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 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's modified Eagle medium (HG-DMEM), β-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific β-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.
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, CD79α 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' 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 β-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.
All experiments were carried out according to the approved guidelines, and all procedures were approved by the Ethical Committee of the Faculty of Pharmacy, The British University in Egypt (BUE), Cairo, Egypt. The Ad-MSC isolation protocol was adopted from Lopez and Spencer, with modifications15.
1. Isolation of Ad-MSCs from rat epididymal fat pads
2. Characterization of Ad-MSCs by immunophenotyping using flow cytometry analyses
3. Assessment of the differentiation potential of isolated Ad-MSCs into various mesenchymal lineages
4. Differentiation of Ad-MSCs into IPCs
5. Dithizone staining
6. Gene expression of β-cell markers by RT-qPCR
cDNA synthesis master mix | Volume (µl) |
5x cDNA synthesis buffer | 4 |
dNTP | 2 |
RNA primer | 1 |
Verso Enzyme Mix | 1 |
RT enhancer | 1 |
Nuclease Free water | Variable |
Total RNA | Variable |
Total Reaction volume | 20 |
Table 1: cDNA synthesis master mix volumes.
RT-qPCR reaction mix | Volume (µl) | Final concentration in 10 µL |
cDNA | 2 | 2 ng/well |
RT-qPCR Forward primer (3 µM) | 1 | 300 nM |
RT-qPCR Reverse primer (3 µM) | 1 | 300 nM |
Nuclease free water | 1 | ——- |
2x SYBR Green master mix | 5 | 1x |
Total reaction volume | 10 |
Table 2: RT-qPCR reaction mixture.
Gene | Forward primer | Reverse primer |
FOXA2 | GAGCCGTGAAGATGGAAGG | ATGTTGCCGGAACCACTG |
PDX-1 | ATCCACCTCCCGGACCTTTC | CCTCCGGTTCTGCTGCGTAT |
NKX6.1 | ACACCAGACCCACATTCTCCG | ATCTCGGCTGCGTGCTTCTT |
MafA | TTCAGCAAGGAGGAGGTCAT | CCGCCAACTTCTCGTATTTC |
Ins-1 | CACCTTTGTGGTCCTCACCT | CTCCAGTGCCAAGGTCTGA |
β-actin | TGGAGAAGATTTGGCACCAC | AACACAGCCTGGATGGCTAC |
Table 3: Forward and reverse primer sequences.
7. Glucose-stimulated insulin secretion
Component | Concentration |
Magnesium Chloride (Anhydrous) | 0.0468 g/L |
Potassium Chloride | 0.34 g/L |
Sodium Chloride | 7.00 g/L |
Sodium Phosphate Dibasic (Anhydrous) | 0.1 g/L |
Sodium Phosphate Monobasic (Anhydrous) | 0.18 g/L |
Sodium Bicarbonate | 1.26 g/L |
Calcium Chloride | 0.2997 g/L |
Table 4: The components used for the KRB buffer preparation.
8. Statistical analysis
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 β-cell stain (Figure 4B, lower panel).
Afterward, the generated IPCs were genetically assessed for the expression of the specific β-cell markers when compared to the uninduced control cells. As shown in Figure 5A–E, the induced cells were able to express various specific β-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 ± 0.88; Final: 12.10 ± 1.27; p < 0.05). As for Pdx-1 (which is considered an early marker of β-cells), it was elevated in both D3 and final differentiated cells, reaching almost 20 fold when compared to control uninduced cells (D3: 22.39 ± 5.14; Final: 17.13 ± 0.342; p < 0.05; Figure 5B). Concerning the other β-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 ± 0.86, Final: 7.97 ± 1.34, p<0.05; MafA: D3: 6.59 ± 0.4, Final: 11.54 ± 2.40, p < 0.05; and Ins1: D3: 27.29 ± 20.27, Final: 318.20 ± 76.09, p < 0.05) (Figure 5C–E). This indicates that these Ad-MSCs can differentiate into IPCs expressing β-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 ± 33 pg/mL; LG: 234 pg/mL ± 32 pg/mL, p < 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.
Figure 1: A schematic presentation of the steps of the protocol used for the isolation and characterization of Ad-MSCs. Generated by Biorender.com. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. 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; β-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. Please click here to view a larger version of this figure.
