The Effects of Hypoxia on U 937 Cell Line in Mesenchymal Stem Cells Co-Culture System

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(4), 645-650 doi: 10.15171/apb.2016.079 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
As well documented, Bone marrow (BM) traditionally contains two systems: hematopoietic cells and the associated supporting stromal part. 1 One of the major sections of BM milieu is Mesenchymal Stem Cells (MSCs). 2,3][6][7][8] Another important factor in BM milieu is physiologic hypoxia.The effects of hypoxia mediated by a significant master key transcription factor is called hypoxia-inducible factor (HIF).HIF, hetrodimeric key transcription factor, contains HIF-α and HIF-β subunits. 9In hypoxia, HIF-α subunits translocate to nucleus and join to HIF-β subunits, 10,11 so heterodimers bind to sequences of HIF target genes, which they affect different aspects of cells biology. 12,13][16][17] Several in vitro studies have been reported that HIF is a powerful factor, which improved survival and differentiation of stem cells. 18,19In particular, HIF-1 caused resistance to chemotherapy and radiation approaches. 20re, we investigated the effects of Umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) on proliferation rate, cell death and some genes expression by U937 cells in hypoxia milieu.

Isolation and Culture of UCB-MSCs
UCB-MSCs were collected from umbilical cords, with informed consent, according to the Institute's human ethical committee guidelines of Tabriz University of Medical Sciences.Cells were cultured in DMEM medium (Gibco, MA, UK) with 10% fetal bovine serum (FBS) (Gibco, MA, UK) and 100 U/ml penicillin as well as 100μg/ml streptomycin (Pen/Strep) (Gibco, MA, UK).Cells were incubated in humidified incubator containing 5% CO 2 at 37°C.After incubation, nonadherent cells were discarded and fresh DMEM medium was added to cells.Then, fibroblastoid cells were verified by flowcytometry for MSCs markers including CD29, CD105 (Positive markers) and CD34, CD45 (Negative markers). 21ell culture Confirmed U937 cells were purchased from the Pasture Institute of Iran.Thereafter, cells were cultured in RPMI-1640 medium (Sigma-Aldrich, USA) with 10% FBS (Gibco, MA, UK) and Pen/Strep (Gibco, UK) and were incubated.During all steps of the experiments, cell viability was checked by trypan-blue staining and it was more than 86% in all experiments.UCB-MSCs were seeded at the density of 2×10 4 cell/well.After 24 hrs, 1×10 5 U937 cells were added to the UCB-MSCs in RPMI-1640 medium with 10% FBS and Pen/Strep for Co-culturing.

Cells treatment
Cobalt chloride (CoCl 2 ) (Sigma, USA) was used to induce hypoxia.CoCl 2 dissolved in RPMI-1640 to adjusted 100 μM.Then U937 cells were treated with 100 μM of CoCl 2 .Hydrogen peroxide (H 2 O 2 ) (Merck, Germany) was used for cell death inducing, so H 2 O 2 was diluted to 100 mM with distillated water as a stock solution and cells were treated with 0.5 mM H 2 O 2 .

MTT Assay
U937 cells were co-cultured with UCB-MSCs and were incubated with 100 μM of CoCl 2 for 96 hrs.After incubation, U937 cells were collected and RPMI-1640, with 5μl MTT solution (0.4 mg/ml), was added to cells pellet and was incubated.Then, Isopropanol/HCl (0.04M) was added and incubated overnight and optical density of solutions were measured by Picodrop (UK) in 540 and 650 nm.

Cell Death Detection with Flowcytometry
U937 cells were incubated with 0.5mM of H 2 O 2 for 24 hrs.Then, live and dead cells were analyzed by forward and side scattering in flowcytometry assay (FACS Calibor, USA). 22,23

Statistical Analysis
Data are shown as means ± SD from three separate experiments.Data were evaluated using GraphPad Prism v 5.00 (GraphPad Software, Inc., La Jolla, CA).Student's t-test (for single comparison) was used.P < 0.05 was regarded statistically significant.

Results and Discussion
In normoxia and hypoxia proliferation of U937 was promoted by UCB-MSCs MTT assay showed that proliferation of U937 was significantly high in co-culture with UCB-MSCs, in normoxic and hypoxic conditions (*P<0.05)(Figure 1 and 2).In this regard, several studies have proved that, MSCs play noticeable roles in proliferation of malignant cells models such as U937 cells.Also, hypoxia shows suppressing effects on proliferation of leukemic cells. 24,25Here, we proved that hypoxia reduces proliferation of U937 cells, but in presence of UCB-MSCs, effects of hypoxia have been suppressed.

Expression of CD49d down regulated in co-cultured U937 cells
U937 cells were lied down on UCB-MSCs and were treated with 100 μM CoCl 2 for 24 hrs.Then, RNAs of U937 cells were extracted, cDNAs were synthesized and RT-PCR was performed.
CD49d is a part of VLA-4 and expresses on monocytes.Naturally, CD49d interacts with VCAM-1 to help migration of monocytes. 27Hypoxia lead to CD49d upregulation, which lead to transmigration of monocytes. 28,29Contrary, our findings showed, CD49d expression was down-regulated in presence of UCB-MSCs (Figure 5) (*P<0.05).

Hypoxia up-regulated CD116 expression
We seeded U937 cells on UCB-MSCs.In the next step, the U937 cells were treated with 100μM CoCl 2 and incubated for 24 hrs.Then, we performed RT-PCR on co-cultured U937 cells.CD116 is receptor of GM-CSF and expresses on various myeloid cells. 30Furthermore, GM-CSF/CD116 has a direct role in proliferation and survival of monocyte lineage. 31,32As shown in Figure 6, CD116 expression shows meaningful up-regulation in hypoxia (*P<0.05).CD11a, CD54 and CD14 status in hypoxia U937 cells were cultured with UCB-MSCs, C.M and 100 μM CoCl 2 for 24 hrs.RT-PCR performed and didn't show any significant changes in CD11a, CD54 and CD14 markers expression (P>0.05)(Figure 7).CD14 has a role in the phagocytic activity of monocytes 33 and CD11a is classical marker of myeloid differentiation that involves in cell adhesion.Additionally, CD11a and CD14 related to differentiation of U937 cells to monocyte/macrophage. 34,35 CD54 up-regulation in high pressure of oxygen in endothelial cells documented by recent studies, 36 while it should be pointed out that hypoxia enhanced CD54 expression, presumably through NF-kB pathway activation, 37 but in our finding CD54 didn't show any trends in all conditions.Combined with previous findings this discrepancy can be due to differences in cell lines and incubation times.

Conclusion
In orchestrate with previous studies, hypoxia suppresses the U937 proliferation in comparison to normoxia.Reciprocally, when U937 co-cultured with the UCB-MSCs, proliferation of U937 was increased.Apoptotic effects of H 2 O 2 on U937 cells were reduced in presence of the UCB-MSCs.Also, CD49d expression was downregulated and CD116 was up-regulated in the presence of UCB-MSCs and hypoxia respectively.Our findings can be useful in clinical applications and provide a new sight into the roles of MSCs in bone marrow cotransplantation efficacy.We suggest that other experiments should be performing on bone marrowderived MSCs or animal models.

Table 1 .
Primers sequences used for RT-PCR