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BACKGROUND AIMS: In 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards. METHODS: Twelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures. RESULTS: Excellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells. CONCLUSIONS: The current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.
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Antígenos CD34/análisis , Conservación de la Sangre/métodos , Sangre Fetal/citología , Antígenos Comunes de Leucocito/análisis , Células Madre/citología , Bioensayo , Almacenamiento de Sangre/métodos , Recuento de Células , Ensayo de Unidades Formadoras de Colonias , Criopreservación/métodos , Citometría de Flujo/métodos , HumanosRESUMEN
Cryopreservation is the most common method for long-term cell storage. Successful cryopreservation of cells depends on optimal freezing conditions, freezer storage and a proper thawing technique to minimize the cellular damage that can occur during the cryopreservation process. These factors are especially critical for sensitive stem cells with a consequential and significant impact on viability and functionality. Until now, slow-freezing has been the routine method of cryopreservation but, more recently rapid-cooling techniques have also been proposed. In this study, an ultra-rapid cooling technique [1] was performed for the first time on human mesenchymal stem cells and the effectiveness evaluated in comparison with the conventional slow-freezing procedure. A thin nylon-membrane carrier was used combined with different cryoprotective agents: dimethyl sulfoxide, ethylene glycol and/or trehalose. Various aspects of the low cryoprotective doses and the ultra-rapid cooling procedure of the human mesenchymal stem cells were examined including: the physical properties of the nylon-support, cells encumbrance, viability, proliferation and differentiation. The expression of cell surface markers and apoptosis were also investigated. The study used an ultra-rapid cooling/warming method and showed an overall cell integrity preservation (83-99%), with no significant differences between dimethyl sulfoxide or ethylene glycol treatment (83-87%) and a substantial cell viability of 68% and 51%, respectively. We confirmed a discrepancy also observed by other authors in cell viability and integrity, which implies that caution is necessary when assessing and reporting cell viability data.
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Criopreservación/métodos , Células Madre Mesenquimatosas/citología , Apoptosis , Supervivencia Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/metabolismo , TiempoRESUMEN
The adrenal gland is a multiendocrine organ with a steroidogenic mesenchymal cortex and an inner catecholamine-producing medulla of neuroendocrine origin. After embryonic development, this plastic organ undergoes a functional postnatal remodeling. Elucidating these complex processes is pivotal for understanding the early bases of functional endocrine disorders and tumors affecting the mature gland. We developed an in vitro human adrenal cell model derived from fetal adrenal specimens at different gestational ages, consisting of neuroendocrine and cortical components and expressing the zona and functional markers of the original fetal organ. These cortical and neuroendocrine progenitor cells retain in vitro an intrinsic gestational-age-related differentiation and functional program. In vitro these cells spontaneously form 3-dimensional structure organoids with a structure similar to the fetal gland. The organoids show morphofunctional features and adrenal steroidogenic factor, steroid acute regulatory, cytochrome-P450-17A1, dosage-sensitive, sex-reversal, adrenal hypoplasia-critical region on chromosome X protein , NOTCH1, and nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3; stem (BMI1, nestin); and chromaffin (chromogranin A, tyrosine hydroxylase) markers similar to those of the populations of origin. This in vitro human adrenal system represents a unique but preliminar model for investigating the pathophysiological processes underlying physiologic adrenal remodeling and pathologic alterations involved in organ hypo- and hyperplasia and cancer.-Poli, G., Sarchielli, E., Guasti, D., Benvenuti, S., Ballerini, L., Mazzanti, B., Armignacco, R., Cantini, G., Lulli, M., Chortis, V., Arlt, W., Romagnoli, P., Vannelli, G. B., Mannelli, M., Luconi, M. Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.
