RESUMEN
BACKGROUND AIMS: End-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tissue (AT), umbilical cord blood (UCB) and bone marrow (BM) would better restore damaged liver. METHODS: AT was obtained from lipo-aspiration, UCB from an Umbilical Cord Blood Bank and BM from a BM Transplantation Unit. AT (collagenase digestion), UCB and BM (Ficoll gradient) were cultured (Dulbecco's modified Eagle's medium, low glucose, FBS) for 3 days. Detached adherent cells, at passage 4, were characterized as MSCs. Genetic stability was investigated by means of telomerase enzyme activity and karyotype. Hepatocyte differentiation protocol was performed with the use of Dulbecco's modified Eagle's medium, hepatocyte growth factor, basic fibroblast growth factor and nicotinamide (7 days); maturation medium (oncostatin, dexamethasone, insulin, transferrin and selenium) was added at 36 days. Hepatogenesis analyses were performed by use of morphology and albumin, AF, tyrosine-aminotransferase and glutamine synthetase gene expression and quantitative reverse transcription-polymerase chain reaction on days 9, 18, 25 and 36. Functionality was assessed through glycogen storage detection, indocyanine green absorption and transplantation procedure. U-MSCs and DHLCs were injected 48 h after induced fulminant hepatitis (intraperitoneal injection of carbon tetrachloride) in SCID/BALB-c mice. Histopathologic analyses were performed on days 7 and 15. Human origin included albumin and CK19 human markers. RESULTS: All MSCs differentiated into functional hepatocyte-like cells, stored glycogen and absorbed indocyanine green. AT-MSC DHLC gene expression was more consistent with a normal hepatogenic-differentiation profile. UCB-MSCs expanded weakly, impairing their use for the transplantation procedure. AT and BM U-MSCs and DHLCs regenerated liver injury equally. Regenerated hepatocytes exhibited human origin. CONCLUSIONS: AT might be the source and U-MSCS the stem cells useful for liver-regenerative therapy.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Hepatitis/terapia , Fallo Hepático Agudo/terapia , Regeneración Hepática/fisiología , Trasplante de Células Madre Mesenquimatosas , Tejido Adiposo/citología , Animales , Biomarcadores , Células de la Médula Ósea/citología , Tetracloruro de Carbono , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Sangre Fetal/citología , Expresión Génica , Glucógeno/metabolismo , Factor de Crecimiento de Hepatocito , Hepatocitos/citología , Hepatocitos/metabolismo , Hepatocitos/trasplante , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos BALB C , Ratones SCIDRESUMEN
We studied the effects of Chlorella vulgaris (CV) on the interaction between stromal and hematopoietic stem cells in normal and Ehrlich ascites tumor (EAT)-bearing mice. Long-term bone marrow culture (LTBMC), cytokine production, spleen mononuclear cells (SMC) proliferation (SCP), colony stimulating activity (CSA), and NK cells activity were evaluated. In tumor bearers, reduced capacity of stromal cell layer to support the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM), concomitantly to decreased numbers of total nonadherent cells in LTBMC and reduced local production of IL-6 and IL-1α, were observed. Presence of the tumor has not altered the number of stromal adherent cells. CV treatment restored the ability of stromal cells from EAT-bearing mice to produce IL-6 and IL-1α, which was consistent with increased number of nonadherent cells and higher ability to display CFU-GM in vitro. EAT growth increased SCP, serum CSA, and IL-10 production and concurrently depressed NK cell activity and the secretion of IL-2, IFN-γ, and TNF-α. Treatment of tumor-bearing mice with CV augmented CSA, SMC proliferation, NK cell activity, and the production of IL-2, IFN-γ, and TNF-α, whereas IL-10 levels where reduced. Our results suggest that CV modulates immunehematopoietic cell activity and disengages tumor-induced suppression of these responses.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Carcinoma de Ehrlich/tratamiento farmacológico , Carcinoma de Ehrlich/inmunología , Chlorella vulgaris , Factores Inmunológicos/uso terapéutico , Mielopoyesis , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Carcinoma de Ehrlich/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Factores Estimulantes de Colonias/sangre , Factores Estimulantes de Colonias/metabolismo , Citocinas/metabolismo , Suplementos Dietéticos , Células Progenitoras de Granulocitos y Macrófagos/citología , Células Progenitoras de Granulocitos y Macrófagos/inmunología , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Fitoterapia , Organismos Libres de Patógenos Específicos , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Células del Estroma/inmunología , Células del Estroma/metabolismoRESUMEN
RATIONALE: several studies demonstrate that hematopoietic tissues are a source of endothelial progenitor cells, which contribute to newly formed blood vessels during tissue repair in adults. However, it is not clear which cell type in these hematopoietic tissues gives rise to endothelial progenitor cells. OBJECTIVE: to identity the origin of endothelial progenitors within the hematopoietic hierarchy and to assess their in vivo revascularization potential. METHODS AND RESULTS: using a single-cell sorting approach and in vitro multilineage differentiation assays, here we show that individual CD34(+)CD45(+)CD133(+)CD38(+) cells from cord blood uniquely have the ability to differentiate into T- and B-lymphoid, myeloid, and endothelial cells. The latter were characterized by the expression of VE-cadherin, KDR, von Willebrand factor, endothelial nitric oxide synthase, the lack of CD45, CD133, and c-fms (colony stimulating factor-1 receptor). Unexpectedly when transplanted into hindlimb ischemic NOD-scid IL2Rγ(null) mice, freshly isolated CD34(+)CD45(+)CD133(+)CD38(+) cells maintained their hematopoietic identity and were rarely found to integrate into host blood vessels. Nevertheless, they significantly improved perfusion, most likely through a paracrine mechanism. On the other hand, CD34(+)CD45(+)CD133(+)CD38(+) cells differentiated in vitro into endothelial cells were able to form vessels in vivo in both Matrigel plug and hindlimb ischemia transplantation assays. CONCLUSIONS: these findings indicate that the CD34(+)CD45(+)CD133(+)CD38(+) cell fraction contains a common progenitor for the hematopoietic and vascular lineages and may represent a valuable cell source for therapeutic applications.
