RESUMEN
Antibiotics, particularly the ß-lactams, are a cornerstone of modern medicine. However, the rise of bacterial resistance to these agents, particularly through the actions of ß-lactamases, poses a significant threat to our continued ability to effectively treat infections. Metallo-ß-lactamases (MBLs) are of particular concern due to their ability to hydrolyze a wide range of ß-lactam antibiotics including carbapenems. For this reason there is growing interest in the development of MBL inhibitors as well as novel antibiotics that can overcome MBL-mediated resistance. Here, we report the synthesis and evaluation of novel conjugates that combine a carbapenem (meropenem or ertapenem) with a recently reported MBL inhibiting indole carboxylate scaffold. These hybrids were found to display potent inhibition against MBLs including NDM-1 and IMP-1, with IC50 values in the low nanomolar range. However, their antibacterial potency was limited. Mechanistic studies suggest that despite maintaining effective MBL inhibiting activity in live bacteria, the new carbapenem/MBL inhibitor conjugates have a reduced ability to engage with the bacterial target of the ß-lactams.
RESUMEN
Background: We aimed to investigate the causal association between TIM-3, an immune checkpoint inhibitor, and anterior uveitis (AU), as well as associated systemic immune diseases. Materials and methods: We performed two-sample Mendelian randomization (MR) analyses to estimate the causal effects of TIM-3 on AU and three associated systemic diseases, namely ankylosing spondylitis (AS), Crohn's disease (CD), and ulcerative colitis (UC). Single-nucleotide polymorphisms (SNPs) associated with AU, AS, CD, and UC were selected as the outcomes: AU GWAS with 2,752 patients with acute AU accompanied with AS (cases) and 3,836 AS patients (controls), AS GWAS with 968 cases and 336,191 controls, CD GWAS with 1,032 cases and 336,127 controls, and UC GWAS with 2,439 cases and 460,494 controls. The TIM-3 dataset was used as the exposure (n = 31,684). Four MR methods, namely, inverse-variance weighting (IVW), MR-Egger regression, weighted median, and weighted mode, were used in this study. Comprehensive sensitivity analyses were conducted to estimate the robustness of identified associations and the potential impact of horizontal pleiotropy. Results: Our studies show that TIM-3 is significantly associated with CD using the IVW method (OR = 1.001, 95% CI = 1.0002-1.0018, P-value = 0.011). We also found that TIM-3 may be a protective factor for AU although these results lacked significance (OR = 0.889, 95% CI = 0.631-1.252, P-value = 0.5). No association was observed between the genetic predisposition to particular TIM-3 and susceptibility to AS or UC in this study. No potential heterogeneities or directional pleiotropies were observed in our analyses. Conclusion: According to our study, a small correlation was observed between TIM-3 expression and CD susceptibility. Additional studies in different ethnic backgrounds will be necessary to further explore the potential roles and mechanisms of TIM-3 in CD.
RESUMEN
OBJECTIVES: To develop a risk prediction model for severe adenovirus pneumonia (AVP) in children, and to explore the appropriate timing for intravenous immunoglobulin (IVIG) therapy for severe AVP. METHODS: Medical data of 1 046 children with AVP were retrospectively analyzed, and a risk prediction model for severe AVP was established using multivariate logistic regression. The model was validated with 102 children with AVP. Then, 75 children aged ≤14 years who were considered at risk of developing severe AVP by the model were prospectively enrolled and divided into three groups (A, B and C) in order of visit, with 25 children in each group. Group A received symptomatic supportive therapy only. With the exception of symptomatic supportive therapy, group B received IVIG treatment at a dose of 1g/(kg·d) for 2 consecutive days, before progressing to severe AVP. With the exception of symptomatic supportive therapy, group C received IVIG treatment at a dose of 1 g/(kg·d) for 2 consecutive days after progressing to severe AVP. Efficacy and related laboratory indicators were compared among the three groups after treatment. RESULTS: Age<18.5 months, underlying diseases, fever duration >6.5 days, hemoglobin level <84.5 g/L, alanine transaminase level >113.5 U/L, and co-infection with bacteria were the six variables that entered into the risk prediction model for severe AVP. The model had an area under the receiver operating characteristic curve of 0.862, sensitivity of 0.878, and specificity of 0.848. The Hosmer-Lemeshow test showed good consistency between the predicted values and the actual observations (P>0.05). After treatment, group B had the shortest fever duration and hospital stay, the lowest hospitalization costs, the highest effective rate of treatment, the lowest incidence of complications, the lowest white blood cell count and interleukin (IL)-1, IL-2, IL-6, IL-8, IL-10 levels, and the highest level of tumor necrosis factor alpha (P<0.05). CONCLUSIONS: The risk model for severe AVP established in this study has good value in predicting the development of severe AVP. IVIG therapy before progression to severe AVP is more effective in treating AVP in children.
