| Background/AimsAdult stem cells are undifferentiated cells that can be easily isolated from infants, children, and adults. They have the ability to divide indefinitely and produce different kinds of cells that maintain the body’s tissues and organs. Because the production of adult stem cells does not require the destruction of an embryo, the use of adult stem cells in research and therapy is not ethically controversial, which paves a new way for the treatment of major human diseases such as cancer. As one of the representatives of adult stem cells and ideal therapeutic agents for the cell-based therapies, mesenchymal stem cells (MSCs) have been applied in basic research and clinical treatment of various malignant diseases. These cells are of stromal origin and may differentiate into a variety of mesenchymal lineage cells, which makes them applicable for damaged tissue repair or regenerative medicine. Besides, MSCs are capable of suppressing the growth of certain tumors, migrating into tumors and chronic inflammatory sites, amenable to genetic modification, and possess a profound immunosuppressive activity as well as hematopoiesis-supportive function. Such versatile properties have prompted their applications in the treatment of solid tumors and leukemia as gene delivery vehicle and immunosuppressant, respectively. Correspondingly, there have also emerged a great deal of interest in the direct interaction between MSCs and cancer cells over the past decade. However, the existing data on the functions of MSCs in cancer development remain controversial and further evaluation is greatly needed.It has been reported that MSCs can be isolated from a great variety of tissues, including bone marrow, umbilical cord, placenta, adipose tissue, etc. Compared with bone marrow-derived MSCs (BM-MSCs), human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have several advantages such as better expandability, painless collection procedures, and lower risk of viral contamination. These advantages enable them to be excellent sources for the hematopoietic and non-hematopoietic cancer treatments. In spite of this, little is known about the actual effects or the direct impact of hUC-MSCs on specific cancer cells.In the present study, hUC-MSCs were prepared and evaluated for their fundamental MSC characteristics. Specifically, the immunomodulatory properties of hUC-MSCs were also verified. Thereafter, they were used to treat cancer cells of hematopoietic and non-hematopoietic origins, followed by the investigation of their influence on the malignant phenotype of these cancer cells as well as the underlying mechanisms. Overall, the findings in this study enrich the knowledge about umbilical cord-derived MSCs and provide some useful clues for the safe development of MSCs as therapeutic tools.MethodsHuman UC-MSC cultures were established from umbilical cords of healthy donors by plastic adherence method. The resulted cells were further expanded and assessed for their morphology, proliferating ability by MTT assay, immunophenotype by flow cytometry, and triple differentiation potentials. The immunomodulatory abilities of hUC-MSCs were determined by evaluating their impacts on lymphocyte activation and T cell subpopulations.For the observation of the effects of hUC-MSCs on solid tumor cells, human lung cancer cell line A549and human hepatocarcinoma cell line BEL7402were, respectively, cultured in the mixture of tumor cell culture medium and hUC-MSC supernatant at different proportions. As controls, tumor cells were grew in the mixture of tumor cell culture medium and hUC-MSC growth medium not containing FBS. After cultured for72h, tumor cells per well were collected and detected by flow cytometry for cell cycle distribution and cell apoptosis. Tumor migration ability was assessed by using the Transwell chambers. The transcription levels of tumor dormancy-associated genes including EphA5, AMOT, OPN and CD73in either tumor cells were evaluated by RT-qPCR, and the expression of EphA5, Bcl-2, Caspase-7, β-Catenin and c-Myc at protein level were detected by Western blotting analysis. On the other hand, the co-culture experiments were performed by direct contact of DiD-labeled hUC-MSCs and DiO-labeled A549or BEL7402tumor cells, followed by confocal observation-and flow cytometry assay.For the observation of the effect of hUC-MSCs on leukemia cells, human T cell leukemia cell line Jurkat or human promyelocytic leukemia cell line HL-60were cultured in direct contact with hUC-MSCs. In some experiments, hUC-MSCs were equipped with human IDO gene via the aid of adenovirus expression system before they were used as stromal cells for Jurkat and HL-60leukemia cells. Spontaneous or drug-induced apoptosis in leukemia cells was detected by flow cytometry after cells were triply stained with CD45-APC/AnnexinV-FITC/PI. Leukemia cell proliferation was examined by CFSE dilution assay. Specifically, expression of Notch pathway-related molecules in hUC-MSCs and Jurkat cell line was confirmed by RT-PCR, immunofluorescence staining and flow cytometry, respectively. Blocking of Jagged1with neutralizing antibody and extra stimulation of Notch signaling by recombinant Jagged1were performed to identify the role of Jagged1in the hUC-MSC-induced drug resistance of Jurkat