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 tissues2,3. The development of NCCs is characterized by a complex series of morphogenetic events that are fine-tuned by various molecular signals. Given the complex regulation of NCCs and their important contributions to numerous structures, the dysregulation of NCC development can commonly lead to congenital birth defects, which account for nearly 30% of all human congenital birth defects. Abnormalities during the neural crest development can lead to cleft lip/palate, flawed nose formation, syndromes, defects such as a defective cardiac outflow tract, or even infant mortality1,4,5,6,7. Understanding the molecular mechanisms of NCC development is important for developing treatments for diseases caused by defects in NCC development. With the use of various in vitro and in vivo approaches8,9,10,11,12,13,14,15, considerable progress has been made in NCC research. In vivo, animal models, including chickens, amphibians, zebrafish, and mice, have been used to investigate NCCs1. Furthermore, human embryos have been used to study the process of NCC migration in early human embryo development16. In vitro, cell models for NCCs, such as human NCCs that originated from patient subcutaneous fat, have been used to investigate Parkinson's disease17. The O9-1 NCC line, which was originally derived from mass cultures of endogenous NCCs isolated from E8.5 mouse embryos18, is a powerful cell model for studying NCCs. Importantly, under non-differentiating culture conditions, O9-1 cells are multipotent stem-like NCCs. However, under varying culture conditions, O9-1 cells can be differentiated into distinguished cell types, such as smooth muscle cells, osteoblasts, chondrocytes, and glial cells18. Given these properties, O9-1 cells have been broadly used for NCC-related studies, such as investigating the molecular mechanism of cranial-facial defects19,20.
Here, detailed protocols are provided for maintaining O9-1 cells, differentiating O9-1 cells into different cell types, and manipulating O9-1 cells by performing gene loss-of-function studies with CRISPR-Cas9 genome editing and small interfering RNA (siRNA)-mediated knockdown technologies. As a representative example, the use of O9-1 cells to study Yap and Taz loss-of-function is described. Yap and Taz are the downstream effectors of the Hippo signaling pathway, which plays a critical role in the cell proliferation, differentiation, and apoptosis. The Hippo pathway has also been shown to be important in the development and homeostasis of several different tissues and organs, as well as in the pathogenesis of different diseases20,21,22,23,24,25,26,27,28. The core components of Hippo signaling include the tumor suppressor sterile 20-like kinases Mst1/2, WW domain-containing Salvador scaffold protein, and the large tumor suppressor homolog (Lats1/2) kinases. Hippo signaling inhibits Yap and Taz activity and promotes their degradation in the cytoplasm. Without repression from Hippo, Yap and Taz can translocate into nuclei and function as transcriptional co-activators. We recently showed that specifically inactivating the Hippo signaling effectors Yap and Taz in mouse NCCs by using the Wnt1cre and Wnt1Cre2SOR drivers resulted in embryonic lethality at E10.5 with severe craniofacial defects20. We have also performed studies using O9-1 cells to investigate the role of Yap and Taz in NCCs. To study Yap and Taz function in NCC proliferation and differentiation, Yap and Taz knockdown cells were generated in O9-1 cells by using siRNA, and Yap knockout cells were generated by using CRISPR-Cas9 genome editing. The same gene loss-of-function strategies can be applied to different target genes in other pathways. In addition, gain-of-function studies and transfection assays can also be applied to O9-1 cells to study gene function and regulation. The protocols described here are intended to be used by investigators as guides for culturing O9-1 cells to maintain multipotent stemness, for differentiating O9-1 cells into other cell types under different culture conditions, and for studying gene function and the molecular mechanisms of NCCs.
Protocol
Representative Results
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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 |
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