Asunto(s)
Infecciones por Adenoviridae , Neumonía Viral , Niño , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Estudios Prospectivos , Estudios Retrospectivos , Infecciones por Adenoviridae/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , AdenoviridaeRESUMEN
Microglia are known to play essential roles in the development, progression and treatment of diverse neurodegenerative diseases in the central nervous system, including the retina, brain and spinal cord. Recently, brain-induced microglia-like cells (iMGs) have been generated from human pluripotent stem cells (hPSCs); however, retinal microglia have yet to be developed in vitro. In this study, by mimicking in vivo microglial development, we established a simplified approach to differentiate hPSCs into high purity (>90%) iMGs. The iMGs express microglia-specific markers, release cytokines upon stimulation, and are capable of phagocytizing bacteria. When co-cultured with three-dimensional human retinal organoids (hROs), iMGs migrated into the hROs, tended to differentiate into resident retinal microglia, and simultaneously induced apoptosis in some neural cells. Notably, the resident iMGs in the hROs formed sparse web-like structures beneath the photoreceptor cell layer, resembling microglia's orientation in human retina. In conclusion, we developed a simplified and efficient method to generate microglia from human pluripotent stem cells, and we report the first derivation of retinaresident microglia in vitro, providing a new source of human retinal microglia for developmental and disease studies and regenerative therapeutics.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Diferenciación Celular , Humanos , Microglía , RetinaRESUMEN
A novel pleuromutilin derivative, 22-(4-(2-(4-nitrophenyl-piperazin-1-yl)-acetyl)-piperazin-1-yl)-22-deoxypleuromutilin (NPDM), was synthesized in our laboratory and proved excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). In this study, more methods were used to further study its preliminary pharmacological effect. The antibacterial efficacy and toxicity of NPDM were evaluated using tiamulin as the reference drug. The in vitro antibacterial activity study showed that NPDM is a potent bactericidal agent against MRSA that induced time-dependent growth inhibition and a concentration-dependent post-antibiotic effect (PAE). Toxicity determination showed that the cytotoxicity of NPDM was slightly higher than that of tiamulin, but the acute oral toxicity study proved that NPDM was a low-toxic compound. In an in vivo antibacterial effect study, NPDM exhibited a better therapeutic effect than tiamulin against MRSA in a mouse thigh infection model as well as a mouse systemic infection model with neutropenia. The 50% effective dose (ED50) of NPDM in a Galleria mellonella infection model was 50.53 mg/kg. The pharmacokinetic properties of NPDM were also measured, which showed that NPDM was a rapid elimination drug in mice.
