Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status

Abstract

BackgroundIn the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions.ResultsMorphologic studies, differentiation and transcriptional profiling experiments were performed on MSC cultured in normoxia (21% O2) versus hypoxia (5% O2) for up to passage 2. Cells at passage 0 and at passage 2 were compared, and those at passage 0 in hypoxia generated fewer and smaller colonies than in normoxia. In parallel, MSC displayed (>4 fold) inhibition of genes involved in DNA metabolism, cell cycle progression and chromosome cohesion whereas transcripts involved in adhesion and metabolism (CD93, ESAM, VWF, PLVAP, ANGPT2, LEP, TCF1) were stimulated. Compared to normoxic cells, hypoxic cells were morphologically undifferentiated and contained less mitochondrias. After this lag phase, cells at passage 2 in hypoxia outgrew the cells cultured in normoxia and displayed an enhanced expression of genes (4-60 fold) involved in extracellular matrix assembly (SMOC2), neural and muscle development (NOG, GPR56, SNTG2, LAMA) and epithelial development (DMKN). This group described herein for the first time was assigned by the Gene Ontology program to "plasticity".ConclusionThe duration of hypoxemia is a critical parameter in the differentiation capacity of MSC. Even in growth promoting conditions, hypoxia enhanced a genetic program that maintained the cells undifferentiated and multipotent. This condition may better reflect the in vivo gene signature of MSC, with potential implications in regenerative medicine.

Concise Review: Optimizing Expansion of Bone Marrow Mesenchymal Stem/Stromal Cells for Clinical Applications

“…MSC proliferation was enhanced by hypoxia in all studies [30,[33][34][35][36][37]. Primary MSCs cultured in hypoxia initially exhibited a lag phase in proliferation, after which they superseded primary MSC growth cultured in normoxia [28,29]. The increase in MSC number can be quite profound.…”

“…Since that observation, enhanced proliferation as a function of hypoxic culture has been observed in a diversity of cell types including pericytes [75], osteoblasts [76], hematopoietic cells [77], and MSCs [28][29][30][31][32][33][34][35][36][37]. MSCs reside in the bone marrow (4%-7% O 2 ) [78] with an oxygen tension approximately half that of arterial blood (8%-15% O 2 ) [79].…”

“…In essence, numerous aspects of the bone marrow niche that delicately regulate MSC behavior [66] are egregiously absent in 2D culture. As such, many endeavors to preserve MSC progenitor potency have focused on techniques to recreate characteristics of the elaborate niche through expansion amid growth factors [19][20][21][22][23][24][25][26], in hypoxia [27][28][29][30][31][32][33][34][35][36][37], and through 3D expansion [38][39][40][41][42][43][44][45][46]. …”

“…Robust proliferation, multilineage potential, and colony-forming efficiency (CFE) can be compromised by 2D culture [13][14][15][16][17][18]. By mimicking the niche, MSC progenitor potency has been enhanced amid growth factors [19][20][21][22][23][24][25][26], in reduced oxygen tension [27][28][29][30][31][32][33][34][35][36][37], and through alternative expansion in 3D [38][39][40][41][42][43][44][45][46]. Ample evidence over the past 30 years has demonstrated that the 2D paradigm compromises the potency of MSCs, yet these classically expanded MSCs are still regularly used in clinical trials.…”

Autophagy - the friendly fire in endothelial cell regeneration. Focus on “Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions”

“…Previous studies have shown that hypoxic conditions are beneficial to stem and progenitor cell growth and survival because the embryonic environment in which these cells are often found are typically under low oxygen tension (2,8). Additionally, the self-degradation of damaged intracellular components, or autophagy, has been shown to have a "protective" effect during times of cellular stress (7).…”