|Year : 2019 | Volume
| Issue : 1 | Page : 10-14
Investigating the role of the histone deacetylases-inhibitor suberanilohydroxamic acid in the differentiation of stem cells into insulin secreting cells
Ibrahim Elsharkawi1, Divyasree Sandeep1, Ahmed El-Serafi2
1 Sharjah Institute for Medical Research, University of Sharjah, Sharjah, UAE
2 Sharjah Institute for Medical Research; Basic Medical Department, College of Medicine, University of Sharjah, Sharjah, UAE; Medical Biochemistry Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
|Date of Web Publication||27-Feb-2019|
Basic Medical Department, College of Medicine, University of Sharjah, Sharjah
Source of Support: None, Conflict of Interest: None
Introduction: The United Arab Emirates has the second incidence of diabetes in the world. The current diabetes management plans are associated with many complications and do not provide a cure. Stem cells offer hope for permanent alleviation of this health problem through the possible differentiation into insulin-secreting cells. The current methods for the differentiation do not produce homogeneous beta-cell populations. Histone deacetylation is an epigenetic silencing mechanism that can render many genes irresponsive to the induction protocols. This study aimed at investigating the effect of the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) on the production of functional beta cells, based on a mesenchymal stem cells model. Methods: MG63 cells were treated for three consecutive days with SAHA, followed by a three-steps of beta cells differentiation protocol, with media-contained retinoic acid, epidermal growth factor, nicotinamide and exendin-4 at different stages. Then, glucose-stimulated insulin secretion was conducted to assess the functional state of the differentiated cells. Results: Pretreating the cells with SAHA enhanced the insulin production and secretion in comparison to the control (PBS) and the vehicle dimethyl sulfoxide, as shown by the immunofluorescence detection of insulin and the transcription factor “PDX1”, as well as an increase in insulin secretion in the media. Gene expression analysis showed that SAHA pretreated cells had more induction of the studied markers when challenged with high glucose level. Conclusion: Such findings indicate a novel approach to enhance the ability of stem cells to differentiate into insulin-producing cells with potential therapeutic implications.
Keywords: Diabetes, epigenetics, histone acetylation, insulin, stem cells
|How to cite this article:|
Elsharkawi I, Sandeep D, El-Serafi A. Investigating the role of the histone deacetylases-inhibitor suberanilohydroxamic acid in the differentiation of stem cells into insulin secreting cells. Hamdan Med J 2019;12:10-4
|How to cite this URL:|
Elsharkawi I, Sandeep D, El-Serafi A. Investigating the role of the histone deacetylases-inhibitor suberanilohydroxamic acid in the differentiation of stem cells into insulin secreting cells. Hamdan Med J [serial online] 2019 [cited 2019 Sep 17];12:10-4. Available from: http://www.hamdanjournal.org/text.asp?2019/12/1/10/236268
| Introduction|| |
Diabetes affects millions around the world which are estimated to be 415 million in 2015. In the United Arab Emirates, over 1 million cases of diabetes were reported in 2015, and the prevalence of diabetes in adults is more than double the international prevalence (19.3% and 8.8% respectively).
In spite of the continuous development in the medical field, there is no current cure for diabetes. The management plan includes insulin injections for Type 1 diabetes and/or oral hypoglycaemic drugs for Type 2. Considering the limitations of the current management strategies that do not provide a final treatment, several diabetes cure-focused researches were established trying to find an ultimate cure. Currently, islet transplantation represents the only treatment for diabetes in spite of many limitations.,,
The possibility of stem cells for differentiation into insulin-secreting cells could represent an inexhaustible source for transplantation and a great hope as a potential permanent cure of diabetic patients. In the embryo, stem cells undergo different development stages to become functional and mature β-cells. Each of these stages is controlled by specific key transcriptional factors such as FOXA2, SOX17, PDX1, NKX6.1, NEUROD1 and MafA.
