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.

<ol>
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All the co-authors declare no conflict of interests associated with this work.

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 the criteria to be defined as MSCs. The used protocol was previously described with slight modifications15. This protocol has been proven to be effective and reproducible. The cells showed fibroblastic-like morphology from day 1 after isolation and continued to expand until they reached a homogenous population. Interestingly, we used the same protocol to isolate human Ad-MSCs from lipo-aspirate with comparable success to rat tissue (data not shown). The only limitation of this process is the chemical digestion using collagenase when put side to side with mechanical digestion in other protocols28. This step also represents a critical measure, as the chemical digestion of the cells can adversely affect their viability29,30. Otherwise, this protocol offers a good start for any researcher who would consider launching an Ad-MSC line of research in their lab. 
The use of MSCs in the treatment of diabetes has opened new pathways and given new hopes for the treatment of diabetes. However, the generation of fully mature IPCs from MSCs is still a matter of debate and challenge31. Currently, there are several protocols for inducing the differentiation of MSCs toward IPCs in the literature. These protocols utilize a plethora of chemical compounds and growth factors for various periods of time20,32,33. These factors mainly depend on the MSC type and source to begin with34,35. Nevertheless, obtaining mature functional IPCs from MSCs is still a matter of debate and research in the field20.
In the presented protocol, we provide a short, relatively simple, efficient way to obtain functional IPCs from Ad-MSCs. The resulting cells showed marked morphological changes with positive DTZ staining, expressed most of the &#946;-cell markers, and showed increased insulin secretion in response to increased glucose concentration challenge. All this evidence confirms the efficiency of this protocol as a &#946;-cell induction protocol from Ad-MSCs. We used this protocol in another type of MSCs, namely human Wharton's jelly MSCs (WJ-MSCs), derived from the umbilical cord, and it actually showed similar results21,36. These outcomes warrant trying our protocol on other types and sources of MSCs, rendering the procedure a universal protocol for the generation of IPCs from MSCs. It is noteworthy to highlight the importance of inducing the cells at early passages, usually between P3-P5, as late passages adversely affect the cells' properties and differentiation abilities17,24.
As mentioned before, attaining fully mature &#946;-cells from MSCs is a matter of debate and far from complete elucidation. However, such protocols as those described here provide a simple, fast means to better understand the underlying mechanisms of the differentiation of Ad-MSCs, or even other types of MSCs, into IPCs. The ability of the added compounds for the induction of the Ad-MSCs to express specific &#946;-cell markers provides a useful tool to study the genetic and epigenetic factors that govern the differentiation of MSCs into IPCs. In addition, the quick, simple protocol allows researchers to study other intrinsic factors that may improve the outcome of such differentiation. These factors may represent either an adjuvant therapy with stem cells or even may provide a therapeutic modality for diabetes. In our lab, we used a similar approach to prove that obestatin, a gut hormone, can be a novel potential factor for the generation of IPCs from WJ-MSCs37.
It is also worth mentioning that the current protocol has two main limitations. First, as mentioned previously, we used chemical digestion by collagenase, which can adversely affect the viability of the cells29,30. Second, we employed the differentiation process in vitro and did not investigate the expected further maturation and differentiation enhancement of the generated IPCs in vivo. It is important to point out that the transplantation of in vitro-differentiated MSCs into IPCs showed a profound induction of the expression of various &#946;-cells markers. This increase reached about 100 fold 12-18 months post transplantation in vivo38. Thus, future studies to further investigate the in vivo maturation of generated IPCs by this simple protocol will be of good value.
In conclusion, we provided a detailed protocol for the isolation and characterization of Ad-MSCs, followed by a relatively simple, fast, and efficient protocol for the generation of IPCs from Ad-MSCs. These protocols not only provide an efficient tool to initiate a stem cell therapy line of research but also can help to develop the ever-growing field of MSC cell therapy for diabetes.

