RESUMEN
Purpose: Retinal ganglion cell (RGC) transplantation is a therapeutic approach to replace irreversibly degenerated RGCs in diseases such as glaucoma. However, the application of primary RGCs is limited by the availability of tissues. The goal of this study was to evaluate whether transplanted mouse embryonic stem cell (mESC)-derived RGCs can integrate into the host retina and form cell connectivity with host cells. Methods: In this study, we prepared small retinal fragments containing RGC as THY1-enhanced green fluorescent protein (EGFP)+ cells from mESCs and placed them near the retinal surface in the air-injected mouse eyes with or without N-methyl-d-aspartate (NMDA)-induced RGC depletion. After transplantation, THY1-EGFP+ cell integration was observed in whole-mounts and with immunostaining for synaptic markers. Results: Transplanted THY1-EGFP+ cells survived for 12 weeks and extended neurites into the inner plexiform layer (IPL) of the host retina. Presumptive synapse formation was identified between grafted RGCs and host bipolar cells. The ratio of transplanted eyes with integration of THY1-EGFP+ neurites in the host IPL was higher in RGC-injured mice compared with healthy controls. Conclusions: This report shows the potential for therapeutic use of pluripotent cell-derived RGCs by grafting the cells in healthy conditions and with an appropriate technical approach.
Asunto(s)
Células Madre Embrionarias de Ratones/trasplante , Neurogénesis/fisiología , Degeneración Retiniana/terapia , Células Ganglionares de la Retina/trasplante , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Glaucoma , Ratones , Degeneración Retiniana/patología , Células Ganglionares de la Retina/citología , Trasplante de Células Madre , Sinapsis/patologíaRESUMEN
Hearing loss is a sensory deprivation that can affect the quality of life. Currently, only rehabilitation devices such as hearing aids and cochlear implants are used, without a definitive cure. However, in chronic hearing-deprived patients, in whom secondary auditory neural degeneration is expected, a relatively poor rehabilitation prognosis is projected. Stem cell therapy for cochlear neural structures would be an easier and feasible strategy compared with cochlear sensory cells. Considering the highly developed cochlear implantation technology, improving cochlear neural health has significant medical and social effects. Stem cell delivery to Rosenthal's canal in an acutely damaged mouse model has been performed and showed cell survival and the possibility of differentiation. The results of stem cell transplantation in chronic auditory neural hearing loss should be evaluated because neural stem cell replacement therapy for chronic (long-term) sensorineural hearing loss is a major target in clinics. In the present study, we established a mouse model that mimicked chronic auditory neural hearing loss (secondary degeneration of auditory neurons after loss of sensory input). Then, mouse embryonic stem cells (mESCs) were transplanted into the scala tympani and survival and distribution of transplanted cells were compared between the acute and chronic auditory neural hearing loss models induced by ouabain or kanamycin (KM), respectively. The mESC survival was similar to the acute model, and perilymphatic distribution of cell aggregates was more predominant in the chronic model. Lastly, the effects of mESC transplantation on neural signal transduction observed in the cochlear nucleus (CN) were compared and a statistical increase was observed in the chronic model compared with other models. These results indicated that after transplantation, mESCs survived in the cochlea and increased the neural signaling toward the central auditory pathway, even in the chronic (secondary) hearing loss mouse model.
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Vías Aferentes/patología , Cóclea/patología , Pérdida Auditiva Sensorineural/terapia , Células Madre Embrionarias de Ratones/trasplante , Neuronas/patología , Enfermedad Aguda , Animales , Umbral Auditivo/fisiología , Enfermedad Crónica , Cóclea/fisiopatología , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Pérdida Auditiva Sensorineural/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
The worldwide rise in prevalence of chronic kidney disease (CKD) demands innovative bio-medical solutions for millions of kidney patients. Kidney regenerative medicine aims to replenish tissue which is lost due to a common pathological pathway of fibrosis/inflammation and rejuvenate remaining tissue to maintain sufficient kidney function. To this end, cellular therapy strategies devised so far utilize kidney tissue-forming cells (KTFCs) from various cell sources, fetal, adult, and pluripotent stem-cells (PSCs). However, to increase engraftment and potency of the transplanted cells in a harsh hypoxic diseased environment, it is of importance to co-transplant KTFCs with vessel forming cells (VFCs). VFCs, consisting of endothelial cells (ECs) and mesenchymal stem-cells (MSCs), synergize to generate stable blood vessels, facilitating the vascularization of self-organizing KTFCs into renovascular units. In this paper, we review the different sources of KTFCs and VFCs which can be mixed, and report recent advances made in the field of kidney regeneration with emphasis on generation of vascularized kidney tissue by cell transplantation.
