RESUMO
Mechanobiology is a rapidly advancing field, with growing evidence that mechanical signaling plays key roles in health and disease. To accelerate mechanobiology-based drug discovery, novel in vitro systems are needed that enable mechanical perturbation of cells in a format amenable to high throughput screening. Here, both a mechanical stretch device and 192-well silicone flexible linear stretch plate were designed and fabricated to meet high throughput technology needs for cell stretch-based applications. To demonstrate the utility of the stretch plate in automation and screening, cell dispensing, liquid handling, high content imaging, and high throughput sequencing platforms were employed. Using this system, an assay was developed as a biological validation and proof-of-concept readout for screening. A mechano-transcriptional stretch response was characterized using focused gene expression profiling measured by RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Gen sequencing. Using articular chondrocytes, a gene expression signature containing stretch responsive genes relevant to cartilage homeostasis and disease was identified. The possibility for integration of other stretch sensitive cell types (e.g., cardiovascular, airway, bladder, gut, and musculoskeletal), in combination with alternative phenotypic readouts (e.g., protein expression, proliferation, or spatial alignment), broadens the scope of high throughput stretch and allows for wider adoption by the research community. This high throughput mechanical stress device fills an unmet need in phenotypic screening technology to support drug discovery in mechanobiology-based disease areas.
RESUMO
Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043-a derivative of angiopoietin-like 3 (ANGPTL3)-as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5ß1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial ( NCT02491281 ; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial ( NCT04864392 ) in patients with knee OA.
Assuntos
Cartilagem Articular , Osteoartrite do Joelho , Humanos , Osteoartrite do Joelho/tratamento farmacológico , Condrócitos , Transdução de Sinais , Angiopoietinas/metabolismo , Angiopoietinas/farmacologia , Angiopoietinas/uso terapêutico , Proteína 3 Semelhante a AngiopoietinaRESUMO
BACKGROUND: Previous studies have reported that adult mesenchymal stem cells (MSCs) tend to gradually lose their stem cell characteristics in vitro when placed outside their niche environment. They subsequently undergo spontaneous differentiation towards mesenchymal lineages after only a few passages. We observed a similar phenomenon with adult tendon stem cells (TSCs) where expression of key tendon genes such as Scleraxis (Scx), are being repressed with time in culture. We hypothesized that an environment able to restore or maintain Scleraxis expression could be of therapeutic interest for in vitro use and tendon cell-based therapies. METHODS: TSCs were isolated from human cadaveric Achilles tendon and expanded for 4 passages. A high content imaging assay that monitored the induction of Scx protein nuclear localization was used to screen ~1000 known drugs. RESULTS: We identified retinoic acid receptor (RAR) agonists as potent inducers of nuclear Scx in the small molecule screen. The upregulation correlated with improved maintenance of tendon stem cell properties through inhibition of spontaneous differentiation rather than the anticipated induction of tenogenic differentiation. Our results suggest that histone epigenetic modifications by RAR are driving this effect which is not likely only dependent on Scleraxis nuclear binding but also mediated through other key genes involved in stem cell self-renewal and differentiation. Furthermore, we demonstrate that the effect of RAR compounds on TSCs is reversible by revealing their multi-lineage differentiation ability upon withdrawal of the compound. CONCLUSION: Based on these findings, RAR agonists could provide a valid approach for maintaining TSC stemness during expansion in vitro, thus improving their regenerative potential for cell-based therapy.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular/efeitos dos fármacos , Receptores do Ácido Retinoico/metabolismo , Tendão do Calcâneo/citologia , Transporte Ativo do Núcleo Celular , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células , Células Cultivadas , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Metilação , Pessoa de Meia-Idade , Ácidos Nicotínicos/farmacologia , Ligamento Patelar/citologia , Processamento de Proteína Pós-Traducional , Receptores do Ácido Retinoico/agonistas , Transdução de Sinais , Ativação TranscricionalRESUMO
Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for transplantation. Here, we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.
