Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue

BMC Veterinary Research Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue Beatriz Ranera Ana Rosa Remacha Samuel lvarez-Arguedas Antonio Romero Francisco Jos Vzquez Pilar Zaragoza Inmaculada Martn-Burriel Clementina Rodellar Background: Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. Results: At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. Conclusions: Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs. Hypoxia; Horse; AT-MSC; BM-MSC; Characterisation - Background In recent years, mesenchymal stem cells (MSCs) have become increasingly utilised in regenerative medicine and tissue engineering applications because of their properties for self-renewal, differentiation and immunoregulation [1]. To study these properties, MSCs must be isolated from their physiological niches and cultured ex vivo. The micro-environment that cells experience in laboratory culture is very different from their native settings; therefore, it is possible that the true in vivo properties of * Correspondence: 1Laboratorio de Gentica Bioqumica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain 3Instituto Aragons de Ciencias de la Salud (IACS), Zaragoza 50009, Spain Full list of author information is available at the end of the article these cells might be modified by artificial culture. One environmental property that is commonly altered by the change of environment is the percentage of oxygen. Traditional incubators are supplied with atmospheric air that contains 20% oxygen (defined as normoxia), which is a not physiologically accurate for any kind of cell. Two common MSC sources are bone marrow and adipose tissue, in which the oxygen tension ranges from 1%-7% [2] and 2%-8% [3], respectively. All nucleated cells are able to sense and respond to the availability of O2 [4]. Rat MSCs modify the expression of molecules involved in cell proliferation and survival when they are exposed to low oxygen tensions that approximate physiological conditions [5]. Hypoxia inducible factor 1 (HIF-1) regulates the expression of many cell cycle molecules, including p21, anti-apoptotic factors, such as Bcl-2 [6], and pro-apoptotic proteins, such as p53 [7]. Consequently, rat MSCs exhibit different proliferation rates when cell expansion under hypoxia and normoxia are compared; however, some controversy exists regarding whether low oxygen tension enhances [8] or suppresses proliferation [9]. Additionally, oxygen plays an important role in the differentiation [10] and maintenance of stemness in MSCs [11]. Due to the inability of tendons and articulations to heal properly, MSC-based therapies have been utilised in horses to treat orthopaedic disorders resulting from sporting endeavours [12,13]. Oxygen levels in cartilage are among the lowest throughout the body [14], and hypoxia appears to be essential for tendon repair [15]. In addition, hypoxic preconditioning improves the therapeutic potential of human MSCs [16]. Taken together, these facts suggest that horse MSCs cultured in hypoxia might constitute a more relevant model for the treatment of injuries in low-oxygen tissues than those currently utilised, which are usually cultured in 20% O2. To improve the methodology for equine stem cell therapy, it is necessary to examine the characteristics and to compare the behaviour of MSCs in normoxic and hypoxic conditions. Specifically, this study contrasts the proliferation kinetics, viability, cell cycle progression, phenotype and stemness of MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) cultured in 5% and 20% O2. BM-MSCs exposed to both oxygen conditions showed similar lag phase (Figure 1A); however, the log phase lasted less in hypoxic BM-MSCs, until day 5, when they reached a growth plateau state, while normoxic BMMSCs continued growing slowing down their proliferation the last day of the culture period. Similarly to BM-MSCs, AT-MSCs at 5% and 20% O2 showed similar lag phase and the log phase ended before in hypoxic than in normoxic AT-MSCs, which went on the log phase until the end of the culture period (Figure 1B). Significantly higher number of AT-MSCs in normoxic cultures was detected on days 5 and 7. Cell cycle To examine the cell cycle progression under both oxygen conditions, cellular DNA content was quantified in the cultures used in the proliferation study for 7 days. Figure 2 shows the proportions of cells in each cell cycle phase observed in BM- and AT-MSCs expanded in normoxia and hypoxia. Cell cycle data obtained for BM-MSC cultures showed that normoxic cells were more active than hypoxic cells from day 2 (Table 1A). Significantly higher percentage of normoxic BM-MSCs was observed in S phase on days 2 and 4, and in G2/M phases on day 2. Supporting this finding, significantly higher proportion of hypoxic BMMSCs in G0/G1 phases was found on days 2, 3 and 4. However, hypoxic and normoxic AT-MSCs did not display any statistically significant difference over the course of the culture period (Table 1B). Comparing normoxic cultures of BM-MSCs and ATMSCs, BM-MSCs displayed a significantly higher percentage of cells in G0/G1 and reduced frequency of cells in S phase compared with AT-MSCs on days 2 and 3. The proportions of cells in each phase of the cell cycle were comparable throughout the remaining time course (Table 1C). Figure 1 Growth kinetic curves of equine MSCs at different oxygen concentrations. Growth kinetics of BM-MSCs (n = 6) (A) and AT-MSCs (n = 6) (B). The Y axis represents the number of cells, and the X axis represents the number of days in culture. Data are represented (...truncated)