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Células Endoteliales/trasplante , Células Madre Embrionarias Humanas/trasplante , Trasplante de Células Madre Mesenquimatosas , Medicina Regenerativa/métodos , Insuficiencia Renal Crónica/terapia , Manejo de Especímenes/métodos , Animales , Humanos , Ratones , Células Madre Embrionarias de Ratones/trasplanteRESUMEN
Careful selection of the host embryo is critical to the efficient production of knockout (KO) mice when injecting mouse embryonic stem (mES) cells into blastocysts. B6(Cg)-Tyrc-2j/J (B6 albino) and C57BL/6NTac (B6NTac) strains of mice are widely used to produce host blastocysts for such procedures. Here, we tested these two strains to identify an appropriate match for modified agouti C57BL/6N (JM8A3.N1) mES cells. When comparing blastocyst yield, super-ovulated B6NTac mice produced more injectable blastocysts per female than B6 albino mice (8.2 vs. 5.4). There was no significant difference in birth rate when injected embryos were transferred to the same pseudopregnant recipient strain. However, the live birth rate was significantly higher for B6NTac blastocysts than B6 albino blastocysts (62.7% vs. 50.2%). In addition, the proportion of pups exhibiting high-level and complete chimerism, as identified by coat color, was also significantly higher in the B6NTac strain. There was no obvious difference in the efficiency of germline transmission (GLT) when compared between B6NTac and B6 albino host embryos (61.5% vs. 63.3% for mES clones; 64.5% vs. 67.9% for genes, respectively), thus suggesting that an equivalent GLT rate could be obtained with only a few blastocyst injections for B6NTac embryos. In conclusion, our data indicate that B6NTac blastocysts are a better choice for the microinjection of JM8A3.N1 mES cells than B6 albino blastocysts.
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Blastocisto/metabolismo , Transferencia de Embrión , Ratones Noqueados/genética , Células Madre Embrionarias de Ratones/trasplante , Animales , Embrión de Mamíferos , Células Germinativas/crecimiento & desarrollo , Ratones , Ratones Noqueados/crecimiento & desarrollo , Microinyecciones , Células Madre Embrionarias de Ratones/citologíaRESUMEN
MiR-140-5p is high expressed in normal fracture healing, but its specific role and mechanism in tissue-to-bone healing are rarely reported. Therefore, this study investigated the effects of miR-140-5p on tissue-to-bone healing. Clone formation experiment, flow cytometry, Alizarin Red S Staining and Oil Red O Staining were performed to investigate the biological characteristics of mouse embryonic bone marrow mesenchymal stem cells C3H10T1/2. MiR-140-5p mimic was transfected into osteogenic medium (OS)-treated C3H10T1/2 cells to investigate the effects of miR-140-5p on osteogenic differentiation. MiR-140-5p transgenic mouse model and the transgenic fracture model were established, and the effects of miR-140-5p on osteogenic differentiation, bone mineral density (BMD) and bone mass of bone tissues were detected by haematoxylin and eosin staining and computed tomography scan. The expressions of osteocalcin, differentiation-related genes (Runx2, ALP, Spp1 and Bglap3) and miR-140-5p were determined by quantitative real-time polymerase chain reaction. C3H10T1/2 cells showed the abilities of forming cloned differentiation of osteogenesis, fat cells, and its phenotypes including CD44, CD90.1 and Sca-1 but excluding CD45 haematopoietic stem cell marker. Overexpression of miR-140-5p promoted the expressions of differentiation-related genes and calcium deposition of OS-treated C3H10T1/2 cells. MiR-140-5p increased the expression of osteocalcin, BMD and bone mass and promoted bone healing of miR-140-5p-transgenic mice with fracture. MiR-140-5p promoted osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post-fracture healing in mice. SIGNIFICANCE OF THE STUDY: C3H10T1/2 cells showed the abilities of forming cloned differentiation of osteogenesis, fat cells and its phenotypes including CD44, CD90.1 and Sca-1 but excluding CD45 haematopoietic stem cell marker. Overexpression of miR-140-5p promoted the expressions of differentiation-related genes and calcium deposition of osteogenic medium-treated C3H10T1/2 cells. MiR-140-5p increased the expression of osteocalcin and bone mineral density and bone mass and promoted bone healing of miR-140-5p-transgenic mice with fracture. Our results showed that miR-140-5p promoted osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post-fracture healing in mice, which may be a therapeutic target for treating fractures and promoting bone healing.
