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"Schaffer, David V"
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هندسة الخلايا الجذعية : أساسيات وممارسات
هذا الكتاب إضافة قيمة للمكتبة العربية، ويعتبر من أمهات الكتب الحديثة في مجال العلوم البيولوجية الطبية، لذا فهو مهم لجميع المختصين والمهتمين بجميع علوم الحياة، إذ إن هندسة وتكنولوجيا الخلايا الجذعية وتطبيقاتها في مجال الطب التجديدي والبحث العلمي هي امتداد طبيعي الثورة القرن الحادي والعشرين في الهندسة الوراثية والتكنولوجيا الحيوية. ويقدم الكتاب شرحًا تفصيليا لآخر التطورات في بحوث الخلايا الجذعية وهندسة الأنسجة وتطبيقاتها الإكلينيكية في الطب التجديدي الحديث من خلال عرض ومناقشة موضوعات مهمة مثل؛ شرح بيولوجيا الخلايا الجذعية وخصائصها وأنواعها ومصادرها، وفهم بيئاتها الميكرونية وسلوكها داخل الكائن الحي، كما يناقش النشأة الجنينية للخلايا الجذعية ومسئوليتها عن إنتاج جميع خلايا جسم الجنين، مع الاحتفاظ بمخزون استراتيجي من الخلايا الجذعية النسيجية لإصلاح أنسجة الجسم التالفة وتجديدها طوال حياة الإنسان.
Book
Engineering adeno-associated viruses for clinical gene therapy
Although gene delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective for numerous clinical gene therapy applications, many challenges remain. Recent advances in AAV vector development through rational design and directed evolution, as well as in the design of novel genetic cargoes, promise to extend clinical successes of AAV-mediated gene therapy.
Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.
Journal Article
fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation
Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 10 ⁷²-fold expansion over 280 d), yield (∼2.0 × 10 ⁷ cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 10 ⁷ dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.
Journal Article
The Coming of Age of Topical Gene Therapy for Dystrophic Epidermolysis Bullosa
Dystrophic epidermolysis bullosa is a genetic disorder in which there is a loss of anchoring fibrils and structural integrity in the superficial layers of the dermis owing to mutations in the gene encoding type VII collagen (
COL7A1
), as explained by Payne in this issue of the
Journal
.
1
This process leads to the development of painful skin blisters along with a range of secondary illnesses.
2
The condition can be inherited in dominant or recessive patterns and in varying degrees of severity. There are currently no approved therapies for the disorder. Gene therapy has made strides in recent years, and in a . . .
Journal Article
The AAV Vector Toolkit: Poised at the Clinical Crossroads
The discovery of naturally occurring adeno-associated virus (AAV) isolates in different animal species and the generation of engineered AAV strains using molecular genetics tools have yielded a versatile AAV vector toolkit. Promising results in preclinical animal models of human disease spurred the much awaited transition toward clinical application, and early successes in phase I/II clinical trials for a broad spectrum of genetic diseases have recently been reported. As the gene therapy community forges ahead with cautious optimism, both preclinical and clinical studies using first generation AAV vectors have highlighted potential challenges. These include cross-species variation in vector tissue tropism and gene transfer efficiency, pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients. A battery of second generation AAV vectors, engineered through rational and combinatorial approaches to address the aforementioned concerns, are now available. This review will provide an overview of preclinical studies with the ever-expanding AAV vector portfolio in large animal models and an update on new lead AAV vector candidates poised for clinical translation.
Journal Article
CRISPR-mediated Activation of Latent HIV-1 Expression
Complete eradication of HIV-1 infection is impeded by the existence of cells that harbor chromosomally integrated but transcriptionally inactive provirus. These cells can persist for years without producing viral progeny, rendering them refractory to immune surveillance and antiretroviral therapy and providing a permanent reservoir for the stochastic reactivation and reseeding of HIV-1. Strategies for purging this latent reservoir are thus needed to eradicate infection. Here, we show that engineered transcriptional activation systems based on CRISPR/Cas9 can be harnessed to activate viral gene expression in cell line models of HIV-1 latency. We further demonstrate that complementing Cas9 activators with latency-reversing compounds can enhance latent HIV-1 transcription and that epigenome modulation using CRISPR-based acetyltransferases can also promote viral gene activation. Collectively, these results demonstrate that CRISPR systems are potentially effective tools for inducing latent HIV-1 expression and that their use, in combination with antiretroviral therapy, could lead to improved therapies for HIV-1 infection.
