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
<ol>
	<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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sources+and+therapeutic+strategies+of+mesenchymal+stem+cells+in+regenerative+medicine.">Sources and therapeutic strategies of mesenchymal stem cells in regenerative medicine.</a> Handbook of Stem Cell Therapy. , 1-28 (2022).</li><li>Jiang, 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=Pluripotency+of+mesenchymal+stem+cells+derived+from+adult+marrow.">Pluripotency of mesenchymal stem cells derived from adult marrow.</a> Nature. 418 (6893), 41-49 (2002).</li><li>De Ugarte, D. 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. F., Fradette, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-tissue+engineering:+Taking+advantage+of+the+properties+of+human+adipose-derived+stem/stromal+cells.">Adipose-tissue engineering: Taking advantage of the properties of human adipose-derived stem/stromal cells.</a> Pathologie Biologie. 57 (4), 309-317 (2009).</li><li>Dominici, 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Gong, W., 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=Mesenchymal+stem+cells+stimulate+intestinal+stem+cells+to+repair+radiation-induced+intestinal+injury.">Mesenchymal stem cells stimulate intestinal stem cells to repair radiation-induced intestinal injury.</a> Cell Death &amp; Disease. 7 (9), 2387 (2016).</li><li>Dai, R., Wang, Z., Samanipour, R., Koo, K. I., Kim, K. <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+stem+cells+for+tissue+engineering+and+regenerative+medicine+applications.">Adipose-derived stem cells for tissue engineering and regenerative medicine applications.</a> Stem Cells International. 2016, 6737345 (2016).</li><li>Ceccarelli, S., Pontecorvi, P., Anastasiadou, E., Napoli, C., Marchese, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effect+of+adipose-derived+stem+cells:+The+cutting+edge+of+clinical+application.">Immunomodulatory effect of adipose-derived stem cells: The cutting edge of clinical application.</a> Frontiers in Cell and Developmental Biology. 8, 236 (2020).</li><li>Karp, J., Leng Teo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+homing:+The+devil+is+in+the+details.">Mesenchymal stem cell homing: The devil is in the details.</a> Cell Stem Cell. 4 (3), 206-216 (2009).</li><li>Andaloussi, S., Mager, I., Breakefield, X. O., Wood, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+Biology+and+emerging+therapeutic+opportunities.">Extracellular vesicles: Biology and emerging therapeutic opportunities.</a> Nature Reviews Drug Discovery. 12 (5), 347-357 (2013).</li><li>Lopez, M. J., Spencer, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+adult+rat+adipose+tissue-derived+stromal+cell+isolation+and+differentiation.">In vitro adult rat adipose tissue-derived stromal cell isolation and differentiation.</a> Methods in Molecular Biology. 702, 37-46 (2011).</li><li>Karnieli, O., Izhar-Prato, Y., Bulvik, S., Efrat, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+insulin-producing+cells+from+human+bone+marrow+mesenchymal+stem+cells+by+genetic+manipulation.">Generation of insulin-producing cells from human bone marrow mesenchymal stem cells by genetic manipulation.</a> Stem Cells. 25 (11), 2837-2844 (2007).</li><li>Yang, Y. K., Ogando, C. R., Wang See, C., Chang, T. Y., Barabino, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+phenotype+and+differentiation+potential+of+human+mesenchymal+stem+cells+aging+in+vitro.">Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro.</a> Stem Cell Research &amp; Therapy. 9 (1), 131 (2018).</li><li>Lee, R. H., 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=Multipotent+stromal+cells+from+human+marrow+home+to+and+promote+repair+of+pancreatic+islets+and+renal+glomeruli+in+diabetic+NOD/scid+mice.">Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (46), 17438-17443 (2006).</li><li>Gao, L. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+mesenchymal+stromal/stem+cells+to+insulin-producing+cells:+Progress+and+challenges.">From mesenchymal stromal/stem cells to insulin-producing cells: Progress and challenges.</a> Stem Cell Reviews and Reports. 16 (6), 1156-1172 (2020).</li><li>Kassem, D. H., Kamal, M. M., El-Kholy, A. E. -. L. G., 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=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. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+expression+levels+of+pluripotency/stem+cell+markers+with+the+differentiation+outcome+of+Wharton's+jelly+mesenchymal+stem+cells+into+insulin+producing+cells.">Association of expression levels of pluripotency/stem cell markers with the differentiation outcome of Wharton's jelly mesenchymal stem cells into insulin producing cells.</a> Biochimie. 127, 187-195 (2016).</li><li>El-Asfar, R. K., Kamal, M. M., Abd El-Razek, R. S., El-Demerdash, E., 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=Obestatin+can+potentially+differentiate+Wharton's+jelly+mesenchymal+stem+cells+into+insulin-producing+cells.">Obestatin can potentially differentiate Wharton's jelly mesenchymal stem cells into insulin-producing cells.</a> Cell and Tissue Research. 372 (1), 91-98 (2018).</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=Insulin-producing+cells+from+adult+human+bone+marrow+mesenchymal+stromal+cells+could+control+chemically+induced+diabetes+in+dogs:+A+preliminary+study.">Insulin-producing cells from adult human bone marrow mesenchymal stromal cells could control chemically induced diabetes in dogs: A preliminary study.</a> Cell Transplantation. 27 (6), 937-947 (2018).</li></ol>

