RESUMEN
BACKGROUND: Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease associated with failure to achieve bone union and recurrent fractures. There is evidence showing that CPT is associated with decreased osteogenesis. Based on the capacity of mesenchymal stromal cells (MSCs) to induce osteogenesis, we develop an osteogenic organoid (OstO) constituted by these cells, and other components of the bone niche, for inducing bone formation in a child diagnosed with CPT. AIM: To evaluate the capacity of an OstO to induce bone formation in a patient with CPT. METHODS: The OstO was fabricated with allogeneic bone marrow MSCs from a healthy donor, collagen microbeads (CM) and PRP clot. The CM and PRP function as extracellular matrix and scaffolds for MSC. The OstO was placed at the site of non-union. Internal and external fixation was placed in the tibia. Radiological evaluation was performed after MSCs transplantation. RESULTS: After 4 months of MSCs transplantation, radiographic imaging showed evidence of osteogenesis at the site of CPT lesion. The tibia showed bone consolidation and complete healing of the non-union CPT lesion after 6 months. Functional improvement was observed after 1 year of MSC transplantation. CONCLUSIONS: The OstO is a bone-like niche which promote osteogenesis in patients with failure in bone formation, such as CPT. To our knowledge, these results provide the first evidence showing CPT healing induced by an OstO constituted by allogeneic MSCs. Future studies incorporating a larger number of patients may confirm these results.
Asunto(s)
Osteogénesis , Seudoartrosis/congénito , Tibia , Niño , Humanos , Tibia/diagnóstico por imagen , Tibia/cirugía , Tibia/anomalías , Regeneración Ósea , Colágeno , Organoides , Diferenciación CelularRESUMEN
BACKGROUND: Under certain clinical and experimental conditions hematopoiesis occurs in other site than bone marrow (BM), such as the liver. Here, we develop a 3D organoid that mimics several components of the hematopoietic niche present during liver extramedullary hematopoiesis. AIM: To evaluate the capacity of a 3D hematopoietic organoid (3D-HO) to function as a hematopoietic like-niche allowing for blood cell production outside of the BM. METHODS: The 3D-HO is constituted by liver sinusoidal endothelial cells (LSEC) as the stromal component, BM isolated from 5-FU treated mice (FU-BMCs), collagen microspheres and plasma clot as scaffolds. The ability of the 3D-HO to support the survival and functionality of FU-BMCs was investigated by using confocal microscopy, histology analysis, flow cytometry, and clonogenic assays. RESULTS: After 15 and 30 days, post-ectopic implantation, histological studies of the 3D-HO showed the presence of cells with myeloid and lymphoid lineage morphology. Flow cytometry analysis of these cells showed the presence of cells expressing hematopoietic stem progenitor cells (HSPC) (Sca-1+/c-Kit+), myeloid (Gr-1+) and lymphoid (B220+ and CD19+) markers. Clonogenic assays showed that cells from the 3D-HO formed hematopoietic colonies. Expression of the Sry gene by cells from the 3D-HO, implanted for 30 days in female mice, indicated that male donor cells persist in this model of extramedullary hematopoiesis. CONCLUSIONS: The 3D-HO constitutes an extramedullary hematopoietic-like niche which supports the survival and functionality of FU-BMCs. It may constitute an efficient model for investigating, in vitro and in vivo, those factors that control hematopoiesis outside BM.
Asunto(s)
Hematopoyesis Extramedular , Masculino , Femenino , Ratones , Animales , Células Endoteliales , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Organoides , Células de la Médula Ósea/metabolismoRESUMEN
Cellular therapy and platelet-rich plasma (PRP) have been used as a treatment for skin wounds. Previous evidence has shown that mesenchymal stromal cells (MSC) may improve skin wound healing. In contrast, contradictory effects have been reported by using PRP treatment on skin wound healing. However, there is evidence that PRP constitutes an excellent scaffold for tissue engineering. In this work, we aim to study the effect of MSC on skin wound healing. We used an experimental murine model of full-thickness wounds. Wounds were treated with human bone marrow-MSC contained in a PRP clot. Untreated or PRP-treated wounds were used as controls. Wound healing was evaluated by macroscopic observation and histological analysis at day 7 post-wounding. Immunohistochemical studies were performed to detect the presence of epithelial progenitor cells (EPC) and the expression of basic fibroblast growth factor (bFGF). MSC/PRP implantation induced a significant wound closure and re-epithelialization as compared with the controls. Increase of CD34+ cells and bFGF was observed in the wounds treated with MSC/PRP. Our results show that MSC included in PRP clot induce cutaneous wound repair by promoting re-epithelialization, migration of EPC and expression of bFGF. PRP alone does not exert a significant effect on wound healing. Our results support the possible clinical use of MSC in PRP scaffold as potential treatment of skin wounds.
