Effect of small molecule hydrogels on proliferation, apoptosis and myocardial differentiation of bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.21.004

Abstract

Abstract:

BACKGROUND: A short-peptide small molecule hydrogel (SMH) developed in the previous study has more obvious advantages than other hydrogels to improve local microenvironment, carry bioactive substances and interfere with stem cell signal transduction pathways.
OBJECTIVE: To explore the effect of SMHs on bone marrow mesenchymal stem cells (BMSCs) proliferation, apoptosis and differentiation into myocardial cells.
METHODS: (1) Passage 9 rat BMSCs in vitro were divided into control group and experimental group, followed by routine culture and culture in SMHs, respectively. At 7 days of culture, cell proliferation and apoptosis were detected. Cells in the two groups were exposed to anaerobic environment for 12 hours, and expression levels of Bcl-2, Bax and Caspase-3 in BMSCs were detected. (2) Passage 9 BMSCs were divided into four groups and then cultured in 5-azacytidine, SMHs, SMHs+5-azacytidine, and L-DMEM (normal control), respectively. After 4 weeks of induction, expression of CTnT, desmin and Cx-43 proteins was detected and expression levels of early cardiac transcription factors, NKX2.5 and GATA-4, were also measured.
RESULTS AND CONCLUSION: (1) Compared with the control group, better proliferation and lower apoptosis of BMSCs were found in the experimental group. Under anaerobic conditions, the number of survival cells was reduced in both groups, but less apoptosis or necrosis was found in the experimental group than the control group (P < 0.05). Moreover, the level of Bcl-2 was higher in the experimental group than the control group (P < 0.01), while the levels of Bax and Caspases-3 protiens were lower in the experimental group than the control group (P < 0.01). (2) NKx2.5 and GATA-4 mRNA expression was found in both 5-azacytidine and SMHs+5-azacytidine groups, and moreover, the mRNA levels of early cardiac transcription factors were significantly higher in the SMHs+5-azacytidine group than in the 5-azacytidine group (P < 0.05). In the normal control group, cTnT expressed negatively, and desmin and Cx-43 expressed weakly. The expression of cTnT, desmin and Cx-43 proteins was higher in the SMHs+5-azacytidine group than in the 5-azacytidine and SMHs groups, while there was no significant difference between the latter two groups. To conclude, SMHs as a culture medium is conducive to the proliferation of BMSCs, reduces cell apoptosis, and promotes myocardial differentiation of BMSCs.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Hydrogels, Mesenchymal Stem Cells, Cell Proliferation, Apoptosis, Tissue Engineering

CLC Number:

R394.2

Chen Guo-qin, Li Jin-liang, Song Ming-cai, Ou Cai-wen. Effect of small molecule hydrogels on proliferation, apoptosis and myocardial differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3299-3305.