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Glándulas Suprarrenales/citología , Diferenciación Celular , Senescencia Celular , Feto/citología , HumanosRESUMEN
PURPOSE: GLP-1 receptor agonists are antidiabetic drugs currently used in the therapy of type 2 diabetes. Despite several in vitro and in vivo animal studies suggesting a beneficial effect of GLP-1 analogues on bone, in humans their skeletal effects are not clear and clinical studies report conflicting results. METHODS: We differentiated human mesenchymal stromal cells (hMSC) toward the adipogenic and the osteoblastic lineages, analysing the effect of Exendin-4 (EXE) before, during and after specific differentiations. RESULTS: We showed EXE ability to act selectively on a sub-population of hMSC characterised by a more stem potential, shifting them from G1 to S/M phase of cell cycle. We observed that EXE pre-treatment promotes both adipogenic and osteoblastic differentiations, possibly determined by an increased number of uncommitted progenitors. In fully differentiated cells, EXE affects mature adipocytes by increasing lipolysis, otherwise not altering osteoblasts metabolic activity. Moreover, the increased expression of osteoprotegerin, a modulator of the RANK/RANKL system, observed during osteogenic induction in presence of EXE, could negatively modulate osteoclastogenesis. CONCLUSIONS: Our data suggest a complex action of EXE on bone, targeting the proliferation of mesenchymal progenitors, the metabolism of mature adipocytes and the modulation of osteoclastogenesis. Thus, an overall positive effect of this molecule on bone quality might be hypothesised.
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Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Exenatida/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Humanos , Lipólisis/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteoprotegerina/metabolismoRESUMEN
The degeneration of cholinergic neurons of the nucleus basalis of Meynert (NBM) in the basal forebrain (BF) is associated to the cognitive decline of Alzheimer's disease (AD) patients. To date no resolutive therapies exist. Cell-based replacement therapy is a strategy currently under consideration, although the mechanisms underlying the generation of stem cell-derived NBM cholinergic neurons able of functional integration remain to be clarified. Since fetal brain is an optimal source of neuronal cells committed towards a specific phenotype, this study is aimed at isolating cholinergic neurons from the human fetal NBM (hfNBMs) in order to study their phenotypic, maturational and functional properties. Extensive characterization confirmed the cholinergic identity of hfNBMs, including positivity for specific markers (such as choline acetyltransferase) and acetylcholine (Ach) release. Electrophysiological measurements provided the functional validation of hfNBM cells, which exhibited the activation of peculiar sodium (INa) and potassium (IK) currents, as well as the presence of functional cholinergic receptors. Accordingly, hfNBMs express both nicotinic and muscarinic receptors, which were activated by Ach. The hfNBMs cholinergic phenotype was regulated by the nerve growth factor (NGF), through the activation of the high-affinity NGF receptor TrkA, as well as by 17-ß-estradiol through a peculiar recruitment of its own receptors. When intravenously administered in NBM-lesioned rats, hfNBMs determined a significant improvement in memory functions. Histological examination of brain sections showed that hfNBMs (labeled with PKH26 fluorescent dye prior to administration) reached the damaged brain areas. The study provides a useful model to study the ontogenetic mechanisms regulating the development and maintenance of the human brain cholinergic system and to assess new lines of research, including disease modeling, drug discovery and cell-based therapy for AD.
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Preservation of implant biocompatibility following peri-implantitis treatments is a crucial issue in odontostomatological practice, being closely linked to implant re-osseointegration. Our aim was to assess the responses of osteoblast-like Saos2 cells and adult human bone marrow-mesenchymal stromal cells (MSCs) to oxidized titanium surfaces (TiUnite®, TiU) pre-treated with a 808 ± 10 nm GaAlAs diode laser operating in non-contact mode, in continuous (2 W, 400 J/cm2; CW) or pulsed (20 kHz, 7 µs, 0.44 W, 88 J/cm2; PW) wave, previously demonstrated to have a strong bactericidal effect and proposed as optional treatment for peri-implantitis. The biocompatibility of TiU surfaces pre-treated with chlorhexidine digluconate (CHX) was also evaluated. In particular, in order to mimic the in vivo approach, TiU surfaces were pre-treated with CHX (0.2%, 5 min); CHX and rinse; and CHX, rinse and air drying. In some experiments, the cells were cultured on untreated TiU before being exposed to CHX. Cell viability (MTS assay), proliferation (EdU incorporation assay; Ki67 confocal immunofluorescence analysis), adhesion (morphological analysis of actin cytoskeleton organization), and osteogenic differentiation (osteopontin confocal immunofluorescence analysis; mineralized bone-like nodule formation) analyses were performed. CHX resulted cytotoxic in all experimental conditions. Diode laser irradiation preserved TiU surface biocompatibility. Notably, laser treatment appeared even to improve the known osteoconductive properties of TiU surfaces. Within the limitations of an in vitro experimentation, this study contributes to provide additional experimental basis to support the potential use of 808 ± 10 nm GaAlAs diode laser at the indicated irradiation setting, in the treatment of peri-implantitis and to discourage the use of CHX.
