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-F442A, and OP9 are useful cellular models to study adipogenesis or lipogenesis⁵, regulatory mechanisms for adipogenesis differ between cell lines and primary cells. The ADSCs in the stromal vascular cell fraction (SVF) isolated directly from adipose tissues and induced to differentiate into adipocytes most likely recapitulate in vivo adipogenesis and lipogenesis⁶. However, the fragility, buoyancy, and the variations in size and immunophenotypes of the ADSCs make their direct isolation challenging. In addition, the different isolation procedures can also significantly affect the phenotype and adipogenic potential ability of these cells⁷, thus emphasizing the need for a protocol that maintains ADSC integrity.

Adipose tissue is typically classified as either the morphologically and functionally distinct white adipose tissue (WAT), or the brown adipose tissue (BAT)⁸, which harbors distinct ADSCs⁹. While ADSCs isolated from perigonadal and inguinal subcutaneous WATs have been characterized in previous studies^9,10,11,12, less is known regarding the ADSCs from PVAT that is mainly composed of BAT¹³.

In a recent study, we found that a portion of the resident ADSCs in the periaortic arch adipose tissue (PAAT) are derived from neural crest cells (NCCs), a transient population of migratory progenitor cells that originate from the ectoderm^14,15. Wnt1-Cre transgenic mice were used for tracing neural crest cell development^16,17. We crossed Wnt1-Cre⁺ mice with Rosa26^RFP/+ mice to generate Wnt-1 Cre^+/-;Rosa26^RFP/+ mice, in which NCCs and their descendants are labeled with red fluorescent protein (RFP) and are easily tracked in vivo and in vitro¹⁵. Here, we describe a method for isolating neural crest derived ADSCs (NC-derived ADSCs, or NCADSCs) from mouse PAAT and induce the NCADSCs to differentiate into white adipocytes or brown adipocytes.

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.

1. Sterilize all surgical tools (e.g., surgical scissors, standard forceps, and microsurgical scissors and forceps) by autoclaving at 121 °C for 30 min.
2. Prepare the digestion medium (High glucose Dulbecco's Modified Eagle Medium [HDMEM] containing 2 mg/mL collagenase type I). Prepare the culture medium (HDMEM containing 10% fetal bovine serum [FBS] and 1% v/v penicillin-streptomycin [PS]). Sterilize using a 0.22 μm syringe filter before use.
3. Sterilize the cell culture reagents using UV, ethanol, filtration, or steam, as appropriate.
4. Prepare a Petri dish with Hanks' Balanced Saline Solution (HBSS) and a 15 mL conical tube with 10 mL of HBSS supplemented with 1% v/v of PS solution. Keep both on ice.
5. Anesthetize the mice¹⁵ with isoflurane and sacrifice by cervical dislocation. Immerse the bodies in a beaker filled with 75% alcohol (200 mL) for 5 min to sterilize the skin surface.
6. Snip and separate the skin on the abdomen and cut along the ventral midline from the pelvis to the neck. Open the abdomen and move the liver to expose the diaphragm¹⁹.
7. Cut the diaphragm and the ribs on both sides of the midline and expose the heart and lungs by peeling back the ribs.
8. Remove the lung and thymus and extract the PAAT along with the aorta and heart.
9. Cut off the aorta at the aortic root to remove the heart. Make a cut between the aortic arch and descending aorta and carefully separate the adipose tissue surrounding both structures and the left and right common carotid arteries from the posterior chest wall. Transfer the tissue to the ice-cold HBSS buffer in the Petri dish.
10. Using sterile forceps, remove as much of the vasculature (e.g., aorta, common carotid arteries, and other small vasculature) and fascia as possible, and transfer the adipose tissue into the 2 mL Eppendorf tubes contain 0.5 mL ice-cold HBSS buffer on ice.

3. Isolation of the SVF

Collect the PAAT of 5-6 mice into one 2 mL microcentrifuge tube containing 1 mL freshly prepared digestion medium and mince the tissue using surgical scissors in an Eppendorf tube at room temperature (RT).