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 ...

The overall goal of this protocol is to isolate and characterize red adipose tissue, dried mesenchymal stem cells and differentiate them towards insulin-producing cells using simple methods. Mesenchymal stem cells have found the ways in the field of genetics. They can be isolated from various sources such as bone marrow, adipose tissue and perinatal tissue.They accept excellent cultural characteristics. They are multi potent and can potentially treat various disease. Adipose tissue provides a rich source of mesenchymal stem cells.These adipose MSCs can be isolated from lipo aspirates easily with high yield compared to other sources like bone marrow. And they can provide a good source for both autologous and allogenic transplantation. In this video are presenting a simple protocol for isolation and characterization of adipose mesenchymal stem cells from rat epididymal fat.This procedure is a simple highly efficient method for isolation. After isolation, we will endure the differentiation of these adipose mesenchymal stem cells into insulin-producing cells using a simple, relatively short protocol. We will start with isolation of adipose mesenchymal stem cells by collagenous digestion of epididymal fat to obtain stronger vascular fraction.Then mesenchymal stem cells will be characterized by their plastic adherence, as well as expression of CD markers and differentiation into multiple mesodermal lineages. Anaesthise the animal, then euthanize using cervical dislocation. Spray all over the body with 70%ethanol and start with excision of the skin and the muscles at lower part of the abdomen.Then expose the testes with the epididymal fat. Gently cut the epididymal fat. Be careful not to cut the blood vessels with the fats.In a bio safety cabinet, cut the fat tissue into small pieces using forceps and scalpel. Transfer the adipose tissue into centrifuge tube containing 10 milliliters sterile PBS. Start washing by tilting the Falcon tube for 45 seconds.Then keep the tube standing for five minutes to separate into two layers. Remove the infranated PBS using a 10 milliliter syringe. Repeat this washing steps four to five times till PBS is clear.After washing, re-suspend adipose tissue in collagenous solution, then incubate in shaking water bath until the tissue is almost homogeneous. Afterwards, vortex the tissue collagenous mixture for 15 seconds, then centrifuge afterwards for five minutes. After centrifugation, you will have three layers.Carefully discard the supernatant over the stromal vascular fraction. First discard the oil layer at the top carefully, followed by the echos layer without disturbing the stromal vascular fraction pellet. Re-suspend the Stromal vascular fraction in sterile BSA and re-centrifuge for five minutes.Whole centrifugation, place complete DMEM F-12 media in 25 centimeter square flask. After centrifugation, discard the supernatant and re-suspend the stromal vascular pellet in complete DMEM F-12 media. And transfer to the culture flask.Place in 37 degree 5%carbon dioxide incubator. Next day, remove unattached cells and replace the media with fresh media. Starting from next day of isolation, The fibroblast-like cells will start to appear.Then gradually as time passes they will increase in number and size. By passaging, cells will become more homogenous. These cells exhibit all mesenchymal stem cell characterization criteria.These cells were differentiated towards insulin-producing cells using this simple protocol containing nicotinamide beta mercaptoethanol and Exendin-4. Throughout the differentiation steps, the cells start to lose their fibroblastic shape and I think cluster like morphology. When used insulin-producing cells show positive crimson red disithone staining, then used insulin-producing cells express various beta cell markers like Pdx-1, mafA and insulin.In addition, induced insulin-producing cells secrete higher levels of insulin in the supernatant when challenged with higher glucose concentration. We provided a detailed, stepwise protocol for isolation and characterization of adipose mesenchymal stem cells. We also provide here a relatively short, simple and efficient protocol for differentiation of adipose mesenchymal stem cells into insulin-producing cells.This provides a gateway for researcher to enter the field of mesenchymal stem cells and study their differentiation into insulin-producing cells.

isolation-rat-adipose-tissue-mesenchymal-stem-cells-for

Research

JoVE Journal

Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...