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 ml of PBS(-) at room temperature (RT) once, in a 50 ml tube and then place the sample in a 10 cm plastic dish.</li>
</ol>
2. Collagenase Digestion
<ol>
	<li>Remove the sample from the PBS(-), then add 10 ml of sterile 0.1% (w/v) collagenase solution (Collagenase type II) to the dish containing the tissue sample.</li>
	<li>Mince the tissue with surgical scissors for approximately 15 min, until it reaches a pureed consistency.</li>
	<li>Subsequently, digest the minced tissue in the 0.1% (w/v) collagenase solution at 37 &#176;C for 30 min (60 rpm) in a 50 ml tube with gentle agitation on a shaker.</li>
	<li>Filter the digested sample through a 100 &#181;m filter into a 50 ml tube. Next, pass 10 ml of DCM (see Table of Materials) supplemented with 2% FBS through the filter.</li>
</ol>
3. Cell Isolation
<ol>
	<li>Centrifuge the filtered cells at 100 x g for 1 min at RT.</li>
	<li>At this point, prepare 5 ml of DCM supplemented with 2% FBS in a 50 ml tube.</li>
	<li>Next, draw up the floating fat cells using a 1,000 &#181;l pipette and add the cells to a 50 ml tube containing medium.</li>
	<li>Subsequently, shake the 50 ml tube gently to mix the DCM medium with the cells.</li>
	<li>Then, centrifuge the tube at 100 x g for 1 min at RT.</li>
	<li>Discard the medium at the bottom of the tube using an 18 G needle and a 20 ml syringe.</li>
	<li>Next, add 10 ml of DCM supplemented with 2% FBS to the collected cells.</li>
	<li>Mix the DCM with the cells by shaking the 50 ml tube gently.</li>
	<li>Centrifuge the tube at 100 x g for 1 min at RT.</li>
</ol>
4. Plating Cells
<ol>
	<li>Add 41 ml of DCM or DMEM supplemented with 20% FBS to a 12.5 cm flask.</li>
	<li>Next, using a 200 &#181;l pipette add 40 &#181;l of fat cells to the flask.</li>
	<li>Then, fill up the flask with DCM or DMEM supplemented with 20% FBS. CRITICAL STEP: At this point, ensure that the cells are spread throughout the medium. Visually check the detached cells and afterwards keep the flask on the lid of a round petri dish to keep it flat.</li>
	<li>Finally, place the flask inside the incubator and leave it undisturbed in a 5% CO2 incubator at 37 &#176;C for 7 days.</li>
	<li>Following a week of incubation, invert the flask and check for DFAT cells. Next, assess DFAT cell generation proficiency as described10 since at this point fibroblast-like cells (DFAT cells) are located in regions distinct from fat cells.</li>
	<li>Add 5 ml of DCM or DMEM supplemented with 20% FBS to the flask following removal of the previous cell culture medium. Allow the DFAT cells to grow for another week after changing the medium.</li>
	<li>Using a cell counter, count the number of DFAT cells generated under various cell culture conditions (DCM vs. DMEM).</li>
</ol>

<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>

The authors have nothing to disclose.

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 to those found in ASCs6. Accordingly, DFAT cells may function similarly to MSCs.
The other group which has successfully generated DFAT cells utilized a DMEM F12 cell culture medium supplemented with 20% calf serum7-8. Although we attempted to produce DFAT cells using a cell culture medium supplemented with 10% FBS, the number of DFAT cells produced with this protocol was significantly lower than when supplemented with 20% FBS (data not shown). Therefore, the FBS concentration and/or its contents is important for either dedifferentiation of DFAT cells and/or proliferation of DFAT cells. As part of a future study, we will attempt to further elucidate the mechanism by which serum content governs DFAT cell generation and proliferation. On the other hand, DCM contains less glucose, fatty acids and other chemicals than the DMEM used in our study. This variability in cell culture medium constituents may modulate the efficiency of both DFAT cell generation and growth. In fact, low glucose concentration slightly improves the efficiency of DFAT cell generation when using DMEM (data not shown). Although the precise mechanisms underlying DCM&#39;s effect on DFAT cell generation and growth are unknown, we postulate here that the concentration of certain medium constituents drives these cellular events. Further studies are needed to clarify this point. Moreover, since we have tested a limited number of DMEM formulations, further experiments are required to compare the efficiency of other DMEM variations (e.g., DMEM containing L-glutamine or pyridoxine) in producing DFAT cells.
Although we are able to produce DFAT cells, the molecular mechanisms governing DFAT cell generation is still unclear. Similar questions surround the production of iPS cells11. As such clinical use of these therapeutic modalities will require further exploration of cell state regulatory mechanisms. Accordingly, we strongly believe that DFAT cells provide a model for elucidating how cells return to a stem cell-like state, free of external gene transductions. DFAT cell production protocols are therefore, being optimized currently for use in future clinical trials.
Ensuring that collagenase treatment and fat cell isolation are both performed properly is a key factor in successfully generating DFAT cells using this protocol. Appropriate use of fine scissors and removal of bubbles during culture also aid in enhancing DFAT cell proliferation.

