RESUMEN
Proliferative vitreoretinopathy (PVR) is a complication of rhegmatogenous retinal detachment and ocular trauma. The disease is characterized by development of membranes that may apply traction to the retina and cause redetachment. Membrane contractions are attributed to myofibroblasts arising from retinal pigment epithelial cells, glia and fibroblasts. The progenitors of myofibrobasts in the lens are Myo/Nog cells that express the skeletal muscle transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. The retina and choroid also contain Myo/Nog cells that respond to stress. We examined preretinal PVR membranes from three ocular trauma patients with retinal detachment for Myo/Nog cells and their expression of muscle proteins. Myo/Nog cells were identified by co-localization of antibodies to the G8 antigen and Noggin. Greater than 80% of all cells in sections from two of three patients expressed both G8 and Noggin. Myo/Nog cells lacked pigment. Alpha smooth muscle actin (α-SMA) and striated myosin II heavy chain were present in the majority of Myo/Nog cells in these two patients. Differentiation of Myo/Nog cells was paralleled by low levels of MyoD. Membrane sections from the third patient consisted mostly of connective tissue with very few cells. A small subpopulation in these sections expressed both G8 and Noggin, and muscle proteins were detected in only a minority of G8-positive (+) cells. In all three patients, greater than 99% of cells with MyoD, α-SMA and striated muscle myosin co-expressed G8. These findings suggest that contractile myofibroblasts in PVR membranes may be derived from differentiating Myo/Nog cells.
Asunto(s)
Proteínas Musculares/biosíntesis , Proteína MioD/biosíntesis , Retina/patología , Vitreorretinopatía Proliferativa/metabolismo , Animales , Humanos , Retina/metabolismo , Vitreorretinopatía Proliferativa/diagnósticoRESUMEN
Myo/Nog cells, named for their expression of MyoD and noggin, enter the eye during early stages of embryonic development. Their release of noggin is critical for normal morphogenesis of the lens and retina. Myo/Nog cells are also present in adult eyes. Single nucleated skeletal muscle cells designated as myofibroblasts arise from Myo/Nog cells in cultures of lens tissue. In this report we document the presence of Myo/Nog cells in the lens, ciliary body and on the zonule of Zinn in mice, rabbits and humans. Myo/Nog cells were rare in all three structures. Their prevalence increased in the lens and ciliary body of rabbits 24â¯h following cataract surgery. Rabbits developed posterior capsule opacification (PCO) within one month of surgery. The number of Myo/Nog cells continued to be elevated in the lens and ciliary body. Myo/Nog cells containing alpha smooth muscle actin and striated muscle myosin were present on the posterior capsule and overlaid deformations in the capsule. Myo/Nog cells also were present on the zonule fibers and external surface of the posterior capsule. These findings suggest that Myo/Nog contribute to PCO and may use the zonule fibers to migrate between the ciliary processes and lens.
Asunto(s)
Proteínas Portadoras/metabolismo , Cuerpo Ciliar/metabolismo , Cristalino/metabolismo , Ligamentos/metabolismo , Proteína MioD/metabolismo , Facoemulsificación , Cápsula Posterior del Cristalino/metabolismo , Actinas/metabolismo , Animales , Opacificación Capsular/metabolismo , Femenino , Fibrilina-1/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/metabolismo , Miosinas/metabolismo , Conejos , Vimentina/metabolismoRESUMEN
Osteoarthritis (OA) is characterized by degeneration of articular cartilage within the joint, inflammation and pain. The purpose of this study was to develop a primary, serum free cell culture system of human osteoarthritic articular chondrocytes (HOACs) with which to study manifestations of the disease process. Joint tissues were obtained from OA patients undergoing total knee arthroplasty (TKA). HOACs isolated from the femoral condyles and tibial plateau of the same side were combined, plated in three-dimensional, alginate beads and cultured for five days in serum, hormone and protein free medium. More living cells were obtained from the femoral condyles than the tibial plateau. The optimal plating density was 2.5 × 106 cells/ml of alginate. The amounts of DNA, RNA, proteoglycans and total collagen were similar in cultures prepared from the sides of least and greatest pathology. More type 1 than type 2 collagen was detected in the medium on days 2 and 5. A greater percentage of type 1 than type 2 collagen was degraded. The inflammatory cytokine interleukin-1 beta was present in the medium and alginate associated matrix. Although variation in the metabolic profiles between subjects was observed, HOACs from all patients continued to reflect the OA phenotype for five days in culture. This serum free, three-dimensional primary culture system of HOACs provides a platform with which to measure clinically relevant endpoints of OA and screen potential disease modifying OA therapeutics.