Asunto(s)
Células Madre Mesenquimatosas , Plasma Rico en Plaquetas , Traumatismos de los Tejidos Blandos , Humanos , Ratones , Animales , Cicatrización de Heridas , Piel/patología , Plasma Rico en Plaquetas/metabolismo , RepitelizaciónRESUMEN
OBJECTIVE: Cartilage damage (CD) in the temporomandibular joint (TMJ) continues being a major problem in maxillofacial field. Evidence suggests that cellular therapy may be used for repairing CD in the TMJ. DESIGN: A murine model of condyle CD (CCD) was generated in the TMJ to evaluate the capacity of mesenchymal stromal cells (MSCs) to induce cartilage regeneration in CCD. A large CCD was surgically created in a condyle head of the TMJ of C57BL/6 mice. Human MSC embedded into preclotted platelet-rich plasma (PRP) were placed on the surface of CCD. As controls, untreated CCD and exposed TMJ condyle (sham) were used. After 6 weeks, animals were sacrificed, and each mandibular condyle was removed and CCD healing was assessed macroscopically and histologically. RESULTS: Macroscopic observation of CCD treated with MSC showed the presence of cartilage-like tissue in the CCD site. Histological analysis showed a complete repair of the articular surface with the presence of cartilage-like tissue and subchondral bone filling the CCD area. Chondrocytes were observed into collagen and glycosaminoglycans extracellular matrix filling the repaired tissue. There was no evidence of subchondral bone sclerosis. Untreated CCD showed denudated osteochondral lesions without signs of cartilage repair. Histological analysis showed the absence of tissue formation over the CCD. CONCLUSIONS: Transplantation of MSC induces regeneration of TMJ-CCD. These results provide strong evidence to use MSC as potential treatment in patients with cartilage lesions in the TMJ.
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Cartílago Articular , Células Madre Mesenquimatosas , Animales , Cartílago Articular/patología , Condrocitos , Humanos , Ratones , Ratones Endogámicos C57BL , Articulación Temporomandibular/cirugíaRESUMEN
The hematopoietic niche is a specialized microenvironment that supports the survival, proliferation and differentiation of hematopoietic stem progenitor cells (HSPCs). Three-dimensional (3D) models mimicking hematopoiesis might allow in vitro and in vivo studies of the hematopoietic (HP) process. Here, we investigate the capacity of a 3D construct based on non-adherent murine bone marrow mononuclear cells (NA-BMMNCs), mesenchymal stromal cells (MSCs) and collagen microspheres (CMs), all embedded into plasma clot (PC) to support in vitro and in vivo hematopoiesis. Confocal analysis of the 3D hematopoietic construct (3D-HPC), cultured for 24 h, showed MSC lining the CM and the NA-BMMNCs closely associated with MSC. In vivo hematopoiesis was examined in 3D-HPC subcutaneously implanted in mice and harvested at different intervals. Hematopoiesis in the 3D-HPC was evaluated by histology, cell morphology, flow cytometry, confocal microscopy and hematopoietic colony formation assay. 3D-HPC implants were integrated and vascularized in the host tissue, after 3 months of implantation. Histological studies showed the presence of hematopoietic tissue with the presence of mature blood cells. Cells from 3D-HPC showed viability greater than 90%, expressed HSPCs markers, and formed hematopoietic colonies, in vitro. Confocal microscopy studies showed that MSCs adhered to the CM and NA-BMMNCs were scattered across the 3D-HPC area and in close association with MSC. In conclusion, the 3D-HPC mimics a hematopoietic niche supporting the survival, proliferation and differentiation of HSPCs, in vivo. 3D-HPC may allow evaluation of regulatory mechanisms involved in hematopoiesis.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Imagenología Tridimensional/métodos , Células Madre Mesenquimatosas/metabolismo , Microesferas , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Ratones , Análisis de SupervivenciaRESUMEN
