A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation

The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/−), or null (−/−) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56140/1/20534_ftp.pd

A Student Team in a University of Michigan Biomedical Engineering Design Course Constructs a Microfluidic Bioreactor for Studies of Zebrafish Development

Abstract The zebrafish is a valuable model for teaching developmental, molecular, and cell biology; aquatic sciences; comparative anatomy; physiology; and genetics. Here we demonstrate that zebrafish provide an excellent model system to teach engineering principles. A seven-member undergraduate team in a biomedical engineering class designed, built, and tested a zebrafish microfluidic bioreactor applying microfluidics, an emerging engineering technology, to study zebrafish development. During the semester, students learned engineering and biology experimental design, chip microfabrication, mathematical modeling, zebrafish husbandry, principles of developmental biology, fluid dynamics, microscopy, and basic molecular biology theory and techniques. The team worked to maximize each person's contribution and presented weekly written and oral reports. Two postdoctoral fellows, a graduate student, and three faculty instructors coordinated and directed the team in an optimal blending of engineering, molecular, and developmental biology skill sets. The students presented two posters, including one at the Zebrafish meetings in Madison, Wisconsin (June 2008).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78149/1/zeb.2008.0572.pd

Mouse Embryonic Stem Cells Differentiated into Neuron-like Cells or Schwann Cell-like Cells for the Development of Strategies to Ameliorate Deafness.

Cochlear prostheses (CI) can restore hearing to patients with extensive sensorineural hearing loss (SNHL) by replacing lost or damaged cochlear mechanosensory hair cells (HC) with an electrode array. For the CI to send meaningful acoustic information to the brain, as large a population of remaining inner ear spiral ganglion neurons (SGN) as possible must become functionally coupled to the CI. SGN-CI coupling would be enhanced by a means of inducing directed neurite extension from the remaining SGN to the CI. Macrophage migration inhibitory factor (MIF) is a neurotrophic cytokine, which is expressed in early embryogenesis in the central and peripheral nervous systems, the eye and inner ear, where it is expressed in the supporting cells that underlie the HC and in all Schwann Cells. We produced a mESC-derived neuron-like population of cells from mESC exposed to recombinant MIF that in many respects resemble SGN. Maturation of these MIF mESC-derived neuron-like cells was enhanced by Docosahexaenoic acid, a fatty acid that helps promote neuronal maturation. Such cells could be used eventually to replace lost of damaged SGN. This laboratory also produced the first embryonic stem cell (ESC) derived model of the Schwann Cell. These Schwann cells produce MIF, which is capable of inducing directional neurite outgrowth and supporting the survival of both early stage statoacoustic ganglion neurons (SAG) and their adult counterparts in the SGN as do all Schwann Cells. We have coated CIs with mESC-MIF-producing “Schwann Cells” and observed directional outgrowth and contact formation between the “Schwann Cell”-coated CI and primary mouse embryonic SAG or adult SGN to bring the neurites closer to the CI for better performance. Thus two different stem cell-based approaches have been used to address two different problems in deafness: improved integration of CI with native SGN and replacement of SGN themselves with a stem cell population molecularly engineered to resemble SGN.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/94096/1/poorna_1.pd

Influence of Hormones and Hormone Metabolites on the Growth of Schwann Cells Derived From Embryonic Stem Cells and on Tumor Cell Lines Expressing Variable Levels of Neurofibromin

A benign human plexiform neurofibroma cell. Note the extensive tubulin networks (red), which become disrupted after addition of 2ME2, an estrogen metabolite implicated in tumor toxicity. The green staining represents S100 staining, indicating the Schwann Cell origin of the tumor cells. Blue is DAPI nuclear staining. See Roth et al., Developmental Dynamics 237:513–524, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58084/1/21537_ftp.pd

Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear

This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system ‘inflammatory’ cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development