RESUMEN

Therapeutic approaches requiring the intravenous injection of autologous or allogeneic mesenchymal stromal cells (MSCs) are currently being evaluated for treatment of a range of diseases, including orthopaedic injuries. An alternative approach would be to mobilise endogenous MSCs into the blood, thereby reducing costs and obviating regulatory and technical hurdles associated with development of cell therapies. However, pharmacological tools for MSC mobilisation are currently lacking. Here we show that ß3 adrenergic agonists (ß3AR) in combination with a CXCR4 antagonist, AMD3100/Plerixafor, can mobilise MSCs into the blood in mice and rats. Mechanistically we show that reversal of the CXCL12 gradient across the bone marrow endothelium and local generation of endocannabinoids may both play a role in this process. Using a spine fusion model we provide evidence that this pharmacological strategy for MSC mobilisation enhances bone formation.

RESUMEN

The cellular microenvironment plays a critical role in the maintenance of bone marrow-derived mesenchymal stem cells (BM-MSCs) and their subsequent cell lineage differentiation. Recent studies suggested that individuals with adipocyte-related metabolic disorders have altered function and adipogenic potential of adipose stem cell subpopulations, primarily BM-MSCs, increasing the risk of heart attack, stroke or diabetes. In this study, we explored the potential therapeutic effect of some of the most abundant non-euphoric compounds derived from the Cannabis sativa plant (or phytocannabinoids) including tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabigerol (CBG), cannabidiolic acid (CBDA) and cannabigerolic acid (CBGA), by analysing their pharmacological activity on viability of endogenous BM-MSCs as well as their ability to alter BM-MSC proliferation and differentiation into mature adipocytes. We provide evidence that CBD, CBDA, CBGA and THCV (5 µM) increase the number of viable BM-MSCs; whereas only CBG (5 µM) and CBD (5 µM) alone or in combination promote BM-MSCs maturation into adipocytes via distinct molecular mechanisms. These effects were revealed both in vitro and in vivo. In addition, phytocannabinoids prevented the insulin signalling impairment induced by palmitate in adipocytes differentiated from BM-MSCs. Our study highlights phytocannabinoids as a potential novel pharmacological tool to regain control of functional adipose tissue in unregulated energy homeostasis often occurring in metabolic disorders including type 2 diabetes mellitus (T2DM), aging and lipodystrophy.

Asunto(s)

Adipogénesis/efectos de los fármacos , Cannabinoides/farmacología , Cannabis/química , Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Células de la Médula Ósea/citología , Cannabinoides/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Endocannabinoides/metabolismo , Metabolismo Energético/efectos de los fármacos , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Insulina/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Triglicéridos/metabolismo

RESUMEN

BACKGROUND: The nuclear Peroxisome Proliferator Activated Receptors (PPARs) are ligand-activated transcription factors playing a fundamental role in energy homeostasis and metabolism. Consequently, functional impairment or dysregulation of these receptors lead to a variety of metabolic diseases. While some phytocannabinoids (pCBs) are known to activate PPARγ, no data have been reported so far on their possible activity at PPARα. METHODS: The putative binding modes of pCBs into PPARα/γ Ligand Binding Domains were found and assessed by molecular docking and molecular dynamics. Luciferase assays validated in silico predictions whereas the biological effects of such PPARα/γ ligands were assessed in HepG2 and 3T3L1 cell cultures. RESULTS: The in silico study identified cannabigerolic acid (CBGA), cannabidiolic acid (CBDA) and cannabigerol (CBG) from C. sativa as PPARα/γ dual agonists, suggesting their binding modes toward PPARα/γ isoforms and predicting their activity as full or partial agonists. These predictions were confirmed by luciferase functional assays. The resulting effects on downstream gene transcription in adipocytes and hepatocytes were also observed, establishing their actions as functional dual agonists. CONCLUSIONS: Our work broadens the activity spectrum of CBDA, CBGA and CBG by providing evidence that these pCBs act as dual PPARα/γ agonists with the ability to modulate the lipid metabolism. GENERAL SIGNIFICANCE: Dual PPARα/γ agonists have emerged as an attractive alternative to selective PPAR agonists to treat metabolic disorders. We identified some pCBs as dual PPARα/γ agonists, potentially useful for the treatment of dyslipidemia and type 2 diabetes mellitus.