Asunto(s)
Cartílago Articular/citología , Condrocitos/metabolismo , Osteoartritis/metabolismo , Cultivo Primario de Células , Proteoglicanos/metabolismo , Colágeno/metabolismo , Colágeno Tipo II/metabolismo , Medio de Cultivo Libre de Suero , Matriz Extracelular/metabolismo , HumanosRESUMEN
Posterior capsule opacification (PCO) occurs in some adults and most children following cataract surgery. The fibrotic form of PCO arises, in part, from migratory, contractile myofibroblasts that deform the lens capsule and impair vision. In short-term cultures of human anterior lens tissue, myofibroblasts emerge from Myo/Nog cells that are identified with the G8 monoclonal antibody and by their expression of the MyoD transcription factor and bone morphogenetic protein inhibitor noggin. In this study, we tested the hypothesis that targeted depletion of Myo/Nog cells with the G8 monoclonal antibody (mAb) conjugated to three-dimensional DNA nanocarriers intercalated with doxorubicin (G8:3DNA:Dox) would prevent the accumulation of myofibroblasts in long-term, serum- and growth factor-free cultures of human lens tissue obtained by capsulorhexis. The mAb:nanocarrier complex was internalized into acidic compartments of the cell. G8:3DNA:Dox killed nearly all Myo/Nog cells without affecting the lens epithelial cells. In 30-day cultures, all G8-positive cells expressed noggin, and subpopulations had synthesized MyoD, sarcomeric myosin, and alpha smooth muscle actin (α-SMA). Myo/Nog cells responded to scratching of the lens epithelium by accumulating around the edges of the wound. Treatment with two doses of G8:3DNA:Dox completely eliminated G8+/α-SMA+ cells throughout the explant. These experiments demonstrate that Myo/Nog cells are the source of myofibroblasts in long-term cultures of anterior human lens tissue and mAb:3DNA nanocarriers specifically and effectively deliver cytotoxic cargo to a subpopulation of cells without off-target effects. G8:3DNA:Dox has the potential to reduce PCO following cataract surgery.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , ADN/administración & dosificación , Doxorrubicina/administración & dosificación , Cristalino/efectos de los fármacos , Miofibroblastos/efectos de los fármacos , Nanoestructuras/administración & dosificación , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/metabolismo , ADN/metabolismo , Doxorrubicina/metabolismo , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Cristalino/citología , Cristalino/metabolismo , Masculino , Persona de Mediana Edad , Miofibroblastos/metabolismo , Técnicas de Cultivo de ÓrganosRESUMEN
Myo/Nog cells are essential for eye development in the chick embryo and respond to injury in adult tissues. These cells express mRNA for the skeletal muscle specific transcription factor MyoD, the bone morphogenetic protein (BMP) inhibitor Noggin and the cell surface protein recognized by the G8 monoclonal antibody (mAb). In this study, we determined that Myo/Nog cells are present in low numbers in the retina of the mouse eye. G8-positive Myo/Nog cells were distinguished from neuronal, Müller and microglial cells that were identified with antibodies to calretinin, Chx10, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1, respectively. In the neonatal retina, the number of Myo/Nog cells increased in parallel with cell death induced by transient exposure to hyperoxia. In this model of retinopathy of prematurity, depletion of Myo/Nog cells by intravitreal injection of the G8 mAb and complement increased cell death. These findings demonstrate that Myo/Nog cells are a distinct population of cells, not previously described in the retina, which increases in response to retinal damage and mitigate hypoxia-induced cell death.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteína MioD/metabolismo , Estrés Oxidativo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Retinopatía de la Prematuridad/metabolismo , Animales , Muerte Celular , Humanos , Células Fotorreceptoras de Vertebrados/patología , Retina/patología , Retinopatía de la Prematuridad/diagnósticoRESUMEN