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Células Endoteliales , Sangre Fetal/citología , Células Madre/citología , Antígeno AC133 , ADP-Ribosil Ciclasa 1 , Animales , Antígenos CD , Antígenos CD34 , Diferenciación Celular , Linaje de la Célula , Células Clonales/citología , Células Endoteliales/citología , Glicoproteínas , Humanos , Antígenos Comunes de Leucocito , Células Progenitoras Linfoides/citología , Glicoproteínas de Membrana , Ratones , Ratones SCID , Células Progenitoras Mieloides/citología , Péptidos , Trasplante de Células MadreRESUMEN
The effects of a dry extract of the roots of Angelica sinensis (Oliv.) Diels (ASE) on the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM) in normal and Listeria monocytogenes-infected mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in infected mice. Prophylactic administration of ASE (10, 25, and 50 mg/kg) stimulated marrow myelopoiesis in a dose-dependent manner and reduced spleen colony formation to control values. The dose of 50 mg/kg ASE was the optimal biologically active dose in infected mice, and this dose schedule significantly increased survival of mice infected with a lethal dose of L. monocytogenes, with survival rate up to 30%. Investigation of the production of colony-stimulating factors revealed a dose-dependent increased colony-stimulating activity in the serum of infected mice, with higher response produced by the 50 mg/kg dose. Notably, no effects were observed with the 100 mg/kg dose, compared with infected nontreated controls. Further studies to investigate the production of factors such as inteferon-γ and tumor necrosis factor-α demonstrated increased levels of both cytokines in mice infected with L. monocytogenes and treated with 50 mg/kg ASE. We propose that ASE indirectly modulates immune activity and probably disengages Listeria-induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (colony-stimulating factors, interferon-γ, and tumor necrosis factor-α).
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Medicamentos Herbarios Chinos/uso terapéutico , Factores Inmunológicos/uso terapéutico , Inmunomodulación/efectos de los fármacos , Listeria monocytogenes , Listeriosis/inmunología , Mielopoyesis/efectos de los fármacos , Mielopoyesis/inmunología , Angelica sinensis , Animales , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Factores Estimulantes de Colonias/sangre , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/farmacología , Células Progenitoras de Granulocitos y Macrófagos/efectos de los fármacos , Factores Inmunológicos/farmacología , Interferón gamma/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Listeriosis/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Organismos Libres de Patógenos Específicos , Bazo/citología , Bazo/efectos de los fármacos , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In this study, Chlorella vulgaris (CV) was examined for its chelating effects on the ability of bone marrow stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice, using the long-term bone marrow culture (LTBMC). In addition, the levels of interleukin (IL)-6, an important hematopoietic stimulator, as well as the numbers of adherent and non-adherent cells were also investigated. Mice were gavage treated daily with a single 50mg/kg dose of CV for 10 days, concomitant to continuous offering of 1300ppm lead acetate in drinking water. We found that CV up-modulates the reduced ability of stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice and restores both the reduced number of non-adherent cells and the ability of stromal cells from these mice to produce IL-6. Monitoring of lead poisoning demonstrated that CV treatment significantly reduced lead levels in blood and tissues, completely restored the normal hepatic ALA levels, decreased the abnormally high plasma ALA and partly recovered the liver capacity to produce porphyrins. These findings provide evidence for a beneficial use of CV for combination or alternative chelating therapy to protect the host from the damage induced by lead poisoning.