RESEARCH

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Blood Res (2024) 59:43

Published online December 20, 2024

https://doi.org/10.1007/s44313-024-00051-5

© The Korean Society of Hematology

Bone marrow mesenchymal stem cell exosomes suppress JAK/STAT signaling pathway in acute myeloid leukemia in vitro

Sahar Jalilivand1†, Maryam Nabigol1†, Mehdi Bakhtiyaridovvombaygi2,3 and Ahmad Gharehbaghian1*

1 Department of Laboratory Hematology and Blood Bank, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Student Research Committee, Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Hematopoietic Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Correspondence to : *Ahmad Gharehbaghian
gharehbaghian@hotmail.com; gharehbaghian@sbmu.ac.ir

Sahar Jalilivand and Maryam Nabigol contributed equally to this work.

Received: September 10, 2024; Accepted: December 4, 2024

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Abstract

IntroductionDespite advances in the treatment of acute myeloid leukemia (AML), refractory forms of this malignancy and relapse remain common. Therefore, development of novel, synergistic targeted therapies are needed urgently. Recently, mesenchymal stem cells (MSCs) have been shown to be effective in treating various diseases, with most of their therapeutic outcomes attributed to their exosomes. In the current study, we investigated the effects of bone marrow mesenchymal stem cell (BM-MSC) exosomes on the expression of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling genes involved in AML pathogenesis.
Materialand Methods Exosomes were isolated from BM-MSCs and confirmed using transmission electron microscopy, dynamic light scattering, and flow cytometry. Subsequently, the exosome concentration was estimated using the bicinchoninic acid assay, and HL-60 cells were cocultured with 100 μg/mL of BM-MSC exosomes. Finally, the JAK2, STAT3, and STAT5 expression levels were analyzed using qRT-PCR.
ResultsThe exosome characterization results confirmed that most isolated nanoparticles exhibited a round morphology, expressed CD9, CD63, and CD81, which are specific protein markers for exosome identification, and ranged between 80 and 100 nm in diameter. Furthermore, qRT-PCR analysis revealed a significant downregulation of JAK2, STAT3, and STAT5 in HL-60 cells treated with 100 μg/mL of BM-MSC exosomes.
ConclusionSince JAK/STAT signaling contributes to AML survival, our findings suggest that the downregulation of JAK/STAT genes by BM-MSC exosomes in leukemic cells may aid in designing a potent therapeutic strategy for AML treatment.

Keywords Bone marrow mesenchymal stem cell, Exosome, Acute myeloid leukemia, JAK2, STAT3, STAT5

Article

RESEARCH

Blood Res 2024; 59():

Published online December 20, 2024 https://doi.org/10.1007/s44313-024-00051-5

Copyright © The Korean Society of Hematology.

Bone marrow mesenchymal stem cell exosomes suppress JAK/STAT signaling pathway in acute myeloid leukemia in vitro

Sahar Jalilivand1†, Maryam Nabigol1†, Mehdi Bakhtiyaridovvombaygi2,3 and Ahmad Gharehbaghian1*

1 Department of Laboratory Hematology and Blood Bank, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Student Research Committee, Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Hematopoietic Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Correspondence to:*Ahmad Gharehbaghian
gharehbaghian@hotmail.com; gharehbaghian@sbmu.ac.ir

Sahar Jalilivand and Maryam Nabigol contributed equally to this work.

Received: September 10, 2024; Accepted: December 4, 2024

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Abstract

IntroductionDespite advances in the treatment of acute myeloid leukemia (AML), refractory forms of this malignancy and relapse remain common. Therefore, development of novel, synergistic targeted therapies are needed urgently. Recently, mesenchymal stem cells (MSCs) have been shown to be effective in treating various diseases, with most of their therapeutic outcomes attributed to their exosomes. In the current study, we investigated the effects of bone marrow mesenchymal stem cell (BM-MSC) exosomes on the expression of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling genes involved in AML pathogenesis.
Materialand Methods Exosomes were isolated from BM-MSCs and confirmed using transmission electron microscopy, dynamic light scattering, and flow cytometry. Subsequently, the exosome concentration was estimated using the bicinchoninic acid assay, and HL-60 cells were cocultured with 100 μg/mL of BM-MSC exosomes. Finally, the JAK2, STAT3, and STAT5 expression levels were analyzed using qRT-PCR.
ResultsThe exosome characterization results confirmed that most isolated nanoparticles exhibited a round morphology, expressed CD9, CD63, and CD81, which are specific protein markers for exosome identification, and ranged between 80 and 100 nm in diameter. Furthermore, qRT-PCR analysis revealed a significant downregulation of JAK2, STAT3, and STAT5 in HL-60 cells treated with 100 μg/mL of BM-MSC exosomes.
ConclusionSince JAK/STAT signaling contributes to AML survival, our findings suggest that the downregulation of JAK/STAT genes by BM-MSC exosomes in leukemic cells may aid in designing a potent therapeutic strategy for AML treatment.

Keywords: Bone marrow mesenchymal stem cell, Exosome, Acute myeloid leukemia, JAK2, STAT3, STAT5

Fig 1.

Figure 1.The identification and confirmation of BM-MSCs. A In passage 2, BM-MSCs demonstrated fibroblast-like morphology under an inverted microscope. B The flow cytometry was used to check the immune phenotype of BM-MSCs. BM-MSCs lacked CD45, CD34, and CD14, but expressed CD105, CD90, and CD73

Fig 2.

Figure 2.The identification and confirmation of BM-MSC exosomes. A The size of the isolated exosomes was determined using the DLS technique. The exosome sizes varied from 80–100 nm. B Morphology of isolated exosomes under TEM. The BM-MSC exosomes demonstrated a spherical-shaped morphology. C The expression of the CD63, CD81, and CD9 exosome-specific surface markers in BM-MSC exosomes was examined using flow cytometry

Fig 3.

Figure 3.The impact of BM-MSC exosomes on the JAK/STAT gene expression of HL-60 cells. JAK2, STAT3, and STAT5 levels were notably reduced in HL-60 cells following treatment with 100 μg/mL of exosomes. The expression levels were normalized using the GAPDH housekeeping gene. Statistical significance was observed (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) compared to the control group (n = 3)

Table 1 . This table provides the primer sequences used in this study.

GenesprimerProduct sizeTMSequence (5’– > 3’)
JAK2Forward15259.4GGC AAT GAC AAA CAA GGA CAG
Reverse58.4AAG GAG GGG CGT TGA TTT AC
STAT3Forward19960.3AGG AGG CAT TCG GAA AGT ATTG
Reverse60.3GGT TCA GCA CCT TCA CCA TTAT
STAT5Forward17759.4CTG GCT AAA GCT GTT GAT GGA
Reverse61.3TAC ATG GTC AGG GTT CTG TGG
GAPDHForward7059.5ATG GGG AAG GTG AAG GTC G
Reverse60.5TAA AAG CAG CCC TGG TGA CC

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