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Clorhexidina/farmacología , Láseres de Semiconductores , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Titanio/farmacología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/efectos de la radiación , Fluorescencia , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de la radiación , Osteoblastos/efectos de los fármacos , Osteoblastos/efectos de la radiación , Osteogénesis/efectos de los fármacos , Osteogénesis/efectos de la radiación , Propiedades de SuperficieRESUMEN
BACKGROUND: Clinical grade processing of harvested bone marrow is required in various clinical situations, particularly in the management of ABO mismatching in allogeneic haematopoietic stem cell transplantation (HSCT) and in regenerative medicine. MATERIAL AND METHODS: We report a single-centre experience using a fully automated, clinical grade, closed system (Sepax, Biosafe, Switzerland). From 2003 to 2015, 125 procedures were performed in our laboratory, including buffy-coat production for HSCT (n=58), regenerative medicine in an orthopaedic setting (n=54) and density-gradient separation in a trial for treatment of critical limb ischaemia (n=13). RESULTS: Buffy coat separation resulted in a median volume reduction of 85% (range, 75-87%), providing satisfactory red blood cell depletion (69%, range 30-88%) and a median recovery of CD34 cells of 96% (range, 81-134%) in the setting of allogeneic HSCT. Significantly greater volume reduction (90%; range, 90-92%) and red blood cell depletion (88%; range, 80-93%) were achieved by the new SmartRedux software released for Sepax2, validated in the last eight allogeneic HSCT. The density gradient separation programme resulted in complete red blood cell depletion associated with high CD34 recovery (69%; range, 36-124%). No reactions related to the quality of the product were reported. Time to engraftment following allogeneic HSCT was in the normal range. No cases of microbiological contamination related to the manipulation were reported. DISCUSSION: Clinical grade, automated bone marrow manipulation with Sepax was shown to be effective, giving operator-independent results and could be used for a broad range of clinical applications.
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Separación Celular/métodos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células de la Médula Ósea/citología , Separación Celular/instrumentación , Extremidades/irrigación sanguínea , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Isquemia/terapia , Medicina Regenerativa , Programas Informáticos , Trasplante Homólogo/métodosRESUMEN
Context: Inflammatory pathways may impair central regulatory networks involving gonadotropin-releasing hormone (GnRH) neuron activity. Studies in humans are limited by the lack of human GnRH neuron cell lines. Objective: To establish an in vitro model of human GnRH neurons and analyze the effects of proinflammatory cytokines. Design: The primary human fetal hypothalamic cells (hfHypo) were isolated from 12-week-old fetuses. Responsiveness to kisspeptin, the main GnRH neurons' physiological regulator, was evaluated for biological characterization. Tumor necrosis factor alpha (TNF-α) was used as a proinflammatory stimulus. Main Outcome Measures: Expression of specific GnRH neuron markers by quantitative reverse transcription-polymerase chain reaction, flow cytometry, and immunocytochemistry analyses; and GnRH-releasing ability and electrophysiological changes in response to kisspeptin. Results: The primary hfHypo showed a high percentage of GnRH-positive cells (80%), expressing a functional kisspeptin receptor (KISS1R) and able to release GnRH in response to kisspeptin. TNF-α exposure determined a specific inflammatory intracellular signaling and reduced GnRH secretion, KISS1R expression, and kisspeptin-induced depolarizing effect. Moreover, hfHypo possessed a primary cilium, whose assembly was inhibited by TNF-α. Conclusion: The hfHypo cells represent a novel tool for investigations on human GnRH neuron biology. TNF-α directly affects GnRH neuron function by interfering with KISS1R expression and ciliogenesis, thereby impairing kisspeptin signaling.