Figures/Tables 3

2.1 小分子水凝胶对骨髓间充质干细胞存活和生长增殖的影响 AO/EB染色显示，实验组骨髓间充质干细胞在小分子水凝胶中生长第5-7天后仍能保持较好的活力，而对照组第5-7天可见数少量凋亡及坏死的细胞；实验组中细胞培养7 d后绿色荧光着色较深，呈亮绿色，表明活力明显较对照组好，存活细胞数目较高，见图1。 MTT检测结果显示，实验组骨髓间充质干细胞在小分子水凝胶中能够保持良好的增殖能力，与对照组相比，5 d前细胞生长曲线较低，但5 d后明显增高(P < 0.05)，见图2。 2.2 小分子水凝胶对缺氧环境下骨髓间充质干细胞凋亡的影响 2.2.1 AnnexinV/PI流式细胞分析细胞凋亡 AnnexinV/Pl双标流式细胞仪检测显示，在缺氧环境中实验组和对照组存活的细胞均有减少，实验组的凋亡或坏死细胞数目明显少于对照组(P < 0.05)，见图3。AO/EB染色显示，绿色染色为存活细胞，黄色为凋亡或坏死细胞，结果显示，实验组存活细胞数量明显多于对照组，而凋亡或坏死的细胞数量明显少于对照组，见图4。 2.2.2 Western-Blot分析细胞凋亡相关蛋白表达从图5A可观察到，经缺氧处理后，实验组及对照组细胞Bcl-2蛋白表达明显高于正常对照组(P < 0.01)，且实验组细胞Bcl-2蛋白表达高于对照组(P < 0.01)。从图5B，C可观察到，经缺氧处理后，对照组细胞Bax、Caspase-3蛋白表达高于正常对照组(P < 0.01)，实验组细胞Bax、Caspase-3蛋白表达低于对照组(P < 0.01)。结果表明实验组在小分子水凝胶培养的骨髓间充质干细胞，在缺氧环境下能通过增加Bcl-2蛋白表达，同时降低Bax、Caspase-3蛋白表达来减少凋亡，提示小分子水凝胶可减少缺氧环境下骨髓间充质质干细胞的凋亡。 2.3 小分子水凝胶对骨髓间充质干细胞向心肌分化的作用 2.3.1 实时荧光定量RT-PCR检测心肌早期转录因子NKx2.5、GATA-4的表达诱导4周后，从扩增图及溶解曲线图中分析5-氮杂胞苷组和实验组各样本中均出现明确的心肌早期分化基因，实验组NKx2.5和GATA-4 mRNA表达高于5-氮杂胞苷组(P < 0.05)，见表1。 2.3.2 Western印迹法检测cTnT蛋白表达诱导4周后，未诱导的在正常对照组中几乎未发现cTnT蛋白表达，5-氮杂胞苷组、凝胶组及实验组中cTnT蛋白均可见阳性表达，实验组cTnT蛋白表达高于5-氮杂胞苷组、凝胶组(P < 0.05)，5-氮杂胞苷组与凝胶组比较差异无显著性意义，见图6。 2.3.3 免疫细胞化学染色检测结果诱导后4周相差显微镜下可见，实验组细胞形态和排列方式均发生明显改变，细胞平行排列，呈簇团状且相邻细胞间出现细胞连接。免疫细胞化学染色结果显示，正常对照组cTnT蛋白表达阴性，desmin及Cx-43蛋白表达很弱；实验组cTnT、desmin及Cx-43蛋白表达较5-氮杂胞苷组、凝胶组明显增强，凝胶组cTnT、desmin及Cx-43蛋白表达与5-氮杂胞苷组相似(图7)。"

References

[1] Bilgimol JC,Ragupathi S,Vengadassalapathy L,et al.Stem cells: An eventual treatment option for heart diseases.World J Stem Cells.2015;7(8):1118-1126.

[2] Faiella W,Atoui R.Therapeutic use of stem cells for cardiovascular disease.Clin Transl Med.2016;5:34.

[3] Der Sarkissian S,Lévesque T,Noiseux N.Optimizing stem cells for cardiac repair: Current status and new frontiers in regenerative cardiology.World J Stem Cells. 2017;9(1):9-25.

[4] Gaffey AC,Chen MH,Venkataraman CM,et al.Injectable shear-thinning hydrogels used to deliver endothelial progenitor cells, enhance cell engraftment, and improve ischemic myocardium.J Thorac Cardiovasc Surg. 2015;150(5):1268-1276.

[5] Boopathy AV,Martinez MD,Smith AW,et al.Intramyocardial Delivery of Notch Ligand-Containing Hydrogels Improves Cardiac Function and Angiogenesis Following Infarction. Tissue Eng Part A.2015;21(17-18):2315-2322.

[6] Mihic A,Cui Z,Wu J,et al.A Conductive Polymer Hydrogel Supports Cell Electrical Signaling and Improves Cardiac Function After Implantation into Myocardial Infarct. Circulation.2015;132(8):772-784.

[7] Günter J,Wolint P,Bopp A,et al.Microtissues in Cardiovascular Medicine: Regenerative Potential Based on a 3D Microenvironment.Stem Cells Int.2016;2016:9098523.

