Browsing by Subject "stem cell"
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Item Applications for human neural stem cell-derived motor neurons in amyotrophic lateral sclerosis: Cell replacement therapy and disease modeling(2010-04-19) Jason Robert Thonhoff; Ping Wu, M.D., Ph.D.; Steven A. Weinman, M.D., Ph.D.; Stanley H. Appel, M.D.; Jin Mo Chung, Ph.D.; Darren F. Boehning, Ph.D.Amyotrophic lateral sclerosis (ALS) is an incurable neurological disease characterized by the selective degeneration of spinal and upper motor neurons. One approach in the development of therapies for ALS is to explore the potential of human fetal neural stem cells (hNSCs) to replace lost motor neurons. The therapeutic efficacy of stem cell transplantation would depend greatly on the survival of grafted stem cell-derived motor neurons in the microenvironment of the spinal cord in ALS. Previously, we reported that hNSCs could be instructed to differentiate into motor neurons both in vitro and in vivo. Here, we report that the transplantation of primed hNSCs into the spinal cords of transgenic ALS rats only slightly delayed disease progression. Morphological analyses of the transplantation sites revealed that the grafted hNSCs differentiated into motor neurons, but were degenerated and showed signs of nitroxidative damage at the disease end-stage. Using an in vitro coculture system, we provided evidence that human mutant SOD1(G93A)-expressing primary microglia, isolated after disease onset, were directly toxic to hNSC-derived motor neurons. Additionally, normal astrocytes not only lost their protective capacity toward hNSC-derived motor neuron survival in vitro, but also exhibited toxic features, when cocultured with mutant SOD1(G93A) microglia. Using inhibitors of inducible nitric oxide synthase and NADPH oxidase as well as scavengers for reactive oxygen and nitrogen species (ROS/RNS), we showed that microglia-generated nitric oxide, superoxide and peroxynitrite, at least, partially contributed to motor neuron loss and astrocyte dysfunction in this coculture paradigm. In summary, ROS/RNS released from overactivated microglia directly damage motor neurons and reduce the neuroprotective capacity of astrocytes, collectively dooming motor neuron survival in ALS. These data provide evidence that treating ALS with motor neuron cell-replacement therapies will not be efficacious unless the toxic milieu created by endogenous overactivated microglia in the spinal cord of ALS is dramatically altered. Outcomes from these studies should aid in the development of novel combined therapies using stem cells to treat patients with ALS.\r\nItem Development of hyaluronic acid – poly(ethylene glycol) hydrogels towards hematopoietic differentiation of mouse embryonic stem cells(2009-08) Erickson, Kathryn Marie; Roy, Krishnendu; Suggs, LauraThe fields of tissue engineering, regenerative medicine, and stem cell engineering are rapidly growing. However, these fields must overcome several obstacles before they can make a significant impact on treating cellular disorders. Two major hurdles that must be addressed are: determining how to control the pluripotency of stem cells and developing systems for high-throughput culture of stem cells. The prospect of using a cell source capable of differentiating into cells of any tissue in the body (embryonic stem cells) has received enormous interest in recent years. The pluripotent attribute of embryonic stem cells seems ideal but developing methods to drive embryonic stem cells to specific lineages, including the hematopoietic lineage, is a complex process dependent on multiple intrinsic and extrinsic factors including chemical, cellular, and environmental signaling. With regards to environmental signaling, the use of three-dimensional culture systems such as scaffolds and hydrogels, have been utilized in an attempt to drive lineage-specific differentiation in a synthetic, biomimetic microenvironment. To determine specific environmental factors responsible for hematopoietic differentiation a systematic biological and engineering process must be implemented. A biodegradable hydrogel composed of the hyaluronic acid, a polysaccharide abundant in the bone marrow microenvironment, and the synthetic polymer, poly(ethylene glycol) was formulated to culture mouse embryonic stem cells (mESCs). Photoencapsulation of mESCs did not significantly decrease cellular viability or proliferation. The FACS data was inconclusive however, from gene expression studies, it was determined that the hydrogel culture system promoted differentiation of mESCs as evidenced by a down-regulation of the gene encoding for stem cell maintenance transcription factor, Oct-3/4. Furthermore, embryoid bodies, necessary for in vitro differentiation were observed in the hydrogel systems. Although an increase in the gene encoding for the cell surface marker, c-kit was up-regulated, the surface marker, sca-1 was not up-regulated. Up-regulation of both c-kit and sca-1 is necessary for the development of hematopoietic progenitor cells. Results indicate that the differentiation of mESCs into the hematopoietic lineage was unsuccessful but differentiation in these hydrogel systems did occur. Future cell marker and gene expression studies are necessary to determine which cell lineage the encapsulated mESCs are differentiating into before the effects of incorporating other environmental, cellular, and chemical factors can be investigated.