Journal Article
Biophysical regulation of epigenetic state and cell reprogramming
Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells’ epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.
Somatic cells can be reprogrammed into induced pluripotent stem cells biochemically through the expression of a few transcription factors. It is now shown that aligned microgrooves or nanofibres on cell-adhesive substrates can promote the reprogramming of somatic cells more efficiently through epigenetic regulation of genes related to pluripotency and the mesenchymal-to-epithelial transition. The findings suggest that the epigenetic state can be regulated by variations in cell morphology.
Journal Article
Optogenetic protein clustering and signaling activation in mammalian cells
A modular optogenetic method for higher-order protein oligomerization uses a single cryptochrome 2-based fusion for rapid, reversible and tunable oligomerization in response to blue light. Inducible aggregation can be used to specifically activate different signaling pathways.
We report an optogenetic method based on
Arabidopsis thaliana
cryptochrome 2 for rapid and reversible protein oligomerization in response to blue light. We demonstrated its utility by photoactivating the β-catenin pathway, achieving a transcriptional response higher than that obtained with the natural ligand Wnt3a. We also demonstrated the modularity of this approach by photoactivating RhoA with high spatiotemporal resolution, thereby suggesting a previously unknown mode of activation for this Rho GTPase.
Journal Article
CRISPR-Cas9-Mediated Genome Editing Increases Lifespan and Improves Motor Deficits in a Huntington’s Disease Mouse Model
Huntington’s disease (HD) is a currently incurable and, ultimately, fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant protein that forms inclusions and selectively destroys neurons in the striatum and other adjacent structures. The RNA-guided Cas9 endonuclease from CRISPR-Cas9 systems is a versatile technology for inducing DNA double-strand breaks that can stimulate the introduction of frameshift-inducing mutations and permanently disable mutant gene function. Here, we show that the Cas9 nuclease from Staphylococcus aureus, a small Cas9 ortholog that can be packaged alongside a single guide RNA into a single adeno-associated virus (AAV) vector, can be used to disrupt the expression of the mutant HTT gene in the R6/2 mouse model of HD following its in vivo delivery to the striatum. Specifically, we found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by a trinucleotide repeat expansion via in vivo genome editing.
Journal Article
Nox2 redox signaling maintains essential cell populations in the brain
A new fluorescent indicator reveals that a ROS-producing NADPH oxidase generates H
2
O
2
, normally associated with pathological conditions such as neurodegeneration, in neural stem cells where it regulates Akt phosphorylation and normal cell proliferation.
Reactive oxygen species (ROS) are conventionally classified as toxic consequences of aerobic life, and the brain is particularly susceptible to ROS-induced oxidative stress and damage owing to its high energy and oxygen demands. NADPH oxidases (Nox) are a widespread source of brain ROS implicated in seizures, stroke and neurodegeneration. A physiological role for ROS generation in normal brain function has not been established, despite the fact that mice and humans lacking functional Nox proteins have cognitive deficits. Using molecular imaging with Peroxyfluor-6 (PF6), a new selective fluorescent indicator for hydrogen peroxide (H
2
O
2
), we show that adult hippocampal stem/progenitor cells (AHPs) generate H
2
O
2
through Nox2 to regulate intracellular growth signaling pathways, which in turn maintains their normal proliferation
in vitro
and
in vivo
. Our results challenge the traditional view that brain ROS are solely deleterious by demonstrating that controlled ROS chemistry is needed for maintaining specific cell populations.
Journal Article