Effect of low-dose decitabine on the biological behavior of bone marrow mesenchymal stem cells derived from patients with immune thrombocytopenia

doi:10.3969/j.issn.1674-8115.2022.06.010

Abstract

Abstract: Objective

·To investigate the effects of low-dose decitabine on the biological behaviors of proliferation and apoptosis of bone marrow mesenchymal stem cells (MSC) derived from the patients with primary immune thrombocytopenia (ITP).

Methods

·Ten patients with ITP onset who were outpatients or inpatients in the Department of Hematology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, from April 2020 to October 2021 were included, and 10 non-ITP patients without bone marrow abnormalities were included as controls (normal control, NC). The bone marrow aspiration specimens were collected, MSCs were isolated, and flow cytometry was used to detect their surface antigenic markers. Differences in MSC proliferation levels between the ITP patients (MSC-ITP) and the controls (MSC-NC) were detected by using the CCK-8 kit and the EdU cell proliferation assay kit, and the changes in cell morphology were detected by using microtubule fluorescent probes. Apoptosis was observed by using Hoechst 33258 staining, and the level of apoptosis was detected by using Annexin V-FITC/PI apoptosis double-staining kit. The optimal concentration and treatment time of decitabine to promote the proliferation of MSC-ITP were explored by treatment with different concentrations and different durations. Western blotting was performed to detect the expression of apoptosis-related proteins and the effect of decitabine.

Results

·Compared with MSC-NC, MSC-ITP showed abnormal morphology, more nuclear fragmentation and crinkling, reduced proliferation level in vitro, and increased basal apoptosis rate. The optimal working concentration of decitabine to stimulate MSC-ITP proliferation was 2.5 μmol/L, and the optimal treatment time was 24 h. After 2.5 μmol/L decitabine treating MSC-ITP for 24 h, cell nuclear morphology was improved, nuclei fragmentation and coalescence were reduced, and apoptosis rate was significantly decreased (P<0.05). Western blotting results showed that BAX, a mitochondrial apoptosis pathway-related protein, cystatin protease 3 (caspase3) and cleaved-caspase3 were reduced in MSC-ITP after treatment with decitabine (P<0.05).

Conclusion

·The MSCs originating from ITP patients' bone marrow have abnormal morphology, reduced proliferation level and increased basal apoptosis rate in vitro; low-dose decitabine can promote the proliferation and inhibit the apoptosis of the cells, which may be mediated through the mitochondrial apoptosis pathway.

Key words: immune thrombocytopenia (ITP), bone marrow mesenchymal stem cell, decitabine, DNA methyltransferase inhibitor, apoptosis

CLC Number:

R558⁺.2

WANG Xinpeng, WANG Junying, CAI Jiayi, FU Wanbing, ZHONG Hua. Effect of low-dose decitabine on the biological behavior of bone marrow mesenchymal stem cells derived from patients with immune thrombocytopenia[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(6): 758-767.

Figures/Tables 7

Tab 1 Clinical information of the ITP patients and the normal controls enrolled in the study

ITP patient	Gender	Age/year	Platelet count/（×10⁹ L^－1）	NC subject	Gender	Age/year	Platelet count/（×10⁹ L^－1）
P1	Female	48	44	N1	Female	60	137
P2	Female	44	34	N2	Female	74	206
P3	Female	70	69	N3	Female	65	169
P4	Female	71	102	N4	Male	31	145
P5	Male	72	61	N5	Female	66	172
P6	Female	84	38	N6	Male	39	151
P7	Male	77	20	N7	Female	51	183
P8	Female	45	48	N8	Male	68	201
P9	Female	85	34	N9	Male	45	195
P10	Male	61	7	N10	Female	75	141

Fig 1 Phenotype of MSCs cultured in vitro identified by flow cytometry

Fig 2 Detection of proliferation and morphology of MSCs derived from ITP patients

Fig 3 Detection of basal apoptosis in MSCs derived from ITP patients

Fig 4 Effect of decitabine on MSC-ITP proliferation under different doses and working hours detected by CCK-8 assay

Fig 5 Apoptosis detection of MSC-ITP after 2.5 μmol/L decitabine treatment for 24 h

Fig 6 Expression levels of apoptosis-related proteins BAX, caspase-3 and cleaved-caspase3 detected by Western blotting