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Feto/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/metabolismo , Kisspeptinas/metabolismo , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células Cultivadas , Femenino , Feto/citología , Feto/efectos de los fármacos , Humanos , Kisspeptinas/genética , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Anal incontinence is a disabling condition that adversely affects the quality of life of a large number of patients, mainly with anal sphincter lesions. In a previous experimental work, in-vitro expanded bone marrow (BM)-derived mesenchymal stem cells (MSC) were demonstrated to enhance sphincter healing after injury and primary repair in a rat preclinical model. In the present article we investigated whether unexpanded BM mononuclear cells (MNC) may also be effective. METHODS: Thirty-two rats, divided into groups, underwent sphincterotomy and repair (SR) with primary suture of anal sphincters plus intrasphincteric injection of saline (CTR), or of in-vitro expanded MSC, or of minimally manipulated MNC; moreover, the fourth group underwent sham operation. At day 30, histologic, morphometric, in-vitro contractility, and functional analysis were performed. RESULTS: Treatment with both MSC and MNC improved muscle regeneration and increased contractile function of anal sphincters after SR compared with CTR (p < 0.05). No significant difference was observed between the two BM stem cell types used. GFP-positive cells (MSC and MNC) remained in the proximity of the lesion site up to 30 days post injection. CONCLUSIONS: In the present study we demonstrated in a preclinical model that minimally manipulated BM-MNC were as effective as in-vitro expanded MSC for the recovery of anal sphincter injury followed by primary sphincter repair. These results may serve as a basis for improving clinical applications of stem cell therapy in human anal incontinence treatment.
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Canal Anal/cirugía , Células de la Médula Ósea/citología , Incontinencia Fecal/terapia , Leucocitos Mononucleares/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Canal Anal/lesiones , Animales , Células de la Médula Ósea/fisiología , Modelos Animales de Enfermedad , Incontinencia Fecal/fisiopatología , Genes Reporteros , Humanos , Leucocitos Mononucleares/fisiología , Leucocitos Mononucleares/trasplante , Masculino , Células Madre Mesenquimatosas/fisiología , Contracción Muscular/fisiología , Ratas , Ratas Endogámicas Lew , Regeneración/fisiología , Esfinterotomía EndoscópicaRESUMEN
Grafting fetal striatal cells into the brain of Huntington's disease (HD) patients has raised certain expectations in the past decade as an effective cell-based-therapy for this devastating condition. We argue that the first requirement for successful transplantation is defining the window of plasticity for the striatum during development when the progenitor cells, isolated from their environment, are able to maintain regional-specific-identity and to respond appropriately to cues. The primary cell culture from human fetal striatal primordium described here consists of a mixed population of neural stem cells, neuronal-restricted progenitors and striatal neurons. These cells express trophic factors, such as BDNF and FGF2. We show that these neurotrophins maintain cell plasticity, inducing the expression of neuronal precursor markers and cell adhesion molecules, as well as promoting neurogenesis, migration and survival. We propose that BDNF and FGF2 play an important autocrine-paracrine role during early striatum development in vivo and that their release by fetal striatal grafts may be relevant in the setting of HD cell therapy.
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Factor Neurotrófico Derivado del Encéfalo/farmacología , Moléculas de Adhesión Celular/metabolismo , Cuerpo Estriado/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células-Madre Neurales/efectos de los fármacos , Antígenos/genética , Antígenos/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Moléculas de Adhesión Celular/genética , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Feto , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuritas/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Antígenos O/genética , Antígenos O/metabolismo , Proteoglicanos/genética , Proteoglicanos/metabolismoRESUMEN
In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.
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Albuminuria/complicaciones , Podocitos/fisiología , Regeneración , Tretinoina/metabolismo , Albuminuria/patología , Animales , Células Cultivadas , Femenino , Glomeruloesclerosis Focal y Segmentaria/etiología , Humanos , Ratones , Ratones SCID , Elementos de Respuesta/fisiología , Tretinoina/farmacologíaRESUMEN
Interferon (IFN)-α and IFN-ß are the central regulators of antiviral immunity but little is known about their roles in viral glomerulonephritis (eg, HIV nephropathy). We hypothesized that IFN-α and IFN-ß would trigger local inflammation and podocyte loss. We found that both IFNs consistently activated human and mouse podocytes and parietal epithelial cells to express numerous IFN-stimulated genes. However, only IFN-ß significantly induced podocyte death and increased the permeability of podocyte monolayers. In contrast, only IFN-α caused cell-cycle arrest and inhibited the migration of parietal epithelial cells. Both IFNs suppressed renal progenitor differentiation into mature podocytes. In Adriamycin nephropathy, injections with either IFN-α or IFN-ß aggravated proteinuria, macrophage influx, and glomerulosclerosis. A detailed analysis showed that only IFN-ß induced podocyte mitosis. This did not, however, lead to proliferation, but was associated with podocyte loss via podocyte detachment and/or mitotic podocyte death (mitotic catastrophe). We did not detect TUNEL-positive podocytes. Thus, IFN-α and IFN-ß have both common and differential effects on podocytes and parietal epithelial cells, which together promote glomerulosclerosis by enhancing podocyte loss while suppressing podocyte regeneration from local progenitors.