Purpose: The present study aimed to determine the dose-response relationship between targeted nanocarriers released from a novel, sustained release formulation and their ability to specifically deplete cells responsible for the development of posterior capsular opacification (PCO) in month-long, dynamic cell cultures. Methods: Injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) triblock copolymer hydrogels were loaded with either a low or a high dose of doxorubicin-loaded antibody-targeted nanocarriers (G8:3DNA:Dox). Human rhabdomyosarcoma cells, selected for their expression of PCO marker brain-specific angiogenesis inhibitor 1 (BAI1), were kept under dynamic media flow and received either a low or high dose of nanocarriers. Cells were fixed and stained at predetermined time points to evaluate targeted depletion of BAI1+ cells. Results: A lower dose of nanocarriers in hydrogel depleted BAI1+ cells at a slower rate than the higher dose, whereas both reached over 90% BAI1+ cellular nonviability at 28 days. Both treatment groups also significantly lowered the relative abundance of BAI1+ cells in the population compared with the control group. Conclusions: Controlled release of a lower dose of nanocarriers can still achieve therapeutically relevant effects in the prevention of PCO, while avoiding potential secondary effects associated with the administration of a higher dose.
Asunto(s)
Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Doxorrubicina , Portadores de Fármacos , Nanopartículas , Humanos , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Nanopartículas/administración & dosificación , Hidrogeles/química , Hidrogeles/administración & dosificación , Catarata/tratamiento farmacológico , Ácidos Nucleicos/administración & dosificación , Línea Celular Tumoral , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma/patología , Polietilenglicoles/química , Polietilenglicoles/administración & dosificación , Sistemas de Liberación de MedicamentosRESUMEN
We investigated an alternative pathway for emergence of the mesenchymal cells involved in epithelial sheet wound healing and a source of myofibroblasts that cause fibrosis. Using a mock cataract surgery model, we discovered a unique subpopulation of polyploid mesenchymal progenitors nestled in small niches among lens epithelial cells that expressed the surface antigen G8 and mRNA for the myogenic transcription factor MyoD. These cells rapidly responded to wounding of the lens epithelium with population expansion, acquisition of a mesenchymal phenotype, and migration to the wound edges where they regulate the wound response of the epithelium. These mesenchymal cells also were a principal source of myofibroblasts that emerged following lens injury and were responsible for fibrotic disease of the lens that occurs following cataract surgery. These studies provide insight into the mechanisms of wound-healing and fibrosis.
Asunto(s)
Células Madre Mesenquimatosas/citología , Cicatrización de Heridas , Animales , Antígenos de Superficie/metabolismo , Diferenciación Celular , Embrión de Pollo , Fibrosis/genética , Fibrosis/metabolismo , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Proteína MioD/genéticaRESUMEN
Herein, we review a unique and versatile lineage composed of Myo/Nog cells that may be beneficial or detrimental depending on their environment and nature of the pathological stimuli they are exposed to. While we will focus on the lens, related Myo/Nog cell behaviors and functions in other tissues are integrated into the narrative of our research that spans over three decades, examines multiple species and progresses from early stages of embryonic development to aging adults. Myo/Nog cells were discovered in the embryonic epiblast by their co-expression of the skeletal muscle-specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin and brain-specific angiogenesis inhibitor 1. They were tracked from the epiblast into the developing lens, revealing heterogeneity of cell types within this structure. Depletion of Myo/Nog cells in the epiblast results in eye malformations arising from the absence of Noggin. In the adult lens, Myo/Nog cells are the source of myofibroblasts whose contractions produce wrinkles in the capsule. Eliminating this population within the rabbit lens during cataract surgery reduces posterior capsule opacification to below clinically significant levels. Parallels are drawn between the therapeutic potential of targeting Myo/Nog cells to prevent fibrotic disease in the lens and other ocular tissues.