BACKGROUND: Skin wounds continue to be a global health problem. Several cellular therapy protocols have been used to improve and accelerate skin wound healing. Here, we evaluated the effect of transplantation of mesenchymal stromal cells (MSC) on the wound re-epithelialization process and its possible relationship with the presence of epithelial progenitor cells (EPC) and the expression of growth factors. METHODS: An experimental wound model was developed in C57BL/6 mice. Human MSCs seeded on collagen membranes (CM) were implanted on wounds. As controls, animals with wounds without treatment or treated with CM were established. Histological and immunohistochemical (IH) studies were performed at day 3 post-treatment to detect early skin wound changes associated with the presence of EPC expressing Lgr6 and CD34 markers and the expression of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF). RESULTS: MSC transplantation enhanced skin wound re-epithelialization, as compared with controls. It was associated with an increase in Lgr6+ and CD34+ cells and the expression of KGF and bFGF in the wound bed. CONCLUSION: Our results show that cutaneous wound healing induced by MSC is associated with an increase in EPC and growth factors. These preclinical results support the possible clinical use of MSC to treat cutaneous wounds.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Repitelización/fisiología , Piel/lesiones , Células Madre Adultas/metabolismo , Animales , Antígenos CD34/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Voluntarios Sanos , Humanos , Masculino , Ratones , Cultivo Primario de Células , Receptores Acoplados a Proteínas G/metabolismo , Piel/citología , Piel/metabolismoRESUMEN
BACKGROUND: Deep dermal and full-thickness burns are not only difficult to treat, but they are also associated with significant morbidity and mortality. Recent reports have proposed the use of mesenchymal stromal cells (MSCs) for inducing tissue repair in burn injuries. OBJECTIVE: We aim to evaluate the effect of allogeneic MSC transplantation on full-thickness burns with delayed healing. MATERIAL AND METHODS: This study includes five patients with AB B/B burns. All patients received conservative treatments, including cleaning, debridement of necrotic tissue, and silver based dressing on the burn wounds. Cryopreserved allogeneic MSCs were thawed and rapidly expanded and used for application in burned patients. MSCs were implanted into preclotted platelet-rich plasma onto the surface of burn wounds. RESULTS: All treated burn wounds showed early granulation tissue and rapid re-epithelialization after MSC transplantation. Healing took between 1 and 5 months after MSC transplantation. Repair of burn wounds was associated with slight discoloration of the regenerated skin without hypertrophic scarring or contractures. CONCLUSION: Our results provide evidence of healing in deep- and full-thickness burns by allogeneic MSC transplantation. Rapid healing of burn patients, after MSC transplantation, improves their quality of life and reduces the length of hospitalization. Future studies incorporating a larger number of patients may confirm the results obtained in this work.
Asunto(s)
Quemaduras , Trasplante de Células Madre Hematopoyéticas , Células Madre Mesenquimatosas , Quemaduras/cirugía , Humanos , Calidad de Vida , Trasplante de Piel , Cicatrización de HeridasRESUMEN
INTRODUCTION: The olfactory neuro-epithelium has an intrinsic capability of renewal during lifetime provided by the existence of globose and horizontal olfactory precursor cells. Additionally, mesenchymal stromal olfactory cells also support the homeostasis of the olfactory mucosa cell population. Under in vitro culture conditions with Dulbecco modified eagle/F12 medium supplemented with 10% fetal bovine serum, tissue biopsies from upper turbinate have generated an adherent population of cells expressing mainly mesenchymal stromal phenotypic markers. A closer examination of these cells has also found co-expression of olfactory precursors and ensheathing cell phenotypic markers. These results were suggestive of a unique property of olfactory mesenchymal stromal cells as potentially olfactory progenitor cells. OBJECTIVE: To study whether the expression of these proteins in mesenchymal stromal cells is modulated upon neuronal differentiation. MATERIALS AND METHODS: We observed the phenotype of olfactory stromal cells under DMEM/F12 plus 10% fetal bovine serum in comparison to cells from spheres induced by serum-free medium plus growth factors inducers of neural progenitors. RESULTS: The expression of mesenchymal stromal (CD29+, CD73+, CD90+, CD45-), horizontal basal (ICAM-1/CD54+, p63+, p75NGFr+), and ensheathing progenitor cell (nestin+, GFAP+) proteins was determined in the cultured population by flow cytometry. The determination of Oct 3/4, Sox-2, and Mash-1 transcription factors, as well as the neurotrophins BDNF, NT3, and NT4 by RT-PCR in cells, was indicative of functional heterogeneity of the olfactory mucosa tissue sample. CONCLUSIONS: Mesenchymal and olfactory precursor proteins were downregulated by serum-free medium and promoted differentiation of mesenchymal stromal cells into neurons and astroglial cells.
