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Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and ex vivo stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Considering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs. Through homozygous knockout (KO) of MMR pathway key proteins MSH2, MSH3, and MSH6, we reveal that MutSα and MutSß determine PE efficiency in an editing size-dependent manner. Notably, MSH2 perturbation disrupted both MutSα and MutSß complexes, dramatically escalating PE efficiency from base mispair to 10 bases, up to 50 folds. Similarly, impaired MutSα by MSH6 KO improved editing efficiency from single to three base pairs, while defective MutSß by MSH3 KO heightened efficiency from three to 10 base pairs. Thus, the size-dependent effect of MutSα and MutSß on prime editing implies that MMR is a vital PE efficiency determinant in hPSCs and highlights the distinct roles of MutSα and MutSß in its outcome.
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Owing to the advances in genome editing technologies, research on human pluripotent stem cells (hPSCs) have recently undergone breakthroughs that enable precise alteration of desired nucleotide bases in hPSCs for the creation of isogenic disease models or for autologous ex vivo cell therapy. As pathogenic variants largely consist of point mutations, precise substitution of mutated bases in hPSCs allows researchers study disease mechanisms with "disease-in-a-dish" and provide functionally repaired cells to patients for cell therapy. To this end, in addition to utilizing the conventional homologous directed repair system in the knock-in strategy based on endonuclease activity of Cas9 (i.e., 'scissors' like gene editing), diverse toolkits for editing the desirable bases (i.e., 'pencils' like gene editing) that avoid the accidental insertion and deletion (indel) mutations as well as large harmful deletions have been developed. In this review, we summarize the recent progress in genome editing methodologies and employment of hPSCs for future translational applications.
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Edición Génica , Células Madre Pluripotentes , Humanos , Edición Génica/métodos , MutaciónRESUMEN
BACKGROUND: The incorporation of co-stimulatory signaling domains into second-generation chimeric antigen receptors (CARs) significantly enhances the proliferation and persistence of CAR-T cells in vivo, leading to successful clinical outcomes. METHODS: To achieve such functional enhancement in transgenic T-cell receptor-engineered T-cell (TCR-T) therapy, we designed a second-generation TCR-T cell in which CD3ζ genes modified to contain the intracellular domain (ICD) of the 4-1BB receptor were selectively inserted into the CD247 locus. RESULTS: This modification enabled the simultaneous recruitment of key adaptor molecules for signals 1 and 2 on TCR engagement. However, the addition of full-length 4-1BB ICD unexpectedly impaired the expression and signaling of TCRs, leading to suboptimal antitumor activity of the resulting TCR-T cells in vivo. We found that the basic-rich motif (BRM) in the 4-1BB ICD was responsible for the undesirable outcomes, and that fusion of minimal tumor necrosis factor receptor-associated factor (TRAF)-binding motifs at the C-terminus of CD3ζ (zBBΔBRM) was sufficient to recruit TRAF2, the key adaptor molecule in 4-1BB signaling, while retaining the expression and proximal signaling of the transgenic TCR. Consequently, TCR-T cells expressing zBBΔBRM exhibited improved persistence and expansion in vitro and in vivo, resulting in superior antitumor activity in a mouse xenograft model. CONCLUSIONS: Our findings offer a promising strategy for improving the intracellular signaling of TCR-T cells and their application in treating solid tumors.
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Neoplasias , Receptores Quiméricos de Antígenos , Animales , Humanos , Ratones , Modelos Animales de Enfermedad , Neoplasias/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T , Ensayos Antitumor por Modelo de Xenoinjerto , Sitios de UniónRESUMEN
In a conventional two-dimensional (2D) culture method, cells are attached to the bottom of the culture dish and grow into a monolayer. These 2D culture methods are easy to handle, cost-effective, reproducible, and adaptable to growing many different types of cells. However, monolayer 2D cell culture conditions are far from those of natural tissue, indicating the need for a three-dimensional (3D) culture system. Various methods, such as hanging drop, scaffolds, hydrogels, microfluid systems, and bioreactor systems, have been utilized for 3D cell culture. Recently, external physical stimulation-based 3D cell culture platforms, such as acoustic and magnetic forces, were introduced. Acoustic waves can establish acoustic radiation force, which can induce suspended objects to gather in the pressure node region and aggregate to form clusters. Magnetic targeting consists of two components, a magnetically responsive carrier and a magnetic field gradient source. In a magnetic-based 3D cell culture platform, cells are aggregated by changing the magnetic force. Magnetic fields can manipulate cells through two different methods: positive magnetophoresis and negative magnetophoresis. Positive magnetophoresis is a way of imparting magnetic properties to cells by labeling them with magnetic nanoparticles. Negative magnetophoresis is a label-free principle-based method. 3D cell structures, such as spheroids, 3D network structures, and cell sheets, have been successfully fabricated using this acoustic and magnetic stimuli-based 3D cell culture platform. Additionally, fabricated 3D cell structures showed enhanced cell behavior, such as differentiation potential and tissue regeneration. Therefore, physical stimuli-based 3D cell culture platforms could be promising tools for tissue engineering.