Figure 5: Relative expression levels of β-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 < 0.05; b: mean is different from Day-3 at p < 0.05; *: mean of LG is different from HG at p < 0.05; the comparison was done using an independent samples t-test. Please click here to view a larger version of this figure.
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 β-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 β-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 β-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 β-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 β-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.
The authors have nothing to disclose.
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.
Albumin, bovine serum Fraction V | MP Biomedicals | ||
Alcian Blue 8GX | Sigma-Aldrich, USA | A3157 | |
Alizarin Red S | Sigma-Aldrich, USA | A5533 | |
Ammonium hydroxide | Fisher Scientific, Germany | ||
Antibody for Rat CD90, FITC | Stem Cell Technologies | 60024FI | |
Bovine serum albumin | Sigma Aldrich | A3912 | |
Calcium Chloride | Fisher Scientific, Germany | ||
CD105 Monoclonal Antibody, FITC | Thermo Fisher Scientific, Invitrogen, USA | MA1-19594 | |
CD34 Polyclonal Antibody | Thermo Fisher Scientific, Invitrogen, USA | PA5-85917 | |
Chloroform | Fisher Scientific, USA | ||
Collagenase type I, powder | Gibco, Thermo Fisher, USA | 17018029 | |
D-Glucose anhydrous, extra pure | Fisher Scientific, Germany | G/0450/53 | |
Dimethyl sulfoxide (DMSO) | Fisher Scientific, Germany | BP231-100 | |
Dithizone staining | Sigma-Aldrich, USA | D5130 | |
DMEM – High Glucose 4.5 g/L | Lonza, Switzerland | 12-604F | |
DMEM – Low Glucose 1 g/L | Lonza, Switzerland | 12-707F | |
DMEM/F12 medium | Lonza, Switzerland | BE12-719F | |
DNAse/RNAse free water | Gibco Thermo Fisher, USA | 10977-035 | |
Ethanol absolute, Molecular biology grade | Sigma-Aldrich, Germany | 24103 | |
Exendin-4 | Sigma-Aldrich, Germany | E7144 | |
Fetal Bovine Serum (FBS) | Gibco Thermo Fisher, Brazil | 10270-106 | |
Formaldehyde 37% | Fisher Scientific | ||
Hydrochloric acid (HCl) | Fisher Scientific, Germany | ||
Isopropanol, Molecular biology grade | Fisher Scientific, USA | BP2618500 | |
L-Glutamine | Gibco Thermo Fisher, USA | 25030-024 | |
Magnesium Chloride (Anhydrous) | Fisher Scientific, Germany | ||
Mesenchymal Stem Cell Functional identification kit | R&D systems Inc., MN, USA | SC006 | |
Nicotinamide | Sigma-Aldrich, Germany | N0636 | |
Oil Red Stain | Sigma-Aldrich, USA | O0625 | |
Penicillin-Streptomycin-Amphotericin | Gibco Thermo Fisher, USA | 15240062 | |
Phosphate buffered saline, 1X, without Ca/Mg | Lonza, Switzerland | BE17-516F | |
Potassium Chloride | Fisher Scientific, Germany | ||
Rat Insulin ELISA Kit | Cloud-Clone Corp., USA | CEA682Ra | |
Sodium Bicarbonate | Fisher Scientific, Germany | ||
Sodium Chloride | Fisher Scientific, Germany | ||
Sodium Phosphate Dibasic (Anhydrous) | Fisher Scientific, Germany | ||
Sodium Phosphate Monobasic (Anhydrous) | Fisher Scientific, Germany | ||
SYBR Green Maxima | Thermo Scientific, USA | K0221 | |
Syringe filter, 0.2 micron | Corning, USA | 431224 | |
TRIzol | Thermo Scientific, USA | 15596026 | |
Trypan blue | Gibco Thermo Fisher, USA | 15250061 | |
Trypsin-Versene-EDTA, 1X | Lonza, Switzerland | CC-5012 | |
Verso cDNA synthesis kit | Thermo Scientific, USA | AB-1453/A | |
β-mercaptoethanol | Sigma-Aldrich, Germany | M3148 |