Introducción. El recambio celular del neuroepitelio olfatorio ocurre durante la vida del individuo gracias a precursores olfatorios. Además, las células mesenquimales del estroma también contribuyen a la homeostasis de la mucosa. Cuando un explante de una biopsia de mucosa se cultiva en un medio esencial mínimo, se genera una población predominante de células adherentes que expresan proteínas típicas de las células mesenquimales del estroma. La coexpresión de marcadores fenotípicos de precursores olfatorios y de células del recubrimiento del nervio olfatorio constituiría una propiedad única de las células mesenquimales del estroma. Objetivo. Determinar si la diferenciación celular de las células mesenquimales hacia fenotipos neurales modula la expresión de los marcadores mesenquimales característicos. Materiales y métodos. Se compararon las células aisladas de la mucosa olfatoria en un medio de cultivo con suplemento de 10 % de suero fetal bovino con esferas generadas en un medio sin suero más factores de crecimiento. Resultados. Se determinó la expresión de proteínas de las células mesenquimales del estroma (CD29+, CD73+, CD90+, CD45-), de las basales horizontales (ICAM-1/CD54+, p63+, p75NGFr+), y de las del recubrimiento del nervio olfatorio (nestin+, GFAP+) en la misma población cultivada. La determinación de Oct 3/4, Sox-2 y Mash-1, así como de las neurotrofinas BDNF, NT3 y NT4, sugirió que las células del estroma son funcionales. La expresión de las proteínas de las células mesenquimales y los precursores olfatorios, disminuyó en las células de las mesenesferas inducidas por ausencia de suero en el medio de cultivo. Conclusión. Las células mesenquimales del estroma de la mucosa olfatoria presentan una tendencia dominante hacia la diferenciación neural.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Mucosa Nasal/citología , Mucosa Olfatoria/citología , Biosíntesis de Proteínas , Adipogénesis , Antígenos de Diferenciación/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Condrogénesis , Medios de Cultivo/farmacología , Medio de Cultivo Libre de Suero/farmacología , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Mucosa Nasal/metabolismo , Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/genética , Nestina/biosíntesis , Nestina/genética , Neuroglía/metabolismo , Neuronas/metabolismo , Mucosa Olfatoria/metabolismo , Osteogénesis , Proteínas Recombinantes/farmacología , Esferoides Celulares , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Cornetes NasalesRESUMEN
Introduction: The olfactory neuro-epithelium has an intrinsic capability of renewal during lifetime provided by the existence of globose and horizontal olfactory precursor cells. Additionally, mesenchymal stromal olfactory cells also support the homeostasis of the olfactory mucosa cell population. Under in vitro culture conditions with Dulbecco modified eagle/F12 medium supplemented with 10% fetal bovine serum, tissue biopsies from upper turbinate have generated an adherent population of cells expressing mainly mesenchymal stromal phenotypic markers. A closer examination of these cells has also found co-expression of olfactory precursors and ensheathing cell phenotypic markers. These results were suggestive of a unique property of olfactory mesenchymal stromal cells as potentially olfactory progenitor cells. Objective: To study whether the expression of these proteins in mesenchymal stromal cells is modulated upon neuronal differentiation. Materials and methods: We observed the phenotype of olfactory stromal cells under DMEM/F12 plus 10% fetal bovine serum in comparison to cells from spheres induced by serum-free medium plus growth factors inducers of neural progenitors. Results: The expression of mesenchymal stromal (CD29+, CD73+, CD90+, CD45-), horizontal basal (ICAM-1/CD54+, p63+, p75NGFr+), and ensheathing progenitor cell (nestin+, GFAP+) proteins was determined in the cultured population by flow cytometry. The determination of Oct 3/4, Sox-2, and Mash-1 transcription factors, as well as the neurotrophins BDNF, NT3, and NT4 by RT-PCR in cells, was indicative of functional heterogeneity of the olfactory mucosa tissue sample. Conclusions: Mesenchymal and olfactory precursor proteins were downregulated by serum-free medium and promoted differentiation of mesenchymal stromal cells into neurons and astroglial cells.
Introducción. El recambio celular del neuroepitelio olfatorio ocurre durante la vida del individuo gracias a precursores olfatorios. Además, las células mesenquimales del estroma también contribuyen a la homeostasis de la mucosa. Cuando un explante de una biopsia de mucosa se cultiva en un medio esencial mínimo, se genera una población predominante de células adherentes que expresan proteínas típicas de las células mesenquimales del estroma. La coexpresión de marcadores fenotípicos de precursores olfatorios y de células del recubrimiento del nervio olfatorio constituiría una propiedad única de las células mesenquimales del estroma. Objetivo. Determinar si la diferenciación celular de las células mesenquimales hacia fenotipos neurales modula la expresión de los marcadores mesenquimales característicos. Materiales y métodos. Se compararon las células aisladas de la mucosa olfatoria en un medio de cultivo con suplemento de 10 % de suero fetal bovino con esferas generadas en un medio sin suero más factores de crecimiento. Resultados. Se determinó la expresión de proteínas de las células mesenquimales del estroma (CD29+, CD73+, CD90+, CD45-), de las basales horizontales (ICAM-1/CD54+, p63+, p75NGFr+), y de las del recubrimiento del nervio olfatorio (nestin+, GFAP+) en la misma población cultivada. La determinación de Oct 3/4, Sox-2 y Mash-1, así como de las neurotrofinas BDNF, NT3 y NT4, sugirió que las células del estroma son funcionales. La expresión de las proteínas de las células mesenquimales y los precursores olfatorios, disminuyó en las células de las mesenesferas inducidas por ausencia de suero en el medio de cultivo. Conclusión. Las células mesenquimales del estroma de la mucosa olfatoria presentan una tendencia dominante hacia la diferenciación neural.