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.

<table border="0" cellpadding="0" cellspacing="0" width="705">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CSTI303-MSC medium&#160;</td>
			<td style="width:143px;">CSTI</td>
			<td style="width:155px;">87-671</td>
			<td style="width:167px;">This medium is defined as DCM in the text</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:243px;">PBS(-)</td>
			<td style="width:143px;">Wako</td>
			<td style="width:155px;">166-23555</td>
			<td style="width:167px;">It does not contain Mg2+ and Ca2+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEM medium</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:155px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal Bovine Serum</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:155px;">172012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Collagenase type II</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:155px;">C-6885</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Scissors</td>
			<td style="width:143px;">Takasago Medical Industry Co., Ltd</td>
			<td style="width:155px;">TKZ-F2194-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Shaker</td>
			<td style="width:143px;">TAITEC</td>
			<td style="width:155px;">Bioshaker V.BR-36</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Falcon Cell Strainer 100um Yellow</td>
			<td style="width:143px;">CORNING LIFE SCIENCES&#160;</td>
			<td style="width:155px;">DL 352360</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Falcon 12.5cm&#178; Rectangular Canted Neck Cell Culture Flask with Blue Vented Screw Cap</td>
			<td style="width:143px;">CORNING LIFE SCIENCES&#160;</td>
			<td style="width:155px;">353107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">18G needle</td>
			<td style="width:143px;">NIPRO</td>
			<td style="width:155px;">02-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">20ml Syringe&#160;</td>
			<td style="width:143px;">NIPRO</td>
			<td style="width:155px;">08-753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Z Series Coulter Counter</td>
			<td style="width:143px;">BECKMAN COULTER</td>
			<td style="width:155px;">383550</td>
			<td></td>
		</tr>
	</tbody>
</table>

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). Using 20% FBS also enhances cell growth when compared to protocols using 10% FBS (data not shown). We further confirmed that DFAT cells produced using DCM have the ability to redifferentiate into adipocytes and differentiate into osteoblasts (Figure 3). The method for the assays is described in our previous study6.
<img alt="Figure 1" src="/files/ftp_upload/54177/54177fig1.jpg" /> 
Figure 1: A schematic depicting the isolation, dedifferentiation, and culture of mature adipocytes. A small piece of adipose tissue was digested with 0.1% collagenase. After centrifugation, unilocular adipocytes were isolated from the floating layer. The isolated cells were cultured in flasks filled with medium containing 20% FBS for 7 days. During culture, the cells attached and flattened to the upper surface of the flasks and then were converted to DFAT cells. Next, the flasks were inverted and DFAT cells were cultured using conventional methods. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54177/54177fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54177/54177fig2.jpg" /> 
Figure 2: DCM enhances DFAT cell generation. (A) DFAT cell colonies following 14 days of culture in DMEM or DCM. Scale bar = 200 &#956;m (B) DFAT cell proliferation quantification in either DMEM or DCM (mean &#177; SD, n = 3). The efficiency of DCM and DMEM in generating DFAT cells was compared. Enhanced cell proliferation is observed with use of DCM. We quantified the proliferation of DFAT cells using a cell counter, as per the manufacturer&#39;s directions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54177/54177fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54177/54177fig3.jpg" /> 
Figure 3: Multilineage potential of DFAT cells. Human DFAT cells generated using DCM were cultured for 3 weeks in the appropriate induction media to evaluate the redifferentiation and differentiation abilities of DFAT cells. The cells were analyzed for osteogenic (ALP and alizarin red S), adipogenic (Oil red-O) capacity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54177/54177fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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7.9K Views.  Nihon University. 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.

Video: An Efficient Method to Obtain Dedifferentiated Fat Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

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Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

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Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

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The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

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We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

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 ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...