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Antivirales/farmacología , Glomerulonefritis/tratamiento farmacológico , Interferón-alfa/farmacología , Interferón beta/farmacología , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Doxorrubicina/toxicidad , Células Epiteliales/efectos de los fármacos , Femenino , Glomerulonefritis/fisiopatología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/fisiopatología , Humanos , Glomérulos Renales/fisiología , Ratones , Ratones SCID , Podocitos/efectos de los fármacos , Regeneración/efectos de los fármacosRESUMEN
Recent studies implicated the existence in adult human kidney of a population of renal progenitors with the potential to regenerate glomerular as well as tubular epithelial cells and characterized by coexpression of surface markers CD133 and CD24. Here, we demonstrate that CD133+CD24+ renal progenitors can be distinguished in distinct subpopulations from normal human kidneys based on the surface expression of vascular cell adhesion molecule 1, also known as CD106. CD133+CD24+CD106+ cells were localized at the urinary pole of Bowman's capsule, while a distinct population of scattered CD133+CD24+CD106- cells was localized in the proximal tubule as well as in the distal convoluted tubule. CD133+CD24+CD106+ cells exhibited a high proliferative rate and could differentiate toward the podocyte as well as the tubular lineage. By contrast, CD133+CD24+CD106- cells showed a lower proliferative capacity and displayed a committed phenotype toward the tubular lineage. Both CD133+CD24+CD106+ and CD133+CD24+CD106- cells showed higher resistance to injurious agents in comparison to all other differentiated cells of the kidney. Once injected in SCID mice affected by acute tubular injury, both of these populations displayed the capacity to engraft within the kidney, generate novel tubular cells, and improve renal function. These properties were not shared by other tubular cells of the adult kidney. Finally, CD133+CD24+CD106- cells proliferated upon tubular injury, becoming the predominating part of the regenerating epithelium in patients with acute or chronic tubular damage. These data suggest that CD133+CD24+CD106- cells represent tubular-committed progenitors that display resistance to apoptotic stimuli and exert regenerative potential for injured tubular tissue.
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Lesión Renal Aguda/patología , Necrosis Tubular Aguda/patología , Túbulos Renales Proximales/citología , Riñón/citología , Células Madre/citología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedades Renales/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones , Ratones SCID , Microscopía Confocal , Regeneración/fisiología , Células Madre/metabolismo , Trasplante HeterólogoRESUMEN
Glomerular diseases account for 90% of end-stage kidney disease. Podocyte loss is a common determining factor for the progression toward glomerulosclerosis. Mature podocytes cannot proliferate, but recent evidence suggests that they can be replaced by renal progenitors localized within the Bowman's capsule. Here, we demonstrate that Notch activation in human renal progenitors stimulates entry into the S-phase of the cell cycle and cell division, whereas its downregulation is required for differentiation toward the podocyte lineage. Indeed, a persistent activation of the Notch pathway induced podocytes to cross the G(2)/M checkpoint, resulting in cytoskeleton disruption and death by mitotic catastrophe. Notch expression was virtually absent in the glomeruli of healthy adult kidneys, while a strong upregulation was observed in renal progenitors and podocytes in patients affected by glomerular disorders. Accordingly, inhibition of the Notch pathway in mouse models of focal segmental glomerulosclerosis ameliorated proteinuria and reduced podocyte loss during the initial phases of glomerular injury, while inducing reduction of progenitor proliferation during the regenerative phases of glomerular injury with worsening of proteinuria and glomerulosclerosis. Taken altogether, these results suggest that the severity of glomerular disorders depends on the Notch-regulated balance between podocyte death and regeneration provided by renal progenitors.