Asunto(s)
Mucosa Olfatoria , Células Madre Mesenquimatosas , HomeostasisRESUMEN
The CXCR4 chemokine receptor plays an essential role in the homing of cells to organs expressing its ligand, CXCL12. CXCR4 expressed on tumor cells might regulate their traffic during metastasis. Here, we investigated whether the activation of CXCR4 on B16 murine melanoma cells regulates biological functions associated with metastasis, in vitro and in vivo. Flow cytometry and PCR analysis showed that B16 constitutively expresses high levels of CXCR4 (CXCR4-B16). Biological assays showed that the activation of CXCR4, by its ligand CXCL12, increases the migration, invasion, and proliferation of CXCR4-B16. AMD3100 significantly inhibited the stimulatory migrating effect induced by CXCL12. Treatment of CXCR4-B16 with CXCL12 increases their adhesion to liver sinusoidal endothelial cell (LSEC) monolayers. LSEC, expressing CXCL12, increased the migration of CXCR4-B16. In a liver metastasis model, CXCR4-B16 metastasis was associated with an increased expression of CXCL12 in LSEC territories. CXCR4-B16 cells were located close to LSEC microenvironments expressing CXCL12. Increased liver metastasis was observed after injecting CXCR4-B16 cells previously treated with CXCL12. Our results provide evidence showing that CXCR4 plays an important role in regulating biological functions associated with B16 liver metastasis.
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Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Receptores CXCR4/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Animales , Línea Celular Tumoral , Quimiocina CXCL12/biosíntesis , Quimiocina CXCL12/farmacología , Femenino , Neoplasias Hepáticas/genética , Melanoma Experimental/genética , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neoplasias Cutáneas/genéticaRESUMEN
PURPOSE: There is evidence showing that mesenchymal stromal cells (MSC) may constitute a potential therapeutic strategy to induce bone regeneration. In this work, we investigate the capacity of autologous bone marrow (BM) MSC loaded on collagen microspheres (CM) and included into autologous platelet-rich plasma (PRP) clots (MSC/CM/PRP) to induce bone formation in patients with nonunion lesions. METHODS: MSC were isolated from BM cells of patients with nonunion lesions. Phenotypical (marker expression) and functional studies (osteogenic differentiation) were performed. MSC were seeded on CM and included into autologous PRP clot (MSC/CM/PRP). The capacity of MSC/CM/PRP to induce bone formation was evaluated in three patients diagnosed with nonunion. RESULTS: MSC loaded on CM/PRP clots maintain their biological functions, in vitro. After three months, post-MSC transplantation, all patients showed evidence of osteogenesis at the site of nonunion. After one year, all patients showed a complete healing of the nonunion. CONCLUSIONS: Our results support the use of autologous MSC transplanted as MSC/CM/PRP for the treatment of nonunion fractures. Future studies incorporating a larger number of patients may confirm the results obtained in this work.
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Regeneración Ósea/efectos de los fármacos , Fracturas no Consolidadas/tratamiento farmacológico , Trasplante de Células Madre Mesenquimatosas/métodos , Cicatrización de Heridas/efectos de los fármacos , Adulto , Anciano de 80 o más Años , Colágeno/metabolismo , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Microesferas , Osteogénesis/efectos de los fármacos , Plasma Rico en Plaquetas/efectos de los fármacosRESUMEN
Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease whose etiology and pathogenesis is unknown. Several evidences suggest that decreased osteogenic capacities, impaired local vascularization, and microenvironment alterations may play a role in the pathogenesis of CPT. Additionally, it is not clear if the pathogenesis of this disease is related to the absence of cells with osteogenic capacity of differentiation. In this work, a two-year-old patient diagnosed with CPT underwent an orthopedic surgery to promote bone union in a pseudoarthrosis lesion. Tissue from CPT lesion was excised, and histological evaluation and tissue culture were performed. Histologic analysis of the soft CPT lesion showed the presence of highly cellular fibrous tissue, vascularization, and abundant extracellular matrix. Fusiform cells of mesenchymal appearance were observed but osteoblasts, osteoclasts, chondrocytes, and adipose cells were not found. There was no evidence of osteogenesis. CPT tissue cultured as explants showed, after one month of culture, evidence of osteogenesis, chondrogenesis, and adipogenesis. Cells isolated from explants of CPT tissue showed a fibroblast-like morphology and expressed the mesenchymal stromal cell (MSC) markers: CD105, CD73, and CD90 (CPT-MSC). Functional analysis showed that CPT-MSC differentiate, in vitro, into osteogenic, chondrogenic, and adipocytic cells. CPT-MSC expressed osteocalcin and agrecan. CPT-MSC produced collagen in the presence of ascorbic acid. MSC from BM of normal individuals were used as control. In summary, our results indicate that CPT tissue contains MSC with osteogenic capacity of differentiation. It is possible that CPT microenvironment may contribute to impair the osteogenic capacity of differentiation of CPT-MSC.