Asunto(s)
Antibacterianos/farmacología , Diterpenos/farmacología , Nitrofenoles/farmacología , Piperazina/farmacología , Compuestos Policíclicos/farmacología , Animales , Línea Celular , Insectos/microbiología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Pruebas de Sensibilidad Microbiana/métodos , Ratas , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , PleuromutilinasRESUMEN
BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly due in large part to age-dependent atrophy of retinal pigment epithelium (RPE) cells. RPE cells form a monolayer located between the choroid and the outer segments of photoreceptors, playing multifarious roles in maintenance of visual function. Allogeneically induced pluripotent stem cell-derived RPE (iPSC-RPE or iRPE) has become a potential approach for providing an abundant source of donors for clinical cell products. Transplantation of iRPE has been proven effective in rescuing impaired retinas in Royal College of Surgeons (RCS) rats after approximately 5 to 6 weeks. Here, we explore the long-term (19 weeks) safety and efficacy of human iRPE cell transplantation in pre-clinical animal models. METHODS: The expression of human RPE-specific markers in iRPE cells was determined using immunofluorescence staining. For the proliferative test, Ki-67 expression was also verified by immunofluorescence and flow cytometric analysis. Then, iRPE cells were transplanted into the subretinal space of immune-deficient NOD/SCID/IL-2Rgcnull (NSG) mice to assess their safety. To evaluate whether the transplanted cells could survive and rescue visual function, we performed color fundus photography, focal electroretinogram and immunostaining after delivering iRPE cells into the subretinal space of RCS rats. RESULTS: Human iRPE cells expressed native RPE-specific markers, such as microphthalmia-associated transcription factor (MiTF), retinal pigment epithelium-specific 65-kDa protein (RPE65) and tight-junction associated structural protein (ZO-1), and their proliferative capacity (Ki-67 expression) was poor after 25 days of induction. A tumorigenicity test revealed no tumor formation or abnormal proliferation in the immunodeficient mice after subretinal injection of 5×105 iRPE cells. The transplanted iRPE cells survived for at least 19 weeks and maintained visual function for 15 weeks. CONCLUSIONS: In the present study, we provided further evidence for the use of human iRPE transplantation to treat retinal degenerative disease in pre-clinical animal models. Therefore, we consider human iRPE cells a promising source of cell replacement therapy for AMD.
RESUMEN
BACKGROUND: Significant progress has been made in cell replacement therapy for neural retinal diseases using retinal cells differentiated from human pluripotent stem cells. Low tumorigenicity and the ability to mature to form synaptic junctions make precursor cells a promising donor source. Here, we attempted to improve the yield of photoreceptor precursor cells in three-dimensional retinal organoids from human embryonic stem cells (hESCs). METHODS: A CRX-tdTomato-tagged hESC line was generated to track retinal precursors in 3D retinal organoids. COCO, a multifunctional antagonist of the Wnt, TGF-ß, and BMP pathways, was employed to 3D organoid differentiation schemes for enhanced photoreceptor precursor cells. Organoid fluorescence intensity measurement was used to monitor retinalization tendency with the number of precursors further checked by flow cytometry. Signature gene expression during organoid differentiation were assessed by qPCR and immunocytochemistry after COCO supplementation. RESULTS: CRX-positive cells can be spatiotemporally tracked by tdTomato without affecting retinalization during retinal organoid differentiation. Fluorescence intensity of organoids, which turned out highly consistent with flow cytometry measurement, allowed us to determine the differentiation efficiency of precursors during organoid culturing directly. Using COCO as an auxiliary supplement, rather than alone, can yield an increased number of photoreceptor precursors in the early stage of organoid differentiation. Over a longer time-frame, photoreceptor precursors enhanced their fate of cones and decreased fate of rods after treatment with COCO. CONCLUSIONS: Tracing with the CRX-reporter system showed that in retinal organoids derived from human pluripotent stem cells, COCO increased the differentiation efficiency of photoreceptor precursors and cones.