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Células de la Médula Ósea/metabolismo , Diferenciación Celular , Curación de Fractura , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Células Madre Embrionarias de Ratones , Osteogénesis , Animales , Línea Celular , Masculino , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/trasplanteRESUMEN
Cellular inflammation following acute myocardial infarction has gained increasing importance as a target mechanism for therapeutic approaches. We sought to investigate the effect of syngeneic cardiac induced cells (CiC) on myocardial inflammation using 18F-FDG PET (Positron emission tomography)-based imaging and the resulting effect on cardiac pump function using cardiac magnetic resonance (CMR) imaging in a mouse model of myocardial infarction. Mice underwent permanent left anterior descending coronary artery (LAD) ligation inducing an acute inflammatory response. The therapy group received an intramyocardial injection of 106 CiC into the border zone of the infarction. Five days after myocardial infarction, 18F-FDG PET was performed under anaesthesia with ketamine and xylazine (KX) to image the inflammatory response in the heart. Flow cytometry of the mononuclear cells in the heart was performed to analyze the inflammatory response. The effect of CiC therapy on cardiac function was determined after three weeks by CMR. The 18F-FDG PET imaging of the heart five days after myocardial infarction (MI) revealed high focal tracer accumulation in the border zone of the infarcted myocardium, whereas no difference was observed in the tracer uptake between infarct and remote myocardium. The CiC transplantation induced a shift in 18F-FDG uptake pattern, leading to significantly higher 18F-FDG uptake in the whole heart, as well as the remote area of the heart. Correspondingly, high numbers of CD11+ cells could be measured by flow cytometry in this region. The CiC transplantation significantly improved the left ventricular ejection function (LVEF) three weeks after myocardial infarction. The CiC transplantation after myocardial infarction leads to an improvement in pump function through modulation of the cellular inflammatory response five days after myocardial infarction. By combining CiC transplantation and the cardiac glucose uptake suppression protocol with KX in a mouse model, we show for the first time, that imaging of cellular inflammation after myocardial infarction using 18F-FDG PET can be used as an early prognostic tool for assessing the efficacy of cardiac stem cell therapies.
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Antígenos CD11/metabolismo , Fluorodesoxiglucosa F18/administración & dosificación , Corazón/diagnóstico por imagen , Células Madre Embrionarias de Ratones/trasplante , Infarto del Miocardio/terapia , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Corazón/fisiopatología , Humanos , Imagen por Resonancia Cinemagnética , Ratones , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/inmunología , Infarto del Miocardio/fisiopatología , Tomografía de Emisión de Positrones , Resultado del Tratamiento , Función Ventricular IzquierdaRESUMEN
Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectoderm-derived surface epithelium. Here, we report means for induction of an LB-like mesenchymal/epithelial complex tissues from murine pluripotent stem cells (PSCs) in vitro. The LB-like tissues selectively differentiate into forelimb- or hindlimb-type mesenchymes, depending on a concentration of retinoic acid. Comparative transcriptome analysis reveals that the LB-like tissues show similar gene expression pattern to that seen in LBs. We also show that manipulating BMP signaling enables us to induce a thickened epithelial structure similar to the apical ectodermal ridge. Finally, we demonstrate that the induced tissues can contribute to endogenous digit tissue after transplantation. This PSC technology offers a first step for creating an artificial limb bud in culture and might open the door to inducing other mesenchymal/epithelial complex tissues from PSCs.