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 to grow as plastic-adherent, fibroblastic-like cells as shown until passage 3, fulfilling the first criterion of MSC characteristics (Figure 2D). These Ad-MSCs showed very good culture characteristics, and this protocol was found to be a relevant, easy, and relatively fast protocol to isolate Ad-MSCs from epididymal fat pads.
The next step was to characterize the isolated Ad-MSCs. According to ISCT, MSCs should follow the three criteria of plastic adherence, expression of mesenchymal CDs with a lack of hematopoietic markers, and the ability to differentiate into adipocytes, osteocytes, and chondrocytes. As shown in Figure 3A, the flow cytometry analysis showed that most of these cells expressed CD90 and CD105 (76.4% and 73.6%, respectively). Meanwhile, they were almost negative for CD34 (0.1%).
Moreover, upon induction of differentiation of these cells, they showed the ability to differentiate into adipocytes, osteocytes, and chondrocytes. As shown in Figure 3B (upper panel), the adipocytes showed Oil Red staining of lipid vacuoles when compared to control uninduced cells. The osteocytes showed characteristic Alizarin Red staining (Figure 3B, middle panel) when compared to control cells. Finally, chondrocyte-induced cells showed blue staining of the extracellular matrix when compared to control uninduced cells (Figure 3B, bottom panel).
These data clearly indicate that isolated cells from adipose tissue not only exhibit good culture characteristics but also exhibit all the criteria proposed for MSCs.
Differentiation of Ad-MSCs into insulin-producing cells (IPCs) 
As shown in Figure 4A, we used a relatively simple, short protocol in order to differentiate Ad-MSCs into IPCs. After the induction of differentiation, the induced IPCs were assessed in several ways. The induced cells showed marked morphological changes. As shown in Figure 4B (upper panel), the induced cells showed rounded, cluster-like morphology when compared to the normal fibroblastic-like morphology of Ad-MSCs. Interestingly, upon staining with dithizone, these clusters showed a crimson stain, which is a characteristic of zinc granules of &#946;-cell stain (Figure 4B, lower panel).
Afterward, the generated IPCs were genetically assessed for the expression of the specific &#946;-cell markers when compared to the uninduced control cells. As shown in Figure 5A-E, the induced cells were able to express various specific &#946;-cell markers, indicating their ability to generate IPCs. As for FOXA2-a definitive endoderm marker (as shown in Figure 5A)-, it was highly expressed at D3 differentiation when compared to control, reaching almost 30 fold and then decreasing to only 10 fold of the control in the final differentiated cells (D3: 28.37 &#177; 0.88; Final: 12.10 &#177; 1.27; p &#60; 0.05). As for Pdx-1 (which is considered an early marker of &#946;-cells), it was elevated in both D3 and final differentiated cells, reaching almost 20 fold when compared to control uninduced cells (D3: 22.39 &#177; 5.14; Final: 17.13 &#177; 0.342; p &#60; 0.05; Figure 5B). Concerning the other &#946;-cell markers, namely NKX6.1, MafA, and insulin-1 (Ins1), they all showed elevation starting from D3 until final differentiation, reaching almost 8 fold, 12 fold, and 300 fold, respectively, when compared to control uninduced cells (NKX6.1: D3: 1.94 &#177; 0.86, Final: 7.97 &#177; 1.34, p&#60;0.05; MafA: D3: 6.59 &#177; 0.4, Final: 11.54 &#177; 2.40, p &#60; 0.05; and Ins1: D3: 27.29 &#177; 20.27, Final: 318.20 &#177; 76.09, p &#60; 0.05) (Figure 5C-E). This indicates that these Ad-MSCs can differentiate into IPCs expressing &#946;-cell markers.
Finally, these cells were assessed for the secretion of insulin when challenged with increasing concentrations of glucose. As shown in Figure 5F, the insulin secreted in the supernatant of the induced IPCs when challenged with 20 mM glucose was significantly higher than that secreted when the cells were challenged with 2 mM glucose (HG: 390 pg/mL &#177; 33 pg/mL; LG: 234 pg/mL &#177; 32 pg/mL, p &#60; 0.05; Figure 5F)
These data confirmed that the used protocol managed to differentiate the Ad-MSCs into IPCs, which was genetically and functionally confirmed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63348/63348fig01.jpg" /> 
Figure 1: A schematic presentation of the steps of the protocol used for the isolation and characterization of Ad-MSCs. Generated by Biorender.com. <a href="https://www.jove.com/files/ftp_upload/63348/63348fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63348/63348fig02.jpg" /> 
Figure 2: Photomicrographs showing the isolated Ad-MSCs. (A) Isolated cells exhibiting plastic-adherent, fibroblast-like morphology start to appear the day following isolation. (B) Over time, these adherent Ad-MSCs (with fibroblast-like morphology) proliferate and increase in number, reaching a more homogenous fibroblast-like population in (C) P1 and (D) P3. <a href="https://www.jove.com/files/ftp_upload/63348/63348fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63348/63348fig3v3.jpg" /> 
Figure 3: Characterization of Ad-MSCs. (A) Flow cytometric analysis of Ad-MSCs shows that these cells are almost negative for CD34 (upper panel), while the majority of cells express CD90 and CD105 (lower panel). Ad-MSCs can differentiate into the three mesenchymal lineages, namely (B) adipocytes (where oil droplets are stained by oil red), (C) osteocytes, stained by alizarin red, and (D) chondrocytes, stained by Alcian Blue (when compared to control uninduced cells). Control: uninduced cells, Diff: differentiated cells. <a href="https://www.jove.com/files/ftp_upload/63348/63348fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63348/63348fig04.jpg" /> 
Figure 4: Differentiation of Ad-MSCs into IPCs. (A) Schematic presentation of the differentiation protocol used to generate IPCs from Ad-MSCs, together with photomicrographs for the cells at each stage during the induction of differentiation towards IPCs.&#160;Upon differentiation, cells lose their fibroblastic morphology and tend to aggregate forming clusters, which tend to detach and grow in suspension media. (B) Photomicrographs show control Ad-MSCs and IPCs generated by the above protocol showing round cluster morphological changes (right panel) when compared to the fibroblast-like morphology of the un-induced Ad-MSCs (left panel), either unstained (upper panel), or DTZ stained (lower panel). 
Control: un-induced cells; IPCs: insulin-producing cells; NA: nicotinamide; &#946;-ME: beta mercaptoethanol; D3: Induced cells at Day 3 during the induction of differentiation towards IPCs; D10: final differentiated IPCs at the end of the induction protocol; Ex-4: exendin-4. <a href="https://www.jove.com/files/ftp_upload/63348/63348fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63348/63348fig5v2.jpg" /> 
Figure 5: Relative expression levels of &#946;-cell markers and GSIS for IPCs. Relative expression levels by qRT-PCR for (A) FOXA2, (B) Pdx-1, (C) NKX6.1, (D) MafA, and (E) Ins-1. (F) Levels of secreted insulin in the supernatant upon challenging the generated IPCs with 2mM glucose (LG) or 20mM glucose (HG). Control: uninduced Ad-MSCs, Day-3: differentiated cells collected at D3; Final: final differentiated IPCs; LG: low glucose; HG: high glucose. a: mean is different from control at p &#60; 0.05; b: mean is different from Day-3 at p &#60; 0.05; *: mean of LG is different from HG at p &#60; 0.05; the comparison was done using an independent samples t-test. <a href="https://www.jove.com/files/ftp_upload/63348/63348fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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

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

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7.0K Views.  Ain Shams University. 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.

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

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 ...