Assuntos
Biotina/análogos & derivados , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Retina/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Biotina/química , Biotina/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células-Tronco Fetais , Imunofluorescência , Humanos , Estrutura Molecular , Compostos de Fenilureia/química , Pirimidinas/química , Ratos , Retina/efeitos dos fármacos , Degeneração Retiniana/tratamento farmacológicoRESUMO
Emerging evidence suggests that some cancers contain a population of stem-like TICs (tumor-initiating cells) and eliminating TICs may offer a new strategy to develop successful anti-cancer therapies. As molecular mechanisms underlying the maintenance of the TIC pool are poorly understood, the development of TIC-specific therapeutics remains a major challenge. We first identified and characterized TICs and non-TICs isolated from a mouse breast cancer model. TICs displayed increased tumorigenic potential, self-renewal, heterogeneous differentiation, and bipotency. Gene expression analysis and immunostaining of TICs and non-TICs revealed that FGFR2 was preferentially expressed in TICs. Loss of FGFR2 impaired self-renewal of TICs, thus resulting in marked decreases in the TIC population and tumorigenic potential. Restoration of FGFR2 rescued the defects in TIC pool maintenance, bipotency, and breast tumor growth driven by FGFR2 knockdown. In addition, pharmacological inhibition of FGFR2 kinase activity led to a decrease in the TIC population which resulted in suppression of breast tumor growth. Moreover, human breast TICs isolated from patient tumor samples were found enriched in a FGFR2+ population that was sufficient to initiate tumor growth. Our data suggest that FGFR2 is essential in sustaining the breast TIC pool through promotion of self-renewal and maintenance of bipotent TICs, and raise the possibility of FGFR2 inhibition as a strategy for anti-cancer therapy by eradicating breast TICs.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , Células-Tronco Neoplásicas/imunologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Antígeno CD24/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Integrina beta1/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Microscopia de Fluorescência , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Transdução de SinaisRESUMO
Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44(+)/CD133(+) prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/metabolismo , Receptores CXCR4/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Benzilaminas , Adesão Celular , Proliferação de Células , Quimiocina CXCL12/metabolismo , Ciclamos , Docetaxel , Compostos Heterocíclicos/farmacologia , Humanos , Masculino , Camundongos , Metástase Neoplásica , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Taxoides/farmacologiaRESUMO
Here we report the design and evaluation of a bifunctional, small molecule switch that induces a targeted immune response against tumors in vivo. A high affinity ligand for prostate specific membrane antigen (PSMA) was conjugated to a hapten that binds dinitrophenyl (DNP)-specific antibodies. When introduced into hu-PBL-NOD/SCID mice previously immunized with a KLH-DNP immunogen, this conjugate induced a targeted antibody-dependent cellular cytotoxicity (ADCC) response to PSMA-expressing tumor cells in a mouse xenograft model. The ability to create a small molecule inducible antibody response against self-antigens using endogenous non-autoreactive antibodies may provide advantages over the autologous immune response generated by conventional vaccines in certain therapeutic settings.
Assuntos
2,4-Dinitrofenol/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , 2,4-Dinitrofenol/química , Animais , Anticorpos Antineoplásicos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/metabolismo , Autoantígenos/imunologia , Vacinas Anticâncer/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Glutamato Carboxipeptidase II/metabolismo , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/patologiaRESUMO
Integration of new neurons into the adult hippocampus has been linked to specific types of learning. Primary cilia were found to be required for the formation of adult neural stem cells (NSCs) in the hippocampal dentate gyrus during development. However, the requirement of cilia in maintenance of adult NSCs is unknown. We developed a genetic mouse model in which fetal/perinatal brain development is unaffected, but adult hippocampal neurogenesis is constantly reduced by conditional ablation of primary cilia in adult GFAP(+) neural stem/progenitor cells. We found that this approach specifically reduces the number of hippocampal amplifying progenitors (also called type 2a cells) without affecting the number of radial NSCs (or type 1 cells). Constant reduction of adult hippocampal neurogenesis produced a delay rather than a permanent deficiency in spatial learning without affecting the retention of long-term memories. Decreased neurogenesis also altered spatial novelty recognition and hippocampus-independent cue conditioning. Here, we propose that adult hippocampal newborn neurons increase the efficiency of generating the new representations of spatial memories and that reduction of adult hippocampal neurogenesis may be biased toward cue-based strategies. This novel mouse model provides evidences that cognitive deficits associated with ciliary defects (ciliopathies) might be, in part, mediated by the deficiency of primary cilia in adult hippocampal stem/progenitor cells.
Assuntos
Células-Tronco Adultas/fisiologia , Proliferação de Células , Cílios/fisiologia , Condicionamento Psicológico/fisiologia , Hipocampo/citologia , Neurogênese/fisiologia , Neurônios/fisiologia , Análise de Variância , Animais , Bromodesoxiuridina/metabolismo , Proteínas de Transporte/genética , Contagem de Células/métodos , Sinais (Psicologia) , Proteínas do Domínio Duplacortina , Comportamento Exploratório/fisiologia , Medo/fisiologia , Feminino , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Marcação In Situ das Extremidades Cortadas/métodos , Proteínas de Filamentos Intermediários/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Nestina , Neurogênese/genética , Neuropeptídeos/metabolismo , Fosfopiruvato Hidratase/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Desempenho Psicomotor/fisiologia , Percepção Espacial/fisiologia , Natação/psicologia , Transferência de Experiência/fisiologiaRESUMO