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Técnicas de Cultivo de Célula/métodos , Esbozos de los Miembros/embriología , Células Madre Embrionarias de Ratones/fisiología , Ingeniería de Tejidos/métodos , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Ectodermo/citología , Ectodermo/metabolismo , Embrión de Mamíferos , Desarrollo Embrionario , Epitelio/metabolismo , Femenino , Miembro Anterior/embriología , Miembro Anterior/trasplante , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Miembro Posterior/embriología , Miembro Posterior/trasplante , Esbozos de los Miembros/trasplante , Masculino , Ratones , Células Madre Embrionarias de Ratones/trasplante , Transducción de Señal/fisiologíaRESUMEN
Neurogenesis in the substantia nigra (SN) has been a controversial issue. Here we report that neurogenesis can be induced in the adult rodent SN by transplantation of embryoid body cells (EBCs) derived from mouse embryonic stem cells. The detection of Sox2+ dividing (BrdU+) putative host neural precursor cells (NPCs) between 1 and 6â¯days post-transplantation (dpt) supported the neurogenic capacity of the adult SN. In agreement with the awakening of NPCs by EBCs, only host cells from implant-bearing SN were able to generate neurosphere-like aggregates in the presence of Egf and Fgf2. Later, at 15 dpt, a significant number of SN Dcx+ neuroblasts were detected. However, a continuous BrdU administration after transplantation showed that only a fraction (about 20-30%) of those host Dcx+ progeny derived from dividing cells and few BrdU+ cells, some of them NeuN+, survived up to 30 dpt. Unexpectedly, 25-30% of Dcx+ or Psa-Ncam+ cells at 15 dpt displayed astrocytic markers such as Gfap and S100b. Using a genetic lineage tracing strategy, we demonstrated that a large proportion of host Dcx+ and/or Tubb3+ neuroblasts originated from Gfap+ cells. Remarkably, new blood vessels formed in association with the neurogenic process that, when precluded, caused a reduction in neuroblast production. Accordingly, two proteins secreted by EBCs, Fgf2 and Vegf, were able to promote the emergence of Dcx+/Psa-Ncam+, Tubb3+ and NeuN+/BrdU+ cells in vivo in the absence of EBCs. We propose that the adult SN is a mostly silent neurogenic niche with the ability to generate new neurons by typical and atypical mechanisms.
Asunto(s)
Células Madre Embrionarias de Ratones/trasplante , Neurogénesis/fisiología , Neuronas/fisiología , Sustancia Negra/fisiología , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Proteína Doblecortina , Factor 2 de Crecimiento de Fibroblastos/farmacología , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Irisin, a newly identified hormone and cardiokine, is critical for modulating body metabolism. New evidence indicates that irisin protects the heart against myocardial ischemic injury. However, whether irisin enhances cardiac progenitor cell (CPC)-induced cardiac repair remains unknown. This study examines the effect of irisin on CPC-induced cardiac repair when these cells are introduced into the infarcted myocardium. Nkx2.5+ CPC stable cells were isolated from mouse embryonic stem cells. Nkx2.5 + CPCs (0.5 × 10 6 ) were reintroduced into the infarcted myocardium using PEGlylated fibrin delivery. The mouse myocardial infarction model was created by permanent ligation of the left anterior descending (LAD) artery. Nkx2.5 + CPCs were pretreated with irisin at a concentration of 5 ng/ml in vitro for 24 hr before transplantation. Myocardial functions were evaluated by echocardiographic measurement. Eight weeks after engraftment, Nkx2.5 + CPCs improved ventricular function as evident by an increase in ejection fraction and fractional shortening. These findings are concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Transplantation of Nkx2.5 + CPCs promoted cardiac regeneration and neovascularization, which were increased with the pretreatment of Nkx2.5 + CPCs with irisin. Furthermore, irisin treatment promoted myocyte proliferation as indicated by proliferative markers Ki67 and phosphorylated histone 3 and decreased apoptosis. Additionally, irisin resulted in a marked reduction of histone deacetylase 4 and increased p38 acetylation in cultured CPCs. These results indicate that irisin promoted Nkx2.5 + CPC-induced cardiac regeneration and functional improvement and that irisin serves as a novel therapeutic approach for stem cells in cardiac repair.