Asunto(s)
Células Madre Embrionarias Humanas , Diferenciación Celular , Cocos , Humanos , Organoides , RetinaRESUMEN
Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1ß secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1ß levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1ß secretion after PVL exposure and controls S. aureus lung burdens.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Exotoxinas/antagonistas & inhibidores , Células Madre Pluripotentes Inducidas/citología , Leucocidinas/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/efectos de los fármacos , Staphylococcus aureus , Animales , Antígeno CD11b/inmunología , Sistemas CRISPR-Cas , Diferenciación Celular , Células Cultivadas , Exotoxinas/deficiencia , Técnicas de Sustitución del Gen , Humanos , Interleucina-1beta/metabolismo , Antígenos Comunes de Leucocito/fisiología , Pulmón/inmunología , Pulmón/microbiología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Fragmentos de Péptidos/inmunología , Neumonía Estafilocócica/inmunología , Subunidades de Proteína , Receptor de Anafilatoxina C5a/deficiencia , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/fisiología , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/fisiologíaRESUMEN
BACKGROUND: Recently, the effects of erector spinae plane block on postoperative pain have become increasingly controversial. This meta-analysis compared the effects of ESP block versus placebo on postoperative analgesia and side effects to determine whether the new technique is a reliable alternative for pain management. METHODS: PubMed, Cochrane Library, Embase, China National Knowledge Infrastructure (CNKI), and Wanfang Database were searched for clinical studies investigating the analgesic effect of ESP block versus placebo. The primary outcomes included the visual analogue scale (VAS) at rest and during movement, as well as the postoperative morphine consumption in 24 h, and the secondary outcome was the rate of postoperative nausea and vomiting (PONV). The choice of using the fixed or random-effects model depended on whether the heterogeneity tested by I2 statistic was more than 50%. Seeking sources of heterogeneity and exploring the effect of clinical details on the final result were performed by subgroup analysis. Additionally, the test for stability of the pooled result was realized by sensitivity analysis. Finally, we evaluated the quality of the evidence for the outcomes. STATA 13.0 software was selected as the main analysis software in the meta-analysis. RESULTS: Eighteen randomized controlled trials (RCTs) comprising 1041 patients were reviewed. This meta-analysis showed that ESP block could significantly reduce patients' pain scores at 1 h, 6 h, 12 h, and 24 h after surgery at rest or during movement; 24-h postoperative morphine consumption; and the incidence of PONV. CONCLUSIONS: ESP block as a novel technique exhibited superior postoperative analgesic effects, reducing the postoperative complications in spinal, thoracic, and abdominal surgeries during the early postoperative period. However, as a new nerve block technique, numerous large-sized RCTs are needed for further research.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Morfina/uso terapéutico , Bloqueo Nervioso/métodos , Manejo del Dolor/métodos , Dolor Postoperatorio/tratamiento farmacológico , Adulto , Femenino , Humanos , Masculino , Dimensión del Dolor , Dolor Postoperatorio/etiología , Náusea y Vómito Posoperatorios/inducido químicamente , Náusea y Vómito Posoperatorios/epidemiología , Periodo Posoperatorio , Ensayos Clínicos Controlados Aleatorios como Asunto , Resultado del TratamientoRESUMEN
BACKGROUND: How to effectively control the postoperative pain of patients is extremely important to clinicians. Transversus abdominis plane (TAP) block is a novel analgesic method reported to greatly decrease postoperative pain. However, in many areas, there still exists a phenomenon of surgeons using wound infiltration (WI) with conventional local anesthetics (not liposome anesthetics) as the main means to decrease postoperative pain because of traditional wisdom or convenience. Here, we compared the analgesic effectiveness of the two different methods to determine which method is more suitable for adult patients. Materials and methods. A systematic review and meta-analysis of randomized controlled trials (RCTs) comparing TAP block and WI without liposome anesthetics in adult patients were performed. Frequently used databases were extensively searched. The main outcomes were postoperative pain scores in different situations (at rest or during movement) and the time until the first use of rescue analgesics. The secondary outcomes were postoperative nausea and vomiting (PONV) incidence and patient satisfaction scores. RESULTS: Fifteen studies with 983 participants met the inclusion criteria and were included in the present study. The heterogeneity in the final analysis regarding the pain score was low to moderate. The major results of the sensitivity analysis were stable. WI had the same analgesic effect as TAP block only at the one-hour postoperative time point (mean difference = -0.32, 95% confidence interval (-0.87, 0.24), P = 0.26) and was associated with a shorter time until the first rescue analgesic and poorer patient satisfaction. CONCLUSION: TAP block results in a more effective and steady analgesic effect than WI with conventional local anesthetics in adult patients from the early postoperative period and obtains higher patient satisfaction.
