Contributions
Abstract: PB2137
Type: Publication Only
Background
The bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donor is one of the most promising sources of cell therapy. We have previously shown that extracellular vesicles (EVs) secreted from BM-MSCs had anti-angiogenic effect rather than direct growth inhibition in multiple myeloma (MM) (Umezu et al. Blood Adv 2017).
Aims
To elucidate the molecular mechanism of BM-MSCs administration in cancer patients, we investigated both EVs and soluble factor mediated effect on multiple myeloma cells using a freshly obtained BM-MSC specimens.
Methods
BMSCs from Russian healthy volunterrs (KNT_D_170714, KNT_L_170714, KNT_80_170825 and KNT_86_170825: KNT cells) are kindly provided by Kintaro cells power Co. (Tokyo, Japan). Fresh bone marrow specimens were collected at Federal Research and Clinical Center of Federal Biomedical Agency of Russia (FRCC FMBA, Moskva, Russia) after obtaining approval of institutional ethical committee. Spectral karyotyping (SKY) , immunophnotyping was done to validate KNT cells. In addition, KNT cells was evaluated after induction using MSC differentiation media, according to the manufacuture's protocol (PromoCell). To analyze the factors secreted by the KNT cells, 90% confluent, passage 2-4 KNT cells in T25 tissue culture flask, were transferred to a serum-free AIM-V medium (Invitrogen) during 48 h. The EVs were isolated from conditioned medium (CM) using a Exoquick-TC (SBI). The size of EVs was confirmed using a NanoSight LM10 (Malvren). CM was fractionated using 3-kDa, 10-kDa and 50-kDa MW cut-off filter units (Millipore). To determine the effect of secretion from KNT cells, we used a MM cell line, RPMI8226. Endothelial tube formation assay was used for validating function of MM cell-endothelial cell (HUVECs) communication.
Results
We first validated KNT cells as follows. The KNT cells had a fibroblast-like morphology in culture regardless of donor's age. Immunophenotyping and karyotyping showed a normal karyotypes. The cells were positive for all MSC markers (CD90, CD73, CD105), but negative for hematopoietic markers (CD34 and CD45). When cultured in appropriate culture conditions KNT cells showed adipogenic or osteogenic potential. The nanoparticle size distribution of EVs secreted from KNT cells (KNT-EVs) were approximately 50 nm. We found that the donor’s age involved the properties of KNT-EVs, We found that KNT-EVs derived from young healthy donors significantly reduced tube formation of HUVECs co-cultured with a MM cell line (RPMI8226), but did not affect the survival of RPMI8226 in vitro. We also found KNT cell-derived CM (KNT-CM) derived from young healthy donors markedly inhibited the growth of RPMI8226 as well as the tube formation of HUVECs. Especially the MM cell growth inhibitory effect was remarkable in the CM fractionated by 50-kDa MW cut-off filter.
Conclusion
In the current study, we used freshly obtained BM-MSCs (KNT cells) instead of commercially avairable BM-MSCs, such as widely used BM-MSCs from LONZA co. Our results indicates that the young healthy donor-derived KNT cell secretome including EVs and other soluble factors have the ability to inhibit MM-induced angiogenesis. In contrast, growth inhibitory effect was only seen at the molecules up to a MW of 50-kDa in KNT-CM. These findings indicated the limitation of EV-based therapy, however, analysis of EVs and fractionated CM may shed light on the complex molecular mechanism of BM-MSC-based therapy.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Angiogenesis, Bone marrow stroma, Microvesicles, Multiple Myeloma
Abstract: PB2137
Type: Publication Only
Background
The bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donor is one of the most promising sources of cell therapy. We have previously shown that extracellular vesicles (EVs) secreted from BM-MSCs had anti-angiogenic effect rather than direct growth inhibition in multiple myeloma (MM) (Umezu et al. Blood Adv 2017).
Aims
To elucidate the molecular mechanism of BM-MSCs administration in cancer patients, we investigated both EVs and soluble factor mediated effect on multiple myeloma cells using a freshly obtained BM-MSC specimens.
Methods
BMSCs from Russian healthy volunterrs (KNT_D_170714, KNT_L_170714, KNT_80_170825 and KNT_86_170825: KNT cells) are kindly provided by Kintaro cells power Co. (Tokyo, Japan). Fresh bone marrow specimens were collected at Federal Research and Clinical Center of Federal Biomedical Agency of Russia (FRCC FMBA, Moskva, Russia) after obtaining approval of institutional ethical committee. Spectral karyotyping (SKY) , immunophnotyping was done to validate KNT cells. In addition, KNT cells was evaluated after induction using MSC differentiation media, according to the manufacuture's protocol (PromoCell). To analyze the factors secreted by the KNT cells, 90% confluent, passage 2-4 KNT cells in T25 tissue culture flask, were transferred to a serum-free AIM-V medium (Invitrogen) during 48 h. The EVs were isolated from conditioned medium (CM) using a Exoquick-TC (SBI). The size of EVs was confirmed using a NanoSight LM10 (Malvren). CM was fractionated using 3-kDa, 10-kDa and 50-kDa MW cut-off filter units (Millipore). To determine the effect of secretion from KNT cells, we used a MM cell line, RPMI8226. Endothelial tube formation assay was used for validating function of MM cell-endothelial cell (HUVECs) communication.
Results
We first validated KNT cells as follows. The KNT cells had a fibroblast-like morphology in culture regardless of donor's age. Immunophenotyping and karyotyping showed a normal karyotypes. The cells were positive for all MSC markers (CD90, CD73, CD105), but negative for hematopoietic markers (CD34 and CD45). When cultured in appropriate culture conditions KNT cells showed adipogenic or osteogenic potential. The nanoparticle size distribution of EVs secreted from KNT cells (KNT-EVs) were approximately 50 nm. We found that the donor’s age involved the properties of KNT-EVs, We found that KNT-EVs derived from young healthy donors significantly reduced tube formation of HUVECs co-cultured with a MM cell line (RPMI8226), but did not affect the survival of RPMI8226 in vitro. We also found KNT cell-derived CM (KNT-CM) derived from young healthy donors markedly inhibited the growth of RPMI8226 as well as the tube formation of HUVECs. Especially the MM cell growth inhibitory effect was remarkable in the CM fractionated by 50-kDa MW cut-off filter.
Conclusion
In the current study, we used freshly obtained BM-MSCs (KNT cells) instead of commercially avairable BM-MSCs, such as widely used BM-MSCs from LONZA co. Our results indicates that the young healthy donor-derived KNT cell secretome including EVs and other soluble factors have the ability to inhibit MM-induced angiogenesis. In contrast, growth inhibitory effect was only seen at the molecules up to a MW of 50-kDa in KNT-CM. These findings indicated the limitation of EV-based therapy, however, analysis of EVs and fractionated CM may shed light on the complex molecular mechanism of BM-MSC-based therapy.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Angiogenesis, Bone marrow stroma, Microvesicles, Multiple Myeloma