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Células Madre Mesenquimatosas/fisiología , Seudoartrosis/congénito , Tibia/citología , Tibia/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Preescolar , Humanos , Masculino , Seudoartrosis/diagnóstico por imagen , Seudoartrosis/cirugía , Radiografía , Tibia/diagnóstico por imagenRESUMEN
The stromal cell derived factor 1 (SDF-1/CXCL12) plays an essential role in the homing of hematopoietic stem and progenitor cells (HSPCs) to bone marrow (BM). It is not known whether SDF-1 may also regulate the homing of HSPCs to the liver during extramedullary hematopoiesis (EMH). Here, we investigated the possible role of SDF-1 in attracting HSPCs to the liver during experimental EMH induced by the hematopoietic mobilizers G-CSF, AMD3100 and phenylhydrazine (PHZ). Mice treated with G-CSF, AMD3100 and PHZ showed a significant increase in the expression of SDF-1 in the liver sinusoidal endothelial cells (LSECs) microenvironments. Liver from mice treated with the hematopoietic mobilizers showed HSPCs located adjacent to the LSEC microenvironments, expressing high levels of SDF-1. An inverse relationship was found between the hepatic SDF-1 levels and those in the BM. In vitro, LSEC monolayers induced the migration of HSPCs, and this effect was significantly reduced by AMD3100. In conclusion, our results provide the first evidence showing that SDF-1 expressed by LSEC can be a major player in the recruitment of HSPCs to the liver during EMH induced by hematopoietic mobilizers.
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Quimiocina CXCL12/genética , Quimiocina CXCL12/fisiología , Hematopoyesis Extramedular , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/fisiología , Hígado/citología , Fenilhidrazinas/farmacología , Animales , Bencilaminas , Médula Ósea/química , Médula Ósea/fisiología , Quimiocina CXCL12/sangre , Ciclamas , Femenino , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Compuestos Heterocíclicos/farmacología , Hígado/química , Hígado/efectos de los fármacos , Hígado/fisiología , Ratones Endogámicos C57BLRESUMEN
Liver sinusoid endothelial cells (LSEC) constitute an in vitro and in vivo microenvironment for the proliferation and differentiation of hematopoietic stem cells (HSC). Previously, we have shown that LSEC support the survival and growth of murine embryonic stem cells (ESC). In this study, we investigated the capacity of LSEC to promote hematopoietic differentiation from the murine ESC cell line, CGR8. Undifferentiated ESC were cultured on LSEC monolayers in the absence of exogenous cytokines. After 10 and 20 days, cells were harvested and examined by their morphology, phenotype and capacity of hematopoietic colony formation. Microscopic observation of LSEC/ESC cocultures showed the presence of cobblestone areas formation, which indicates active hematopoiesis. Morphological analysis of cell from these foci showed the presence of hematopoietic cells at different stages of differentiation. Cells expressing B lymphoid markers (B220 and CD19) were detected by flow cytometry, and clonogenic assays showed the formation of CFU-pre B colonies. Similar results were observed when ESC were cultured with LSEC conditioned media. Myeloid precursors were also detected by the presence of CFU-GM colonies and cells expressing myeloid markers. These results indicate that LSEC provided an in vitro microenvironment mainly for B cell development, but also myeloid differentiation from ESC. Coculture of ESC with LSEC may constitute a very powerful tool to study the mechanisms involved in B cell generation from ESC.
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Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/citología , Hígado/citología , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Hígado/metabolismo , Ratones , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismoRESUMEN
Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors and CD34(+) cells were found, at 43 days of co-culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines.
