Dr. Akhurst's general research area has to do with the TGF-Beta signaling pathway, which is active in many cellular processes. Most notably in the control of cellular proliferation and differentiation, which is why it has been implicated in a variety of cancers and other diseases. HHT, hereditary hemorrhagic telangiectasia is one of these diseases, it has been described as a disease of excessive angiogenesis.
X linked severe combined immunodeficiency (X-SCID), is a recessive disease caused by mutations to the gene IL2Rγ and is characterized by a non-functional immune system. We have designed a method for functionally correcting patient hematopoietic stem cells, at the IL2Rγ locus, by creating a double strand break at the first exon of IL2Rγ and delivering a repair donor using an AAV6 vector. The repair donor contains a functional cDNA of IL2Rγ and a selectable marker, truncated nerve growth factor receptor (tNGFR). Optimizing our genome editing strategy in K562 cells a myelogenous cell line.
Mesenchymal stem cells (MSCs) are adult stem cells that have significant clinical implications due to their multipotent ability to differentiate into the mesodermal lineage, their involvement in tissue repair, and secretion of cytokines that promote healing. However, it is known that MSCs populate a small fraction of cells in the bone marrow, therefore necessitating in-vitro expansion and pooling different MSCs donor banks for treatment.
Understanding human physiology and disease requires model systems that faithfully capture homo and heterotypic cellular interactions. To study human cells in the laboratory, the most optimal system thus far has been the culture of primary cells in a 3-dimensional format, where cellular division and differentiation occurs with spontaneous organization into ultrastructure mimicking that of parent organs. This organotypic culture methodology has been well described for the gastrointestinal tract, but limited studies have been performed on the respiratory system.
Muscular dystrophies are a group of degenerative diseases that arise from genetic mutations that cause a loss of function in the corresponding gene products. Of particular interest is Limb Girdle Muscular Dystrophy Type 2B (LGMD2B), which is caused by mutations in the Dysferlin gene (DYSF). Here, we describe a potential gene therapy approach to deliver the healthy, full-length DYSF coding sequence in a safe and long-lasting manner to muscle fibers. We have injected Dysferlin-negative mice with naked DNA designed to knock-in DYSF using φC31 integrase.
Pluripotent stem cells provide a promising source to derive endothelial progenitor cells (EPs) for the treatment of vascular disease, ischemia, and stroke. We have developed a robust protocol to differentiate human iPSCs into nearly pure EP population (94-97% CD31+ and 78-83% VE-Cadherin+). These EPs also expressed other endothelial markers, vWF and NOS3. Compared to HUVECs, these EPs had higher level of artery marker, HEY1, and lower level of venous marker, Coup-TFII.
Peripheral arterial disease is an ischemic tissue related disorder characterized by a lack of adequate blood flow to the extremities. This occurs when the artery is blocked, typically as a result of atherosclerosis. Mesenchymal stem cells (MSCs) have been shown to be a possible therapeutic for ischemic diseases through their ability to secrete pro-angiogenic signals. Recently, it was shown that exosomes isolated from these MSC are key components in the delivery of pro-angiogenic factors.
Neurogenesis is the process of generating new neurons from undifferentiated neural stem and progenitor cells (NSPCs). Currently, the functional role of the NSPCs in the subgranular zone (SGZ) is largely attributed to the new neurons that NSPCs create. However, Recent study showed the hippocampal progenitors secrete large quantities of the essential growth factor VEGF in vitro and vivo. These findings arise the possibility that NSPCs could be an unexpected source of VEGF in the brain. Interestingly, after a seizure, the VEGF levels highly increase.
Neural Crest Stem Cells (NCSC) are a multipotent population of stem cells that arise from the neural plate border and possess the ability to migrate ventrally throughout the developing embryo. NCSCs retain multipotency prior to migration, during, and after migration, and are capable of differentiating into a variety of cells including cartilage, bone, melanocytes, autonomic neurons, and Schwann cells among others. During development cranial NCSCs migrate into the first and second pharyngeal arch and differentiate into the bones and cartilage that make up the craniofacial structure.
In order to study disease in physiologically relevant cell models, it is important to develop technologies that enable efficient control of gene expression. Using the newly developed CRISPR interference (CRISPRi) technology, we aim to interrogate genes involved in iPSC growth and maintenance at the whole-genome level. CRISPRi utilizes a deactivated Cas9 enzyme with a fused KRABs domain (dCas9-KRAB) for gene silencing. We have generated a genome-wide kinase and phosphatase library of 13,000 single guide RNAs designed to target 2600 genes.