Name: an efficient method to obtain dedifferentiated fat cells
Uploaded: 2016-07-15T13:00:00
Description: Watch this Scientific Journal Video about An Efficient Method to Obtain Dedifferentiated Fat Cells at JoVE.com

This research was supported in part by Program for Creating Start-ups from Advanced Research and Technology (START Program) from the Japan Society for the Promotion of Science (ST261006IP, TM) and by Program for the Strategic Research Foundation at Private Universities (2014-2019) (S1411018, TM) from the Ministry of Education, Sports, Science and Technology.

Samples of human subcutaneous fat were obtained from patients undergoing surgery in the Departments of Plastic Surgery, Urology, Pediatric Surgery and Orthopedic Surgery of Nihon University Itabashi Hospital (Tokyo, Japan). The patients gave written informed consent, and the Ethics Committee of Nihon University School of Medicine approved the study.
1. Tissue Preparation
<ol>
	<li>Bring the tissue sample from the operating room to the laboratory.</li>
	<li>Wash 1-2 g of adipose tissue with 5...

<ol>
	<li>Lanzoni, G., 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=Concise+review:+clinical+programs+of+stem+cell+therapies+for+liver+and+pancreas.">Concise review: clinical programs of stem cell therapies for liver and pancreas.</a> Stem Cells. 31 (10), 2047-2060 (2013).</li><li>de Girolamo, L., 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/stromal+cells:+a+new+"cells+as+drugs"+paradigm.+Efficacy+and+critical+aspects+in+cell+therapy.">Mesenchymal stem/stromal cells: a new "cells as drugs" paradigm. Efficacy and critical aspects in cell therapy.</a> Curr. Pharm. Des. 19 (13), 2459-2473 (2013).</li><li>Lindroos, B., Suuronen, R., Miettinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+adipose+stem+cells+in+regenerative+medicine.">The potential of adipose stem cells in regenerative medicine.</a> Stem Cell. Rev. 7 (2), 269-291 (2011).</li><li>Yan, J., Tie, G., Xu, T. Y., Cecchini, K., Messina, L. M. <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+as+a+treatment+for+peripheral+arterial+disease:+current+status+and+potential+impact+of+type+II+diabetes+on+their+therapeutic+efficacy.">Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type II diabetes on their therapeutic efficacy.</a> Stem Cell. Rev. 9 (3), 360-372 (2013).</li><li>Ringden, O., Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+as+treatment+for+chronic+GVHD.">Mesenchymal stromal cells as treatment for chronic GVHD.</a> Bone Marrow Transplant. 46 (2), 163-164 (2011).</li><li>Matsumoto, T., 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=Mature+adipocyte-derived+dedifferentiated+fat+cells+exhibit+multilineage+potential.">Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.</a> J. Cell. Physiol. 215 (1), 210-222 (2008).</li><li>Lessard, J., 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=Generation+of+human+adipose+stem+cells+through+dedifferentiation+of+mature+adipocytes+in+ceiling+cultures.">Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.</a> J. Vis. Exp. (97), (2015).</li><li>Lessard, J., 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=Characterization+of+dedifferentiating+human+mature+adipocytes+from+the+visceral+and+subcutaneous+fat+compartments:+fibroblast-activation+protein+alpha+and+dipeptidyl+peptidase+4+as+major+components+of+matrix+remodeling.">Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.</a> PLoS One. 10 (3), 0122065 (2015).</li><li>Peng, X., 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=Phenotypic+and+Functional+Properties+of+Porcine+Dedifferentiated+Fat+Cells+during+the+Long-Term+Culture+In+Vitro.">Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro.</a> Biomed. Res. Int. 2015, 673651 (2015).</li><li>Kono, S., Kazama, T., Kano, K., Harada, K., Uechi, M., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+properties+of+feline+dedifferentiated+fat+cells+and+adipose-derived+stem+cells.">Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells.</a> Vet. J. 199 (1), 88-96 (2014).</li><li>Bellin, M., Marchetto, M. C., Gage, F. H., Mummery, 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=Induced+pluripotent+stem+cells:+the+new+patient.">Induced pluripotent stem cells: the new patient.</a> Nat. Rev. Mol. Cell Biol. 13 (11), 713-726 (2012).</li></ol>

Mature adipocytes that undergo in vitro dedifferentiation, a process known as ceiling culture, may revert to a more primitive phenotype and gain proliferative abilities. These cells are referred to as dedifferentiated fat (DFAT) cells. The multilineage differentiation potential of DFAT cells was evaluated. Flow cytometry analysis and gene expression analysis revealed that DFAT cells were highly homogeneous in comparison to ASCs6. In fact, the cell-surface antigen profile of DFAT cells is very similar ...

Cell therapy and tissue engineering are hot topics in the field of regenerative medicine1-5. While these therapeutic modalities hold great promise, several technical issues currently hamper their clinical use. In this regard, as in the generation of iPS cells, all tissue engineering therapies must produce cells free of external gene transductions in order to maintain patient safety. Accordingly, we were the first group to successfully produce human DFAT cells6. Several other research groups have since adopted our method to generate DFAT cells of mammalian origin7-9, further highlighting the usefulness of our model.
Over the course of several studies, we have found that the quality of the cell culture environment may be modified by adjusting the content of the cell medium. This finding has led to an increase in the success rate of DFAT cell production and improved cell quality; both critical factors in efficiently generating cells for future clinical trials. In this regard, an improved DFAT culture medium (DCM, a medium similar to mesenchymal stem cell medium, which contains recombinant human insulin, serum albumin, L-glutamic acid, several fatty acids, and cholesterol) and a method for DFAT cell generation and proliferation was developed (more information about the contents of DCM is available upon request). Using this method high quality DFAT cells were generated with the ability to differentiate into several cell types including adipogenic, osteogenic, and chondrogenic cells. Altogether, this validated cell culture protocol enhances the quality of DFAT cells and may be quite useful for enhancing clinical applications of cell therapy and tissue engineering.

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			<td style="width:167px;">This medium is defined as DCM in the text</td>
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			<td style="width:167px;">It does not contain Mg2+ and Ca2+</td>
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an efficient method to obtain dedifferentiated fat cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

In this study, the method and toolkit for DFAT cell generation was improved (Figure 1). Our method allows us to generate DFAT cells using both DCM and DMEM medium containing 20% FBS (Figure 2A). As such, we compared the efficiency of DCM and DMEM in generating DFAT cells. In this regard, DCM enhanced DFAT cell proliferation by three times when compared to DMEM, regardless of the number of adipocytes (Figure 2A and B). Usi...

Watch this Scientific Journal Video about An Efficient Method to Obtain Dedifferentiated Fat Cells at JoVE.com

An Efficient Method to Obtain Dedifferentiated Fat Cells

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

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