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Diferenciación Celular , Proliferación Celular , Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/metabolismo , Mucosa Olfatoria/citología , Antígenos CD34/genética , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Antígeno CD56/genética , Antígeno CD56/metabolismo , Recuento de Células , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Células Cultivadas , Microambiente Celular , Técnicas de Cocultivo/métodos , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Leucocitos Mononucleares/metabolismo , Linfopoyesis , Células Madre Mesenquimatosas/citología , Mielopoyesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Factores de TiempoRESUMEN
Stromal-derived factor (SDF)-1 is the main regulating factor for trafficking/homing of hematopoietic stem cells (HSC) to the bone marrow (BM). It is possible that this chemokine may also play a fundamental role in regulating the migration of HSC to several organs during extramedullary hematopoiesis. Because liver sinusoidal endothelial cells (LSEC) constitute an extramedullary niche for HSC, it is possible that these cells represent one of the main cellular sources of SDF-1 at the liver. Here, we show that LSEC express SDF-1 at the mRNA and protein level. Biological assays showed that conditioned medium from LSEC (LSEC-CM) stimulated the migration of BM progenitor lineage-negative (BM/Linâ») cells. This effect was significantly reduced by AMD3100, indicating that the SDF-1/CXCR4 axis is involved in the stimulatory migrating effect induced by LSEC-CM. Early localization of HSC in SDF-1-expressing LSEC microenvironment together with increased levels of this chemokine in hepatic homogenates was found in an experimental model of liver extramedullary hematopoiesis. Flow cytometry studies showed that LSEC express the CXCR4 receptor. Functional assays showed that activation of this receptor by SDF-1 stimulated the migration of LSEC and increased the expression of PECAM-1. Our findings suggest that LSEC through the production of SDF-1 may constitute a fundamental niche for regulation of HSC migration to the liver. To our knowledge, this is the first report showing that LSEC not only express and secrete SDF-1, but also its receptor CXCR4.
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Quimiocina CXCL12/metabolismo , Células Endoteliales/metabolismo , Hematopoyesis Extramedular , Células Madre Hematopoyéticas/fisiología , Hígado/citología , Receptores CXCR4/metabolismo , Animales , Adhesión Celular , Movimiento Celular , Células Cultivadas , Quimiocina CXCL12/genética , Medios de Cultivo Condicionados , Femenino , Expresión Génica , Interleucina-6/fisiología , Ratones , Ratones Endogámicos C57BL , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores CXCR4/genética , Factor de Necrosis Tumoral alfa/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Murine embryonic stem cells (muESC) are maintained and expanded in vitro by culturing in the presence of leukemia inhibitory factor (LIF) or by coculturing on murine embryonic fibroblast (MEF). Previously we have shown that liver sinusoidal endothelial cells (LSEC) promote the survival, proliferation and differentiation of hematopoietic stem cells. In the present study we investigated whether LSEC might promote the survival and undifferentiated growth of muESC. For these purposes, muESC (CGR8 cell line) were cultured on LSEC monolayers (muESC/LSEC) or in the presence of conditioned medium from LSEC cultures (muESC/LSEC-CM), both in the absence of LIF. Microscopic observation showed the growth of undifferentiated ESC colonies in both muESC/LSEC or muESC/LSEC-CM cultures. A significant reduction in the growth of undifferentiated ESC colonies was observed when ESC were cultured in LSEC-CM previously incubated with anti-LIF. RT-PCR and Western blot analysis showed that LSEC constitutively express LIF at the mRNA and protein level. At different times of culture, muESC were harvested and analyzed for the expression of embryonic markers (SSEA-1 and Oct-4) and differentiation capacity. Flow cytometry analysis showed the presence of a higher percentage of muESC (>90%) expressing SSEA-1 in muESC/LSEC-CM, as compared with muESC/LSEC cocultures. muESC obtained from both types of cultures formed embryoid bodies in vitro, and form teratomas in testicles of mice. These results provide the first evidence that LSEC support the in vitro survival, self-renewal, undifferentiated growth and differentiation capacity of the muESC CGR8 cell line.
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Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Factor Inhibidor de Leucemia/metabolismo , Hígado/citología , Animales , Anticuerpos Bloqueadores/farmacología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados/farmacología , Cuerpos Embrioides/citología , Cuerpos Embrioides/efectos de los fármacos , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Factor Inhibidor de Leucemia/genética , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , ARN Mensajero/metabolismo , Teratoma/metabolismo , Teratoma/patología , Factores de Transcripción/metabolismoRESUMEN
Las células madres son las unidades naturales a partir de los cuales pueden diferenciarse todos los tipos de células del organismo. El desarrollo de técnicas para el aislamiento, cultivo-expansión y diferenciación de células madres no solo ha permitido avanzar en el conocimiento de la biología de estas células, sino también evaluar su potencial uso en medicina regenerativa. Experimentalmente se ha demostrado que procesos como vasculogénesis, miogénesis, hematopoyesis y neurogénesis pueden generarse a partir de células madres. Sin embargo, no existen evidencias claras, publicadas en revistas biomédicas de alto impacto, que demuestren la efectividad clínica del uso de células madres para regeración de órganos en humanos. El trasplante de médula ósea constituye la única terapia basada en células madres que ha demostrado su efectividad clínica en pacientes. En esta revisión, nosotros discutimos acerca de la biología de las células madres y de las evidencias del uso de estas células en medicina regenerativa.
