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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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+distribution,+and+risk+factor+correlates+of+high+thoracic+periaortic+fat+in+the+Framingham+Heart+Study.">Prevalence, distribution, and risk factor correlates of high thoracic periaortic fat in the Framingham Heart Study.</a> Journal of the American Heart Association. 1 (6), 004200 (2012).</li><li>Police, S. B., Thatcher, S. E., Charnigo, R., Daugherty, A., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+promotes+inflammation+in+periaortic+adipose+tissue+and+angiotensin+II-induced+abdominal+aortic+aneurysm+formation.">Obesity promotes inflammation in periaortic adipose tissue and angiotensin II-induced abdominal aortic aneurysm formation.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 29 (10), 1458-1464 (2009).</li><li>Farmer, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+adipocyte+formation.">Transcriptional control of adipocyte formation.</a> Cell Metabolism. 4 (4), 263-273 (2006).</li><li>Aune, U. 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(109), e53886 (2016).</li><li>Ye, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+functional+characteristics+of+the+thoracic+aorta+perivascular+adipocyte.">Developmental and functional characteristics of the thoracic aorta perivascular adipocyte.</a> Cellular and Molecular Life Sciences. 76 (4), 777-789 (2019).</li><li>Medeiros, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+the+neural+crest:+new+perspectives+from+lamprey+and+invertebrate+neural+crest-like+cells.">The evolution of the neural crest: new perspectives from lamprey and invertebrate neural crest-like cells.</a> Wiley Interdisciplinary Reviews. Developmental Biology. 2 (1), 1-15 (2013).</li><li>Fu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Crest+Cells+Differentiate+Into+Brown+Adipocytes+and+Contribute+to+Periaortic+Arch+Adipose+Tissue+Formation.">Neural Crest Cells Differentiate Into Brown Adipocytes and Contribute to Periaortic Arch Adipose Tissue Formation.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. , (2019).</li><li>Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K., McMahon, A. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zfp423+expression+identifies+committed+preadipocytes+and+localizes+to+adipose+endothelial+and+perivascular+cells.">Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells.</a> Cell Metabolism. 15 (2), 230-239 (2012).</li><li>Sowa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+stromal+cells+contain+phenotypically+distinct+adipogenic+progenitors+derived+from+neural+crest.">Adipose stromal cells contain phenotypically distinct adipogenic progenitors derived from neural crest.</a> PLoS One. 8 (12), 84206 (2013).</li><li>Billo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+of+adipocytes+by+the+neural+crest.">The generation of adipocytes by the neural crest.</a> Development. 134 (12), 2283-2292 (2007).</li><li>Thelen, K., Ayala-Lopez, N., Watts, S. 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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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

