Name: isolation of rat adipose tissue mesenchymal stem cells for differentiation into insulin-producing cells
Uploaded: 2022-08-29T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells at JoVE.com

We acknowledge Dr. Rawda Samir Mohamed, MSc, Veterinarian Specialist, Faculty of Pharmacy, The British University of Egypt (BUE) for helping with the dissection of the rats.
We also would like to acknowledge and appreciate the efforts of the Faculty of Mass Communication, The British University in Egypt (BUE) for the production and editing of the video of this manuscript.
We would like to thank Miss Fatma Masoud, MSc, Assistant Lecturer of English, The British University in Egypt (BUE) for the revision and English language proofreading of the manuscript.
This work was partially funded by the Center for Drug Research and Development (CDRD), Faculty of Pharmacy, The British University in Egypt (BUE), Cairo, Egypt.

All experiments were carried out according to the approved guidelines, and all procedures were approved by the Ethical Committee of the Faculty of Pharmacy, The British University in Egypt (BUE), Cairo, Egypt. The Ad-MSC isolation protocol was adopted from Lopez and Spencer, with modifications15.
1. Isolation of Ad-MSCs from rat epididymal fat pads
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	<li>Use male Sprague-Dawley rats weighing 250-300 g that are not more than 1 month of age (two per...