Asunto(s)

Cannabinoides/análisis , Cannabinoides/aislamiento & purificación , PPAR alfa/agonistas , PPAR gamma/agonistas , Fitoquímicos , Células 3T3-L1 , Animales , Células COS , Cannabinoides/química , Cannabinoides/farmacología , Chlorocebus aethiops , Biología Computacional , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Ratones , Modelos Moleculares , Simulación del Acoplamiento Molecular , PPAR alfa/química , PPAR alfa/metabolismo , PPAR gamma/química , PPAR gamma/metabolismo , Fitoquímicos/análisis , Fitoquímicos/química , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Unión Proteica , Elementos de Respuesta/efectos de los fármacos

RESUMEN

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)-a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin-is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus, a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea. Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

Asunto(s)

Factores Biológicos/farmacología , Caulerpa/metabolismo , Enfermedades de los Peces/etiología , Indoles/toxicidad , Especies Introducidas , Perciformes/fisiología , Receptores Activados del Proliferador del Peroxisoma/agonistas , Animales , Factores Biológicos/metabolismo , Simulación por Computador , Ecotoxicología , Enfermedades de los Peces/metabolismo , Cadena Alimentaria , Indoles/metabolismo , Ligandos , Modelos Biológicos , Receptores Activados del Proliferador del Peroxisoma/metabolismo

RESUMEN

Regenerative medicine using mesenchymal stem cells for the purposes of tissue repair has garnered considerable public attention due to the potential of returning tissues and organs to a normal, healthy state after injury or damage has occurred. To achieve this, progenitor cells such as pericytes and bone marrow-derived mesenchymal stem cells can be delivered exogenously, mobilised and recruited from within the body or transplanted in the form organs and tissues grown in the laboratory from stem cells. In this review, we summarise the recent evidence supporting the use of endogenously mobilised stem cell populations to enhance tissue repair along with the use of mesenchymal stem cells and pericytes in the development of engineered tissues. Finally, we conclude with an overview of currently available therapeutic options to manipulate endogenous stem cells to promote tissue repair.

Asunto(s)

Células Madre Mesenquimatosas/fisiología , Pericitos/fisiología , Regeneración , Ingeniería de Tejidos , Animales , Movimiento Celular , Fibrosis/patología , Fibrosis/terapia , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/efectos de los fármacos , Neovascularización Fisiológica , Pericitos/efectos de los fármacos , Pericitos/trasplante

RESUMEN

Methods for lineage tracing of stem cell progeny in human tissues are currently not available. We describe a technique for detecting the expansion of a single cell's progeny that contain clonal mitochondrial DNA (mtDNA) mutations affecting the expression of mtDNA-encoded cytochrome c oxidase (COX). Because such mutations take up to 40 years to become phenotypically apparent, we believe these clonal patches originate in stem cells. Dual-color enzyme histochemistry was used to identify COX-deficient cells, and mutations were confirmed by microdissection of single cells with polymerase chain reaction sequencing of the entire mtDNA genome. These techniques have been applied to human intestine, liver, pancreas, and skin. Our results suggest that the stem cell niche is located at the base of colonic crypts and above the Paneth cell region in the small intestine, in accord with dynamic cell kinetic studies in animals. In the pancreas, exocrine tissue progenitors appeared to be located in or close to interlobular ducts, and, in the liver, we propose that stem cells are located in the periportal region. In the skin, the origin of a basal cell carcinoma appeared to be from the outer root sheath of the hair follicle. We propose that this is a general method for detecting clonal cell populations from which the location of the niche can be inferred, also affording the generation of cell fate maps, all in human tissues. In addition, the technique allows analysis of the origin of human tumors from specific tissue sites.