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Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Nefritis Lúpica/metabolismo , Podocitos/metabolismo , Receptores Notch/metabolismo , Células Madre/metabolismo , Animales , Estudios de Casos y Controles , Ciclo Celular , Muerte Celular , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Doxorrubicina , Femenino , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Nefritis Lúpica/patología , Ratones , Ratones SCID , Podocitos/efectos de los fármacos , Podocitos/patología , Proteinuria/metabolismo , Proteinuria/patología , Receptores Notch/antagonistas & inhibidores , Receptores Notch/genética , Índice de Severidad de la Enfermedad , Células Madre/efectos de los fármacos , Células Madre/patología , Factores de Tiempo , TransfecciónRESUMEN
Glomerular injury can involve excessive proliferation of glomerular epithelial cells, resulting in crescent formation and obliteration of Bowman's space. The origin of these hyperplastic epithelial cells in different glomerular disorders is controversial. Renal progenitors localized to the inner surface of Bowman's capsule can regenerate podocytes, but whether dysregulated proliferation of these progenitors contributes to crescent formation is unknown. In this study, we used confocal microscopy, laser capture microdissection, and real-time quantitative reverse transcriptase-PCR to demonstrate that hypercellular lesions of different podocytopathies and crescentic glomerulonephritis consist of three distinct populations: CD133(+)CD24(+)podocalyxin (PDX)(-)nestin(-) renal progenitors, CD133(+)CD24(+)PDX(+)nestin(+) transitional cells, and CD133(-)CD24(-)PDX(+)nestin(+) differentiated podocytes. In addition, TGF-beta induced CD133(+)CD24(+) progenitors to produce extracellular matrix, and these were the only cells to express the proliferation marker Ki67. Taken together, these results suggest that glomerular hyperplastic lesions derive from the proliferation of renal progenitors at different stages of their differentiation toward mature podocytes, providing an explanation for the pathogenesis of hyperplastic lesions in podocytopathies and crescentic glomerulonephritis.
Asunto(s)
Células Madre Adultas/patología , Glomerulonefritis/patología , Glomérulos Renales/patología , Podocitos/patología , Antígeno AC133 , Células Madre Adultas/clasificación , Células Madre Adultas/metabolismo , Antígenos CD/metabolismo , Cápsula Glomerular/metabolismo , Cápsula Glomerular/patología , Antígeno CD24/metabolismo , Diferenciación Celular , Proliferación Celular , Matriz Extracelular/patología , Glomerulonefritis/clasificación , Glomerulonefritis/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Glicoproteínas/metabolismo , Humanos , Hiperplasia , Proteínas de Filamentos Intermediarios/metabolismo , Glomérulos Renales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Péptidos/metabolismo , Fenotipo , Podocitos/clasificación , Podocitos/metabolismo , Sialoglicoproteínas/metabolismoRESUMEN
Depletion of podocytes, common to glomerular diseases in general, plays a role in the pathogenesis of glomerulosclerosis. Whether podocyte injury in adulthood can be repaired has not been established. Here, we demonstrate that in the adult human kidney, CD133+CD24+ cells consist of a hierarchical population of progenitors that are arranged in a precise sequence within Bowman's capsule and exhibit heterogeneous potential for differentiation and regeneration. Cells localized to the urinary pole that expressed CD133 and CD24, but not podocyte markers (CD133+CD24+PDX- cells), could regenerate both tubular cells and podocytes. In contrast, cells localized between the urinary pole and vascular pole that expressed both progenitor and podocytes markers (CD133+CD24+PDX+) could regenerate only podocytes. Finally, cells localized to the vascular pole did not exhibit progenitor markers, but displayed phenotypic features of differentiated podocytes (CD133-CD24-PDX+ cells). Injection of CD133+CD24+PDX- cells, but not CD133+CD24+PDX+ or CD133-CD24- cells, into mice with adriamycin-induced nephropathy reduced proteinuria and improved chronic glomerular damage, suggesting that CD133+CD24+PDX- cells could potentially treat glomerular disorders characterized by podocyte injury, proteinuria, and progressive glomerulosclerosis.