Asunto(s)
Opacificación Capsular , Cristalino , Animales , Conejos , Cristalino/metabolismo , Opacificación Capsular/metabolismo , Músculo Esquelético/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulación de la Expresión GénicaRESUMEN
Purpose: Myo/Nog cells are the source of myofibroblasts in the lens and synthesize muscle proteins in human epiretinal membranes (ERMs). In the current study, we examined the response of Myo/Nog cells during ERM formation in a mouse model of proliferative vitreoretinopathy (PVR). Methods: PVR was induced by intravitreal injections of gas and ARPE-19 cells. PVR grade was scored by fundus imaging, optical coherence tomography, and histology. Double label immunofluorescence localization was performed to quantify Myo/Nog cells, myofibroblasts, and leukocytes. Results: Myo/Nog cells, identified by co-labeling with antibodies to brain-specific angiogenesis inhibitor 1 (BAI1) and Noggin, increased throughout the eye with induction of PVR and disease progression. They were present on the inner surface of the retina in grades 1/2 PVR and were the largest subpopulation of cells in grades 3 to 6 ERMs. All α-SMA-positive (+) cells and all but one striated myosin+ cell expressed BAI1 in grades 1 to 6 PVR. Folds and areas of retinal detachment were overlain by Myo/Nog cells containing muscle proteins. Low numbers of CD18, CD68, and CD45+ leukocytes were detected throughout the eye. Small subpopulations of BAI1+ cells expressed leukocyte markers. ARPE-19 cells were found in the vitreous but were rare in ERMs. Pigmented cells lacking Myo/Nog and muscle cell markers were present in ERMs and abundant within the retina by grade 5/6. Conclusions: Myo/Nog cells differentiate into myofibroblasts that appear to contract and produce retinal folds and detachment. Targeting BAI1 for Myo/Nog cell depletion may be a pharmacological approach to preventing and treating PVR.
Asunto(s)
Membrana Epirretinal , Vitreorretinopatía Proliferativa , Animales , Ratones , Humanos , Vitreorretinopatía Proliferativa/metabolismo , Membrana Epirretinal/metabolismo , Miofibroblastos/metabolismo , Retina/metabolismo , Proteínas Musculares/metabolismoRESUMEN
Cells that express MyoD mRNA, the G8 antigen and the bone morphogenetic protein (BMP) inhibitor noggin (Nog) are present in the epiblast before gastrulation. Ablation of "Myo/Nog" cells in the blastocyst results in an expansion of canonical BMP signaling and prevents the expression of noggin and follistatin before and after the onset of gastrulation. Once eliminated in the epiblast, they are neither replaced nor compensated for as development progresses. Older embryos lacking Myo/Nog cells exhibit severe axial malformations. Although Wnts and Sonic hedgehog are expressed in ablated embryos, skeletal muscle progenitors expressing Pax3 are missing in the somites. Pax3+ cells do emerge adjacent to Wnt3a+ cells in vitro; however, few undergo skeletal myogenesis. Ablation of Myo/Nog cells also results in ectopically placed cardiac progenitors and cardiomyocytes in the somites. Reintroduction of Myo/Nog cells into the epiblast of ablated embryos restores normal patterns of BMP signaling, morphogenesis and skeletal myogenesis, and inhibits the expression of cardiac markers in the somites. This study demonstrates that Myo/Nog cells are essential regulators of BMP signaling in the early epiblast and are indispensable for normal morphogenesis and striated muscle lineage specification.