Asunto(s)

Linaje de la Célula , ADN Mitocondrial/genética , Células Epiteliales/citología , Células Clonales , Complejo IV de Transporte de Electrones/genética , Humanos , Inmunohistoquímica , Mutación , Nicho de Células Madre/citología

RESUMEN

Markers of gastrointestinal (GI) stem cells remain elusive. We employed synchrotron Fourier-transform infrared (FTIR) microspectroscopy to derive mid-infrared (IR) spectra along the length of human GI crypts. Tissue sections (10-µm thick) were floated onto BaF2 windows and image maps were acquired of small intestine and large bowel crypts in transmission mode with an aperture of ≤10 µm×10 µm. Counting upwards in a step-size (≤10 µm) fashion from the crypt base, IR spectra were extracted from the image maps and each spectrum corresponding to a particular location was identified. Spectra were analyzed using principal component analysis plus linear discriminant analysis. Compared to putative crypt base columnar/Paneth cells, those assigned as label-retaining cells were chemically more similar to putative large bowel stem cells and, the small intestine transit-amplifying cells were closest to large bowel transit-amplifying cells; interestingly, the base of small intestine crypts was the most chemically-distinct. This study suggests that in the complex cell lineage of human GI crypts, chemical similarities as revealed by FTIR microspectroscopy between regions putatively assigned as stem cell, transit-amplifying and terminally-differentiated facilitates identification of cell function.

Asunto(s)

Intestino Grueso/citología , Intestino Delgado/citología , Espectroscopía Infrarroja por Transformada de Fourier , ADN/química , Análisis Discriminante , Humanos , Intestino Grueso/química , Intestino Delgado/química , Modelos Biológicos , Análisis de Componente Principal , ARN/química , Células Madre/química , Células Madre/citología , Sincrotrones

RESUMEN

UNLABELLED: We have used immunohistochemical and histochemical techniques to identify patches of hepatocytes deficient in the enzyme cytochrome c oxidase, a component of the electron transport chain and encoded by mitochondrial DNA (mtDNA). These patches invariably abutted the portal tracts and expanded laterally as they spread toward the hepatic veins. Here we investigate, using mtDNA mutations as a marker of clonal expansion, the clonality of these patches. Negative hepatocytes were laser-capture microdissected and mutations identified by polymerase chain reaction sequencing of the entire mtDNA genome. Patches of cytochrome c oxidase-deficient hepatocytes were clonal, suggesting an origin from a long-lived cell, presumably a stem cell. Immunohistochemical analysis of function and proliferation suggested that these mutations in cytochrome c oxidase-deficient hepatocytes were nonpathogenic. CONCLUSION: These data show, for the first time, that clonal proliferative units exist in the human liver, an origin from a periportal niche is most likely, and that the trajectory of the units is compatible with a migration of cells from the periportal regions to the hepatic veins.

Asunto(s)

Linaje de la Célula , Complejo IV de Transporte de Electrones/metabolismo , Hepatocitos/enzimología , Hígado/citología , Nicho de Células Madre/citología , Análisis Mutacional de ADN , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Humanos , Inmunohistoquímica

RESUMEN

Complex biomolecules absorb in the mid-infrared (lambda = 2-20 microm), giving vibrational spectra associated with structure and function. We used Fourier transform infrared (FTIR) microspectroscopy to "fingerprint" locations along the length of human small and large intestinal crypts. Paraffin-embedded slices of normal human gut were sectioned (10 microm thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected using globar (15 microm x 15 microm aperture) FTIR microspectroscopy in reflection mode, synchrotron (

Asunto(s)

Biomarcadores/análisis , Mucosa Intestinal/citología , Espectroscopía Infrarroja por Transformada de Fourier , Células Madre/citología , Humanos , Inmunohistoquímica , Análisis de Componente Principal

RESUMEN

In the twenty-first century, diabetic patients are likely to be one of the major beneficiaries from the advancement of regenerative medicine through cellular therapies. Though the existence of a specific self-renewing stem cell within the pancreas is still far from clear, a surprising variety of cells within the pancreas can differentiate towards a beta-cell phenotype: ductular cells, periductular mesenchymal cells and beta-cells themselves can all give rise to new beta-cells. Extra-pancreatic adult somatic stem cells, in particular, those originating from bone marrow may also be capable of differentiating to beta-cells, though equally well the beneficial effects of bone marrow cells may reside in their contribution to the damaged islet vasculature. Forced expression of the beta-cell-specific transcription factor Pdx1 in hepatocytes also holds promise as a therapeutic strategy to increase insulin levels in diabetic individuals. Embryonic stem (ES) cells are clearly another possible source for generating beta-cells, but ES cells are beyond the scope of this review, which focuses on adult stem and progenitor cells capable of producing beta-cells. Despite considerable endeavour, we still have much to learn in the field of pancreatic regeneration prior to any clinically applicable therapy based upon adult stem cells.

