Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability,motility, and tropism to brain cancer

Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM.Mesenchymal stem cells (MSCs) are multipotent cells, isolated from the bone marrow, adipose tissue, and muscle, among others. They are clonally expansive, with the capacity to differentiate into osteocytes, adipocytes, and chondrocytes.^{1, 2} MSCs are widely studied for their regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer therapeutic potential.^{3, 4, 5} MSCs can serve as vehicles for delivering effective targeted therapy to primary brain cancer and metastatic cancer.^{6, 7, 8}Notwithstanding aggressive treatment of primary brain cancer (glioblastoma (GBM)) with surgical resection, chemotherapy, and radiotherapy, the median survival following diagnosis is 14.6 months.^{9, 10, 11, 12, 13, 14, 15} GBM-targeted therapy using neural stem cells and MSCs as vehicles for therapeutic agents is a promising strategy.¹⁶ MSCs seem to be the ideal stem cells, as they are autologous, easily collected, and easily re-implanted.^{17, 18} The most commonly used MSCs are bone marrow-derived MSCs (BM-MSCs) and human adipose-derived MSCs (hAMSCs). Compared with BM-MSCs, hAMSCs are easier to obtain.^{19, 20}Despite the potential utility of hAMSCs, their use is hampered by their low concentration within tissues.^{21, 22} Thus, in vitro expansion of hAMSCs is necessary. Compared with BM-MSCs, hAMSCs are more genetically and morphologically stable in long-term culture.^{19, 20} Nevertheless, current culturing conditions for both BM-MSCs and hAMSCs show a progressive decrease in viability and proliferative ability, and an increase in senescence ratio for these stem cells with time.^{23, 24, 25, 26, 27, 28, 29} Typically, hAMSCs are cultured under ambient conditions with 21% oxygen in vitro.³⁰ However, physiologically, hAMSCs exist at much lower oxygen tensions, between 1 and 14%.^{31, 32} As a result of the limitations of culturing under normoxia, we investigated the effect of hypoxia on intraoperatively obtained hAMSCs by assessing proliferation, survival, differentiation, tumor formation, tumor tropism, and migration in vitro and in vivo in a rodent model with a human brain cancer. hAMSCs have been reported to transform into tumor-associated fibroblasts (TAFs), which can potentially support tumor growth and promote malignant phenotypes.^{33, 34} Yet, no studies have reported on the changes that may occur in hypoxia-cultured hAMSCs after they are exposed to brain cancer, both in vitro and in vivo. An understanding of the effects of hypoxia on hAMSCs³⁵ is critical for its potential therapeutic applications including in the treatment of brain tumors, stroke, neuro-degenerative diseases such as multiple sclerosis, and dementia (Figure 1a).Open in a separate windowFigure 1Primary human adipose-derived cells cultured in hypoxia (hAMSCs-H) and normoxia (hAMSCs-N) are both MSCs but normoxia-cultured cells show increased signs of senescence, such as increased area and elongated morphology, compared with hypoxia-cultured cells. (a) hAMSCs were isolated from human fat tissue and cultured in hypoxic (1.5% oxygen) or normoxic (21% oxygen) conditions in vitro. The viability, mobility, tumor tropism, safety, and tumorigenic potential were subsequently compared in vitro and in vivo. (b) Differentiation assay. hAMSCs were cultured in control media and differentiation media for 3 weeks, 10 days after the second passage. Three different stains were performed to assess differentiation capabilities (scale bar, 100 μm). (c) Flow cytometric analysis was performed to confirm the absence of CD31-, CD34-, and CD45-positive cells in both cell cultures. In addition, primary hAMSC cultures expressed high levels of CD73, CD90, and CD105, both in hypoxic and normoxic culture conditions at day 10 after passage 2. (d) Representative images of cell morphologies of hAMSCs on 2D surface (scale bar, 200 μm). (e) Schematic of 3D-nanopatterned surface used to assess morphology and motility. (f) Images of cell morphologies of hAMSCs on 3D-nanopatterned surface (scale bar, 200 μm). (g–j) The length, width, area, and length-to-width ratio were measured and compared after cell aligned on the nanopattern surface. Error bars represent S.E.M. *P<0.05, **P<0.01, N.S., not significant