Asunto(s)
Músculos Abdominales/cirugía , Anestésicos Locales/uso terapéutico , Bloqueo Nervioso/métodos , Dolor Postoperatorio/etiología , Adulto , Analgésicos , Anestesia/métodos , Bases de Datos Factuales , Humanos , Dimensión del Dolor , Dolor Postoperatorio/terapia , Náusea y Vómito Posoperatorios , Ensayos Clínicos Controlados Aleatorios como Asunto , Resultado del TratamientoRESUMEN
Although an increasing number of disease genes have been identified, the exact cellular mechanisms of retinitis pigmentosa (RP) remain largely unclear. Retinal organoids (ROs) derived from the induced pluripotent stem cells (iPSCs) of patients provide a potential but unvalidated platform for deciphering disease mechanisms and an advantageous tool for preclinical testing of new treatments. Notably, early-onset RP has been extensively recapitulated by patient-iPSC-derived ROs. However, it remains a challenge to model late-onset disease in a dish due to its chronicity, complexity, and instability. Here, we generated ROs from late-onset RP proband-derived iPSCs harboring a PDE6B mutation. Transcriptome analysis revealed a remarkably distinct gene expression profile in the patient ROs at differentiation day (D) 230. Changes in the expression genes regulating cGMP hydrolysis prompted the elevation of cGMP levels, which was verified by a cGMP enzyme-linked immunosorbent assay (ELISA) in patient ROs. Furthermore, significantly higher cGMP levels in patient ROs than in control ROs at D193 and D230 might lead to impaired formation of synaptic connections and the connecting cilium in photoreceptor cells. In this study, we established the first late-onset RP model with a consistent phenotype using an in vitro cell culture system and provided new insights into the PDE6B-related mechanism of RP.
RESUMEN
As exosomes have been playing an increasingly important role in the diagnosis, treatment and prognosis of diseases, the analysis of exosome contents becomes more crucial. Therefore, the development of a cost-effective and simple exosome separation method that achieves high purity is urgently needed, and it is vital for further research in cancer. In this work, we constructed a DNA-AuNP-based satellite network which integrates low-speed centrifugal exosome isolation, detection and protein analysis. The rolling circle amplification (RCA) reaction is used to produce a long-chain DNA hairpin structure comprising a plurality of functional domains, such as CD63 aptamer sequences, linker sequences, and spacer sequences with complementary base pairs to form a hairpin structure. When the CD63 aptamers bind to exosomes, the hairpin structure changes its conformation, exposing the linker sequences (AuNP binding sequence). Then the probe on the surface of AuNPs combines with the long-chain DNA by the toehold-mediated strand displacement reaction, releasing the fluorescent labeled complementary probe as the detection signal and simultaneously forming the DNA-AuNP-based satellite network. Thus, exosomes can be isolated by low-speed centrifugation. The formation of the DNA-AuNP-based satellite network was confirmed by transmission electron microscopy and confocal fluorescence microscopy. In addition, we established a standard curve for exosome detection which showed good linearity of the fluorescence ratio vs. log(exosome concentration). LC/MS for protein profiling of the captured exosomes demonstrated that our method has potential application in the field of exosome research.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN de Neoplasias/química , Exosomas/metabolismo , Colorantes Fluorescentes/química , Oro/química , Nanopartículas del Metal/química , Exosomas/química , Células Hep G2 , HumanosRESUMEN
An increasing number of studies have found that circulating exosomes play a vital role in the occurrence and metastasis of cancer. Therefore, a direct, sensitive and specific method for detection of tumor exosomes will contribute to the diagnosis and prognosis of cancer. In this work, we take advantage of the facile adaptability of aptamers to design an exosome quantitative method, which converts an exosome capture event to nucleic acid detection. With the help of a hairpin DNA cascade reaction (HDCR) and easy accessibility of DNA dendrimer self-assembly, dual signal amplification was achieved. A CD63 aptamer linked via a DNA probe to magnetic beads acts as the capture component. In the presence of target exosomes, aptamers identify and combine with exosomes, releasing the DNA probe as a trigger to initiate the HDCR (the first signal amplification process) by opening hairpin DNA (HP1) bound to gold nanoparticles (AuNPs). Fluorescently-labeled DNA dendrimers concatenate with HP1 as the second signal amplification stage to increase the signal-to-noise ratio. Under the optimal conditions, our method achieved a good linear response for HepG2 cell-derived exosomes in a concentration range from 1.75 × 103 to 7.0 × 106 particles per µL with a detection limit of 1.16 × 103 particles per µL. It also shows a good performance for detection of exosomes in biological samples.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN de Neoplasias/genética , Dendrímeros/química , Exosomas/genética , Neoplasias Hepáticas/genética , Nanopartículas del Metal/química , Aptámeros de Nucleótidos/genética , Sondas de ADN , ADN de Neoplasias/química , Oro/química , Voluntarios Sanos , Células Hep G2 , Humanos , Límite de Detección , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/patologíaRESUMEN
Four new diarylheptanoids, (1S, 3R, 5R, 6R)-1, 5-epoxy-3, 6 dihydroxy-1-(4-hydroxy-3, 5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl) heptane (1), (1R, 3R, 5S)-1, 5-epoxy-3-acetoxy-1-(4, 5-dihydroxy-3-methoxyphenyl)-7-(3, 4- hydroxyphenyl) heptane (2), (3R, 5S, 6R, 7S)-3, 6-epoxy-7-hydroxyl-1-(4-hydroxyphenyl)-7-(3-methoxy-4-hydroxyphenyl) heptane (3), (E)-3-keto-1-(3-methoxy-4-hydroxyphenyl)-7-(4, 5-dihydroxy-3-methoxyphenyl)-4- heptene (4), were isolated from Rhizoma Zingiberis, and their structures were determined based on HR-ESI-MS and extensive spectroscopic techniques (UV, IR, 1D-NMR and 2D-NMR). Compounds 1-4 exhibited no cytotoxicity against HepG2 cell lines.
