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

Abstract

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.

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

“…2B) or CNTF (Fig. 2C) similarly took on a neuron-like morphology after 48 hours, as described in our previous studies (Roth et al, 2007;Roth et al, 2008).…”

“…The D3 mouse embryonic stem cell (mESC) line (S6) was cultured as described (Roth et al, 2007;Roth et al, 2008) to assess the effects of MIF bioactivity on differentiation. Control media included 1 µg/l ciliary neurotrophic factor (CNTF) (R&D Systems), 1 µg/l nerve growth factor (NGF) (Chemicon, Temecula, CA, USA), or various concentrations (1 pg/ml to 500 ng/ml) of rmMIF.…”

The Development of Cutaneous Neurofibromas

“…The cells were first cultured under conditions described to favor stem cell proliferation, 19 and NFPs were found to express nestin, alpha 4 integrin, SSEA1, CD9, and E-cadherin (Figure 2, H-L), all of which have been shown to be expressed by multipotent cells at different stages of their cellular differentiation. 27,28 In addition, the presence of nestin protein was demonstrated by Western transfer analysis (not shown). Although a subpopulation of cells in neurofibromas and NFPs express similar multipotency markers, these results do not provide direct evidence that the tumor cells and cultured cells are identical or related.…”

Characterization of Neurofibromas of the Skin and Spinal Roots in a Mouse Model

“…like cells and SC-like cells, (Roth et al, 2007) the latter of which more closely approximate the initiating tumor cell type in NF1. SC-like differentiated mES cells will be referred to as D3SC (NF1ϩ/ϩ), SKOSC (NF1ϩ/Ϫ) and DKOSC (NF1Ϫ/Ϫ), depending on the number of NF1 alleles expressed.…”