Name: isolation, culture, and adipogenic induction of neural crest original adipose-derived stem cells from periaortic adipose tissue
Uploaded: 2020-03-02T12:30:00
Description: Watch this Scientific Journal Video about Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue at JoVE.com

National Key R&#38;D Program of China (2018YFC1312504), National Natural Science Foundation of China (81970378, 81670360, 81870293), and Science and Technology Commission of Shanghai Municipality (17411971000, 17140902402) provided the funds for this study.

The animal protocol has been reviewed and approved by the Animal Care Committee of Shanghai Jiao Tong University.
1. Generation of Wnt-1 Cre+/-;Rosa26RFP/+ Mice
<ol>
	<li>Cross Wnt-1 Cre+/- mice16 with Rosa26RFP/+ mice18 to generate Wnt-1 Cre+/-;Rosa26RFP/+ mice. House mice under a 12 h light/dark cycle in a pathogen-free facility at 25 &#176;C and 45% humidity until they are 4&#...

<ol>
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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+/-;Rosa26RFP/+ 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<sup cla...

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 disease2,3, 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 studies9,10,11,12, 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 ectoderm14,15. Wnt1-Cre transgenic mice were used for tracing neural crest cell development16,17. We crossed Wnt1-Cre+ mice with Rosa26RFP/+ mice to generate Wnt-1 Cre+/-;Rosa26RFP/+ 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.

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	<tbody>
		<tr>
			<td>4% PFA</td>
			<td>BBI life sciences</td>
			<td>E672002-0500</td>
			<td>Lot #: EC11FA0001</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>ABCONE (China)</td>
			<td>A47902</td>
			<td>1% working concentration</td>
		</tr>
		<tr>
			<td>Anti-cebp/&#945;</td>
			<td>ABclonal</td>
			<td>A0904</td>
			<td>1:1000 working concentration</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked</td>
			<td>CST</td>
			<td>7076</td>
			<td>1:5000 working concentration</td>
		</tr>
		<tr>
			<td>Anti-perilipin</td>
			<td>Abcam</td>
			<td>AB61682</td>
			<td>1 &#956;g/mL working concentration; lot #: GR66486-54</td>
		</tr>
		<tr>
			<td>Anti-PPARy</td>
			<td>SANTA CRUZ</td>
			<td>sc-7273</td>
			<td>0.2 &#956;g/mL working concentration</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked</td>
			<td>CST</td>
			<td>7074</td>
			<td>1:5000 working concentration</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin</td>
			<td>CST</td>
			<td>2146</td>
			<td>1:1000 working concentration</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>VWR life sciences</td>
			<td>0332-100G</td>
			<td>50 mg/mL working concentration; lot #: 0536C008</td>
		</tr>
		<tr>
			<td>Collagenase, Type I</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
			<td>0.1 &#181;M working concentration</td>
		</tr>
		<tr>
			<td>Erythrocyte Lysis Buffer</td>
			<td>Invitrogen</td>
			<td>00-4333</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Corning</td>
			<td>R35-076-CV</td>
			<td>50 mg/mL working concentration; lot #: R2040212FBS</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>HDMEM</td>
			<td>Gelifesciences</td>
			<td>SH30243.01</td>
			<td>Lot #: AD20813268</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td>0.5 mM working concentration</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I3536</td>
			<td>1 &#956;g/mL working concentration</td>
		</tr>
		<tr>
			<td>Microsurgical forceps</td>
			<td>Suzhou Mingren Medical Equipment Co.,Ltd. (China)</td>
			<td>MR-F201A-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissor</td>
			<td>Suzhou Mingren Medical Equipment Co.,Ltd. (China)</td>
			<td>MR-H121A</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O solution</td>
			<td>Sigma-Aldrich</td>
			<td>O1516</td>
			<td>0.3% working concentration</td>
		</tr>
		<tr>
			<td>PBS (Phosphate buffered saline)</td>
			<td>ABCONE (China)</td>
			<td>P41970</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript RT reagent Kit</td>
			<td>TAKARA</td>
			<td>RR047A</td>
			<td>Lot #: AK4802</td>
		</tr>
		<tr>
			<td>RNeasy kit</td>
			<td>TAKARA</td>
			<td>9767</td>
			<td>Lot #: AHF1991D</td>
		</tr>
		<tr>
			<td>Rosa26RFP/+ mice</td>
			<td>JAX</td>
			<td>No.007909</td>
			<td>C57BL/6 backgroud; male and female</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td>1 &#956;M working concentration</td>
		</tr>
		<tr>
			<td>Standard forceps</td>
			<td>Suzhou Mingren Medical Equipment Co.,Ltd. (China)</td>
			<td>MR-F424</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Suzhou Mingren Medical Equipment Co.,Ltd. (China)</td>
			<td>MR-S231</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Premix Ex Taq</td>
			<td>TAKARA</td>
			<td>RR420A</td>
			<td>Lot #: AK9003</td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2877</td>
			<td>10 nM working concentration</td>
		</tr>
		<tr>
			<td>Wnt1-Cre+;PPAR&#947;flox/flox mice</td>
			<td>JAX</td>
			<td>No.009107</td>
			<td>C57BL/6 backgroud; male and female</td>
		</tr>
	</tbody>
</table>

isolation, culture, and adipogenic induction of neural crest original adipose-derived stem cells from periaortic adipose tissue

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+/-;Rosa26RFP/+ 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&#8211;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.

Using the protocol described above, we obtained ~0.5&#8211;1.0 x 106 ADSCs from 5&#8211;6 Wnt-1 Cre+/-;Rosa26RFP/+ 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&#8211;90% confluency after 7&#8211;8 days of culture, and the NCADSCs had an expanded fibro...

Watch this Scientific Journal Video about Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue at JoVE.com

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

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+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

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