Cell and Tissue Journal

Abstract Introduction: Mesenchymal stem cells (MSCs) are  important cellular components in the microenvironment of various types of cancers, including multiple myeloma (MM); their dynamic interactions with malignant cells can control the tumor microenvironment (TME) to favor the progression, drug resistance, and survival of cancer cells. It is shown that not only are MM cells influenced by the environmental cells, but also malignant cells affect the behavior of other cells in the TME. Secreted signaling factors are important players in this scenario. Accordingly, MSCs derived from TME, affected by cancer cells, are different from normal MSCs; therefore, in cases of disease modeling and drug screening studies, optimization of MSCs culture condition and their priming with cancer cell-derived secretome are crucial considerations to become more similar to the patient-derived MSCs. In this regard, conditioned medium, which is derived from the supernatant culture medium of cultured cells, contains cell-secreted factors and can be used as a source of cell secretome.
Aims: This study attempted to evaluate the effect of conditioned medium derived from the myeloma cell line U266 (U266-CM) on the proliferation, cell cycle, and differentiation of bone marrow-derived MSCs (BMMSCs).
Materials and methods: U266 cells were cultured in RPMI-1640 complete culture medium, and their conditioned medium was collected after two days of culture and stored. Since the basal culture medium of the U266 cell line (RPMI-1640) was different from the basal culture medium of BMMSCs (DMEM), in the first step, the impact of changing the culture medium from DMEM to RPMI-1640 on the proliferation and viability of BMMSCs was evaluated. In the next step, the effects of U266-CM treatment on the proliferation, cell cycle, and differentiation of BMMSCs into osteoblasts and adipocytes were assessed. Alkaline phosphatase activity assessment and Alizarin red staining were performed to evaluate the osteogenic differentiation, and Oil red O staining was carried out to assess the adipogenic differentiation. BMMSCs cultured in DMEM complete media were considered as control group.
Results: Changing the culture media from DMEM complete media to RPMI-1640 complete media affected the viability and proliferation rate of BMMSCs; however, up to 48 hours, the viability and proliferation rate of cells cultured in both culture media were maintained. Therefore, 48 hours was selected as the optimum incubation time of BMMSCs with U266-CM. Treatment with U266-CM decreased the proliferation rate of BMMSCs, assessed by cell counting and cell cycle analysis. Moreover, BMMSCs cultured in U266-CM showed slightly increased osteogenic differentiation and maintained their adipogenic propensity.
Discussion: Our results showed that CM derived from the MM cell line affected the proliferation rate and the differentiation potential of BMMSCs. These observations highlighted the importance of a more similar recapitulation of BMMSCs culture condition to the TME, for disease modeling and drug screening studies. Further experiments, including providing dynamic interactions of MSCs with MM cells in direct or indirect co-culture systems, are required to better recapitulate TME conditions for MSC priming. Moreover, for mechanistic analysis, evaluation of the secreted factors and assessment of expressed transcripts and proteins are highly recommended for future studies.
Conclusion: In summary, U266-CM influenced the proliferation rate and the differentiation potential of BMMSCs.