Unfortunately, the proportion of the differentiated cells, according to previously used protocols, is insufficient and the cells can be immature. These cells either do not properly respond to glucose stimulation, fail to express β cell markers, co-express other hormones, need a long difficult process or combine >1 of these defects.,, There have been multiple reports suggesting that the stem cells differentiation is controlled by epigenetic factors.
Suberanilohydroxamic acid (SAHA) is a histone deacetylases inhibitor (HDAC-inhibitor) agent that binds to zinc ions in the active site of HDAC enzymes and disturb its function, which render the chromatin to loosen state and the genes more available for transcription.
In this study, we investigated the role of the epigenetic modifier SAHA in the differentiation of stem cells into insulin-secreting cells.
| Materials and Methods|| |
The MG63 cell line (American Type Culture Collection;) has been extensively used for their mesenchymal stem cells (MSCs) characteristics.,, The cells were cultured in the Dulbecco's modified Eagle's medium (DMEM) media (Sigma-Aldrich) contained 25 mM glucose and supplemented with 10% foetal bovine serum (Himedia), 100 μg/ml streptomycin (Sigma-Aldrich), 100 I.U./ml penicillin (Sigma-Aldrich), 200 mM L-Glutamine (Gibco) and 10 mM HEPES (Himedia) in humidified air with 5% CO2, at 37°C.
Cell treatment protocol
MG63 cells were cultured until reached 50% confluent, then serum starved for 24 h and divided into three groups. Each group was incubated with complete DMEM (25 mM glucose) and treated daily with either 1 μM SAHA (Sigma-Aldrich) for three consecutive days, an equivalent amount of dimethyl sulfoxide (DMSO) as a vehicle or PBS (Himedia) as control.
Cell differentiation protocol
Differentiation was carried out according to a previously described, 3-step protocol, using retinoic acid (Sigma-Aldrich), epidermal growth factor (EGF) (Sigma-Aldrich), nicotinamide (Sigma-Aldrich) and exendin-4 (Sigma-Aldrich).
Glucose stimulated insulin secretion assay
Two glucose concentrations (5.5 mM, 16.5 mM) were used. The cells incubated in serum-free DMEM containing 5.5 mM glucose for 4 h at 37°C. Then complete DMEM media with 5.5 mM or 16.5 mM glucose were added for 4 h. The final media were collected and frozen at −20°C until they were assayed for insulin content with ELISA.
The fixed cells on coverslips were permeabilised using 0.05% Triton X-100 (Sigma-Aldrich), saturated with 3% BSA (Sigma-Aldrich) and incubated with primary rabbit anti-human insulin monoclonal antibody (1:500) (Abcam) or anti-human PDX1 monoclonal antibody (1:500) (Abcam). Subsequently, the cells were incubated with a secondary antibody, goat anti-rabbit IgG (1:800) (Abcam). Nuclei were counterstained with DAPI (Invitrogen).
Total RNA was extracted for all the three groups using the all-in-one Purification Kit (Norgen Biotek) according to the manufacturer's instructions. Briefly, the cell pellets were lysed and applied to a spin column attached to a collecting tube, after multiple steps of washing and drying the membrane, large RNA was released by applying a specific RNA elution solution to the column.
Reverse transcription (RT) was performed with the total RNA using GoScript RT system (Promega) according to the manufacturer's protocol. cDNA concentration was determined by Nanodrop 2000 spectrophotometer (Thermo-Fisher Scientific) and diluted accordingly.
Quantitative reverse transcription-polymerase chain reaction analysis
RT-polymerase chain reaction (RT-PCR) reaction included 2X SYBR Green Master mix (Promega) and 5–6 pM of each the forward and reverse primers (Macrogen), as well as 100 ng of cDNA. The final reaction volume was 20 μl. The reaction was performed in triplicates using the real-time PCR thermal cycler “Rotor-gene Q” (Qiagen) under the following conditions: 95°C for 2 min followed by 40 cycles of 95°C for 15 s then 60°C for 30 s and 60°C for 30 s. The relative expression levels of the target genes were normalised to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. The figures represent the relative fold change between the gene expression before and after glucose stimulation in correspondence to the control group. The sequences of the primers are enlisted in [Table 1].