Asunto(s)
Blastocisto/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/fisiología , Morfogénesis , Músculo Estriado/citología , Proteína MioD/fisiología , Transducción de Señal , Animales , Secuencia de Bases , Linaje de la Célula , Embrión de Pollo , Cartilla de ADN , Hibridación in SituRESUMEN
Murine and human skin were examined for the presence of Myo/Nog cells that were originally discovered in the chick embryo by their expression of MyoD mRNA, noggin and the G8 antigen. Myo/Nog cells are the primary source of noggin in telogen hair follicles. They are scarce within the interfollicular dermis and absent in the epidermis. Within 24 h following epidermal abrasion, Myo/Nog cells increase in number in the follicles and appear in the wound. Myo/Nog cells are also recruited to the stroma of tumors formed from v-Ras-transformed keratinocytes (Ker/Ras). Human squamous cell carcinomas and malignant melanomas contain significantly more Myo/Nog cells than basal cell carcinomas. Myo/Nog cells are distinct from macrophages, granulocytes and cells expressing alpha smooth muscle actin in the tumor stroma. Myo/Nog cells may be modulators of skin homoeostasis and wound healing, and potential diagnostic and therapeutic targets in skin cancer.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteína MioD/metabolismo , Neoplasias Cutáneas/metabolismo , Piel/metabolismo , Heridas y Lesiones/metabolismo , Animales , Carcinoma Basocelular/metabolismo , Carcinoma de Células Escamosas/metabolismo , Humanos , Melanoma/metabolismo , RatonesRESUMEN
The epiblast of the chick embryo contains cells that express MyoD mRNA but not MyoD protein. We investigated whether MyoD-positive (MyoDpos) epiblast cells are stably committed to the skeletal muscle lineage or whether their fate can be altered in different environments. A small number of MyoDpos epiblast cells were tracked into the heart and nervous system. In these locations, they expressed MyoD mRNA and some synthesized MyoD protein. No MyoDpos epiblast cells differentiated into cardiac muscle or neurons. Similar results were obtained when MyoDpos cells were isolated from the epiblast and microinjected into the precardiac mesoderm or neural plate. In contrast, epiblast cells lacking MyoD differentiated according to their environment. These results demonstrate that the epiblast contains both multipotent cells and a subpopulation of cells that are stably committed to the skeletal muscle lineage before the onset of gastrulation. Stable programming in the epiblast may ensure that MyoDpos cells express similar signaling molecules in a variety of environments.
Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/citología , Proteína MioD/genética , Animales , Técnicas de Cultivo de Célula , Embrión de Pollo , Músculo Esquelético/metabolismo , ARN Mensajero/metabolismoRESUMEN
Purpose: To compare a novel, sustained release formulation and a bolus injection of a targeted nanocarrier for the ability to specifically deplete cells responsible for the development of posterior capsule opacification (PCO) in week-long, dynamic cell cultures. Methods: A novel, injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymer hydrogel was engineered for the sustained release of targeted, nucleic acid nanocarriers loaded with cytotoxic doxorubicin (G8:3DNA:Dox). Human rhabdomyosarcoma (RD) cells were used due to their expression of brain-specific angiogenesis inhibitor 1 (BAI1), a specific marker for the myofibroblasts responsible for PCO. Under constant media flow, nanocarriers were injected into cell cultures as either a bolus or within the hydrogel. Cells were fixed and stained every other day for 7 days to compare targeted depletion of BAI1+ cells. Results: The formulation transitions to a gel at physiological temperatures, is optically clear, noncytotoxic, and can release G8:3DNA:Dox nanocarriers for up to 4 weeks. In RD cell cultures, G8:3DNA:Dox nanocarriers specifically eliminated BAI1+ cells. The bolus nanocarrier dose showed significantly reduced cell depletion overtime, while the sustained release of nanocarriers showed increased cell depletion over time. By day 7, <2% of BAI1+ cells were depleted by the bolus injection and 74.2% BAI1+ cells were targeted by the sustained release of nanocarriers. Conclusions: The sustained release of nanocarriers from the hydrogel allows for improved therapeutic delivery in a dynamic system. This method can offer a more effective and efficient method of prophylactically treating PCO after cataract surgery.