Asunto(s)
Glomérulos Renales/metabolismo , Riñón/citología , Podocitos/metabolismo , Regeneración , Antígeno AC133 , Animales , Antígenos CD/biosíntesis , Cápsula Glomerular/metabolismo , Antígeno CD24/biosíntesis , Femenino , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glicoproteínas/biosíntesis , Humanos , Riñón/metabolismo , Glomérulos Renales/patología , Ratones , Ratones SCID , Péptidos , Podocitos/patología , Proteinuria/metabolismo , Células MadreRESUMEN
Recently, we have identified a population of renal progenitor cells in human kidneys showing regenerative potential for injured renal tissue of SCID mice. We demonstrate here that among all known chemokine receptors, human renal progenitor cells exhibit high expression of both stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7. In SCID mice with acute renal failure (ARF), SDF-1 was strongly up-regulated in resident cells surrounding necrotic areas. In the same mice, intravenously injected renal stem/progenitor cells engrafted into injured renal tissue decreased the severity of ARF and prevented renal fibrosis. These beneficial effects were abolished by blocking either CXCR4 or CXCR7, which dramatically reduced the number of engrafting renal progenitor cells. However, although SDF-1-induced migration of renal progenitor cells was only abolished by an anti-CXCR4 antibody, transendothelial migration required the activity of both CXCR4 and CXCR7, with CXCR7 being essential for renal progenitor cell adhesion to endothelial cells. Moreover, CXCR7 but not CXCR4 was responsible for the SDF-1-induced renal progenitor cell survival. Collectively, these findings suggest that CXCR4 and CXCR7 play an essential, but differential, role in the therapeutic homing of human renal progenitor cells in ARF, with important implications for the development of stem cell-based therapies.
Asunto(s)
Lesión Renal Aguda/metabolismo , Quimiocina CXCL12/metabolismo , Riñón/citología , Células Madre Multipotentes/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Animales , Línea Celular , Movimiento Celular , Células Cultivadas , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Riñón/metabolismo , Riñón/patología , Ratones , Ratones SCID , ARN Mensajero/metabolismo , Receptores CXCR/genética , Receptores CXCR4/genética , Rabdomiólisis/complicaciones , Rabdomiólisis/metabolismo , Rabdomiólisis/patologíaRESUMEN
Bone marrow-and adult kidney-derived stem/progenitor cells hold promise in the development of therapies for renal failure. Here is reported the identification and characterization of renal multipotent progenitors in human embryonic kidneys that share CD24 and CD133 surface expression with adult renal progenitors and have the capacity for self-renewal and multilineage differentiation. It was found that these CD24+CD133+ cells constitute the early primordial nephron but progressively disappear during nephron development until they become selectively localized to the urinary pole of Bowman's capsule. When isolated and injected into SCID mice with acute renal failure from glycerol-induced rhabdomyolysis, these cells regenerated different portions of the nephron, reduced tissue necrosis and fibrosis, and significantly improved renal function. No tumorigenic potential was observed. It is concluded that CD24+CD133+ cells represent a subset of multipotent embryonic progenitors that persist in human kidneys from early stages of nephrogenesis. The ability of these cells to repair renal damage, together with their apparent lack of tumorigenicity, suggests their potential in the treatment of renal failure.
Asunto(s)
Embrión de Mamíferos/citología , Regeneración , Insuficiencia Renal/patología , Células Madre/citología , Antígeno AC133 , Enfermedad Aguda , Animales , Antígenos CD/biosíntesis , Antígeno CD24/biosíntesis , Glicoproteínas/biosíntesis , Humanos , Túbulos Renales/metabolismo , Ratones , Ratones SCID , Microscopía Confocal , Nefronas/patología , Péptidos , Insuficiencia Renal/metabolismo , Rabdomiólisis/patología , Rabdomiólisis/terapiaRESUMEN
PF-4/CXCL4 is a member of the CXC chemokine family, which is mainly produced by platelets and known for its pleiotropic biological functions. Recently, the proteic product of a nonallelic variant gene of CXCL4 was isolated from human platelets and named as CXCL4L1. CXCL4L1 shows only 4.3% amino acid divergence in the mature protein, but exhibits a 38% amino acid divergence in the signal peptide region. We hypothesized that this may imply a difference in the cell type in which CXCL4L1 is expressed or a difference in its mode of secretion. In different types of transfected cells, CXCL4 and CXCL4L1 exhibited a distinct subcellular localization and a differential regulation of secretion, CXCL4 being stored in secretory granules and released in response to protein kinase C activation, whereas CXCL4L1 was continuously synthesized and secreted through a constitutive pathway. A protein kinase C-regulated CXCL4 secretion was observed also in lymphocytes, a cell type expressing mainly CXCL4 mRNA, whereas smooth muscle cells, which preferentially expressed CXCL4L1, exhibited a constitutive pathway of secretion. These results demonstrate that CXCL4 and CXCL4L1 exhibit a distinct subcellular localization and are secreted in a differentially regulated manner, suggesting distinct roles in inflammatory or homeostatic processes.