leukemia cell line.ResultsFibroblast-like cells were successfully isolated from human umbilical cord tissues using the direct plastic adherence method. The cells formed whirlpool-like arrays when a confluent monolayer had developed. The flow cytometry analysis demonstrated that the hUC-MSCs showed good homogeneity and expressed MSC markers CD73, CD90, CD105, CD44and CD29, but were negative for CD34, CD45, HLA-DR and CD14. The same cells showed multilineage differentiation potential, as assessed by culturing in adipogenic, osteogenic or chondrogenic medium. They were able to suppress the production of IFN-y by OKT3-activated PBMC, promote the differentiation CD4+CD25+CD127low Treg cells and down-regulate the ratio of CD4+T cells versus CD8+T cells.The hUC-MSCs inhibited in vitro migration of A549and BEL7402tumor cells, arrested these tumor cells in the Go/G1or S phase of the cell cycle, and induced their apoptosis independently of direct cell contact. RT-qPCR analysis indicated that the hUC-MSCs elevated the transcription of EphA5gene in these tumor cells. Western blotting data showed that the expressions of Bcl-2, pro-form Caspase-7, β-Catenin and c-Myc were all downregulated in the tumor cells treated with the hUC-MSC conditioned medium. By direct contact with tumor cells, the hUC-MSCs surrounded and then invaded into tumor cell clones, resulting in the loosening of the tumor clones. Of note, cell fusion between hUC-MSCs and tumor cells occurred spontaneously during co-culture.By contrast, hUC-MSCs were able to maintain the viability of the Jurkat or HL-60leukemia cells by preventing them from apoptosis. Compared with control cells cultured alone, Jurkat cells that were cultured on the monolayer of hUC-MSCs showed an improved cell morphology and underwent far less apoptosis induced by dexamethasone. It was also observed that forced IDO expression in hUC-MSCs abolished the anti-apoptotic effect of hUC-MSCs on these leukemia cells and enhanced their leukemia-growth-inhibitory effect. Both hUC-MSCs and Jurkat cells expressed Jagged1. Furthermore, co-culture with hUC-MSCs led to a significant upregulation of Jagged1, Notch1and CD28in Jurkat cells, indicating that the Notch1/Jagged1pair may play a role in the autonomous or reciprocal activation of Notch signaling in these leukemia cells. Blocking Jagged1using the neutralizing antibody restored drug-induced apoptosis in the Jurkat cells that were co-cultured with hUC-MSCs, and also increased the drug sensitivity of the Jurkat cells that were cultured alone, while direct incubation with exogenously recombinant Jagged1 produced the same protective effects in Jurkat cells as those induced by hUC-MSCs.ConclusionsWe established a tissue adherence method-based protocol to isolate MSCs from human umbilical cords with stable yields. Both the fundamental MSC characteristics and immunomodulatory properties of these human umbilical cord-derived cells have been confirmed, ensuring their usability in further studies. Our findings revealed that the hUC-MSCs differently influenced cancer cells of hematopoietic and non-hematopoietic origin through multiple mechanisms. They produced contact-independent inhibition on growth, migration and survival of A549and BEL7402tumor cells, showing a potential to induce dormancy of these tumor cells. Tumor dormancy-associated gene EphA5and Wnt/β-Catenin pathway were involved in malignant phenotype suppression on the above mentioned tumor cells induced by hUC-MSC conditioned medium. In addition, the hUC-MSCs restrained the development of tumor colony and the mechanism might include cell fusion between hUC-MSCs and tumor cells. Specifically, we observed a dual impact of hUC-MSCs on leukemia cells. They inhibited the proliferation of Jurkat and HL-60leukemia cells and also prohibited their death. It could be inferred that such proliferation inhibition could confer leukemia cells a better survival because proliferating cells are more vulnerable to apoptotic stimuli. This mechanism would preserve the self-renewal ability of cancer cells and thus sustain the malignant process. The hUC-MSCs also induced drug resistance of Jurkat leukemia cell line. Jaggedl, one of the Notch ligands, contributes to the hUC-MSC-induced survival and the self-maintenance of Jurkat cells, making it a potential target for the treatment of human T cell leukemia. Interestingly, forced IDO expression in hUC-MSCs abolished their anti-apoptotic effect on leukemia cells of T cell origin and non-lymphoid origin, accompanied by the improvement of their abilities to inhibit leukemia cell growth. These results suggested that equipping MSCs with IDO could be one of the reasonable strategies to reverse their cancer-supportive effect unfavorable for clinical applications. Taken together, our data provide new insights into how hUC-MSCs modulate the malignant phenotype of different cancer cells in vitro and advance understanding of the role of MSCs in carcinogenesis. It established a suitable model to evaluate the interactions between hUC-MSCs and other types of cancer cells. Importantly, it shed light on clinical therapeutic trials using hUC-MSCs for lung cancer and hepatocarcinoma suppression, but also cautioned the potential risk when applying them in the context of hematological malignancies. |