1. Transfer the mix into 50 mL tubes containing 9 mL of the digestion medium. Homogenize the tissues by pipetting up and down with a 1 mL pipette 10x.
2. Incubate the tubes at 37 °C with constant shaking at 100 rpm for 30–45 min and check every 5–10 min to prevent overdigestion. This is critical to improving cell viability and yield.
   NOTE: Good tissue digestion will result in a homogenous, light yellow adipose tissue that is visible to the naked eye upon gently swirling the tube.
3. Stop the digestion by adding 5 mL of HDMEM containing 10% FBS and 1% v/v PS at RT and mix well by pipetting.
4. Centrifuge the cell suspension at 500 x g for 5 min at RT. The SVF will be visible as a brownish pellet. Carefully aspirate the floating adipocytes and decant the remaining supernatant without disturbing the SVF. Dissolve the SVF pellet in 10 mL of culture medium and filter through a 70 µm cell strainer.
5. Centrifuge the cell suspension at 500 x g for 5 min, remove the supernatant, and gently resuspend the pellet in 5 mL of erythrocyte lysis buffer in a 15 mL conical tube for 10 min at RT.
6. Stop the reaction by adding 10 mL of 1x PBS containing 1% FBS. Centrifuge the cell suspension at 500 x g for 5 min at 4 °C, remove the supernatant, and resuspend the pellet in 10 mL of 1x PBS containing 1% FBS.
7. Centrifuge the cells again at 500 x g for 5 min at 4 °C. Remove the supernatant and resuspend the pellet in 5 mL of culture medium in a 15 mL conical tube at 4 °C.
8. After a final round of centrifugation (500 x g for 5 min at 4 °C), resuspend the pelleted cells in 5 mL of FACS buffer (PBS containing 10% FBS, 100 units/mL DNA I, and 1% v/v PS) on ice, and count the cells with a hemocytometer.

4. Isolation of NCADSCs by FACS

1. Set up and optimize the cell sorter following the instruction manual. Select the 100 µm nozzle, sterilize the collection tubes, install the required collection device, and set up the side streams²⁰.
2. A 561 nm yellow/green laser and optical filter 579/16 are recommended for sorting RFP⁺ cells. Perform the compensation using the negative control and the single-stained positive controls. See Figure 2A for the gating scheme.
3. Filter the cells through a 40 µm strainer, centrifuge at 500 x g for 5 min, and resuspend the cells in 2 mL of FACS buffer at a density of 0.5–1 x 10⁷/mL. Transfer the cells to clearly labeled 5 mL round bottom polystyrene tubes, and load into the sorter.
4. Run the experimental sample tube at 4 °C, turn on the deflection plates, and sort into a 15 mL conical tube precoated with RPMI containing 1% FBS and 1% v/v PS.
   NOTE: Protect the samples from strong light to minimize RFP quenching.

5. Culture of NCADSCs

1. Plate the sorted cells at a density of 5,000 cells/cm² in a 12 well culture plate in complete culture medium and incubate at 37 °C in a humid atmosphere with 5% CO₂ for 20–24 h.
2. Remove the culture medium, wash the cells with prewarmed (37 °C) PBS to remove cell debris and add fresh culture medium.
3. Once the cells are 80–90% confluent, digest the monolayer using a 0.25% trypsin EDTA solution at 37 °C in an incubator for 3–5 min, and neutralize with 2 mL of culture medium.
4. Centrifuge the harvested cells for 15 min at 250 x g at RT, remove the supernatant, and resuspend the cells in 1 mL of culture medium. Count the cells with a hemocytometer.
5. Seed the cells in a 12 well culture plate at the density of 5,000 cells/cm².
6. Resuspend the remaining cells in culture medium containing 10% DMSO, freeze, and store in liquid nitrogen.

6. Adipogenic Induction of NCADSCs

1. Induce adipogenic differentiation of the NCADSCs at 80–90% confluency and standard culture conditions²¹.
2. For brown adipogenic induction, first treat the cultured cells with brown adipogenic induction medium (HDMEM, 10% FBS, 1% v/v PS, 0.5 mM/L IBMX, 0.1μM/L dexamethasone, 1 μM/L rosiglitazone, 10 nmol/L triiodothyronine, and 1 μg/mL insulin) for 2 days. Wash the cells with PBS 2x and replace with fresh medium (HDMEM, 10% FBS, 1% v/v PS, 1 μM/L rosiglitazone, 10 nmol/L triiodothyronine, and 1 μg/mL insulin). Change this medium every 2 days for a total of 3–5x.
3. For white adipogenic induction, first treat the cells with white adipogenic induction medium (HDMEM, 10% FBS, 1% v/v PS, 0.5 mM/L IBMX, 0.1 μM/L dexamethasone, and 1 μg/mL insulin) for 2 days. Wash the cells with PBS 2x and replace with fresh medium (HDMEM, 10% FBS, 1% v/v PS, and 1 μg/mL insulin). Change this medium every 2 days for a total of 3-5x.
4. Analyze the adipogenic cells as appropriate.
   NOTE: Be gentle when washing the cells with PBS. Differentiated adipocytes can easily wash away.

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 fibro...

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

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