia , Células Cultivadas , Células Clonais/metabolismo , Células Clonais/patologia , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo
14.
Nature ; 512(7512): 78-81, 2014 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-25043017

RESUMO

Myeloproliferative neoplasms (MPNs) are diseases caused by mutations in the haematopoietic stem cell (HSC) compartment. Most MPN patients have a common acquired mutation of Janus kinase 2 (JAK2) gene in HSCs that renders this kinase constitutively active, leading to uncontrolled cell expansion. The bone marrow microenvironment might contribute to the clinical outcomes of this common event. We previously showed that bone marrow nestin(+) mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibres regulate normal HSCs. Here we demonstrate that abrogation of this regulatory circuit is essential for MPN pathogenesis. Sympathetic nerve fibres, supporting Schwann cells and nestin(+) MSCs are consistently reduced in the bone marrow of MPN patients and mice expressing the human JAK2(V617F) mutation in HSCs. Unexpectedly, MSC reduction is not due to differentiation but is caused by bone marrow neural damage and Schwann cell death triggered by interleukin-1ß produced by mutant HSCs. In turn, in vivo depletion of nestin(+) cells or their production of CXCL12 expanded mutant HSC number and accelerated MPN progression. In contrast, administration of neuroprotective or sympathomimetic drugs prevented mutant HSC expansion. Treatment with ß3-adrenergic agonists that restored the sympathetic regulation of nestin(+) MSCs prevented the loss of these cells and blocked MPN progression by indirectly reducing the number of leukaemic stem cells. Our results demonstrate that mutant-HSC-driven niche damage critically contributes to disease manifestation in MPN and identify niche-forming MSCs and their neural regulation as promising therapeutic targets.


Assuntos
Células-Tronco Hematopoéticas/patologia , Transtornos Mieloproliferativos/patologia , Neoplasias/patologia , Fibras Nervosas/patologia , Nicho de Células-Tronco , Sistema Nervoso Simpático/patologia , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Agonistas de Receptores Adrenérgicos beta 3/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Progressão da Doença , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-1beta/metabolismo , Janus Quinase 2/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Camundongos , Transtornos Mieloproliferativos/tratamento farmacológico , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fibras Nervosas/efeitos dos fármacos , Nestina/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Receptores Adrenérgicos beta 3/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/patologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiopatologia
15.
Cytotherapy ; 15(6): 673-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23522868

RESUMO

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive properties. Nevertheless, it has been previously reported that MSCs might also trigger the immune response. We studied whether MSCs may act as carriers, capturing antigens that can be endocytosed by antigen-presenting cells later on. METHODS: We measured the cellular uptake of mannose receptor-mediated fluid phase macropinocytosis, assessed as cellular uptake of fluorescein isothiocyanate-dextran, and PKH-67-labeled cell lysates as a surrogate marker for antigen capture among dendritic cells (DCs, positive control), T lymphocytes (negative control) and MSCs. RESULTS: All experiments confirmed that MCSs displayed pinocytic and endocytic capacities, which were lower than those observed for DCs but significantly higher than those observed for T cells. We also demonstrated that MSCs release previously endocytosed antigens, which subsequently can be captured by DCs. CONCLUSIONS: MSCs have the ability to capture and release antigens.


Assuntos
Endocitose , Antígenos HLA-D/metabolismo , Células-Tronco Mesenquimais/citologia , Pinocitose , Células Apresentadoras de Antígenos/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Antígenos HLA-D/imunologia , Humanos , Imunossupressores , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Multipotentes , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
16.
Haematologica ; 97(8): 1218-24, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22371183

RESUMO

UNLABELLED: Background Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cell-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze DICER1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS, a DICER1-related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. DESIGN AND METHODS: Mesenchymal stromal cells from 33 bone marrow samples were evaluated. DICER, DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. RESULTS: Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower DICER1 (0.65±0.08 vs. 1.91±0.57; P=0.011) and DROSHA (0.62±0.06 vs. 1.38±0.29; P=0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs. 1.15±0.28; P=0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR-222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. Conclusions This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where DICER1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.