<ol>
	<li>Hmadcha, A., Martin-Montalvo, A., Gauthier, B. R., Soria, B., Capilla-Gonzalez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+mesenchymal+stem+cells+for+cancer+therapy.">Therapeutic potential of mesenchymal stem cells for cancer therapy.</a> Frontiers in Bioengineering and Biotechnology. 8, 43 (2020).</li><li>Kamal, M., Kassem, D., Haider, K. H., Haider, K. 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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=Differential+expression+of+stem+cell+mobilization-associated+molecules+on+multi-lineage+cells+from+adipose+tissue+and+bone+marrow.">Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow.</a> Immunology Letters. 89 (2-3), 267-270 (2003).</li><li>Mosna, F., Sensebe, L., Krampera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+marrow+and+adipose+tissue+mesenchymal+stem+cells:+A+user's+guide.">Human bone marrow and adipose tissue mesenchymal stem cells: A user's guide.</a> Stem Cells and Development. 19 (10), 1449-1470 (2010).</li><li>Camara, B. O. S., 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=Differentiation+of+canine+adipose+mesenchymal+stem+cells+into+insulin-producing+cells:+Comparison+of+different+culture+medium+compositions.">Differentiation of canine adipose mesenchymal stem cells into insulin-producing cells: Comparison of different culture medium compositions.</a> Domestic Animal Endocrinology. 74, 106572 (2021).</li><li>Ren, 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=Isolation,+expansion,+and+differentiation+of+goat+adipose-derived+stem+cells.">Isolation, expansion, and differentiation of goat adipose-derived stem cells.</a> Research in Veterinary Science. 93 (1), 404-411 (2012).</li><li>Vallee, M., Cote, J. 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R., 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=Overexpression+of+apelin+in+Wharton's+jelly+mesenchymal+stem+cell+reverses+insulin+resistance+and+promotes+pancreatic+&#946;+cell+proliferation+in+type+2+diabetic+rats.">Overexpression of apelin in Wharton's jelly mesenchymal stem cell reverses insulin resistance and promotes pancreatic &#946; cell proliferation in type 2 diabetic rats.</a> Stem Cell Research &amp; Therapy. 9 (1), 339 (2018).</li><li>Ghoneim, M. A., Refaie, A. F., Elbassiouny, B. L., Gabr, M. M., Zakaria, M. 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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exendin-4+enhances+the+differentiation+of+Wharton's+jelly+mesenchymal+stem+cells+into+insulin-producing+cells+through+activation+of+various+&#946;-cell+markers.">Exendin-4 enhances the differentiation of Wharton's jelly mesenchymal stem cells into insulin-producing cells through activation of various &#946;-cell markers.</a> Stem Cell Research &amp; Therapy. 7, 108 (2016).</li><li>Yang, Z., Li, K., Yan, X., Dong, F., Zhao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+diabetic+retinopathy+by+engrafted+human+adipose-derived+mesenchymal+stem+cells+in+streptozotocin+diabetic+rats.">Amelioration of diabetic retinopathy by engrafted human adipose-derived mesenchymal stem cells in streptozotocin diabetic rats.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 248 (10), 1415-1422 (2010).</li><li>Zhang, N., Li, J., Luo, R., Jiang, J., Wang, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+mesenchymal+stem+cells+induce+angiogenesis+and+attenuate+the+remodeling+of+diabetic+cardiomyopathy.">Bone marrow mesenchymal stem cells induce angiogenesis and attenuate the remodeling of diabetic cardiomyopathy.</a> Experimental and Clinical Endocrinology &amp; Diabetes. 116 (2), 104-111 (2008).</li><li>Zhao, A. G., Shah, K., Freitag, J., Cromer, B., Sumer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+potential+of+early-+and+late-passage+adipose-derived+mesenchymal+stem+cells+cultured+under+hypoxia+and+normoxia.">Differentiation potential of early- and late-passage adipose-derived mesenchymal stem cells cultured under hypoxia and normoxia.</a> Stem Cells International. 2020, 8898221 (2020).</li><li>Krishnamurthy, H., Cram, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+flow+cytometry.">Basics of flow cytometry.</a> Applications of Flow Cytometry in Stem Cell Research and Tissue. , 1-12 (2010).