Stem cells are the natural units, which can differentiate into all cell types. The development of techniques for isolation, culture. expansion and differentiation of stem cells not only has allowed to know about the biology of these cells, but also to evaluate their potential for application in regenerative medicine. Experimentally, it has been shown that process such as vasculogenesis, myogenesis, hematopotesis and neurogenesis can be derived from stem cells. However, there is no clear evidence, published in high impact biomedical journals, demostrating the clinical benefits of stem cells for organ regeneration in humans. Bone marrow transplant constitutes the only clinical therapy based on stem cells with clinical effectiveness. In this review, we discuss biological and therapeutic evidence of stem cells in generative medicine.
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Humanos , Femenino , Embarazo , Células Madre/citología , Medicina Regenerativa/métodos , Biología MolecularRESUMEN
Although there is evidence suggesting that statins may exert an endothelial protecting effect, recent in vitro data have shown that these compounds may induce endothelial cells (EC) apoptosis. We previously reported that the Fas-death receptor may induce apoptosis of the liver sinusoid endothelial cells (LSEC), and that TNF-alpha increases the susceptibility of these cells to suffer Fas-mediated apoptosis. Based on this evidence, in this study, we investigated the effect of simvastatin on Fas-mediated LSEC apoptosis. Simvastatin induced a significant reduction in LSEC viability, in a dose dependent manner, under serum-containing or serum-free conditions. This effect was prevented by mevalonate and GGPP, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. The simvastatin effect on LSEC death was not associated with increased activation of caspase-3. We found that simvastatin increased the susceptibility of LSEC death mediated by Fas. Further, simvastatin increased LSEC-apoptosis induced by Fas and TNF-alpha. Mevalonate and GGPP partially prevented simvastatin-induced sensitization to LSEC death mediated by Jo2 and TNF-alpha, but not Jo2 alone. Simvastatin did not induce up-regulation of the Fas on the LSEC. Our results provide evidence of simvastatin in modulating Fas-mediated apoptosis in endothelial cells. These results may have clinical implications in those clinical conditions associated with high levels of FasL and TNF-alpha.
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Anticolesterolemiantes/farmacología , Apoptosis/efectos de los fármacos , Células Endoteliales , Hígado/citología , Simvastatina/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/metabolismo , Animales , Anticuerpos Antinucleares/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular , Células Cultivadas , Cicloheximida/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Activación Enzimática/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Humanos , Ácido Mevalónico/metabolismo , Ratones , Fosfatos de Poliisoprenilo/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismoRESUMEN
Although the bone marrow (BM) microenvironment is the main inducer niche of early B lymphopoiesis during the adult life, other extramedullar microenvironments, such as the liver, may also have potential for supporting B-cell development. Previously, we reported that murine liver sinusoidal endothelial cells (LSECs) support in vitro and in vivo hematopoietic stem cell (HSC) proliferation and myeloid differentiation. In the present study, we investigated the capacity of LSEC to promote B lymphopoiesis from BM progenitor lineage-negative (Lin(-)) cells. Murine BM Lin(-) cells were co-cultured with LSEC, in the absence of exogenous cytokines. B cells were characterized by flow cytometry and cytokine expression by RT-PCR. We show that BM Lin(-) cells differentiated to early B-lymphoid progenitors (B220(+)) and subsequently to mature (CD19(+)) B cells. Functional studies showed the presence of a high number of non-adherent cells (NACs), collected from lipopolysaccharide (LPS)-treated Lin(-)/LSEC co-cultures, expressing IgM on their surface (sIgM). Colony formation from NAC was observed in the presence of IL-7 (CFU-IL-7). LSEC constitutively express IL-7, Flt-3L, and SCF at the mRNA level, and VCAM-1 on their surface, which may explain the capacity of these cells to promote B lymphopoiesis. These data demonstrate that LSEC promote all stages of B lymphopoiesis. To our knowledge, this is the first report that LSEC constitute an in vitro microenvironment for B lymphopoiesis. Further studies will establish whether LSEC can serve in vivo as a B-lymphopoietic niche under physiological or pathological condition, or when HSC are mobilized.