Assuntos
RNA Helicases DEAD-box/genética , Regulação Neoplásica da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Síndromes Mielodisplásicas/genética , Proteínas/genética , Ribonuclease III/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/metabolismo
17.
Transfusion ; 52(5): 1086-91, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22023454

RESUMO

BACKGROUND: Posttransplant cytopenias are a severe complication after allogeneic stem cell transplantation (allo-SCT) and their origin is often multifactorial or unknown. They are frequently refractory to standard therapy, which may include steroids and/or immunoglobulins. Mesenchymal stem cells (MSCs) are an attractive therapeutic tool in the allo-SCT setting for the ability to enhance engraftment as well as acting as immunosuppressants for graft-versus-host disease. There is no prior experience in the literature of the use of MSCs to treat cytopenias after allo-SCT. CASE REPORTS: In this work we report for the first time four cases of refractory posttransplant cytopenias (three patients with thrombocytopenia and one with neutropenia) that were treated with MSCs from a third-party donor. MSCs were expanded from 100 mL of marrow obtained under standard good manufacturing practice conditions. Most patients received more than one cell dose, and median dose of MSCs administered was 1 × 10(6) /kg. RESULTS: All patients recovered normal blood counts, with a mean follow-up of 12.5 months. There were no adverse events related to MSC administration. CONCLUSION: MSC therapy may contribute to the recovery of refractory posttransplant peripheral cytopenias in patients undergoing allo-SCT.


Assuntos
Transplante de Células-Tronco Mesenquimais , Neutropenia/cirurgia , Trombocitopenia/cirurgia , Adulto , Transplante de Células-Tronco Hematopoéticas , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Transplante Homólogo
18.
PLoS One ; 6(10): e26241, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22028841

RESUMO

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34(+) progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (i.v.) or intrabone (i.b.) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.


Assuntos
Células da Medula Óssea/citologia , Microambiente Celular , Hematopoese , Células-Tronco Mesenquimais/citologia , Adulto , Animais , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Pessoa de Meia-Idade , Osteopontina/genética , Osteopontina/metabolismo
19.
Eur Spine J ; 20 Suppl 3: 353-60, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21779858

RESUMO

INTRODUCTION: In the last few years, great interest has been focused on tissue engineering as a potential therapeutic approach for musculoskeletal diseases. The role of metallic implants for spinal fusion has been tested in preclinical and clinical settings. Titanium and tantalum have excellent biocompatibility and mechanical properties and are being used in this situation. On the other hand, the therapeutic role of mesenchymal stem cells (MSC) is extensively explored for their multilineage differentiation into osteoblasts. OBJECTIVES: In vitro comparison of titanium and tantalum as MSCs scaffolds. MATERIAL AND METHODS: In the present study, we have compared the in vitro expansion capacity, viability, immunophenotype (both explored by flow cytometry) and multi-differentiation ability of MSC cultured in the presence of either titanium or tantalum fragments. The adherence of MSC to either metal was demonstrated by electron microscopy. RESULTS: Both metals were able to carry MSC when transferred to new culture flasks. In addition, our study shows that culture of MSC with titanium or tantalum improves cell viability and maintains all their biological properties, with no significant differences regarding the metal employed. CONCLUSION: This would support the use of these combinations for clinical purposes, especially in the spinal fusion and reconstruction setting.


Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Osseointegração/fisiologia , Fusão Vertebral/instrumentação , Tantálio/farmacologia , Tecidos Suporte/química , Titânio/farmacologia , Adulto , Apoptose/fisiologia , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Sobrevivência Celular/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
20.
Haematologica ; 96(7): 1072-6, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21393326

RESUMO

This trial evaluated the feasibility and efficacy of the infusion of mesenchymal stem cells expanded using human serum for the treatment of refractory acute or chronic graft-versus-host disease. Twenty-eight expansions were started. In 22, a minimum of more than 1 x 106 mesenchymal stem cells/kg were obtained after a median of 26 days; this threshold was not obtained in the remaining cases. Ten patients received cells for the treatment of refractory or relapsed acute graft-versus-host disease and 8 for chronic disease. One patient treated for acute graft-versus-host disease obtained a complete response, 6 had a partial response and 3 did not respond. One of the chronic patients achieved complete remision, 3 a partial response, and 4 did not respond. The current study supports the use of this approach in less heavily treated patients for both acute and chronic graft-versus-host disease. The trial has been registered at ClinicalTrials.gov: identifier NCT00447460.


Assuntos
Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Soro , Adulto , Idoso , Proliferação de Células , Células Cultivadas , Técnicas de Cultura , Feminino , Doença Enxerto-Hospedeiro/mortalidade , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Transplante Homólogo , Resultado do Tratamento , Adulto Jovem
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