TrendMD

References 32

1	SEMPLE J W, PROVAN D, GARVEY M B, et al. Recent progress in understanding the pathogenesis of immune thrombocytopenia[J]. Curr Opin Hematol, 2010, 17(6): 590-595.
2	REEMS J A, PINEAULT N, SUN S J. In vitro megakaryocyte production and platelet biogenesis: state of the art[J]. Transfus Med Rev, 2010, 24(1): 33-43.
3	GLENNIE S, SOEIRO I, DYSON P J, et al. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells[J]. Blood, 2005, 105(7): 2821-2827.
4	TABERA S, PÉREZ-SIMÓN J A, DÍEZ-CAMPELO M, et al. The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes[J]. Haematologica, 2008, 93(9): 1301-1309.
5	TASSO R, ILENGO C, QUARTO R, et al. Mesenchymal stem cells induce functionally active T-regulatory lymphocytes in a paracrine fashion and ameliorate experimental autoimmune uveitis[J]. Invest Ophthalmol Vis Sci, 2012, 53(2): 786-793.
6	MA J, NING Y N, XU M, et al. Thalidomide corrects impaired mesenchymal stem cell function in inducing tolerogenic DCs in patients with immune thrombocytopenia[J]. Blood, 2013, 122(12): 2074-2082.
7	PÉREZ-SIMÓN J A, TABERA S, SARASQUETE M E, et al. Mesenchymal stem cells are functionally abnormal in patients with immune thrombocytopenic purpura[J]. Cytotherapy, 2009, 11(6): 698-705.
8	ZHANG D L, LI H Y, MA L, et al. The defective bone marrow-derived mesenchymal stem cells in patients with chronic immune thrombocytopenia[J]. Autoimmunity, 2014, 47(8): 519-529.
9	王君颖, 李昕, 殷婷玉, 等. 免疫性血小板减少症患者来源的骨髓间充质细胞对巨核细胞生物学行为的影响[J]. 上海交通大学学报(医学版), 2018, 38(6): 616-623.
	WANG J Y, LI X, YIN T Y, et al. Effects of bone marrow mesenchymal cells from immune thrombocytopenia patients on the biological behaviors of megakaryocytes[J]. J Shanghai Jiao Tong Univ (Med Sci), 2018, 38(6): 616–625.
10	XIAO J H, ZHANG C R, ZHANG Y C, et al. Transplantation of adipose-derived mesenchymal stem cells into a murine model of passive chronic immune thrombocytopenia[J]. Transfusion, 2012, 52(12): 2551-2558.
11	ZHANG P, ZHANG G, LIU X, et al. Mesenchymal stem cells improve platelet counts in mice with immune thrombocytopenia[J]. J Cell Biochem, 2019. DOI: 10.1002/jcb.28405.
12	WANG X H, YIN X G, SUN W, et al. Intravenous infusion umbilical cord-derived mesenchymal stem cell in primary immune thrombocytopenia: a two-year follow-up[J]. Exp Ther Med, 2017, 13(5): 2255-2258.
13	LEE S, KIM H S, ROH K H, et al. DNA methyltransferase inhibition accelerates the immunomodulation and migration of human mesenchymal stem cells[J]. Sci Rep, 2015, 5: 8020.
14	LIU S Y, SHAN N N. DNA methylation plays an important role in immune thrombocytopenia[J]. Int Immunopharmacol, 2020, 83: 106390.
15	ASSIS R I F, WIENCH M, SILVÉRIO K G, et al. RG108 increases NANOG and OCT4 in bone marrow-derived mesenchymal cells through global changes in DNA modifications and epigenetic activation[J]. PLoS One, 2018, 13(12): e0207873.
16	OH Y S, JEONG S G, CHO G W. Anti-senescence effects of DNA methyltransferase inhibitor RG108 in human bone marrow mesenchymal stromal cells[J]. Biotechnol Appl Biochem, 2015, 62(5): 583-590.
17	OH Y S, KIM S H, CHO G W. Functional restoration of amyotrophic lateral sclerosis patient-derived mesenchymal stromal cells through inhibition of DNA methyltransferase[J]. Cell Mol Neurobiol, 2016, 36(4): 613-620.
18	ZHOU H, HOU Y, LIU X N, et al. Low-dose decitabine promotes megakaryocyte maturation and platelet production in healthy controls and immune thrombocytopenia[J]. Thromb Haemost, 2015, 113(5): 1021-1034.
19	RODEGHIERO F, STASI R, GERNSHEIMER T, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group[J]. Blood, 2009, 113(11): 2386-2393.
20	LAMBERT M P, GERNSHEIMER T B. Clinical updates in adult immune thrombocytopenia[J]. Blood, 2017, 129(21): 2829-2835.
21	SHEN Y, NILSSON S K. Bone, microenvironment and hematopoiesis[J]. Curr Opin Hematol, 2012, 19(4): 250-255.
22	SONG Y, WANG Y T, HUANG X J, et al. Abnormalities of the bone marrow immune microenvironment in patients with immune thrombocytopenia[J]. Ann Hematol, 2016, 95(6): 959-965.
23	TAO Y L, SONG D X, ZHANG F Y, et al. Transplantation of bone-marrow-derived mesenchymal stem cells into a murine model of immune thrombocytopenia[J]. Blood Coagul Fibrinolysis, 2017, 28(8): 596-601.
24	LI H Y, XUAN M, YANG R C. DNA methylation and primary immune thrombocytopenia[J]. Semin Hematol, 2013, 50(Suppl 1): S116-S126.
25	WANG J H, YI Z H, WANG S Y, et al. The effect of decitabine on megakaryocyte maturation and platelet release[J]. Thromb Haemost, 2011, 106(2): 337-343.
26	HAN P P, HOU Y, ZHAO Y J, et al. Low-dose decitabine modulates T cell homeostasis and restores immune tolerance in immune thrombocytopenia[J]. Blood, 2021, 138(8): 674-688.
27	WANG Y, WANG F X, WEN S P, et al. Artesunate-enhanced apoptosis of human high-risk myelodysplastic cells induced by the DNA methyltransferase inhibitor decitabine[J]. Oncol Lett, 2015, 9(6): 2449-2454.
28	ZHANG G, GAO X H, ZHAO X Y, et al. Decitabine inhibits the proliferation of human T-cell acute lymphoblastic leukemia molt4 cells and promotes apoptosis partly by regulating the PI3K/AKT/mTOR pathway[J]. Oncol Lett, 2021, 21(5): 340.
29	WANG L, AMOOZGAR Z, HUANG J, et al. Decitabine enhances lymphocyte migration and function and synergizes with CTLA-4 blockade in a murine ovarian cancer model[J]. Cancer Immunol Res, 2015, 3(9): 1030-1041.
30	RÜGER B M, BUCHACHER T, GIUREA A, et al. Vascular morphogenesis in the context of inflammation: self-organization in a fibrin-based 3D culture system[J]. Front Physiol, 2018, 9: 679.
31	ELMORE S. Apoptosis: a review of programmed cell death[J]. Toxicol Pathol, 2007, 35(4): 495-516.
32	FERRI K F, KROEMER G. Mitochondria: the suicide organelles[J]. BioEssays, 2001, 23(2): 111-115.

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