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Fibronectinas/farmacología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/trasplante , Infarto del Miocardio/cirugía , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/trasplante , Regeneración , Trasplante de Células Madre/métodos , Función Ventricular Izquierda , Animales , Apoptosis , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis , Proteína Homeótica Nkx-2.5/genética , Proteína Homeótica Nkx-2.5/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Células Madre Embrionarias de Ratones/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Neovascularización Fisiológica , Recuperación de la Función , Volumen Sistólico , Remodelación VentricularRESUMEN
Cardiac diseases have become the leading cause of death worldwide. Developing efficient strategies to treat such diseases is of great urgency. Stem cell-based regeneration medicine offers a novel approach for heart repair. However, low retention and poor survival rate of engrafted cells limit its applications. Nanomaterials have shown great potentials in addressing above issues due to nanoparticles-bio interactions. Therefore, combining nanomaterials and stem cell therapy is of great interest and significance for heart repair. Herein, we provide a comprehensive understanding of the applications of four types of nanomaterials (nanogels, polymeric nanomaterials, inorganic nanomaterials and exosomes) in stem cell therapy for myocardial repair. In addition, we launch an initial discussion on current problems and more importantly, possible solutions for myocardial repair.
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Corazón/crecimiento & desarrollo , Infarto del Miocardio/terapia , Nanopartículas/administración & dosificación , Ingeniería de Tejidos , Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Exosomas/química , Exosomas/genética , Corazón/fisiopatología , Humanos , Ratones , Células Madre Embrionarias de Ratones/trasplante , Infarto del Miocardio/patología , Miocitos Cardíacos/trasplante , Nanopartículas/químicaRESUMEN
The pluripotency of embryonic stem cells (ESCs) is maintained by core pluripotency transcription factors, cofactors and several signaling pathways. RBM14 is a component of the para-speckle complex, which has been implicated in multiple important biological processes. The role of RBM14 in ESCs and lineage differentiation remains to be elucidated. In the present study, we provided evidence that RBM14 plays important roles in maintaining pluripotency and in the early differentiation of ESCs. RBM14 was demonstrated to be expressed in mouse embryonic stem cells (mESCs) and localized in the nucleus. RBM14 expression was depleted in mESCs using clustered regularly interspaced short palindromic repeats (CRISPR) technology. Our results also showed that RBM14 depletion altered the gene expression profiles of mESCs. In particular, pluripotency-associated genes and genes involved in the Wnt and TGF-ß signaling pathways were downregulated in RBM14 knockout mESCs. Furthermore, RBM14 was found to be essential for mesoderm development in vitro and in vivo. The specific effects of RBM14 depletion were verified by conducting a rescue experiment. Our findings demonstrated that RBM14 not only plays an important role in maintaining the pluripotency of mESCs but is also indispensable for mesoderm development.
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Mesodermo/embriología , Mesodermo/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción/metabolismo , Animales , Sistemas CRISPR-Cas , Diferenciación Celular , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Embrionarias de Ratones/trasplante , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcriptoma , Factor de Crecimiento Transformador beta/metabolismo , Vía de Señalización WntRESUMEN
A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab')2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2-Rac axis in PC differentiation and IgG antibody responses.
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Diferenciación Celular/inmunología , Proteínas Activadoras de GTPasa/fisiología , Inmunidad Humoral , Inmunoglobulina G/metabolismo , Células Plasmáticas/fisiología , Traslado Adoptivo , Animales , Membrana Celular/inmunología , Femenino , Factores de Intercambio de Guanina Nucleótido , Inmunoglobulina G/inmunología , Sinapsis Inmunológicas/inmunología , Sinapsis Inmunológicas/metabolismo , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Células Madre Embrionarias de Ratones/trasplante , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Quimera por Trasplante , Proteínas de Unión al GTP rac/inmunología , Proteínas de Unión al GTP rac/metabolismoRESUMEN
The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor ß2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1-/- mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.
