Generation of Mouse Notochord Cell Lines for the Study of Notochord Function during Intervertebral Disk Development
Authors: Au T. Y. , Chan D. , Cheah K. 
Published: June 2012

Introduction

Degeneration of intervertebral disks (IVDs) is the most common cause of back pain. Herniation of disk material into vertebral column and the reduction of disk thickness are the two prominent clinical features. Our understanding of the mechanisms regulating the processes of IVD development, maintenance, and degeneration is still limited. Therefore, there is no effective biological treatment for IVD degeneration. Nucleus pulposus (NP) is the core of IVD and it is derived from the notochord. However, the biological properties of the NP cell and its precursor, the notochord (NC cell), are not well understood. Here, we aim to establish an in vitro culturing system for maintenance of NP and NC cells. This can help to identify the characteristics (individual growth factors, secrete molecules and matrix components that constitute the unique extracellular environment of the notochord and the IVD) of both cell types and develop cell-based therapy for IVD degeneration.

Materials and Methods

We have generated a mouse model ( Foxa2mNE-Cre/Z/EG double transgenic mouse) to tag the notochord cells and its descendants by GFP, by which notochord lineages can be isolated at different embryonic and postnatal stages in the animal by fluorescence-activated cell sorting (FACS). The enhancer element responsible for Foxa2 expression in the node and notochord has previously been localized to a 520 bp fragment, the minimal notochord element (mNE) (Sasaki and Hogan, 1996). We have utilized this mNE of Foxa2to generate transgenic mice expressing the Crerecombinase ( Cre) gene in the node and notochord. The notochord descendants were labeled after crossing Foxa2mNE-Cre mice with the reporter strain, Z/EG (Novak et al., 2000 ). Enhanced green fluorescent protein (EGFP) signal was detected in the node and the notochord from E8.0 and continued at 1 year of age. EGFP tagged notochord cells were isolated from (a) E8.5 and (b) E9.5 Foxa2mNE-Cre/Z/EG embryos by FACS. The sorted cells were immortalized by SV40 large T antigen.

Results

The established cell lines proliferate and expand in adherent culture. They maintain the in vivo notochord characteristics including the following: (1) Expression of notochord specific transcription factors (c) Foxa2 and (d) Brachyury. (2) Secretion of extracellular matrix component, (e) type II collagen. (3) Sonic hedgehog, (f) Shh, a crucial signaling molecule secreted by the notochord for patterning surrounding tissues is also detected in the cell line.

Conclusion

We have established an initial protocol for the isolation and immortalization of notochord cells. We aim to use these cell lines initially to determine the gene and protein expression profiles, then by induction and ablation of signals to study the impact on NCC fate and their differentiation. This study will contribute to our understanding of notochord biology and their potential for intervertebral disk repair. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Novak, A., Guo,C., Yang,W., Nagy,A., and Lobe,C.G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 2000;28, 147–155 Sasaki, H. and Hogan,B.L. Enhancer analysis of the mouse HNF-3 beta gene: regulatory elements for node/notochord and floor plate are independent and consist of multiple sub-elements. Genes Cells 1996;1:59–72