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. 2014 Dec 11;5(12):e1567.
doi: 10.1038/cddis.2014.521.

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

Y Feng  1 , M Zhu  2 , S Dangelmajer  3 , Y M Lee  3 , O Wijesekera  3 , C X Castellanos  3 , A Denduluri  3 , K L Chaichana  3 , Q Li  3 , H Zhang  4 , A Levchenko  5 , H Guerrero-Cazares  3 , A Quiñones-Hinojosa  3
Affiliations

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

Y Feng et al. Cell Death Dis. .

Erratum in

Abstract

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.

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Conflict of interest statement

The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Primary 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
Figure 2
Figure 2
Primary hAMSCs cultured in hypoxia grow faster, have higher viability, and can be passaged for more generations in vitro. (a) hAMSCs-H had shorter P1 and P2 passage times. The passage time of P0 was calculated once cells reached 80% confluence, after 2 g of fat tissue was digested and seeded at a density of 0.6 × 104/cm2 in hypoxic or normoxic conditions separately. The passage time of P1 and P2 was determined when 80% confluence was achieved. (b) Passage doubling time assay. hAMSCs-H had a constant PDT (<4 days) for up to 20 passages. PDT=τ Ln (2)/Ln (Nτ/N0), where τ=time from plating to counting the cells, Nτ=number of cells when counted and N0=initial number of cells. (c) MTT assay was used to determine the effects of hypoxia on the proliferative capacity of hAMSCs. (d and e) Ki-67 immunostaining was performed to quantify the number of proliferating cells. Representative images (scale bar, 100 μm) and proliferating percentage of hAMSCs are shown. (f and g) Flow cytometric apoptosis analysis for Annexin V–FITC-positive and PI-negative cells. Representative histograms and quantification are shown. (h and i) PI staining was used to test the cell cycle and necrosis percentage of the cells. Representative histograms and quantification are shown. Error bars represent S.E.M. *P<0.05, **P<0.01, N.S., not significant
Figure 3
Figure 3
Hypoxia-cultured primary human adipose-derived mesenchymal stem cells (hAMSCs-H) retain a greater proliferation capacity compared with normoxia-cultured primary hAMSCs (hAMSCs-N) when exposed to GBM media. hAMSCs-H maintain stem cell characteristics when exposed to GBM media. (a) Representative MRI of GBM from a patient. (b) Schema showing the collection of GBM CM and culture of hAMSCs in filtered GBM CM for proliferation and migration assays. (c) MTT assay was used to determine the effects of hypoxic conditions on the proliferative capacity of primary hAMSCs in GBM CM. In GBM CM, hAMSCs-H showed greater proliferation at day 10 and 15 compared with hAMSCs-N. (d) Ki-67 immunostaining was performed to quantify the number of proliferating cells in GBM CM. Proliferative capacities of hAMSCs-H and hAMSCs-N are shown in GBM CM (normalized to hAMSC-N proliferative capacity in control media). In GBM CM, hAMSCs-H had a greater proportion of proliferating cells than hAMSCs-N. (e) Differentiation assay. hAMSCs were cultured in control media, differentiation media, and GBM CM for 3 weeks, 10 days after the second passage. Three stainings were performed to assess the differentiation capabilities (scale bar, 100 μm). Both hAMSCs-N and hAMSCs-H maintained tri-lineage differentiation capability in GBM CM. (f) Flow cytometric analysis for CD31, CD34, CD45, CD73, CD90, and CD105 in hAMSC-N and hAMSC-H cultures after exposure to GBM CM for 20 days. hAMSCs-H maintained MSC phenotypic markers in GBM CM (negative for CD31, CD34, and CD45, and positive for CD73, CD90, and CD105). Error bars represent S.E.M. Error bars represent S.E.M. *P<0.05, **P<0.01, N.S., not significant
Figure 4
Figure 4