Asunto(s)

Diabetes Mellitus Tipo 1/tratamiento farmacológico , Células Secretoras de Insulina/trasplante , Trasplante de Islotes Pancreáticos/métodos , Animales , Células Epiteliales/fisiología , Humanos , Mesodermo/fisiología , Páncreas/citología , Trasplante de Células Madre

RESUMEN

RATIONALE: The participation of circulating precursor cells in the development of experimental pulmonary fibrosing lesions in mice has been recently demonstrated. OBJECTIVES: This study analyzes whether circulating, bone marrow-derived, fibroblastic precursor cells contribute to the development of fibrosing lesions in human lungs, especially bronchiolitis obliterans. METHODS: The occurrence of in situ microchimerism in bronchiolitis obliterans lesions of human lung allografts (n = 12) as well as of autologous lung tissue from patients post-bone marrow transplantation (n = 2) was analyzed using laser-assisted microdissection after immunohistochemical labeling of leukocytes followed by short tandem repeat-polymerase chain reaction-based genotyping. Combined immunofluorescence and fluorescence in situ hybridization for sex chromosomes was performed for independent confirmation in cases with appropriate sex mismatch (n = 2). MEASUREMENTS AND MAIN RESULTS: The bronchiolitis obliterans lesions of all 12 lung transplant patients contained considerable numbers of recipient-derived fibroblasts (mean, 32%). The fibrosing pulmonary lesions of the two bone marrow-transplanted patients also displayed clear in situ microchimerism. The in situ detection methodology confirmed these results, although to a lower degree (6-16%). CONCLUSIONS: These data clearly demonstrate the involvement of circulating fibroblastic precursor cells in the development of human fibrosing lung lesions and provide evidence that these cells are most probably bone marrow derived. These results may open new venues regarding the prevention of fibrosis in lung transplants and potentially in other organs.

Asunto(s)

Trasplante de Médula Ósea/efectos adversos , Bronquiolitis Obliterante/patología , Rechazo de Injerto/patología , Trasplante de Pulmón/efectos adversos , Fibrosis Pulmonar/patología , Quimera por Trasplante/genética , Adulto , Trasplante de Médula Ósea/inmunología , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/inmunología , Niño , Femenino , Fibroblastos/inmunología , Rechazo de Injerto/genética , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Trasplante de Pulmón/inmunología , Masculino , Donantes de Tejidos , Trasplante Homólogo

RESUMEN

RATIONALE: The participation of circulating precursor cells in the development of experimental pulmonary fibrosing lesions in mice has been recently demonstrated. OBJECTIVES: This study analyzes whether circulating, bone marrow-derived fibroblastic precursor cells contribute to the development of fibrosing lesions in human lungs, especially bronchiolitis obliterans. METHODS: The occurrence of in situ-microchimerism in bronchiolitis obliterans lesions of human lung allografts (n = 12) as well as of autologous lung tissue from patients post bone marrow-transplantation (n = 2) was analyzed using laser-assisted microdissection after immunohistochemical labeling of leukocytes followed by STR-PCR-based genotyping. Combined immunofluorescence and fluorescence in situ hybridization for sex chromsomes was performed for independent confirmation in cases with appropriate sex mismatch (n = 2). MEASUREMENTS AND MAIN RESULTS: The bronchiolitis obliterans lesions of all twelve lung transplant patients contained considerable numbers of recipient-derived fibroblasts (mean: 32 %). The fibrosing pulmonary lesions of the two bone marrow-transplanted patients displayed also clear in situ-microchimerism. The in situ detection methodology confirmed these results, although to a lower degree (6-16%). CONCLUSIONS: These data clearly demonstrate the involvement of circulating fibroblastic precursor cells in the development of human fibrosing lung lesions and provide evidence that these cells are most probably bone marrow-derived. These results may open new venues regarding the prevention of fibrosis in lung transplants and potentially other organs.

Asunto(s)

Trasplante de Médula Ósea/efectos adversos , Bronquiolitis Obliterante/patología , Fibroblastos/trasplante , Trasplante de Pulmón/patología , Biopsia , Bronquiolitis Obliterante/etiología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Quimera por Trasplante