PURPOSE: The cancer stem cell hypothesis predicts that standard prostate cancer monotherapy eliminates bulk tumor cells but not a tumor-initiating cell population, eventually leading to relapse. Many studies have sought to determine the underlying differences between bulk tumor and cancer stem cells. EXPERIMENTAL DESIGN: Our previous data suggest that the PTEN/PI3K/AKT pathway is critical for the in vitro maintenance of CD133(+)/CD44(+) prostate cancer progenitors and, consequently, that targeting PI3K signaling may be beneficial in treatment of prostate cancer. RESULTS: Here, we show that inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 leads to a decrease in the population of CD133(+)/CD44(+) prostate cancer progenitor cells in vivo. Moreover, the combination of the PI3K/mTOR modulator NVP-BEZ235, which eliminates prostate cancer progenitor populations, and the chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors in a prostate cancer xenograft model than monotherapy. CONCLUSION: This combination treatment ultimately leads to the expansion of cancer progenitors with a PTEN E91D mutation, suggesting that the analysis of PTEN mutations could predict therapeutic response to the dual therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sistemas de Liberação de Medicamentos/métodos , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Imidazóis/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , PTEN Fosfo-Hidrolase/genética , Peptídeos/metabolismo , Neoplasias da Próstata/patologia , Quinolinas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In the developing mouse retina, multipotent retinal progenitor cells (RPCs) give rise to specific retinal cell types at different times, but the molecular mechanisms regulating how RPCs change over time remain unclear. In the Drosophila neuroblast lineage, the zinc finger transcription factor Hunchback (Hb) is both necessary and sufficient to specify early-born neuronal identity. We show here that Ikaros, a mouse ortholog of Hb, is expressed in all early embryonic RPCs, which then give rise to Ikaros-negative RPCs at later stages in the lineage. Remarkably, misexpression of Ikaros in late RPCs is sufficient to confer competence to generate early-born neurons. Conversely, Ikaros mutant mice have reduced numbers of early-born cell types, whereas late-born cell types are not affected. These results suggest a model in which Ikaros expression is both necessary and sufficient to confer early temporal competence to RPCs and raise the possibility that a similar strategy might be used to control the sequential order of cell birth in other parts of the nervous system.
Assuntos
Fator de Transcrição Ikaros/fisiologia , Retina/citologia , Retina/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Humanos , Fator de Transcrição Ikaros/biossíntese , Fator de Transcrição Ikaros/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Retina/embriologiaAssuntos
Moléculas de Adesão Celular/fisiologia , Proteínas de Ciclo Celular/fisiologia , Polaridade Celular/fisiologia , Doenças Desmielinizantes/fisiopatologia , Proteínas de Membrana/fisiologia , Bainha de Mielina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/fisiologia , Moléculas de Adesão Celular/química , Células Cultivadas/fisiologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Técnicas de Cocultura , Gânglios Espinais/citologia , Humanos , Invertebrados/fisiologia , Camundongos , Esclerose Múltipla/fisiopatologia , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , Receptores de Fator de Crescimento Neural/fisiologia , Células de Schwann/fisiologia , Vertebrados/fisiologiaRESUMO
Cell polarity is critical in various cellular processes ranging from cell migration to asymmetric cell division and axon and dendrite specification. Similarly, myelination by Schwann cells is polarized, but the mechanisms involved remain unclear. Here, we show that the polarity protein Par-3 localizes asymmetrically in Schwann cells at the axon-glial junction and that disruption of Par-3 localization, by overexpression and knockdown, inhibits myelination. Additionally, we show that Par-3 directly associates and recruits the p75 neurotrophin receptor to the axon-glial junction, forming a complex necessary for myelination. Together, these results point to a critical role in the establishment of cell polarity for myelination.
Assuntos
Proteínas de Transporte/metabolismo , Polaridade Celular , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Células de Schwann/fisiologia , Motivos de Aminoácidos , Animais , Axônios/química , Axônios/ultraestrutura , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Proteínas de Transporte/análise , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Técnicas de Cocultura , Gânglios Espinais/ultraestrutura , Junções Intercelulares/química , Camundongos , Proteínas do Tecido Nervoso/química , Estrutura Terciária de Proteína , Ratos , Receptores de Fatores de Crescimento/química , Células de Schwann/citologia , Células de Schwann/ultraestruturaRESUMO
Natural cell death is critical for normal development of the nervous system, but the extracellular regulators of developmental cell death remain poorly characterized. Here, we studied the role of the CNTF/LIF signaling pathway during mouse retinal development in vivo. We show that exposure to CNTF during neonatal retinal development in vivo retards rhodopsin expression and results in an important and specific deficit in photoreceptor cells. Detailed analysis revealed that exposure to CNTF during retinal development causes a sharp increase in cell death of postmitotic rod precursor cells. Importantly, we show that blocking the CNTF/LIF signaling pathway during mouse retinal development in vivo results in a significant reduction of naturally occurring cell death. Using retroviral lineage analysis, we demonstrate that exposure to CNTF causes a specific reduction of clones containing only rods without affecting other clone types, whereas blocking the CNTF/LIF receptor complex causes a specific increase of clones containing only rods. In addition, we show that stimulation of the CNTF/LIF pathway positively regulates the expression of the neuronal and endothelial nitric oxide synthase (NOS) genes, and blocking nitric oxide production by pre-treatment with a NOS inhibitor abolishes CNTF-induced cell death. Taken together, these results indicate that the CNTF/LIF signaling pathway acts via regulation of nitric oxide production to modulate developmental programmed cell death of postmitotic rod precursor cells.