The hAMSC media promotes GBM differentiation in vitro. The proliferation and migration abilities of GBM remain unchanged in hAMSC media. In vivo, GBM cells exposed to primary hAMSCs-H show increased differentiation into astrocytic lineage (increased GFAP levels and decreased Nestin levels). (a) MTT assay was used to determine the effects of hAMSC media on the proliferative capacity of GBM. No difference in GBM proliferative capacity between hAMSC-H or hAMSC-N media and control media was observed. (b) Ki-67 immunostaining was performed to quantify the number of proliferating GBM cells in hAMSC media. No difference in proportion of proliferating GBM cells was found between control media, hAMSC-N CM, and hAMSC-H CM. (c) Immunostaining for Nestin, GFAP, and Tuj1 was performed on GBM cells in AMSC CM. (d–f) Quantification of Nestin, GFAP, and Tuj1 markers for GBM cells in hAMSC CM. GBM showed greater differentiation in hAMSC-H CM than in control media as shown by decreased nestin, increased GFAP, and increased Tuj1 expression. (g) Mice brain sections were immunostained for Nestin, GFAP, and Tuj1, to test the differentiation of GBM276 cells in vivo. GBM276 cells in mice injected with hAMSCs-H showed greater differentiation toward an astrocytic lineage compared with those in mice injected with hAMSCs-N. Scale bars, 100 μm. (h–j) Quantification of GFAP+/human nuclei+, Nestin+, GFAP+, and Tuj1+ cells
Figure 5
Figure 5
Primary hAMSCs are not tumorigenic and do not transform into TAFs in vitro or in vivo. (a) hAMSCs were cultured in GBM CM or control media for 2 weeks and western blottings (GAPDH served as a control) were performed to quantify TAF markers (vimentin and sm-actin). (b) Schematic of the experiment where PBS (n=6), hAMSCs-H (n=7), or hAMSCs-N (n=8) (both of hAMSCs groups labeled with GFP/Luciferase) were injected into mice. The mice (n=3 in each group) were separated and killed at day 10. Bioluminescence for the rest of mice in each group was checked on day 1, 7, 14, and 28, then killed at day 60. (c–f) Live animal imaging of hAMSCs. Bioluminescent radiance was maintained between day 1 and day 7 for hAMSCs-H (P=0.165), whereas a significant decrease occurred in hAMSCs-N (P<0.01). (g and h) Vimentin, sm-actin, GFP, and human nuclear stains for hypoxia and normoxia groups. No hAMSCs were seen at day 60 either in hAMSCs-N or hAMSCs-H group. No positive stains for hAMSC markers (GFP/Ki-67 with human nuclei) or TAF markers (vimentin and sm-actin) were observed on day 60. Error bars represent SEM. *P<0.05, **P<0.01, N.S., not significant
Figure 6
Figure 6
Hypoxia enhances migration ability of primary hAMSCs. (a) 2D-Scratch test. The plates with different groups of hAMSCs were scratched for 5 mm margin space and the borderline of the scratch was marked immediately. Then, the photographs were captured with bright-field microscopy at the endpoint of 24 h (scale bar, 200 μm). (b) Comparison of migration proportion between hAMSCs-H and hAMSCs-N in zones I, II, and III. (c) Representative tracks of hAMSCs-H and hAMSCs-N group. Cell migration was quantified using time-lapse microscopy on 3D nanopattern surface. Images were automatically recorded for 6 h at 10-min intervals. (d–f) Speed, persistence, and distance were assessed by tracking 75 cells in each group. Error bars represent S.E.M. *P<0.05, **P<0.01, N.S., not significant
Figure 7
Figure 7
The tropism of primary hAMSCs-H to GBM CM in vitro and GBM in vivo is increased. (a) Schematic of a Boyden chamber transwell assay. (b and c) Representative images and quantification graphs shown for a Boyden chamber transwell assay. hAMSCs (2 × 104) were seeded in the top well, while either GBM CM or control media was placed in the bottom well. After 24-h incubation, cells on the bottom were stained and quantified. (d) Schematic of the in vivo experiment. (e) Mice brain sections were immunostained for GFP and human nuclei to test the tropism of the primary hAMSCs for GBM276 tumor bulk in vivo. hAMSCs-H have enhanced tropism toward tumor bulk compared with hAMSCs-N. Scale bars, 100 μm. (f) Quantification of percentage of GFP+/human nuclei+ cells per field. Error bars represent S.E.M. *P<0.05, **P<0.01, N.S., not significant
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