Improved survival of mesenchymal stromal cell after hypoxia preconditioning: role of oxidative stress

Abstract

Aims: To investigate the mechanisms underlying the beneficial effect of hypoxia preconditioning (HPC) on mesenchymal stromal cells (MSCs) and optimize novel non-invasive methods to assess the effect of biological interventions aimed to increased cell survival.

Main methods: MSCs from rat femur, with or without HPC, were exposed to hypoxic conditions in cell culture (1% O(2) for 24h) and cell survival (by the LDH release assay and Annexin-V staining) was measured. Oxidant status (conversion of dichloro-fluorescein-DCF- and dihydro-ethidium-DHE-, protein expression of oxidant enzymes) was characterized, together with the mobility pattern of cells under stress. Furthermore, cell survival was assessed non-invasively using state-of-the-art molecular imaging.

Key findings: Compared to controls, Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subunit 67(phox): 0.05 ± 0.01AU and 0.48 ± 0.02AU, respectively, p<0.05) and in the amount of ROS (DCF: 13 ±1 and 42 ± 3 RFU/μg protein, respectively, p<0.05) which led to a decrease in stem cell viability. Hypoxia preconditioning preserved cell biology, as evidenced by preservation of oxidant status (16 ± 1 RFU/μg protein, p<0.05 vs. hypoxia), and cell viability. Most importantly, the beneficial effect of HPC can be assessed non-invasively using molecular imaging.

Significance: HPC preserves cell viability and function, in part through preservation of oxidant status, and its effects can be assessed using state-of-the-art molecular imaging. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.

Fig. 1

A, representative histology of rat mesenchymal stromal cells (MSCs) over time showing how, with increasing passages, cell population becomes more purified and devoid of hematopoietic stem cells (HSCs) and macrophages (rounded cells in passage 1). B, Fluorescent Activated Cell Sorting (FACS) analysis of MSCs (at passage 5) that are CD45⁻ (thus not of hematopoietic origin), CD11b/c⁻ (thus not macrophages) and CD90⁺ and CD29⁺, as characteristic for MSCs. C, Double staining FACS analysis: left, all CD29⁺ cells were stained for CD90 and right, all CD90⁺ cells were stained for CD29.

Fig. 2

Cell survival was assessed by the LDH release assay (panel A) and the FACS staining for Annexin-V (panel B) under hypoxic conditions (1% O₂, 4% CO₂, 95%N for 24 h), in cells with and without hypoxic preconditioning (HPC). In panel C, cell viability was assessed (using the LDH assay) with longer exposure to hypoxia. Hypoxia resulted in decreased survival of MSCs, while HPC cells had increased survival compared to untreated cells. Longer hypoxic exposure resulted in further decreases in cell viability. *p<0.05 compared to control and Hypoxia + HPC.

Fig. 3

Assessment of oxidative stress. A, top, Representative fluorescence staining of the oxidative stress conversion of 2′,7′-dichlorodihydrofluorescein diacetate (DCHFDA); bottom, Fluorescence quantification of the presence of DCF (expressed as fluorescence/µg protein). B, top, Representative fluorescence staining of the oxidative stress conversion of Dihydroethidium (DHE); bottom, Quantification of the percent area where ethidium staining was present, normalized by the number of nuclei in each microscopic field analyzed. Hypoxia led to increased oxidative stress (increased ethidium and DCF) which was prevented by HPC. * p<0.05 compared to control, Hypoxia + HPC, and Hypoxia + HPC + catalase/superoxide dismutase (SOD).

Fig. 4

Assessment of oxidative stress. Protein expression bands and densitometric analysis of NAD(P)H subunits p67^phox and p47^phox, the endogenous scavenger enzymes catalase and manganese–SOD (Mn–SOD). Under hypoxic conditions, there was increased protein expression of NAD(P)H p67^phox and p47^phox and a decrease of the expression of catalase while the Mn–SOD remained unaltered. All of these resulted in an increase in the presence of reactive oxygen species suggesting a pro-oxidant state, what was prevented by HPC. *p<0.05 compared to control and HPC, ¥ p<0.05 compared to control and hypoxia.

Fig. 5

Survival and apoptosis. Protein expression bands and densitometric analysis of the survival genes survivin, phosphorylated Akt/total Akt (top), phospho/non-phospho ERK 1–2 and bcl-2/bax (middle), and p38MAPK and PI3K/PTEN (bottom). Under hypoxia, there was a decrease of survivin and a mild increase in the phosphorylated Akt/total Akt ratio, changes that were prevented by HPC. Hypoxia also decreased the ratio of ERK 1–2 and increased the protein expression of bcl-2/bax. Lastly, hypoxia modulated the expression of p38MAPK and PI3K/PTEN. *p<0.05 compared to control and Hypoxia + HPC, ¥ p<0.05 compared to control.

Fig. 6

Cell mobility. Top, representative mobility of MSCs under control (left), hypoxia (middle), and hypoxia + HPC (right). Bottom, quantification of the mobility of MSCs under these conditions. Under hypoxia, there is increased mobility of MSCs, which is prevented by HPC. Red square demarcates the area where cells were cleared at baseline. *p<0.05 compared to control and Hypoxia + HPC.

Fig. 7

Molecular imaging of cell survival in hypoxia. Cell survival assessment was based on reporter gene-light emission and detection using Bioluminescence Imaging and a charge coupled device camera. Hypoxia leads to a decrease in cell survival (assessed in ph/s/cm²/sr) and HPC preserved cell survival (in a prolonged hypoxic challenge) compared to untreated cells. These studies corroborated the results obtained using the LDH release assay (more traditional method for the assessment of cell viability) but most importantly, demonstrate that reporter genes can be used to assess cell survival in-vivo, underscoring the importance of translating these monitoring strategies to the living subject. *p<0.05 compared to control and hypoxia + HPC.