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Acústica , Ingeniería de Tejidos , Ingeniería de Tejidos/métodos , Técnicas de Cultivo Tridimensional de Células , Diferenciación Celular , Fenómenos MagnéticosRESUMEN
The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.
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Leber congenital amaurosis (LCA), an inherited retinal degeneration, causes severe visual dysfunction in children and adolescents. In patients with LCA, pathogenic variants, such as RPE65, are evident in specific genes, related to the functions of retinal pigment epithelium and photoreceptors. In contrast to the original Cas9, base editing tools can correct pathogenic substitutions without generation of DNA double-stranded breaks (DSBs). In this study, dual adeno-associated virus (AAV) vectors containing split adenine base editors (ABEs) with trans-splicing intein were prepared for in vivo base editing in retinal degeneration of 12 (rd12) mice, an animal model of LCA, possessing a nonsense mutation of C to T transition in the Rpe65 gene (p.R44X). Subretinal injection of AAV-ABE in retinal pigment epithelial cells of rd12 mice resulted in an A to G transition. The on-target editing was sufficient for recovery of wild-type mRNA, RPE65 protein, and light-induced electrical responses from the retina. Compared with our previous therapeutic editing strategies using Cas9 and prime editing, or with the gene transfer strategy shown in the current study, our results suggest that, considering the editing efficacy and functional recovery, ABEs could be a strong, reliable method for correction of pathogenic variants in the treatment of LCA.
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Precise genome editing of human pluripotent stem cells (hPSCs) is crucial not only for basic science but also for biomedical applications such as ex vivo stem cell therapy and genetic disease modeling. However, hPSCs have unique cellular properties compared to somatic cells. For instance, hPSCs are extremely susceptible to DNA damage, and therefore Cas9-mediated DNA double-strand breaks (DSB) induce p53-dependent cell death, resulting in low Cas9 editing efficiency. Unlike Cas9 nucleases, base editors including cytosine base editor (CBE) and adenine base editor (ABE) can efficiently substitute single nucleotides without generating DSBs at target sites. Here, we found that the editing efficiency of CBE was significantly lower than that of ABE in human embryonic stem cells (hESCs), which are associated with high expression of DNA glycosylases, the key component of the base excision repair pathway. Sequential depletion of DNA glycosylases revealed that high expression of uracil DNA glycosylase (UNG) not only resulted in low editing efficiency but also affected CBE product purity (i.e., C to T) in hESCs. Therefore, additional suppression of UNG via transient knockdown would also improve C to T base substitutions in hESCs. These data suggest that the unique cellular characteristics of hPSCs could determine the efficiency of precise genome editing.
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There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.
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Proteína 9 Asociada a CRISPR/genética , ADN/genética , ARN/genética , Imagen Individual de Molécula/métodos , Emparejamiento Base , Proteína 9 Asociada a CRISPR/química , Proteína 9 Asociada a CRISPR/metabolismo , Clonación Molecular , ADN/química , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Modelos Moleculares , Mutación , Hibridación de Ácido Nucleico , Conformación Proteica , Ingeniería de Proteínas/métodos , ARN/química , ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Ribonucleoprotein (RNP) complex-mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector-mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM-targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.
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Edición Génica , Ribonucleoproteínas , Animales , Sistemas CRISPR-Cas , Citosina/metabolismo , ADN/genética , Ratones , ARN , Ribonucleoproteínas/genéticaRESUMEN
An efficient gene-editing technique for use in human pluripotent stem cells (hPSCs) has great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs remains as a major technical hurdle. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to the high expression of SLC35F2 in these cells. Knockout of SLC35F2 with CRISPR-Cas9, or depletion with siRNAs, made the hPSCs highly resistant to YM155. Simultaneous editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled the survival of genome-edited hPSCs as a result of temporary YM155 resistance, thereby achieving an enriched selection of clonal populations with gene knockout or knock-in. This precise and efficient genome editing approach took as little as 3 weeks and required no cell sorting or the introduction of additional genes, to be a more feasible approach for gene editing in hPSCs due to its simplicity.
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Células Madre Pluripotentes , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Resistencia a Medicamentos/genética , Edición Génica , Genoma Humano , HumanosRESUMEN
The CRISPR-Cas system has undoubtedly revolutionized the genome editing field, enabling targeted gene disruption, regulation, and recovery in a guide RNA-specific manner. In this review, we focus on currently available gene recovery strategies that use CRISPR nucleases, particularly for the treatment of genetic disorders. Through the action of DNA repair mechanisms, CRISPR-mediated DNA cleavage at a genomic target can shift the reading frame to correct abnormal frameshifts, whereas DNA cleavage at two sites, which can induce large deletions or inversions, can correct structural abnormalities in DNA. Homology-mediated or homology-independent gene recovery strategies that require donor DNAs have been developed and widely applied to precisely correct mutated sequences in genes of interest. In contrast to the DNA cleavage-mediated gene correction methods listed above, base-editing tools enable base conversion in the absence of donor DNAs. In addition, CRISPR-associated transposases have been harnessed to generate a targeted knockin, and prime editors have been developed to edit tens of nucleotides in cells. Here, we introduce currently developed gene recovery strategies and discuss the pros and cons of each.
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Sistemas CRISPR-Cas/genética , Genes , ADN/genética , División del ADN , Reparación del ADN/genética , Edición GénicaRESUMEN
Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.
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Aterosclerosis/terapia , Células Madre Mesenquimatosas , Nanopartículas , Animales , Arterias Carótidas , Células Endoteliales , Humanos , Ligadura , Ratones , PorcinosRESUMEN
Electrical stimulation (ES) is known to affect the wound healing process by modulating skin cell behaviors. However, the conventional clinical devices that can generate ES for promoting wound healing require patient hospitalization due to large-scale of the extracorporeal devices. Herein, we introduce a disposable photovoltaic patch that can be applied to skin wound sites to control cellular microenvironment for promoting wound healing by generating ES. In vitro experiment results show that exogenous ES could enhance cell migration, proliferation, expression of extracellular matrix proteins, and myoblast differentiation of fibroblasts which are critical for wound healing. Our disposable photovoltaic patches were attached to the back of skin wound induced mice. Our patch successfully provided ES, generated by photovoltaic energy harvested from the organic solar cell under visible light illumination. In vivo experiment results show that the patch promoted cutaneous wound healing via enhanced host-inductive cell proliferation, cytokine secretion, and protein synthesis which is critical for wound healing process. Unlike the current treatments for wound healing that engage passive healing processes and often are unsuccessful, our wearable photovoltaic patch can stimulate regenerative activities of endogenous cells and actively contribute to the wound healing processes.
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Microambiente Celular , Terapia por Estimulación Eléctrica/métodos , Fototerapia/métodos , Cicatrización de Heridas , Animales , Línea Celular , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , RatonesRESUMEN
Cell therapy has been suggested as a treatment modality for ischemic diseases, but the poor survival and engraftment of implanted cells limit its therapeutic efficacy. To overcome such limitation, we used electrical stimulation (ES) derived from a wearable solar cell for inducing angiogenesis in ischemic tissue. ES enhanced the secretion of angiogenic growth factors and the migration of mesenchymal stem cells (MSCs), myoblasts, endothelial progenitor cells, and endothelial cells in vitro. In a mouse ischemic hindlimb model, ES generated by a solar cell and applied to the ischemic region promoted migration of MSCs toward the ischemic site and upregulated expression of angiogenic paracrine factors (vascular endothelial, basic fibroblast, and hepatocyte growth factors; and stromal cell-derived factor-1α). Importantly, solar cell-generated ES promoted the formation of capillaries and arterioles at the ischemic region, attenuated muscle necrosis and fibrosis, and eventually prevented loss of the ischemic limb. Solar cell ES therapy showed higher angiogenic efficacy than conventional MSC therapy. This study shows the feasibility of using solar cell ES as a novel treatment for therapeutic angiogenesis.
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Vascular endothelial growth factor receptor (VEGFR) and matrix metalloproteinase (MMP) are up-regulated in ischemic tissue and play pivotal roles in promoting angiogenesis. The purpose of the present study was to evaluate two fluorophore-conjugated peptide probes specific to VEGFR and MMP for dual-targeted in vivo monitoring of angiogenesis in a murine model of hindlimb ischemia. To this end, VEGFR-Probe and MMP-Probe were developed by conjugating distinct near-infrared fluorophores to VEGFR-binding and MMP substrate peptides, respectively. VEGFR-Probe exhibited specific binding to VEGFR on HUVECs, and self-quenched MMP-Probe produced strong fluorescence intensity in the presence of MMPs in vitro. Subsequently, VEGFR-Probe and MMP-Probe were successfully utilized for time course in vivo visualization of VEGFR or MMP, respectively. Simultaneous visualization provided information regarding the spatial distribution of these proteins, including areas of co-localization. This dual-targeted in vivo imaging approach will be useful for understanding the detailed mechanism of angiogenesis and for evaluating therapeutic angiogenesis.
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Colorantes Fluorescentes/farmacología , Miembro Posterior/irrigación sanguínea , Isquemia/metabolismo , Imagen Óptica , Péptidos/farmacología , Animales , Femenino , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Miembro Posterior/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Isquemia/patología , Ratones , Péptidos/síntesis química , Péptidos/químicaRESUMEN
Stem cell-conditioned medium (CM), which contains angiogenic factors that are secreted by stem cells, represents a potential therapy for ischemic diseases. Along with stem cells, tumor cells also secrete various angiogenic factors. Here, tumor cells as a cell source of CM for therapeutic angiogenesis was evaluated and the therapeutic efficacy of tumor cell CM in mouse hindlimb ischemia models was demonstrated. CM obtained from a human fibrosarcoma HT1080 cell line culture was compared with CM obtained from a human bone marrow-derived mesenchymal stem cell (MSC) culture. HT1080 CM contained higher concentrations of angiogenic factors compared with MSC CM, which was attributable to the higher cell density that resulted from a much faster growth rate of HT1080 cells compared with MSCs. For use in in vitro and in vivo angiogenesis studies, HT1080 CM was diluted such that HT1080 CM and MSC CM would have the same cell number basis. The two types of CMs induced the same extent of human umbilical vein endothelial cell (HUVEC) proliferation in vitro. The injection of HT1080 CM into mouse ischemic limbs significantly improved capillary density and blood perfusion compared with the injection of fresh medium. Although the therapeutic outcome of HT1080 CM was similar to that of MSC CM, the preparation of CM by tumor cell line culture would be much more efficient due to the faster growth and unlimited life-time of the tumor cell line. These data suggest the potential application of tumor cell CM as a therapeutic modality for angiogenesis and ischemic diseases. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:456-464, 2016.
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Medios de Cultivo Condicionados/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Sarcoma/tratamiento farmacológico , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/química , Femenino , Humanos , Ratones , Ratones Desnudos , Neovascularización Patológica/patología , Sarcoma/patologíaRESUMEN
Gold nanoparticles (AuNPs) have been extensively studied for photothermal cancer therapy because AuNPs can generate heat upon near-infrared irradiation. However, improving their tumor-targeting efficiency and optimizing the nanoparticle size for maximizing the photothermal effect remain challenging. We demonstrate that mesenchymal stem cells (MSCs) can aggregate pH-sensitive gold nanoparticles (PSAuNPs) in mildly acidic endosomes, target tumors, and be used for photothermal therapy. These aggregated structures had a higher cellular retention in comparison to pH-insensitive, control AuNPs (cAuNPs), which is important for the cell-based delivery process. PSAuNP-laden MSCs (MSC-PSAuNPs) injected intravenously to tumor-bearing mice show a 37-fold higher tumor-targeting efficiency (5.6% of the injected dose) and 8.3 °C higher heat generation compared to injections of cAuNPs after irradiation, which results in a significantly enhanced anticancer effect.
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Agregación Celular , Sistemas de Liberación de Medicamentos , Oro/uso terapéutico , Células Madre Mesenquimatosas/citología , Nanopartículas del Metal/uso terapéutico , Neoplasias/terapia , Animales , Femenino , Oro/administración & dosificación , Oro/farmacocinética , Hipertermia Inducida/métodos , Nanopartículas del Metal/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fototerapia/métodosRESUMEN
At high concentrations, manganese (Mn) promotes cellular neurodevelopment but causes toxicity. Here, we report that Mn ion at high concentrations can be delivered to pheochromocytoma 12 (PC12) cells using gold nanoparticles (AuNPs) to enhance cellular neurodevelopment without toxicity. Mn(2+) release from AuNPs was designed to be pH-responsive so that low pH condition of the cell endosomes can trigger in situ release of Mn(2+) from AuNPs after cellular uptake of Mn-incorporated AuNPs (MnAuNPs). Due to the differences in reduction potentials of Mn and Au, only Mn ionized and released while Au remained intact when MnAuNPs were uptaken by cells. Compared to PC12 cells treated with a high concentration of free Mn(2+), PC12 cells treated with an equal concentration of MnAuNPs resulted in significantly enhanced cellular neurodevelopment with decreased apoptosis and necrosis. Treatment with a high concentration of free Mn(2+) led to an abrupt consumption of a large amount of ATP for the intracellular transport of Mn(2+) through the ion channel of the cell membrane and to mitochondrial damage caused by the high intracellular concentration of Mn(2+), both of which resulted in cell necrosis and apoptosis. In contrast, MnAuNP-treated cells consumed much smaller amount of ATP for the intracellular transport of MnAuNPs by endocytosis and showed pH-triggered in situ release of Mn(2+) from the MnAuNPs in the endosomes of the cells, both of which prevented the cell death caused by ATP depletion and mitochondrial damage. To our knowledge, this is the first report on the use of AuNPs as a vehicle for pH-responsive, intracellular delivery of metal ion, which may open a new window for drug delivery and clinical therapy.
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Diferenciación Celular , Sistemas de Liberación de Medicamentos , Oro/química , Manganeso/química , Nanopartículas del Metal/química , Neuronas/citología , Adenosina Trifosfato/química , Animales , Apoptosis , Membrana Celular/metabolismo , Endocitosis , Concentración de Iones de Hidrógeno , Iones , Ácido Láctico/química , Mitocondrias/metabolismo , Mitocondrias/patología , Necrosis , Neuronas/efectos de los fármacos , Células PC12/citología , Células PC12/efectos de los fármacos , RatasRESUMEN
Implantation of ex vivo expanded and osteogenically differentiated mesenchymal stem cells (MSCs) for bone regeneration has drawbacks for clinical applications, such as poor survival of implanted cells and increased treatment expenses. As a new approach for bone regeneration that can circumvent these limitations, we propose dual delivery of substance P (SP) and bone morphogenetic protein-2 (BMP-2) to facilitate endogenous stem cell recruitment to bone defects by SP and subsequent in situ osteogenic differentiation of those cells by BMP-2. A heparin-conjugated fibrin (HCF) gel enabled dual delivery with fast release of SP and slow release of BMP-2, which would be ideal for prompt recruitment of endogenous stem cells in the first stage and time-consuming osteogenic differentiation of the recruited stem cells in the second stage. The HCF gels with SP and/or BMP-2 were implanted into mouse calvarial defects for 8 weeks. Local delivery of SP to the calvarial defects using HCF gel was more effective in recruiting MSCs to the calvarial defects than intraperitoneal or intravenous administration of SP. Many of the cells recruited by SP underwent osteogenic differentiation through local delivery of BMP-2. The efficacy of in vivo bone regeneration was significantly higher in the SP/BMP-2 dual delivery group. The dual delivery of SP and BMP-2 using the HCF gel therefore has potential as an effective bone regeneration strategy.