</li><li>Habib, S. A., Kamal, M. M., El-Maraghy, S. A., Senousy, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exendin-4+enhances+osteogenic+differentiation+of+adipose+tissue+mesenchymal+stem+cells+through+the+receptor+activator+of+nuclear+factor-kappa+B+and+osteoprotegerin+signaling+pathway.">Exendin-4 enhances osteogenic differentiation of adipose tissue mesenchymal stem cells through the receptor activator of nuclear factor-kappa B and osteoprotegerin signaling pathway.</a> Journal of Cellular Biochemistry. , (2022).</li><li>Qi, 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-derived+mesenchymal+stem+cells+from+obese+mice+prevent+body+weight+gain+and+hyperglycemia.">Adipose-derived mesenchymal stem cells from obese mice prevent body weight gain and hyperglycemia.</a> Stem Cell Research &amp; Therapy. 12 (1), 277 (2021).</li><li>Tiryaki, T., Conde-Green, A., Cohen, S. R., Canikyan, S., Kocak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3-step+mechanical+digestion+method+to+harvest+adipose-derived+stromal+vascular+fraction.">A 3-step mechanical digestion method to harvest adipose-derived stromal vascular fraction.</a> Plastic and Reconstructive Surgery - Global Open. 8 (2), 2652 (2020).</li><li>Alstrup, T., Eijken, M., Bohn, A. B., Moller, B., Damsgaard, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adipose+tissue-derived+stem+cells:+Enzymatic+digestion+in+combination+with+mechanical+distortion+to+increase+adipose+tissue-derived+stem+cell+yield+from+human+aspirated+fat.">Isolation of adipose tissue-derived stem cells: Enzymatic digestion in combination with mechanical distortion to increase adipose tissue-derived stem cell yield from human aspirated fat.</a> Current Protocols in Stem Cell Biology. 48 (1), 68 (2019).</li><li>Taghizadeh, R. R., Cetrulo, K. J., Cetrulo, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+impacts+the+quantity+and+quality+of+native+mesenchymal+stem/stromal+cells+derived+during+processing+of+umbilical+cord+tissue.">Collagenase impacts the quantity and quality of native mesenchymal stem/stromal cells derived during processing of umbilical cord tissue.</a> Cell Transplantation. 27 (1), 181-193 (2018).</li><li>Kamal, M. M., Kassem, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+Wharton's+jelly+mesenchymal+stem+cells+for+diabetes:+Achievements+and+challenges.">Therapeutic potential of Wharton's jelly mesenchymal stem cells for diabetes: Achievements and challenges.</a> Frontiers in Cell and Developmental Biology. 8, 16 (2020).</li><li>Gabr, M. 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=From+human+mesenchymal+stem+cells+to+insulin-producing+cells:+Comparison+between+bone+marrow-+and+adipose+tissue-derived+cells.">From human mesenchymal stem cells to insulin-producing cells: Comparison between bone marrow- and adipose tissue-derived cells.</a> BioMed Research International. 2017, 3854232 (2017).</li><li>Xin, 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=Insulin-producing+cells+differentiated+from+human+bone+marrow+mesenchymal+stem+cells+in+vitro+ameliorate+streptozotocin-induced+diabetic+hyperglycemia.">Insulin-producing cells differentiated from human bone marrow mesenchymal stem cells in vitro ameliorate streptozotocin-induced diabetic hyperglycemia.</a> PLoS One. 11 (1), 0145838 (2016).</li><li>Kassem, D. H., Kamal, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+efficacy+of+umbilical+cord-derived+stem+cells+for+diabetes+mellitus:+A+meta-analysis+study.">Therapeutic efficacy of umbilical cord-derived stem cells for diabetes mellitus: A meta-analysis study.</a> Stem Cell Research &amp; Therapy. 11 (1), 484 (2020).</li><li>El-Demerdash, R. F., Hammad, L. N., Kamal, M. M., El Mesallamy, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+Wharton's+jelly+and+cord+blood+as+a+source+of+mesenchymal+stem+cells+for+diabetes+cell+therapy.">A comparison of Wharton's jelly and cord blood as a source of mesenchymal stem cells for diabetes cell therapy.</a> Regenerative Medicine. 10 (7), 841-855 (2015).</li><li>Kassem, D. H., Kamal, M. M., El-Kholy, A. E. -. L. G., El-Mesallamy, H. 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In this protocol, we managed to present a detailed protocol for the isolation of Ad-MSCs from rat epididymal fat and the differentiation of these Ad-MSCs into IPCs. In fact, rat epidydimal fat is an easily attainable source of adipose tissue for obtaining Ad-MSCs and does not require any special equipment, neither for collection nor for processing15,26,27. The isolated Ad-MSCs showed excellent culture expansion and exhibited all...

Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are among the most widely used cell types for regenerative medicine1,2. They are classified as adult stem cells and characterized by multilineage differentiation potential and self-renewal capacity3. MSCs can be isolated and obtained from various sources, including adipose tissue, bone marrow, peripheral blood, umbilical cord tissue and blood, hair follicles, and teeth4,5.
The isolation of stem cells from adipose tissue is seen as both appealing and promising due to their easy access, rapid expansion in vitro, and high yield6. Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be isolated from different species such as humans, bovines, mice, rats, and, more recently, goats7. It has been proven that Ad-MSCs are now potential candidates for tissue engineering and gene/cell therapy that can even be used to develop an autologous alternative for the long-term repair of soft tissue injury or defects7,8.
The International Society for Cell and Gene Therapy (ISCT) has defined three minimum criteria that must be exhibited by MSCs for full characterization9. First, they must be plastic adherent. Second, MSCs should express mesenchymal stem cell surface markers such as CD73, CD90, and CD105 and lack expression of the hematopoietic markers CD45, CD34, CD14 or CD11b, CD79&#945; or CD19, and HLA-DR. Finally, MSCs should exhibit the ability to differentiate into the three mesenchymal lineages: adipocytes, osteocytes, and chondrocytes. Interestingly, MSCs can also differentiate into other lineages such as neuronal cells, cardiomyocytes, hepatocytes, and epithelial cells10,11.
In fact, MSCs possess unique properties that enable them to be applied as potential therapeutic agents in regenerative therapy for different diseases. MSCs can secrete soluble factors to induce an immunomodulatory environment that provides therapeutic benefits12. In addition, MSCs can migrate toward sites of injury and tumor microenvironments to deliver targeted therapy; however, the mechanisms are not fully elucidated13. In addition, MSCs have the ability to secrete exosomes, extracellular vesicles in the nanoscale that carry a cargo of non-coding RNAs, protein, and soluble factors, which lately emerged as a novel mechanism of the MSCs&#39; therapeutic potential in various diseases14.
More importantly, MSCs have generated marked attention for their potential to differentiate into insulin-producing cells (IPCs), either by genetic modification15,16 or through utilizing various extrinsic-inducing factors within the culture media in vitro17. The IPC induction period varies greatly, as it depends on the used induction protocol and the utilized extrinsic factors. The process of differentiation can last from days to months, and it requires a combination of exogenous-inducing factors that must be added and/or withdrawn in different stages. Many of these factors that have been used for endocrine pancreatic differentiation are biologically active compounds that have been shown to promote the proliferation or differentiation/neogenesis of insulin-secreting &#946;-cells and/or increase the insulin content of IPCs18,19,20,21. It is noteworthy here that MSCs have also been reported to have therapeutic effects in diabetes and its complications via several mechanisms, including their secretome, as well as a wide array of immuno-modulatory actions22,23,24.
In this protocol, we present a detailed stepwise protocol for the isolation and characterization of Ad-MSCs from rat epididymal fat, followed by a simple, relatively short protocol for the generation of IPCs from Ad-MSCs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Albumin, bovine serum Fraction V</td>
			<td>MP Biomedicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A3157</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody for Rat CD90, FITC</td>
			<td>Stem Cell Technologies</td>
			<td>60024FI</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A3912</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD105 Monoclonal Antibody, FITC</td>
			<td>Thermo Fisher Scientific, Invitrogen, USA</td>
			<td>MA1-19594</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 Polyclonal Antibody</td>
			<td>Thermo Fisher Scientific, Invitrogen, USA</td>
			<td>PA5-85917</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I, powder</td>
			<td>Gibco, Thermo Fisher, USA</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose anhydrous, extra pure</td>
			<td>Fisher Scientific, Germany</td>
			<td>G/0450/53</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific, Germany</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithizone staining</td>
			<td>Sigma-Aldrich, USA</td>
			<td>D5130</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - High Glucose 4.5 g/L</td>
			<td>Lonza, Switzerland</td>
			<td>12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - Low Glucose 1 g/L</td>
			<td>Lonza, Switzerland</td>
			<td>12-707F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>Lonza, Switzerland</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse/RNAse free water</td>
			<td>Gibco Thermo Fisher, USA</td>
			<td>10977-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute, Molecular biology grade</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>24103</td>
			<td></td>
		</tr>
		<tr>
			<td>Exendin-4</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>E7144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Thermo Fisher, Brazil</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, Molecular biology grade</td>
			<td>Fisher Scientific, USA</td>
			<td>BP2618500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco Thermo Fisher, USA</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride (Anhydrous)</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mesenchymal Stem Cell Functional identification kit</td>
			<td>R&#38;D systems Inc., MN, USA</td>
			<td>SC006</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red Stain</td>
			<td>Sigma-Aldrich, USA</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Amphotericin</td>
			<td>Gibco Thermo Fisher, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 1X, without Ca/Mg</td>
			<td>Lonza, Switzerland</td>
			<td>BE17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Insulin ELISA Kit</td>
			<td>Cloud-Clone Corp., USA</td>
			<td>CEA682Ra</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic (Anhydrous)</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic (Anhydrous)</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green Maxima</td>
			<td>Thermo Scientific, USA</td>
			<td>K0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, 0.2 micron</td>
			<td>Corning, USA</td>
			<td>431224</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Thermo Scientific, USA</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco Thermo Fisher, USA</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene-EDTA, 1X</td>
			<td>Lonza, Switzerland</td>
			<td>CC-5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Verso cDNA synthesis kit</td>
			<td>Thermo Scientific, USA</td>
			<td>AB-1453/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>M3148</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of rat adipose tissue mesenchymal stem cells for differentiation into insulin-producing cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Isolation and characterization of Ad-MSCs 
As shown in Figure 2, the isolated cells from adipose tissue showed a heterogeneous population of rounded and fibroblast-like cells starting from the next day of isolation (Figure 2A). 4 days post isolation, the fibroblast cells started to increase in number and size and grow as a homogenous population by passage 1 (Figure 2B,C). These cells continued ...

Watch this Scientific Journal Video about Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells at JoVE.com

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

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