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Células Madre Embrionarias de Ratones/trasplante , Células Fotorreceptoras Retinianas Conos/citología , Degeneración Retiniana/terapia , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Factor Nuclear 6 del Hepatocito/metabolismo , Factor Inhibidor de Leucemia/farmacología , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/citología , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Opsinas/metabolismo , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Factores de Transcripción Otx/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Notch/antagonistas & inhibidores , Receptores Notch/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Degeneración Retiniana/patología , Transducción de Señal , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant and nonmalignant diseases. However, chronic graft-versus-host disease (cGVHD) remains a significant cause of late morbidity and mortality after allogeneic HSCT. cGVHD often manifests as autoimmune syndrome. Thymic epithelial cells (TECs) play a critical role in supporting negative selection and regulatory T-cell (Treg) generation. Studies have shown that damage in TECs is sufficient to induce cGVHD. We have previously reported that mouse embryonic stem cells (mESCs) can be selectively induced to generate thymic epithelial progenitors (TEPs) in vitro. When transplanted in vivo, mESC-TEPs further develop into TECs that support T-cell development. We show here that transplantation of donor-origin mESC-TEPs into cGVHD recipients induces immune tolerance to both donor and host antigens and prevents the development of cGVHD. This is associated with more TECs and Tregs. Our results suggest that embryonic stem cell-derived TEPs may offer a new tool to control cGVHD. Stem Cells Translational Medicine 2017;6:121-130.
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Células Epiteliales/trasplante , Enfermedad Injerto contra Huésped/prevención & control , Células Madre Embrionarias de Ratones/trasplante , Trasplante de Células Madre , Timo/citología , Animales , Antígenos/metabolismo , Recuento de Células , Diferenciación Celular , Enfermedad Crónica , Células Epiteliales/citología , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Tolerancia Inmunológica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Linfocitos T Reguladores/citología , Timocitos/citologíaRESUMEN
Motoneurons derived from embryonic stem cells can be transplanted in the tibial nerve, where they extend axons to functionally innervate target muscle. Here, we studied spontaneous muscle contractions in these grafts 3 mo following transplantation. One-half of the transplanted grafts generated rhythmic muscle contractions of variable patterns, either spontaneously or in response to brief electrical stimulation. Activity generated by transplanted embryonic stem cell-derived neurons was driven by glutamate and was modulated by muscarinic and GABAergic/glycinergic transmission. Furthermore, rhythmicity was promoted by the same transmitter combination that evokes rhythmic locomotor activity in spinal cord circuits. These results demonstrate that there is a degree of self-assembly of microcircuits in these peripheral grafts involving embryonic stem cell-derived motoneurons and interneurons. Such spontaneous activity is reminiscent of embryonic circuit development in which spontaneous activity is essential for proper connectivity and function and may be necessary for the grafts to form functional connections with muscle.NEW & NOTEWORTHY This manuscript demonstrates that, following peripheral transplantation of neurons derived from embryonic stem cells, the grafts are spontaneously active. The activity is produced and modulated by a number of transmitter systems, indicating that there is a degree of self-assembly of circuits in the grafts.
Asunto(s)
Neuronas Motoras/fisiología , Células Madre Embrionarias de Ratones/fisiología , Red Nerviosa/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/cirugía , Potenciales de Acción/efectos de los fármacos , Animales , Estimulación Eléctrica/métodos , Embrión de Mamíferos , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/trasplante , Músculo Esquelético/inervación , Neurotransmisores/farmacologíaRESUMEN
Wnt signaling plays an important role in the self-renewal and differentiation of stem cells. The secretion of Wnt ligands requires Evi (also known as Wls). Genetically ablating Evi provides an experimental approach to studying the consequence of depleting all redundant Wnt proteins, and overexpressing Evi enables a nonspecific means of increasing Wnt signaling. We generated Evi-deficient and Evi-overexpressing mouse embryonic stem cells (ESCs) to analyze the role of autocrine Wnt production in self-renewal and differentiation. Self-renewal was reduced in Evi-deficient ESCs and increased in Evi-overexpressing ESCs in the absence of leukemia inhibitory factor, which supports the self-renewal of ESCs. The differentiation of ESCs into cardiomyocytes was enhanced when Evi was overexpressed and teratoma formation and growth of Evi-deficient ESCs in vivo were impaired, indicating that autocrine Wnt ligands were necessary for ESC differentiation and survival. ESCs lacking autocrine Wnt signaling had mitotic defects and showed genomic instability. Together, our study demonstrates that autocrine Wnt secretion is important for the survival, chromosomal stability, differentiation, and tumorigenic potential of ESCs.