Asunto(s)
Diarilheptanoides/aislamiento & purificación , Rizoma/química , Zingiber officinale/química , Diarilheptanoides/química , Diarilheptanoides/farmacología , Células Hep G2 , HumanosRESUMEN
Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases, such as age-related macular degeneration (AMD), Stargardt's disease, retinitis pigmentosa (RP) and glaucoma. These diseases are all characterized by the degeneration of one or two retinal cell types that cannot regenerate spontaneously in humans. Aberrant retinal pigment epithelial (RPE) cells can be observed through optical coherence tomography (OCT) in AMD patients. In RP patients, the morphological and functional abnormalities of RPE and photoreceptor layers are caused by a genetic abnormality. Stargardt's disease or juvenile macular degeneration, which is characterized by the loss of the RPE and photoreceptors in the macular area, causes central vision loss at an early age. Loss of retinal ganglion cells (RGCs) can be observed in patients with glaucoma. Once the retinal cell degeneration is triggered, no treatments can reverse it. Transplantation-based approaches have been proposed as a universal therapy to target patients with various concomitant diseases. Both the replacement of dead cells and neuroprotection are strategies used to rescue visual function in animal models of retinal degeneration. Diverse retinal cell types derived from pluripotent stem cells, including RPE cells, photoreceptors, RGCs and even retinal organoids with a layered structure, provide unlimited cell sources for transplantation. In addition, mesenchymal stem cells (MSCs) are multifunctional and protect degenerating retinal cells. The aim of this review is to summarize current findings from preclinical and clinical studies. We begin with a brief introduction to retinal degenerative diseases and cell death in diverse diseases, followed by methods for retinal cell generation. Preclinical and clinical studies are discussed, and future concerns about efficacy, safety and immunorejection are also addressed.
Asunto(s)
Células Madre Pluripotentes/trasplante , Degeneración Retiniana/terapia , Trasplante de Células Madre/métodos , Animales , Estudios Clínicos como Asunto , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Myelin-associated glycoprotein (MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase (ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.
RESUMEN
Retinitis pigmentosa (RP) is an irreversible, inherited retinopathy in which early-onset nyctalopia is observed. Despite the genetic heterogeneity of RP, RPGR mutations are the most common causes of this disease. Here, we generated induced pluripotent stem cells (iPSCs) from three RP patients with different frameshift mutations in the RPGR gene, which were then differentiated into retinal pigment epithelium (RPE) cells and well-structured retinal organoids possessing electrophysiological properties. We observed significant defects in photoreceptor in terms of morphology, localization, transcriptional profiling, and electrophysiological activity. Furthermore, shorted cilium was found in patient iPSCs, RPE cells, and three-dimensional retinal organoids. CRISPR-Cas9-mediated correction of RPGR mutation rescued photoreceptor structure and electrophysiological property, reversed the observed ciliopathy, and restored gene expression to a level in accordance with that in the control using transcriptome-based analysis. This study recapitulated the pathogenesis of RPGR using patient-specific organoids and achieved targeted gene therapy of RPGR mutations in a dish as proof-of-concept evidence.