Our simple and practical methodology can be used to obtain abundant ADSCS for studying PVAT adipogenesis in vitro and to the test normal drugs against obesity and related cardiovascular diseases. The advantage of this method is the inherent fluorescence of the NCADSCs of biotin you need to label the cells with flou-chrome conjugated antibodies for FACS, or magnetic activated cell sorting. This method can be used to acquire abundant ADSC derived from neural crest cells from the ectoderm to study PVAT adipogenesis, or lipogenesis in vitro.Using young mice and having experience with cautery and induced adipogenesis means 3T3 cells is critical to the success of this protocol. Demonstrating the procedure will be Yiding Qi, a grad student, and Lian Xu, a technician from my laboratory. For PAAT dissection, after sterilizing the mouse carcass in 75%ethanol for five minutes, use scissors to snip and separate the skin on the abdomen.Cut along the ventral midline from pelvis to the neck, and open the abdomen. Move the liver to expose the diaphragm, and cut the diaphragm and the ribs on both sides of the midline. Peel back the ribs to expose the heart and lungs.And remove the lungs and the thymus. Extract the PAAT along with the aorta and heart into a petri dish. And cut off the aorta at the aortic root to remove the heart.Then, make a cut between the aortic arch and descending aorta. Carefully separate the adipose tissue surrounding both structures, and the left and right common carotid arteries from the posterior chest wall. It is important to separate the PAAT from the root and as a larger vascular vessels carefully, is the presence of vascular wall cells can affect FACS efficiency.Place the tissue into a petri dish containing ice cold HBSS buffer. And use sterile forceps to remove as much of the vasculature and fascia as possible. Then, transfer the adipose tissue into a two milliliter Eppendorf tube, containing point five milliliters of ice cold HBSS buffer on ice.When all of the PAAT has been collected, transfer the harvested adipose tissue into a new two milliliter micro-centrifuge tube containing one milliliter of freshly prepared digestion medium. And use surgical scissors to mince the tissues. Transfer the tissue slurry into a 50 millimeter tube containing nine milliliters of fresh digestion medium.And use a one milliliter pipette to triturate the tissues 10 times. When a homogenous solution has been obtained, incubate the tube for 30 to 45 minutes at 37 degrees Celsius and 100 revolutions per minute, checking every five to 10 minutes to prevent overdigestion. When a light yellow homogenous adipose tissue solution can be observed upon gentle swirling of the tube, stop the digestion with five milliliters of HDMEM supplemented with 10%fetal bovine serum.After a thorough mixing, centrifuge the adipose tissue sample. The stromal vascular cell fraction will be visible as a brownish pellet. Re-suspend the pellet in 10 milliliters of culture medium, and filter the cell suspension through a 70 micrometer cell strainer.Collect the cells by another centrifugation. And gently re-suspend the pellet in five milliliters of erythrocytes lysis buffer. Transfer the suspension to a 15 milliliter tube.After 10 minutes, stop the reaction with two centrifugations in 10 milliliters of PBS, supplemented with 1%fetal bovine serum. After the second centrifugation, re-suspend the pellet in five milliliters of culture medium on ice. And collect the cells with a final centrifugation.Then, re-suspend the cells in five milliliters of FACS buffer for counting. To set up an NCADSC culture, after their isolation by FACS according to standard protocols, place the cells at a five times 10 to the third cells per centimeters squared concentration in each well, a twelve well culture plate. Incomplete culture medium at 37 degrees Celsius and 5%carbon dioxide for 20 to 24 hours.The next day, wash the cells with 37 degrees Celsius PBS, and feed the culture with fresh culture medium. When the culture reaches 80 to 90%confluency, treat the cells with two milliliters of 0.25%trypsin EDTA per well for three to five minutes at 37 degrees Celsius. When the cells have been detached from the bottom of the plate, neutralize the enzymes with two milliliters of cultured medium, and collect the harvested cells by centrifugation.Re-suspend the pellet in one milliliter of fresh culture medium for counting. And seed the cells in a new 12 well culture plate in a five times 10 to the third cells per centimeters squared concentration. Then, re-suspend the remaining cells in culture medium supplemented with 10%dimethyl sulfoxide for frozen storage in liquid nitrogen.To induce adipogenic differentiation, when the cells reach an 80 to 90%confluency, treat the cells with brown or white adipogenic induction medium for two days as appropriate. At the end of the incubation, gently wash the cells two times with PBS before adding the appropriate fresh adipogenic induction medium to each well, and returning the cells to the cell culture incubator for an additional three to five days of incubation. Cultured NCADSCs reach an 80 to 90%confluency after seven to eight days of culture.And demonstrate an expanded fiberglass like morphology. To further confirm their adipogenic potential, oil red staining of the differentiated NCADSCs can be performed to detect mature adipocytes. Typically, mature adipocytes are observed after eight days of white or brown adipogenic induction with over 60%of the NCADSCs exhibiting adipogenic differentiation.Note that NCADSCs demonstrate a greatly reduced adipogenic potential after passaging. Immunoblotting and quantitative real-time PCR reveal that the expression of adipocyte specific relative proteins and genes significantly increases in adipogenically differentiated NCADSCs after eight days of white adipogenic induction. Similarly, the expression of adipocyte specific genes, and brown adipocyte specific genes, also significantly increases after eight days of brown adipogenic induction.Since most NCADSC isolation protocol is simple, economical, and doesn't require the use of antibodies, for that, the strong fluid reasons intensity of IFP can improve the FACS nourishment efficiency.

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Research

JoVE Journal

Developmental Biology

5.9K visualizações.  Shanghai Jiao Tong University. Nossa metodologia simples e prática pode ser usada para obter ADSCS abundante para estudar adipogênese PVAT in vitro e para testar drogas normais contra a obesidade e doenças cardiovasculares relacionadas. A vantagem deste método é a fluorescência inerente dos NCADSCs de biotina que você precisa para rotular as células com anticorpos conjugados flou-cromados para FACS, ou classificação celular ativada magnética. Este método pode ser usado para adquirir ADSC abundante derivado de c...