Measurement of insulin secretion
The culture supernatant was collected after glucose stimulation and used to measure the secreted insulin by the human insulin ELISA kit (Abcam). The incubation and washing steps were performed according to the manufacturer's protocol, and the final colour was read immediately at 450 nm by Varioskan Flash multimode reader (Thermo Fisher Scientific).
| Results|| |
Suberanilohydroxamic acid enhanced the production of insulin and PDX1 at the cellular level
The cells, which received PBS or vehicle, had a basal level of insulin and PDX1 production under standard glucose concentration [Figure 1]. In SAHA pretreated cells, both markers were further expressed in comparison to the other two groups. These results suggested that the vehicle (DMSO) had no additive effect over the control (PBS) on the classical differentiation protocol, while SAHA induced the expression of the studied markers.
|Figure 1: Immunodetection of insulin (a-c) and PDX-1 (d-f) has showed basal expression of both markers in control (a and d) and vehicle (b and e) groups. Suberanilohydroxamic acid pretreated cells showed increased intensity of insulin in the cytoplasm (c) and PDX-1 in the nucleus (f). Photos were taken at ×60|
Click here to view
Gene expression analysis
On the molecular level [Figure 2], SAHA has enhanced the relative gene expression of insulin in response to high glucose challenge by 248% in comparison to the control and 161% to the vehicle. Similarly, SAHA induced the signalling molecules NKX6.1, MafA, SOX17 and FOXA2 and by 304%, 321%, 285% and 246% in response to glucose stimulation as well as GCK by 342%. All the markers were statistically higher than the vehicle (P < 0.05).
|Figure 2: Relative changes of gene expression upon high glucose challenge. Suberanilohydroxamic acid pretreated cells upregulated all the studied markers in comparison to the vehicle. *P < 0.05, **P < 0.01|
Click here to view
Suberanilohydroxamic acid enhanced basal insulin secretion but not in response to high glucose stimulation
As shown in [Figure 3], insulin secretion was evident in all studied groups. However, for both PBS and DMSO treated cells, there was no statistically significant difference between the insulin secretion before (8.5 and 9.8 μlU/ml) or after high glucose stimulation (11.5 and 14.4 μlU/ml). Interestingly, SAHA treated cells secreted almost double amount of insulin (17.5 μlU/ml) than PBS and vehicle. However, these cells did not respond appropriately to the high glucose challenge, as their insulin secretion showed no statistical difference than the nonstimulated cells.
|Figure 3: Insulin secretion in the media was measured by ELISA. At the standard glucose level, suberanilohydroxamic acid enhanced the production of insulin in comparison to the control by 205% and vehicle by 178%, while there was no statistically significant difference between the control and vehicle. After high dose glucose stimulation, there was no significant difference between any of the groups. *P < 0.05|
Click here to view
| Discussion|| |
Diabetes mellitus represents a major public health concern that affects a large number of patients worldwide. The current management plan includes lifelong insulin injection and/or oral hypoglycaemic agents, while there is no current cure for diabetes. Diabetes can lead to many complications due to the high level of blood glucose and the subsequent glycosylation reaction for many proteins and the consequent disturbance of the lipids metabolism. The control of the disease and the management of the complications added economic burden to the diabetic patients, families as well as the healthcare system. The direct annual cost of diabetes worldwide was >827 billion US dollars in 2016, according to the World Health Organisation global report on diabetes.
Pancreatic or pancreatic islets transplantation represents the only hope for physiological glycaemic control. However, the number of donors is limited and the transplantation-related complications are still major obstacles. Therefore, finding an alternative source of insulin-secreting cells may solve this dilemma. Regenerative medicine is an emerging branch of medicine which aims at replacing lost or failed organs, based on the stem cells technology.,
In this study, we aimed at generating insulin-secreting cells that would help diabetic patients and function as the normal beta cells in response to changing glucose levels in the blood. The main target was to provide a feasible source for insulin-secreting cells that may replace the islets or pancreatic transplantation. Furthermore, successful autologous stem cells differentiation into insulin-secreting cells represents an attractive target, as these cells would lack the immune response which can be triggered by the allogenic pancreatic and islets transplantation.
