Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

Summary

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre^+/-;Rosa26^RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

Abstract

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.

In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre^+/-;Rosa26^RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP⁺ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3–5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

Introduction

The prevalence of obesity is increasing worldwide, which increases the risk of related chronic diseases, including cardiovascular disease and diabetes¹. PVAT surrounds blood vessels and is a major source of endocrine and paracrine factors involved in vasculature function. Clinical studies show that high PVAT content is an independent risk factor of cardiovascular disease^2,3, and its pathological function depends on the phenotype of the constituent adipose-derived stem cells (ADSCs)⁴.

Although ADSC cell lines like the murine 3T3-L1, 3T3....

Protocol

The animal protocol has been reviewed and approved by the Animal Care Committee of Shanghai Jiao Tong University.

1. Generation of Wnt-1 Cre^+/-;Rosa26^RFP/+ Mice

1. Cross Wnt-1 Cre^+/- mice¹⁶ with Rosa26^RFP/+ mice¹⁸ to generate Wnt-1 Cre^+/-;Rosa26^RFP/+ mice. House mice under a 12 h light/dark cycle in a pathogen-free facility at 25 °C and 45% humidity until they are 4–8 weeks old.

2. Dissection of the PAAT

NOTE: See Figure 1

Results

Using the protocol described above, we obtained ~0.5–1.0 x 10⁶ ADSCs from 5–6 Wnt-1 Cre^+/-;Rosa26^RFP/+ mice (48 weeks old, male or female).

The flow chart of collection of PAAT from mice is presented in Figure 1. The morphology of the NCADSCs was similar to the ADSC from other mice adipose tissues. The cultured NCADSCs reached 80–90% confluency after 7–8 days of culture, and the NCADSCs had an expanded fibroblast-like morphology (<.......

Discussion

In this study, we present a reliable method for the isolation, culture, and adipogenic induction of NCADSCs extracted from the PVAT of Wnt-1 Cre^+/-;Rosa26^RFP/+ transgenic mice designed to produce RFP⁺ ADSCs. Previous reports show that there is no significant difference in the expression of general multipotent mesenchymal stem cells (MSCs) markers in NCADSCs and non NCADSCs²², and that NCADSCs have a strong potential to differentiate into adipocytes in vitro

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% PFA	BBI life sciences	E672002-0500	Lot #: EC11FA0001
Agarose	ABCONE (China)	A47902	1% working concentration
Anti-cebp/α	ABclonal	A0904	1:1000 working concentration
Anti-mouse IgG, HRP-linked	CST	7076	1:5000 working concentration
Anti-perilipin	Abcam	AB61682	1 μg/mL working concentration; lot #: GR66486-54
Anti-PPARy	SANTA CRUZ	sc-7273	0.2 μg/mL working concentration
Anti-rabbit IgG, HRP-linked	CST	7074	1:5000 working concentration
Anti-β-Tubulin	CST	2146	1:1000 working concentration
BSA	VWR life sciences	0332-100G	50 mg/mL working concentration; lot #: 0536C008
Collagenase, Type I	Gibco	17018029
Dexamethasone	Sigma-Aldrich	D4902	0.1 µM working concentration
Erythrocyte Lysis Buffer	Invitrogen	00-4333
FBS	Corning	R35-076-CV	50 mg/mL working concentration; lot #: R2040212FBS
HBSS	Gibco	14025092
HDMEM	Gelifesciences	SH30243.01	Lot #: AD20813268
IBMX	Sigma-Aldrich	I7018	0.5 mM working concentration
Insulin	Sigma-Aldrich	I3536	1 μg/mL working concentration
Microsurgical forceps	Suzhou Mingren Medical Equipment Co.,Ltd. (China)	MR-F201A-1
Microsurgical scissor	Suzhou Mingren Medical Equipment Co.,Ltd. (China)	MR-H121A
Oil Red O solution	Sigma-Aldrich	O1516	0.3% working concentration
PBS (Phosphate buffered saline)	ABCONE (China)	P41970
Penicillin-Streptomycin	Gibco	15140122
PrimeScript RT reagent Kit	TAKARA	RR047A	Lot #: AK4802
RNeasy kit	TAKARA	9767	Lot #: AHF1991D
Rosa26RFP/+ mice	JAX	No.007909	C57BL/6 backgroud; male and female
Rosiglitazone	Sigma-Aldrich	R2408	1 μM working concentration
Standard forceps	Suzhou Mingren Medical Equipment Co.,Ltd. (China)	MR-F424
Surgical scissor	Suzhou Mingren Medical Equipment Co.,Ltd. (China)	MR-S231
SYBR Premix Ex Taq	TAKARA	RR420A	Lot #: AK9003
Triiodothyronine	Sigma-Aldrich	T2877	10 nM working concentration
Wnt1-Cre+;PPARγflox/flox mice	JAX	No.009107	C57BL/6 backgroud; male and female