Asunto(s)
Opacificación Capsular , Hidrogeles , ADN , Preparaciones de Acción Retardada , Doxorrubicina , Humanos , Ácido Láctico , PolietilenglicolesRESUMEN
MyoD mRNA is expressed in a subpopulation of cells within the embryonic epiblast. Most of these cells are incorporated into somites and synthesize Noggin. Ablation of MyoD-positive cells in the epiblast subsequently results in the herniation of organs through the ventral body wall, a decrease in the expression of Noggin, MyoD, Myf5, and myosin in the somites and limbs, and an increase in Pax-3-positive myogenic precursors. The addition of Noggin lateral to the somites compensates for the loss of MyoD-positive epiblast cells. Skeletal muscle stem cells that arise in the epiblast are utilized in the somites to promote muscle differentiation by serving as a source of Noggin.
Asunto(s)
Diferenciación Celular/fisiología , Embrión de Mamíferos/citología , Embrión no Mamífero , Epitelio/fisiología , Músculo Esquelético/citología , Proteína MioD/fisiología , Animales , Proteínas Portadoras/metabolismo , Embrión de Pollo , Embrión de Mamíferos/fisiología , Epitelio/anatomía & histología , Extremidades , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Morfogénesis , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Proteína MioD/genética , Factor 5 Regulador Miogénico/metabolismo , Miosinas/metabolismo , Factores de Transcripción Paired Box/metabolismo , Somitos/metabolismo , Células Madre/química , Células Madre/citologíaRESUMEN
Focal brain injury in the form of a needlestick (NS) results in cell death and induces a self-protective response flanking the lesion. Myo/Nog cells are identified by their expression of bone morphogenetic protein inhibitor Noggin, brain-specific angiogenesis inhibitor 1 (BAI1) and the skeletal muscle specific transcription factor MyoD. Myo/Nog cells limit cell death in two forms of retinopathy. In this study, we examined the acute response of Myo/Nog cells to a NS lesion that extended from the rat posterior parietal cortex to the hippocampus. Myo/Nog cells were identified with antibodies to Noggin and BAI1. These cells were the primary source of both molecules in the uninjured and injured brain. One day after the NS, the normally small population of Myo/Nog cells expanded approximately eightfold within a 1 mm area surrounding the lesion. Myo/Nog cells were reduced by approximately 50% along the lesion with an injection of the BAI1 monoclonal antibody and complement. The number of dying cells, identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), was unchanged at this early time point in response to the decrease in Myo/Nog cells. However, increasing the number of Myo/Nog cells within the lesion by injecting BAI1-positive (+) cells isolated from the brains of other animals, significantly reduced cell death and increased the number of NeuN+ neurons compared to brains injected with phosphate buffered saline or exogenous BAI1-negative cells. These findings demonstrate that Myo/Nog cells rapidly react to injury within the brain and increasing their number within the lesion is neuroprotective.
RESUMEN
A subpopulation of cells expresses MyoD mRNA and the cell surface G8 antigen in the epiblast prior to the onset of gastrulation. When an antibody to the G8 antigen was applied to the epiblast, labeled cells were later found in the ocular primordia and muscle and non-muscle forming tissues of the eyes. In the lens, retina and periocular mesenchyme, G8-positive cells synthesized MyoD mRNA and the bone morphogenetic protein inhibitor Noggin. MyoD expressing cells were ablated in the epiblast by labeling them with the G8 MAb and lysing them with complement. Their ablation in the epiblast resulted in eye defects, including anopthalmia, micropthalmia, altered pigmentation and malformations of the lens and/or retina. The right eye was more severely affected than the left eye. The asymmetry of the eye defects in ablated embryos correlated with differences in the number of residual Noggin producing, MyoD-positive cells in ocular tissues. Exogenously supplied Noggin compensated for the ablated epiblast cells. This study demonstrates that MyoD expressing cells serve as a Noggin delivery system to regulate the morphogenesis of the lens and optic cup.