Despite the intensive researches in this field, the generated differentiated cells according to previously reported protocols are not completely satisfactory. A recent study, compared three protocols for the differentiation of MSCs into insulin-secreting cells, reached to a conclusion that the used protocols were not very efficient and further development was required. Therefore, the authors suggested the modification of the current protocols to achieve more efficient differentiation. Other research groups showed better differentiation of insulin-secreting cells, but the process was very long and difficult to be routinely applied.
Epigenetic mechanisms, like histone acetylation, are well known to be involved in the stem cell differentiation., In this study, we used an epigenetic modifying agent to improve the responsiveness of the cells to the differentiation protocols. The basic concept is activating the genome, rendering the target genes more accessible for transcription factors and other signalling molecules activated by the differentiation protocol components. SAHA is a HDAC-inhibitor. Similar derivatives were used effectively in improving the differentiation of stem cells into several lineages including neurogenic, osteogenic, chondrogenic lineages.,
The differentiation of stem cells in our study was carried out according to a three-steps differentiation protocol, which required relatively fewer reagents and involves fewer steps than classical protocols. This protocol depended on providing the crucial elements that enhance stem cell differentiation into the endocrine lineage. Retinoic acid, nicotinamide, EGF, and exendin-4 were applied to the culture media through three consequent steps, over 16 days. Retinoic acid induces the expression of transcription factors that control endocrine cell differentiation, such as PDX-1 and Ngn3 that are expressed in the foregut endoderm. Nicotinamide and EGF induces maturation of the pancreatic endocrine cells and sustains PDX1 expression. Exendin-4 is a potent glucagon-like peptide-1 agonist that increases insulin and PDX1 expression., Unfortunately, the original protocol produced immature insulin-producing cells.
In our system, control group PBS showed similar results to Gao et al. 2008. There was evidence of insulin synthesis (by immunofluorescence), secretion (by ELISA) and gene expression (by RT-PCR). Our data suggested that pretreating the cells with SAHA, the HDAC-inhibitor, could enhance the process of insulin production. In addition, the cells were able to respond to the high glucose challenge by inducing the genes coding for the various molecules involved in the insulin production as well as the insulin gene. The latter can be induced by the direct action of SAHA or under the effect of an upregulated signalling molecule, which can be one of the reported upregulated genes. Epigenetic regulation of insulin production and secretion can be achieved through multiple approaches. For example, PDX1 can stimulate insulin gene expression by hyperacetylating histone H4 at the insulin gene promoter. Nevertheless, all four histone acetyltransferases are important for insulin gene expression. Therefore, pretreating the cells with SAHA showed enhancement of insulin production, as showed by immunofluorescence, but the cells appeared to be at an earlier stage of maturation regarding insulin secretion in response to glucose stimulation.
SOX17 has multiple roles in beta cells development and function. While the classical role was in the endoderm lineage development, some reports showed that knocking down SOX17 did not influence the pancreatic development, but it affects insulin trafficking and secretion.,, In our system, SAHA has enhanced the gene expression of SOX17 in response to high glucose stimulation in comparison to the vehicle and control.
The transcription factor Nkx6.1 is important for β-cells development and insulin production, through the regulation of a gene network involved in insulin production and processing. In our study, SAHA pretreated cells showed the highest NKX6.1 expression.
MafA is considered as a key marker that is expressed only in mature β-cells, and it regulates glucose-stimulated insulin secretion as well as drives for beta cells proliferation in the postnatal life., Different cell groups followed the same pattern of increasing the expression of this marker when they were in high glucose. In addition, MafA has a role in the regulation of many pancreatic β-cells associated genes such as the glucose transporter Type 2 (GLUT2) and GCK. GLUT2 is the main transporter of glucose through the β-cells plasma membrane while GCK is the enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate and regulates the carbohydrate metabolism. Moreover, in beta cells, it serves as a glucose sensor, amplifying insulin secretion as blood glucose rises. The gene expression for this enzyme was upregulated in response to high glucose in SAHA pretreated cells.