Asunto(s)
Comunicación Autocrina , Proliferación Celular/genética , Inestabilidad Genómica , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Wnt/genética , Animales , Diferenciación Celular/genética , Autorrenovación de las Células , Supervivencia Celular/genética , Células Cultivadas , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Embrionarias de Ratones/trasplante , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización WntRESUMEN
Heparan sulfate proteoglycans (HSPGs), ubiquitous components of mammalian cells, play important roles in development and homeostasis. These molecules are located primarily on the cell surface and in the pericellular matrix, where they interact with a multitude of macromolecules, including many growth factors. Manipulation of the enzymes involved in biosynthesis and modification of HSPG structures alters the properties of stem cells. Here, we focus on the involvement of heparanase (HPSE), the sole endo-glucuronidase capable of cleaving of HS, in differentiation of embryonic stem cells into the cells of the neural lineage. Embryonic stem (ES) cells overexpressing HPSE (Hpse-Tg) proliferated more rapidly than WT ES cells in culture and formed larger teratomas in vivo. In addition, differentiating Hpse-Tg ES cells also had a higher growth rate, and overexpression of HPSE in NSPCs enhanced Erk and Akt phosphorylation. Employing a two-step, monolayer differentiation, we observed an increase in HPSE as wild-type (WT) ES cells differentiated into neural stem and progenitor cells followed by down-regulation of HPSE as these NSPCs differentiated into mature cells of the neural lineage. Furthermore, NSPCs overexpressing HPSE gave rise to more oligodendrocytes than WT cultures, with a concomitant reduction in the number of neurons. Our present findings emphasize the importance of HS, in neural differentiation and suggest that by regulating the availability of growth factors and, or other macromolecules, HPSE promotes differentiation into oligodendrocytes.
Asunto(s)
Glucuronidasa/genética , Glucuronidasa/metabolismo , Células Madre Embrionarias de Ratones/citología , Oligodendroglía/citología , Teratoma/patología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/trasplante , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Teratoma/genética , Teratoma/metabolismoRESUMEN
Regeneration of skeletal muscle relies on the presence of satellite cells. Satellite cells deficiency accompanying some degenerative diseases is the reason for the search for the "replacement cells" that can be used in the muscle therapies. Due to their unique properties embryonic stem cells (ESCs), as well as myogenic cells derived from them, are considered as a promising source of therapeutic cells. Among the factors crucial for the specification of myogenic precursor cells is Pax7 that sustains proper function of satellite cells. In our previous studies we showed that ESCs lacking functional Pax7 are able to form myoblasts in vitro when differentiated within embryoid bodies and their outgrowths. In the current study we showed that ESCs lacking functional Pax7, cultured in vitro in monolayer in the medium supplemented with horse serum and 5azaC, expressed higher levels of factors associated with myogenesis, such as Pdgfra, Pax3, Myf5, and MyoD. Importantly, skeletal myosin immunolocalization confirmed that myogenic differentiation of ESCs was more effective in case of cells lacking Pax7. Our in vivo studies showed that ESCs transplanted into regenerating skeletal muscles were detectable at day 7 of regeneration and the number of Pax7-/- ESCs detected was significantly higher than of control cells. Our results support the concept that lack of functional Pax7 promotes proliferation of differentiating ESCs and for this reason more of them can turn into myogenic lineage.
Asunto(s)
Azacitidina/farmacología , Células Madre Embrionarias de Ratones , Músculo Esquelético/fisiología , Factor de Transcripción PAX7/deficiencia , Regeneración/efectos de los fármacos , Trasplante de Células Madre , Animales , Femenino , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/trasplante , Regeneración/genéticaRESUMEN
Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells.
Asunto(s)
Líquido Amniótico , Antígenos de Diferenciación/biosíntesis , Células Madre Embrionarias de Ratones , Nicho de Células Madre/fisiología , Líquido Amniótico/citología , Líquido Amniótico/metabolismo , Animales , Femenino , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/trasplanteRESUMEN
Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked ß-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation.