RESUMO
There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.
Assuntos
Brônquios/citologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/terapia , Células Epiteliais/transplante , Edição de Genes/métodos , Brônquios/metabolismo , Brônquios/transplante , Diferenciação Celular , Células Cultivadas , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Mutação , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
It has previously been shown that engineered zinc finger nucleases (ZFNs) can be packaged into adeno-associated viruses (AAVs) and delivered intravenously into mice, non-human primates, and most recently, humans to induce highly efficient therapeutic genome editing in the liver. Lipid nanoparticles (LNPs) are synthetic delivery vehicles that enable repeat administration and are not limited by the presence of preexisting neutralizing antibodies in patients. Here, we show that mRNA encoding ZFNs formulated into LNP can enable >90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene, at mRNA doses 10-fold lower than has ever been reported. Additionally, co-delivering mRNA-LNP containing ZFNs targeted to intron 1 of the ALB locus with AAV packaged with a promoterless human IDS or FIX therapeutic transgene can result in high levels of targeted integration and subsequent therapeutically relevant levels of protein expression in mice. Finally, we show repeat administration of ZFN mRNA-LNP after a single AAV donor dose results in significantly increased levels of genome editing and transgene expression compared to a single dose. These results demonstrate LNP-mediated ZFN mRNA delivery can drive highly efficient levels of in vivo genome editing and can potentially offer a new treatment modality for a variety of diseases.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Edição de Genes/métodos , Nanopartículas/administração & dosagem , RNA Mensageiro/administração & dosagem , Nucleases de Dedos de Zinco/administração & dosagem , Animais , Células Cultivadas , Dependovirus/genética , Feminino , Técnicas de Inativação de Genes , Vetores Genéticos , Hepatócitos/metabolismo , Íntrons/genética , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pré-Albumina/genética , Pró-Proteína Convertase 9/genética , RNA Mensageiro/genética , Transgenes/genética , Nucleases de Dedos de Zinco/farmacologiaRESUMO
Neural stem cells (NSC) in two regions of the adult mammalian brain--the subventricular zone (SVZ) and hippocampus--continuously generate new neurons, enabled by a complex repertoire of factors that precisely regulate the activation, proliferation, differentiation, and integration of the newborn cells. A growing number of studies also report low-level neurogenesis in regions of the adult brain outside these established neurogenic niches--potentially via NSC recruitment or activation of local, quiescent NSCs--under perturbations such as ischemia, cell death, or viral gene delivery of proneural growth factors. We have explored whether implantation of engineered biomaterials can stimulate neurogenesis in normally quiescent regions of the brain. Specifically, recombinant versions of factors found within the NSC microenvironment, Sonic hedgehog, and ephrin-B2 were conjugated to long polymers, thereby creating highly bioactive, multivalent ligands that begin to emulate components of the neurogenic niche. In this engineered biomaterial microenvironment, new neuron formation was observed in normally non-neurogenic regions of the brain, the striatum, and the cortex, and combining these multivalent biomaterials with stromal cell-derived factor-1α increased neuronal commitment of newly divided cells seven- to eightfold in these regions. Additionally, the decreased hippocampal neurogenesis of geriatric rodents was partially rescued toward levels of young animals. We thus demonstrate for the first time de novo neurogenesis in both the cortex and striatum of adult rodents stimulated solely by delivery of synthetic biomaterial forms of proteins naturally found within adult neurogenic niches, offering the potential to replace neurons lost in neurodegenerative disease or injury as an alternative to cell implantation.
Assuntos
Materiais Biocompatíveis/farmacologia , Encéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Animais , Materiais Biocompatíveis/química , Encéfalo/citologia , Encéfalo/metabolismo , Efrina-B2/química , Efrina-B2/genética , Efrina-B2/metabolismo , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Imuno-Histoquímica , Ventrículos Laterais/citologia , Ventrículos Laterais/efeitos dos fármacos , Ventrículos Laterais/metabolismo , Camundongos , Microscopia Confocal , Neostriado/citologia , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , RatosRESUMO
Between 1990 and 2007, 15 southern white (Ceratotherium simum simum) and black (Diceros bicornis) rhinoceroses on average were killed illegally every year in South Africa. Since 2007 illegal killing of southern white rhinoceros for their horn has escalated to >950 individuals/year in 2013. We conducted an ecological-economic analysis to determine whether a legal trade in southern white rhinoceros horn could facilitate rhinoceros protection. Generalized linear models were used to examine the socioeconomic drivers of poaching, based on data collected from 1990 to 2013, and to project the total number of rhinoceroses likely to be illegally killed from 2014 to 2023. Rhinoceros population dynamics were then modeled under 8 different policy scenarios that could be implemented to control poaching. We also estimated the economic costs and benefits of each scenario under enhanced enforcement only and a legal trade in rhinoceros horn and used a decision support framework to rank the scenarios with the objective of maintaining the rhinoceros population above its current size while generating profit for local stakeholders. The southern white rhinoceros population was predicted to go extinct in the wild <20 years under present management. The optimal scenario to maintain the rhinoceros population above its current size was to provide a medium increase in antipoaching effort and to increase the monetary fine on conviction. Without legalizing the trade, implementing such a scenario would require covering costs equal to approximately $147,000,000/year. With a legal trade in rhinoceros horn, the conservation enterprise could potentially make a profit of $717,000,000/year. We believe the 35-year-old ban on rhinoceros horn products should not be lifted unless the money generated from trade is reinvested in improved protection of the rhinoceros population. Because current protection efforts seem to be failing, it is time to evaluate, discuss, and test alternatives to the present policy.
Assuntos
Comércio , Conservação dos Recursos Naturais , Política Ambiental/legislação & jurisprudência , Perissodáctilos , Animais , Modelos Teóricos , Dinâmica Populacional , Fatores Socioeconômicos , África do SulRESUMO
BACKGROUND: α1-antitrypsin deficiency is most commonly caused by a mutation in exon-7 of SERPINA1 (SA1-ATZ), resulting in hepatocellular accumulation of a misfolded variant (ATZ). Human SA1-ATZ-transgenic (PiZ) mice exhibit hepatocellular ATZ accumulation and liver fibrosis. We hypothesized that disrupting the SA1-ATZ transgene in PiZ mice by in vivo genome editing would confer a proliferative advantage to the genome-edited hepatocytes, enabling them to repopulate the liver. METHODS: To create a targeted DNA break in exon-7 of the SA1-ATZ transgene, we generated 2 recombinant adeno-associated viruses (rAAV) expressing a zinc-finger nuclease pair (rAAV-ZFN), and another rAAV for gene correction by targeted insertion (rAAV-TI). PiZ mice were injected i.v. with rAAV-TI alone or the rAAV-ZFNs at a low (7.5×1010vg/mouse, LD) or a high dose (1.5×1011vg/mouse, HD), with or without rAAV-TI. Two weeks and 6 months after treatment, livers were harvested for molecular, histological, and biochemical analyses. RESULTS: Two weeks after treatment, deep sequencing of the hepatic SA1-ATZ transgene pool showed 6%±3% or 15%±4% nonhomologous end joining in mice receiving LD or HD rAAV-ZFN, respectively, which increased to 36%±12% and 36%±12%, respectively, 6 months after treatment. Two weeks postinjection of rAAV-TI with LD or HD of rAAV-ZFN, repair by targeted insertion occurred in 0.10%±0.09% and 0.25%±0.14% of SA1-ATZ transgenes, respectively, which increased to 5.2%±5.0% and 33%±13%, respectively, 6 months after treatment. Six months after rAAV-ZFN administration, there was a marked clearance of ATZ globules from hepatocytes, and resolution of liver fibrosis, along with reduction of hepatic TAZ/WWTR1, hedgehog ligands, Gli2, a TIMP, and collagen content. CONCLUSIONS: ZFN-mediated SA1-ATZ transgene disruption provides a proliferative advantage to ATZ-depleted hepatocytes, enabling them to repopulate the liver and reverse hepatic fibrosis.
Assuntos
Edição de Genes , Nucleases de Dedos de Zinco , Humanos , Animais , Camundongos , Cirrose Hepática/genética , Cirrose Hepática/terapia , Hepatócitos , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Inosine monophosphate dehydrogenase (IMPDH) is considered as the limiting enzyme of thiopurine metabolism for the formation of 6-thioguanine nucleotides (6-TGN). No data are available on the influence of RBC IMPDH activity on the metabolism of thiopurine drugs in individuals with IBD. The aims of this study were as follows: (a) to carry out a phenotypic study of RBC IMPDH activity in adults and children treated or not with azathioprine (AZA) for autoimmune diseases, and (b) to investigate the relationship between the activities of IMPDH, thiopurine metabolites, inosine triphosphatase (ITPA) and thiopurine methyltransferase (TPMT). IMPDH activity was determined in 97 adults and 67 children treated or not with AZA. 6-Thioguanine nucleotides (6-TGN), 6-methylmercaptopurine nucleotide (6-MeMPN) levels, and ITPA as well as TPMT activities were measured in RBCs by HPLC. Using the Gaussian mixture model, distribution of IMPDH activity was evaluated. Influence of age, sex and AZA treatment on IMPDH activity was also assessed. A bimodal distribution in IMPDH activity was found with 87% of patients exhibiting normal activity and 13% of patients with high activity. No influence of age, sex and AZA therapy was found. There is no relationship between TPMT, ITPA and IMPDH activities. A negative correlation between IMPDH activity and 6-MeMPN was shown in adults and children (rs = -0.335 P = 0.014 and rs = -0.383 P = 0.012, respectively). Our results suggest that AZA-treated patients exhibiting lower IMPDH activity could have higher Me-6MPN levels with higher risk of hepatotoxicity. We demonstrated that RBC matrix could be an interesting alternative to lymphocyte matrix to monitor thiopurine metabolites and enzyme activity.
Assuntos
Doenças Autoimunes/sangue , Doenças Autoimunes/tratamento farmacológico , Azatioprina/uso terapêutico , Eritrócitos/enzimologia , IMP Desidrogenase/sangue , Metiltransferases/sangue , Pirofosfatases/sangue , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Doenças Autoimunes/enzimologia , Azatioprina/efeitos adversos , Criança , Pré-Escolar , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
We developed extended wear silicone hydrogel soft contact lenses that deliver ophthalmic drugs for an extended period of time ranging from weeks to months. Silicone hydrogels comprising of N,N-dimethylacrylamide, 3-methacryloxypropyltris(trimethylsiloxy)silane, bis-alpha,omega-(methacryloxypropyl) polydimethylsiloxane, 1-vinyl-2-pyrrolidone, and ethylene glycol dimethacrylate were prepared with varying ratios of monomers and transport of three different ophthalmic drugs, timolol, dexamethasone, and dexamethasone 21-acetate was explored. All the silicone hydrogels of 0.1 mm thickness exhibit diffusion limited transport and extended release varying 20 days up to more than three months depending on the compositions of hydrophobic and hydrophilic components of silicone hydrogels. Also, there are multiple time scales in transport of at least certain molecules, which is perhaps due to the complex microstructure of these gels. The mechanical and physical properties of lenses such as ion permeability, equilibrium water content, transparency, and surface contact angles of some of the gels are suitable for contact lens application.
Assuntos
Anti-Inflamatórios/administração & dosagem , Lentes de Contato de Uso Prolongado , Dexametasona/administração & dosagem , Elastômeros de Silicone , Timolol/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Hidrogel de Polietilenoglicol-Dimetacrilato , Fatores de TempoRESUMO
Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Marcação de Genes/métodos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Camundongos , Camundongos SCIDRESUMO
Despite their preclinical promise, few recombinant growth factors have been fully developed into effective therapies, in part, due to the short interval of therapeutic activity after administration. To address this problem, we developed nanoscale polymer conjugates for multivalent presentation of therapeutic proteins that enhance the activation of targeted cellular responses. As an example of this technology, we conjugated multiple Sonic hedgehog (Shh) proteins onto individual hyaluronic acid biopolymers to generate multivalent protein clusters at defined ratios (i.e., valencies) that yield enhanced Shh pathway activation at equivalent concentrations relative to unconjugated Shh. In this study, we investigated whether these multivalent conjugates (mvShh) could be used to improve the therapeutic function of Shh. We found that a single treatment with mvShh significantly accelerated the closure of full-thickness wounds in diabetic (db/db) mice compared to either an equivalent dose of unconjugated Shh or the vehicle control. Furthermore, we identified specific indicators of wound healing in fibroblasts and endothelial cells (i.e., transcriptional activation and cell migration) that were activated by mvShh in vitro and at concentrations approximately an order of magnitude lower than the unconjugated Shh. Taken together, our findings suggest that mvShh conjugates exhibit greater potency to activate the Shh pathway, and this multivalency advantage improves its therapeutic effect to accelerate wound closure in a diabetic animal model. Our strategy of multivalent protein presentation using nanoscale polymer conjugates has the potential to make a significant impact on the development of protein-based therapies by improving their in vivo performance.
Assuntos
Diabetes Mellitus/patologia , Proteínas Hedgehog/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
We demonstrate that Magnetic Particle Imaging (MPI) enables monitoring of cellular grafts with high contrast, sensitivity, and quantitativeness. MPI directly detects the intense magnetization of iron-oxide tracers using low-frequency magnetic fields. MPI is safe, noninvasive and offers superb sensitivity, with great promise for clinical translation and quantitative single-cell tracking. Here we report the first MPI cell tracking study, showing 200-cell detection in vitro and in vivo monitoring of human neural graft clearance over 87 days in rat brain.
Assuntos
Rastreamento de Células , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita , Animais , Diferenciação Celular , Rastreamento de Células/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Ferro/metabolismo , Imageamento por Ressonância Magnética/métodos , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e RotulagemRESUMO
The activation of cellular signaling cascades, critical for regulating cell function and fate, often involves changes in the organization of receptors in the cell membrane. Using synthetic multivalent ligands to control the nanoscale organization of cellular receptors into clusters is an attractive approach to elicit desired downstream cellular responses, since multivalent ligands can be significantly more potent than their corresponding monovalent ligands. Synthetic multivalent ligands can serve as both versatile biological tools and potent nanoscale therapeutics, for example in applications to harness them to control stem cell fate in vitro and in vivo. Here we describe the use of recombinant protein expression and bioconjugate chemistry to synthesize multivalent ligands that have the potential to regulate cell signaling in a variety of cell types.
Assuntos
Ácido Hialurônico/metabolismo , Polímeros/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Animais , Galinhas , Ligantes , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients.
Assuntos
Materiais Biocompatíveis/metabolismo , Neurônios Dopaminérgicos/citologia , Células-Tronco Embrionárias/citologia , Neurônios GABAérgicos/citologia , Proteínas Hedgehog/metabolismo , Animais , Materiais Biocompatíveis/química , Diferenciação Celular , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Embrionárias/metabolismo , Neurônios GABAérgicos/metabolismo , Proteínas Hedgehog/química , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology. Cellular signal transduction of growth factor and morphogen cues (which have critical roles in regulating cell function and fate) often begins with such multivalent binding of ligands, either secreted or cell-surface-tethered to target cell receptors, leading to receptor clustering. Cellular mechanisms that orchestrate ligand-receptor oligomerization are complex, however, so the capacity to control multivalent interactions and thereby modulate key signalling events within living systems is currently very limited. Here, we demonstrate the design of potent multivalent conjugates that can organize stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induce signalling in neural stem cells and promote their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organization of the receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhance human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates are therefore powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Efrina-B2/farmacologia , Nanoconjugados/química , Células-Tronco Neurais/citologia , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Células Cultivadas , Humanos , Ligantes , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Receptores da Família Eph/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
Stem cells reside within most tissues throughout the lifetimes of mammalian organisms. To maintain their capacities for division and differentiation and thereby build, maintain, and regenerate organ structure and function, these cells require extensive and precise regulation, and a critical facet of this control is the local environment or niche surrounding the cell. It is well known that soluble biochemical signals play important roles within such niches, and a number of biophysical aspects of the microenvironment, including mechanical cues and spatiotemporally varying biochemical signals, have also been increasingly recognized to contribute to the repertoire of stimuli that regulate various stem cells in various tissues of both vertebrates and invertebrates. For example, biochemical factors immobilized to the extracellular matrix or the surface of neighboring cells can be spatially organized in their placement. Furthermore, the extracellular matrix provides mechanical support and regulatory information, such as its elastic modulus and interfacial topography, which modulate key aspects of stem cell behavior. Numerous examples of each of these modes of regulation indicate that biophysical aspects of the niche must be appreciated and studied in conjunction with its biochemical properties.
Assuntos
Nicho de Células-Tronco/fisiologia , Células-Tronco/citologia , Animais , Linhagem da Célula , Módulo de Elasticidade , Matriz Extracelular/metabolismo , Humanos , Células-Tronco/metabolismo , Propriedades de SuperfícieRESUMO
Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs, progenitors, and, ultimately, neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, although a few endogenous factors are known to regulate neuronal differentiation. Astrocytes have been implicated, but their role in juxtacrine (that is, cell-cell contact dependent) signaling in NSC niches has not been investigated. We found that ephrin-B2 presented from rodent hippocampal astrocytes regulated neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies revealed a previously unknown role for ephrin-B2 in instructing neuronal differentiation. In addition, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activated ß-catenin in vitro and in vivo independently of Wnt signaling and upregulated proneural transcription factors. Ephrin-B2(+) astrocytes therefore promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications.
Assuntos
Astrócitos/fisiologia , Efrina-B2/fisiologia , Hipocampo/fisiologia , Neurogênese/fisiologia , Animais , Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Efrina-B2/biossíntese , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Receptor EphB4/biossíntese , Receptor EphB4/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Regulação para Cima , beta Catenina/metabolismoRESUMO
Multiple extracellular factors have been shown to modulate adult hippocampal neural progenitor cell (NPC) proliferation and self-renewal, and we have previously shown that Akt is an important mediator of the effects of these extracellular factors on NPC proliferation and differentiation. However, very little work has investigated how and whether Akt is involved in maintaining the multipotency of these cells. Here we demonstrate that Akt promotes expression of Sox2, a core transcription factor important for the self-renewal of NPCs. Retroviral-mediated overexpression of wild-type Akt increased Sox2 protein expression, particularly under conditions that promote cell differentiation, whereas Akt inhibition decreased Sox2. Similarly, quantitative reverse transcription (RT)-PCR in differentiating cultures indicated that Akt rescued Sox2 mRNA to levels present under conditions that promote cell proliferation. Additionally, pharmacological inhibition of Akt did not affect Sox2 protein levels in cells constitutively expressing Sox2 from a retroviral vector, indicating that Akt does not affect Sox2 protein stability. Further, in contrast to Akt overexpression, Sox2 overexpression does not increase NPC viable cell number or proliferation yet does inhibit differentiation. Collectively, these results indicate that Akt promotes cell proliferation and maintenance of a multipotent state via two downstream paths.
Assuntos
Proliferação de Células , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Western Blotting/métodos , Diferenciação Celular , Feminino , Imunofluorescência , Vetores Genéticos , Células HEK293 , Hipocampo/citologia , Humanos , Células-Tronco Neurais/citologia , Fosforilação , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleosídeos/farmacologiaRESUMO
This research pertains to a new class of liquid bandage polymers which are promising for assisting advanced wound healing by serving as substrates to promote cell viability and proliferation. Amphiphilic nitrogen-containing polymer poly (3-methacryloyloxypropyltris (trimethylsiloxy)silane-co-N-isopropylacrylamide) (poly (TRIS-co-NIPAM)) was synthesized and investigated with further comparison to several different wound care polymers including commercialized 3M Nexcare No Sting Liquid Bandage. Cell viability on different polymers was tested on fetal human skin fibroblasts (HSFs) and neonatal human epidermal keratinocytes (HEKs). Test results were quantified by Sulforhodamine B (SRB) in vitro cytotoxicity assay. It was demonstrated that both HSFs and HEKs survive better on the poly (TRIS-co-NIPAM) film as the cell seeding substrate compared to other candidate polymer formulations, as well as to the commercial 3M No Sting Liquid Bandage polymer. Thus we conclude that wound healing could be accelerated by this new class of liquid bandage polymer, particularly for early-stage wounds..