Published: October 9th, 2018
O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.
Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.
Neural crest cells (NCCs) are multipotent stem-like cells with a remarkable migratory ability and transient existence during embryonic development. NCCs originate between the surface ectoderm and the neural tube and migrate to other parts of the embryo during embryonic development1. Based on their functional domains, NCCs can be classified into several different types, including cranial, trunk, vagal, sacral, and cardiac NCCs. In addition, NCCs can differentiate into multiple cell lineages, such as smooth muscle cells, bone cells, and neurons, and give rise to various tissues2,3. The de....
1. Preparation Before O9-1 Cell Culture
NOTE: Basal media used for O9-1 cell culture must have been conditioned by Sandos inbred mice thioguanine/ouabain-resistant (STO) mouse fibroblast cells; therefore, STO cells need to be obtained and prepared as described below before starting O9-1 cell culture.
The goal of our knockdown and knockout experiments was to study the effects of Yap and Taz loss-of-function in O9-1 cells. Before the knockdown and knockout experiments, we have to make sure that prepare for basal media and culture O9-1 cells as described above (for example, basement membrane matrix needs to cover the whole plate as shown in Figure 1, and O9-1 cells recovered from liquid nitrogen as shown in Figure 2
The NCC is a versatile and key contributor to different tissues and organs during embryonic morphogenesis.The O9-1 cell line maintains its potential to differentiate into many different cell types and mimics the in vivo characteristics of NCCs, making it a useful in vitro tool for studying gene function and molecular regulation in NCCs. The different status of O9-1 cells may correspond to different neural crest progeny in vivo, depending on the culture conditions of O9-1 cells. O9-1 cells can b.......
Nicole Stancel, Ph.D., ELS, of Scientific Publications at the Texas Heart Institute, provided editorial support. We also thank the following funding sources: the American Heart Association's National Center Scientist Development Grant (14SDG19840000 to J. Wang), the 2014 Lawrence Research Award from the Rolanette and Berdon Lawrence Bone Disease Program of Texas (to J. Wang), and the National Institutes of Health (DE026561 and DE025873 to J. Wang, DE016320 and DE019650 to R. Maxson).....
|Active STO feeder cells
|Also available in mitomycin C-inactivated form, catalog # ATCC 56-X
|O9-1 mouse cranial neural crest cell line
|DMEM, high glucose, no glutamine
|DMEM, high glucose
|FBS (fetal bovine serum)
|Penicillin - streptomycin
|L-glutamine 200mM (100X)
|Gelatin from porcine skin
|Trypsin-EDTA 0.25% in HBSS
|DPBS (Dulbecco's phosphate buffered saline) without calcium or magnesium
|MEM non-essential amino acids (MEM NEAA) 100X
|Sodium pyruvate (100mM)
|ESGRO leukemia inhibitory factor (LIF) 106 unit/ml
|Recombinant human fibroblast growth factor-basic (rhFGF-basic)
|Thermo Fisher Scientific
|Opti-MEM I (1X)
|Minimum essential medium, alpha 1X with Earle's salts, ribonucleosides, deoxyribonucleosides, & L-glutamine
|ON-TARGETplus Wwtr1 siRNA
|ON-TARGETplus Non-targeting Pool
|ON-TARGETplus Yap1 siRNA
|FCS (fetal calf serum)
|ITS (insulin-transferrin-selenium )
|BMP2 (bone morphogenetic protein 2)
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