The collective evidence suggested that SAHA enhanced the insulin production as could be observed by immunohistochemistry as well as ELISA. These cells responded to the high glucose stimulation by overexpressing many transcription factors in the insulin production pathway. The cells could be at an earlier stage of development that needed more time to respond by further enhancement of the insulin gene expression and consequently regulated secretion of insulin into the media. Further studies that assess the secretion at different time points, over a longer duration, could help in explaining this part of our findings.
| Conclusion|| |
SAHA represents a potential agent to improve stem cells differentiation into insulin-secreting cells, when used according to our protocol. Further investigation is recommended to improve the cell response to increasing concentration of glucose. In addition, the effect of pretreatment with SAHA on other beta cells differentiation protocols could also be considered.
Financial support and sponsorship
This work is financially supported by Sheikh Hamdan Bin Rashid Al Maktoum Award for Medical Sciences through grant No (MRG-6112013-2014). Lab workflow is supported by the University of Sharjah.
Conflicts of interest
There are no conflicts of interest.
| References|| |
IDF Diabetes Atlas, International Diabetes Federation. Report No.: 7; 2015. Available from: http://www.diabetesatlas.org
. [Last accessed on 2017 Sep 13].
Staels W, De Groef S, Bussche L, Leuckx G, Van de Casteele M. Making ß(-like)-cells from exocrine pancreas. Diabetes Obes Metab 2016;18 Suppl 1:144-51.
Vieira A, Courtney M, Druelle N, Avolio F, Napolitano T, Hadzic B, et al.
ß-Cell replacement as a treatment for type 1 diabetes: An overview of possible cell sources and current axes of research. Diabetes Obes Metab 2016;18 Suppl 1:137-43.
Bouwens L. Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Microsc Res Tech 1998;43:332-6.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al.
Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-7.
Kushner JA, MacDonald PE, Atkinson MA. Stem cells to insulin secreting cells: Two steps forward and now a time to pause? Cell Stem Cell 2014;15:535-6.
Gabr MM, Zakaria MM, Refaie AF, Khater SM, Ashamallah SA, Ismail AM, et al.
Generation of insulin-producing cells from human bone marrow-derived mesenchymal stem cells: Comparison of three differentiation protocols. Biomed Res Int 2014;2014:832736.
Hrvatin S, O'Donnell CW, Deng F. Differentiated human stem cells resemble fetal, not adult, β cells. Proc Natl Acad Sci USA 2014;111:3038-43.
Pagliuca FW, Millman JR, Gürtler M. Generation of functional human pancreatic B cells in vitro
. Cell 2014;159:428-39
Sharma S, Gurudutta G. Epigenetic regulation of hematopoietic stem cells. Int J Stem Cells 2016;9:36-43.
Tessarz P, Kouzarides T. Histone core modifications regulating nucleosome structure and dynamics. Nat Rev Mol Cell Biol 2014;15:703-8.
Tirino V, Desiderio V, d'Aquino R, De Francesco F, Pirozzi G, Graziano A, et al.
Detection and characterization of CD133 cancer stem cells in human solid tumours. PLoS One 2008;3:e3469.
Chen X, Hu C, Zhang W. Metformin inhibits the proliferation, metastasis, and cancer stem-like sphere formation in osteosarcoma MG63 cells in vitro
. Tumour Biol 2015;36:9873-83.
Yan GN, Lv YF, Guo QN. Advances in osteosarcoma stem cell research and opportunities for novel therapeutic targets. Cancer Lett 2016;370:268-74.
Gao F, Wu DQ, Hu YH, Jin GX, Li GD, Sun TW, et al. In vitro
cultivation of islet-like cell clusters from human umbilical cord blood-derived mesenchymal stem cells. Transl Res 2008;151:293-302.
Schaffer AE, Taylor BL, Benthuysen JR, Liu J, Thorel F, Yuan W, et al.
Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity. PLoS Genet 2013;9:e1003274.
Bonal C, Herrera PL. Genes controlling pancreas ontogeny. Int J Dev Biol 2008;52:823-35.
Eto K, Nishimura W, Oishi H, Udagawa H, Kawaguchi M, Hiramoto M, et al.
MafA is required for postnatal proliferation of pancreatic ß-cells. PLoS One 2014;9:e104184.
Sinner D, Kordich J, Spence J, Opoka R, Rankin S, Lin S-C, et al
. Sox17 and Sox4 Differentially Regulate β-Catenin/T-Cell Factor Activity and Proliferation of Colon Carcinoma Cells. Mol Cell Biol 2007;27:7802-15.
D'Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 2005; 23:1534-41.
Srinageshwar B, Maiti P, Dunbar GL, Rossignol J. Role of epigenetics in stem cell proliferation and differentiation: Implications for treating neurodegenerative diseases. Int J Mol Sci 2016;17. pii: E199.
Boheler KR, Czyz J, Tweedie D, Yang HT, Anisimov SV, Wobus AM, et al.
Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res 2002;91:189-201.
Selvaraj V, Jiang P, Chechneva O, Lo UG, Deng W. Differentiating human stem cells into neurons and glial cells for neural repair. Front Biosci (Landmark Ed) 2012;17:65-89.
Wu H, Sun YE. Epigenetic regulation of stem cell differentiation. Pediatr Res 2006;59 (4 Pt 2):21R-5R.
El-Serafi AT. Epigenetic modifiers and stem cell differentiation. Stem Cells and Cancer Stem Cells. Netherlands: Springer; 2012. p. 147-54.
Alexanian AR. Epigenetic modifiers promote efficient generation of neural-like cells from bone marrow-derived mesenchymal cells grown in neural environment. J Cell Biochem 2007;100:362-71.
El-Serafi AT, Oreffo RO, Roach HI. Epigenetic modifiers influence lineage commitment of human bone marrow stromal cells: Differential effects of 5-aza-deoxycytidine and trichostatin A. Differentiation 2011;81:35-41.
Micallef SJ, Janes ME, Knezevic K, Davis RP, Elefanty AG, Stanley EG. Retinoic acid induces Pd×1-positive endoderm in differentiating mouse embryonic stem cells. Diabetes 2005;54:301-5.
Zhou Y, Mack DL, Williams JK, Mirmalek-Sani SH, Moorefield E, Chun SY, et al.
Genetic modification of primate amniotic fluid-derived stem cells produces pancreatic progenitor cells in vitro
. Cells Tissues Organs 2013;197:269-82.
Fujimoto K, Polonsky KS. Pd×1 and other factors that regulate pancreatic beta-cell survival. Diabetes Obes Metab 2009;11 Suppl 4:30-7.
Zhao Q, Yang Y, Hu J, Shan Z, Wu Y, Lei L. Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells. Arch Med Sci 2016;12:199-207.
Wang HW, Breslin MB, Lan MS. Pdx-1 modulates histone H4 acetylation and insulin gene expression in terminally differentiated alpha-TC-1 cells. Pancreas 2007;34:248-53.
Sampley ML, Ozcan S. Regulation of insulin gene transcription by multiple histone acetyltransferases. DNA Cell Biol 2012;31:8-14.
Kanai-Azuma M, Kanai Y, Gad JM, Tajima Y, Taya C, Kurohmaru M, et al.
Depletion of definitive gut endoderm in So×17-null mutant mice. Development 2002;129:2367-79.
Harrelson Z, Kaestner KH, Evans SM. Foxa2 mediates critical functions of prechordal plate in patterning and morphogenesis and is cell autonomously required for early ventral endoderm morphogenesis. Biol Open 2012;1:173-81.
Jonatan D, Spence JR, Method AM, Kofron M, Sinagoga K, Haataja L, et al.
Sox17 regulates insulin secretion in the normal and pathologic mouse ß cell. PLoS One 2014;9:e104675.
[Figure 1], [Figure 2], [Figure 3]