[8] Panda NC,Zuckerman ST,Mesubi OO,et al.Improved conduction and increased cell retention in healed MI using mesenchymal stem cells suspended in alginate hydrogel.J Interv Card Electrophysiol.2014;41(2):117-127.

[9] Yang G,Xiao Z,Ren X,et al.Enzymatically crosslinked gelatin hydrogel promotes the proliferation of adipose tissue-derived stromal cells.PeerJ.2016;4:e2497.

[10] Yang G,Xiao Z,Ren X,et al.Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels.Sci Rep.2017;7:41781.

[11] Lawrence PG,Patil PS,Leipzig ND,et al.Ionically Cross-Linked Polymer Networks for the Multiple-Month Release of Small Molecules.ACS Appl Mater Interfaces. 2016;8(7):4323-4335.

[12] Xu G,Wang X,Deng C,et al.Injectable biodegradable hybrid hydrogels based on thiolated collagen and oligo(acryloyl carbonate)-poly(ethylene glycol)-oligo(acryloyl carbonate) copolymer for functional cardiac regeneration.Acta Biomaterialia.2015;15:55-:64.

[13] Deng B,Shen L,Wu Y,et al.Delivery of alginate-chitosan hydrogel promotes endogenous repair and preserves cardiac function in rats with myocardial infarction.J Biomed Mater Res A.2015;103(3):907-918.

[14] 牡丹,原晖华,顾蓉,等.整合素连接激酶过表达促进猪骨髓间充质干细胞向心肌样细胞分化[J].中国动脉硬化杂志,2016,24(9): 875-881.

[15] Chen G,Chen J,Liu Q,et al.Enzymatic Formation of a Meta-stable Supramolecular Hydrogel for 3D Cell Culture. RSC Adv.2015;(5):30675-30678.

[16] Liang Z,Liu C,Li L,et al.Double-Network Hydrogel with Tunable Mechanical Performance and Biocompatibility for the Fabrication of Stem Cells-Encapsulated Fibers and 3D Assemble. Sci Rep.2016;6:33462.

[17] Lowenthal J,Gerecht S.Stem cell-derived vasculature: A potent and multidimensional technology for basic research, disease modeling, and tissue engineering.Biochem Biophys Res Commun.2016;473(3):733-742.

[18] Chaudhuri O,Gu L,Klumpers D,et al.Hydrogels with tunable stress relaxation regulate stem cell fate and activity.Nat Mater. 2016;15(3):326-334.

[19] Günter J,Wolint P,Bopp A,at al.Microtissues in Cardiovascular Medicine: Regenerative Potential Based on a 3D Microenvironment.Stem Cells Int.2016;2016:9098523.

[20] Chen G,Hua Y,Ou C,et al.Nanostructure formation-induced fluorescence turn-on for selectively detecting protein thiols in solutions, bacteria and live cells.Chem Commun.2015; 51(53): 10758-10761.

[21] Truskey GA.Advancing cardiovascular tissue engineering. Version 1.F1000Res.2016;5:F1000 Faculty Rev-1045.

[22] McGann CL,Akins RE,Kiick KL.Resilin-PEG hybrid hydrogels yield degradable elastomeric scaffolds with heterogeneous microstructure.Biomacromolecules.2016;17(1):128-140.

[23] McGann CL,Dumm RE,Jurusik AK,et al.Thiol-ene Photocrosslinking of Cytocompatible Resilin-like Polypeptide- PEG Hydrogels.Macromol Biosci.2016; 16(1):129-138.

[24] Masumoto H,Nakane T,Tinney JP,et al.The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages.Sci Rep.2016;6:29933.

[25] Yang G,Xiao Z,Ren X,et al.Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels.Sci Rep.2017;7:41781.

[26] Lister Z,Rayner KJ,Suuronen EJ.How Biomaterials Can Influence Various Cell Types in the Repair and Regeneration of the Heart after Myocardial Infarction.Front Bioeng Biotechnol. 2016;4:62.