Culturing and Manipulation of O9-1 Neural Crest Cells

Summary

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.

Abstract

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.

Introduction

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 tissues^2,3. The de....

Protocol

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.

1. Active STO cell culture
   1. Prepare media for culturing active STO cells by making complete Dulbecco's modified Eagle's media (DMEM) with 7% fetal bovi.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Active STO feeder cells	ATCC	ATCC CRL-1503	Also available in mitomycin C-inactivated form, catalog # ATCC 56-X
O9-1 mouse cranial neural crest cell line	Millipore Sigma	SCC049
DMEM, high glucose, no glutamine	Gibco	11960-044
DMEM, high glucose	Hyclone	SH30243.01
FBS (fetal bovine serum)	Millipore Sigma	ES-009-B
Penicillin - streptomycin	Gibco	15140-122
L-glutamine 200mM (100X)	Gibco	25030-081
Gelatin from porcine skin	Sigma	G1890
Trypsin-EDTA 0.25% in HBSS	Genesee Scientific	25-510
DPBS (Dulbecco's phosphate buffered saline) without calcium or magnesium	Lonza	17-512F
MEM non-essential amino acids (MEM NEAA) 100X	Gibco	11140-050
Sodium pyruvate (100mM)	Gibco	11360-070
2-Mercaptoethanol	Sigma	M-7522
ESGRO leukemia inhibitory factor (LIF) 106 unit/ml	Millipore Sigma	ESG1106
Recombinant human fibroblast growth factor-basic (rhFGF-basic)	R&D Systems	233-FB-025
Mitomycin C	Roche	10107409001
Matrigel matrix	Corning	356234
DMSO (dimethylsulfoxide)	Millipore Sigma	MX1458-6
Lipofectamine RNAiMAX	Thermo Fisher Scientific	13778-075
Opti-MEM I (1X)	Gibco	31985-070
Minimum essential medium, alpha 1X with Earle's salts, ribonucleosides, deoxyribonucleosides, & L-glutamine	Corning	10-022-CV
ON-TARGETplus Wwtr1 siRNA	Dharmacon	L-041057
ON-TARGETplus Non-targeting Pool	Dharmacon	D-001810
ON-TARGETplus Yap1 siRNA	Dharmacon	L-046247
FCS (fetal calf serum)
ITS (insulin-transferrin-selenium )
TGF-b3
Ascorbic acid
BMP2 (bone morphogenetic protein 2)
Dexamethasone
B-27 supplement