RESUMO
The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Células HEK293 , Humanos , RNA/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins α5 and ß1, while the osteoclasts upregulated integrins α2 and ß1. There was significant contrast in responses on the silk matrices between osteogenic cells versus undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing, physiological state, and the ability to better understand the role of different cell types is critical to overall regenerative outcomes.
Assuntos
Materiais Biocompatíveis/metabolismo , Teste de Materiais/métodos , Osteogênese , Proteínas/metabolismo , Seda/química , Células Cultivadas , Humanos , Integrinas/genética , Integrinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Regulação para CimaRESUMO
Glioblastoma multiforme (GBM) is the most common type of primary malignant brain cancer and has a very poor prognosis. A subpopulation of cells known as GBM stem-like cells (GBM-SC) have the capacity to initiate and sustain tumor growth and possess molecular characteristics similar to the parental tumor. GBM-SCs are known to be enriched in hypoxic niches and may contribute to therapeutic resistance. Therefore, to identify genetic determinants important for the proliferation and survival of GBM stem cells, an unbiased pooled shRNA screen of 10,000 genes was conducted under normoxic as well as hypoxic conditions. A number of essential genes were identified that are required for GBM-SC growth, under either or both oxygen conditions, in two different GBM-SC lines. Interestingly, only about a third of the essential genes were common to both cell lines. The oxygen environment significantly impacts the cellular genetic dependencies as 30% of the genes required under hypoxia were not required under normoxic conditions. In addition to identifying essential genes already implicated in GBM such as CDK4, KIF11, and RAN, the screen also identified new genes that have not been previously implicated in GBM stem cell biology. The importance of the serum and glucocorticoid-regulated kinase 1 (SGK1) for cellular survival was validated in multiple patient-derived GBM stem cell lines using shRNA, CRISPR, and pharmacologic inhibitors. However, SGK1 depletion and inhibition has little effect on traditional serum grown glioma lines and on differentiated GBM-SCs indicating its specific importance in GBM stem cell survival.Implications: This study identifies genes required for the growth and survival of GBM stem cells under both normoxic and hypoxic conditions and finds SGK1 as a novel potential drug target for GBM. Mol Cancer Res; 16(1); 103-14. ©2017 AACR.
Assuntos
Neoplasias Encefálicas/enzimologia , Glioblastoma/enzimologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Células-Tronco Neoplásicas/enzimologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Células-Tronco Neoplásicas/patologia , Interferência de RNA , Análise de SobrevidaRESUMO
The growth factor and cytokine regulated transcription factor STAT3 is required for the self-renewal of several stem cell types including tumor stem cells from glioblastoma. Here we show that STAT3 inhibition leads to the upregulation of the histone H3K27me2/3 demethylase Jmjd3 (KDM6B), which can reverse polycomb complex-mediated repression of tissue specific genes. STAT3 binds to the Jmjd3 promoter, suggesting that Jmjd3 is a direct target of STAT3. Overexpression of Jmjd3 slows glioblastoma stem cell growth and neurosphere formation, whereas knockdown of Jmjd3 rescues the STAT3 inhibitor-induced neurosphere formation defect. Consistent with this observation, STAT3 inhibition leads to histone H3K27 demethylation of neural differentiation genes, such as Myt1, FGF21, and GDF15. These results demonstrate that the regulation of Jmjd3 by STAT3 maintains repression of differentiation specific genes and is therefore important for the maintenance of self-renewal of normal neural and glioblastoma stem cells.
Assuntos
Neoplasias Encefálicas/enzimologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/enzimologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células-Tronco Neoplásicas/enzimologia , Fator de Transcrição STAT3/fisiologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Glioblastoma/patologia , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
Dental tissue engineering efforts have yet to identify scaffolds that instruct the formation of bioengineered teeth of predetermined size and shape. Here we investigated whether extracellular matrix (ECM) molecules present in natural tooth scaffolds can provide insight on how to achieve this goal. We describe methods to effectively decellularize and demineralize porcine molar tooth buds, while preserving natural ECM protein gradients. Natural tooth ECM composition was assessed using histological and immunohistochemical (IHC) analyses of fibrillar and basement membrane proteins. Our results showed that Collagen I, Fibronectin, Collagen IV, and Laminin gradients were detected in natural tooth tissues, and retained in decellularized samples. Second harmonic generation (SHG) image analysis and 3D reconstructions were used to show that natural tooth tissue exhibited higher collagen fiber density, and less oriented and less organized collagen fibers, as compared to decellularized tooth tissue. We also found that reseeded decellularized tooth scaffolds exhibited distinctive collagen content and organization as compared to decelluarized scaffolds. Our results show that SHG allows for quantitative assessment of ECM features that are not easily characterized using traditional histological analyses. In summary, our results demonstrate the potential for natural decellularized molar tooth ECM to instruct dental cell matrix synthesis, and lay the foundation for future use of biomimetic scaffolds for dental tissue engineering applications.
Assuntos
Matriz Extracelular/metabolismo , Germe de Dente/citologia , Germe de Dente/metabolismo , Animais , Membrana Basal/metabolismo , Células Cultivadas , DNA/metabolismo , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Imageamento Tridimensional , Imuno-Histoquímica , Microscopia , Dente Molar/citologia , Dinâmica não Linear , Sus scrofa , Alicerces TeciduaisRESUMO
Tailoring tissue engineering strategies to match patient- and tissue-specific bone regeneration needs offers to improve clinical outcomes. As a step toward this goal, osteogenic outcomes and metabolic parameters were assessed when varying inputs into the bone formation process. Silk protein scaffolds seeded with human mesenchymal stem cells in osteogenic differentiation media were used to study in vitro osteogenesis under varied conditions of amino acid (lysine and proline) concentration and oxygen level. The cells were assessed to probe how the microenvironment impacted metabolic pathways and thus osteogenesis. The most favorable osteogenesis outcomes were found in the presence of low (5%) oxygen combined with high lysine and proline concentrations during in vitro cultivation. This same set of culture conditions also showed the highest glucose consumption, lactate synthesis, and certain amino acid consumption rates. On the basis of these results and known pathways, a holistic metabolic model was derived which shows that lysine and proline supplements as well as low (5%) oxygen levels regulate collagen matrix synthesis and thereby rates of osteogenesis. This study establishes early steps toward a foundation for patient- and tissue-specific matches between metabolism, repair site, and tissue engineering approaches toward optimized bone regeneration.
Assuntos
Aminoácidos/farmacologia , Hipóxia Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese/efeitos dos fármacos , Seda/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Aminoácidos/química , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de VarreduraRESUMO
Mesenchymal condensation is a pre-requisite of chondrogenesis during embryonic development. The current understanding of chondrogenesis is limited in terms of chondrogenic condensation mechanisms. In particular, the role of matrix stiffness on homotypic cell-cell interactions leading to the establishment of distinct aggregated chondrogenic morphology from mesenchymal cells is unclear. An in vitro biomaterials-based model to assess the interactions of matrix stiffness on chondrogensis is described herein, where by sensing subtle variation in morphology and stiffness of nanofibrous silk protein matrixes human mesenchymal stem cells migrated and assumed aggregated morphologies, mimicking early stage chondrogenesis. This simple in vitro model system has potential to play a significant role to gain insight into underlying mechanisms of mesenchymal condensation steps during chondrogenesis, integrating concepts of developmental biology, biomaterials and tissue engineering.