Asunto(s)
Proteínas Portadoras/metabolismo , Ojo/metabolismo , Proteína MioD/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Embrión de Pollo , Ojo/citología , Ojo/embriología , Oftalmopatías/embriología , Oftalmopatías/metabolismo , Oftalmopatías/patología , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación in Situ , Cristalino/anomalías , Cristalino/efectos de los fármacos , Cristalino/metabolismo , Morfogénesis , Proteína MioD/genética , Factores de TiempoRESUMEN
Myo/Nog cells were discovered in the chick embryo epiblast. Their expression of MyoD reflects a commitment to the skeletal muscle lineage and capacity to differentiate into myofibroblasts. Release of Noggin by Myo/Nog cells is essential for normal morphogenesis. Myo/Nog cells rapidly respond to wounding in the skin and eyes. In this report, we present evidence suggesting that Myo/Nog cells phagocytose tattoo ink in tissue sections of human skin and engulf cell corpses in cultures of anterior human lens tissue and magnetic beads injected into the anterior chamber of mice in vivo. Myo/Nog cells are distinct from macrophages in the skin and eyes indicated by the absence of labeling with an antibody to ionized calcium binding adaptor molecule 1. In addition to their primary roles as regulators of BMP signaling and progenitors of myofibroblasts, Myo/Nog cells behave as nonprofessional phagocytes defined as cells whose primary functions are unrelated to phagocytosis but are capable of engulfment.
Asunto(s)
Miofibroblastos/citología , Fagocitos/citología , Células Madre/citología , Animales , Proteínas Portadoras/metabolismo , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Femenino , Humanos , Cristalino/citología , Cristalino/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína MioD/metabolismo , Miofibroblastos/metabolismo , Fagocitos/metabolismo , Fagocitosis , Conejos , Piel/citología , Piel/metabolismo , Células Madre/metabolismoRESUMEN
The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.
Asunto(s)
Proteínas Angiogénicas/biosíntesis , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Sustitución de Aminoácidos , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Encéfalo/citología , Proteínas Portadoras/análisis , Linaje de la Célula , Epítopos/inmunología , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas del Ojo/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Desarrollo de Músculos , Proteína MioD/análisis , Especificidad de Órganos , Conformación Proteica , Dominios Proteicos , Conejos , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Secuencias Repetitivas de Aminoácido , Piel/citología , Especificidad de la Especie , Tatuaje , Adulto JovenRESUMEN
Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway.
Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula , Embrión de Pollo/anatomía & histología , Epitelio/fisiología , Músculo Esquelético/embriología , Proteína MioD/metabolismo , Células Madre Pluripotentes/fisiología , Animales , Biomarcadores , Proteína Morfogenética Ósea 4 , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Proteínas Morfogenéticas Óseas/metabolismo , Cadherinas/metabolismo , Proteínas Portadoras , Células Cultivadas , Embrión de Pollo/fisiología , Medios de Cultivo Condicionados , Proteínas del Citoesqueleto/metabolismo , Epitelio/anatomía & histología , Separación Inmunomagnética , Músculo Esquelético/citología , Proteína MioD/genética , Células Madre Pluripotentes/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Transactivadores/metabolismo , beta CateninaRESUMEN
The osteopathic undergraduate medical education standards have evolved over the past 2 decades to require undergraduate medical student participation in research and scholarly activity. The authors' objective was to review those evolving standards and develop a model for introducing the principles and practice of research that combines core content with experiential learning. They identified fundamental topics pertinent to the research process and herein provide their recommendations for incorporating these topics into the curriculum as self-study, online modules, and team-based and active learning exercises. The authors' proposed educational model would provide an avenue for osteopathic medical schools to meet the requirements for scholarly activity. Increasing students' and residents' knowledge of the research process will lay the foundation for their engagement is research and scholarly activity and their practice of evidence-based medicine.