Should hypoxia preconditioning become the standardized procedure for bone marrow MSCs preparation for clinical use?

This is the authors' abstract. We don't add key points for this paper.

In recent years, mesenchymal stem/stromal cells (MSCs), especially those isolated from bone marrow (BM-MSCs), have been utilized to treat a diverse range of diseases [1]. Many of the clinical results are encouraging although the clinical criteria for MSCs preparation have not been well-defined. In bone marrow, the niche of MSCs is thought to be immersed in a hypoxic microenvironment (low oxygen tension) [2]; it is thus hypothesized that hypoxia (1–5% O2), rather than normoxia (21% O2)—a classical condition during in vitro culture, can better preserve the biological behavior of MSCs from this tissue. In vitro cell expansion under hypoxic conditions has been shown to favor proliferation, viability, colony-forming efficiency and migration ability, although maintaining immunophenotype and cell cycle and enhancing the radio-resistance potential [3-8]. Importantly, hypoxia preconditioning for 48 hours was demonstrated to enhance the survival and retention of MSCs in immune deficient mice after intramuscular injection [9]. Nevertheless, before considering hypoxia preconditioning as a standardized BM-MSCs preparation procedure, one important question that remains to be answered is the effect of hypoxia on the genomic stability of BM-MSCs. Genomic instability may lead to malignant transformation, thus significantly impacting the safety of BM-MSCs after long-term expansion. In a recent paper published in this journal by Bigot and coauthors, hypoxia was reported to preserve the genomic stability of BM-MSCs after long-term culture in platelet lysate (PL) [10]. The authors found that mimicking the niche microenvironment with hypoxia could promote the DNA damage response, DNA repair and mitochondrial DNA stability of BM-MSCs cultured with PL; in comparison, adipose-derived stromal cells (ADSCs) cultured in the same conditions possessed lower DNA damage response and DNA repair ability. There is substantial evidence indicating that the oxygen tension of bone marrow and adipose tissue is considerably lower than 21% O2 [2, 11]. Nevertheless, the severe hypoxia (1% O2) used in the study may not accurately reflect the oxygen tension in the bone marrow and in the adipose tissue. In mice, the lowest oxygen concentration in bone marrow is much closer to the 1% O2 used in this study, whereas that in adipose tissue is equivalent to 6.5% O2 [2, 12]. The difference in oxygen tension in the stromal cell niche microenvironment may result in a different response of cells to 1% O2 (including genomic stability). In addition, different PL concentrations were selected for BM-MSCs and ADSCs culture systems, which may lead to experimental bias, but at the same time warranting further investigations on the use of different culture protocols required for MSCs isolated from different sources. However, except for the issues discussed above, the authors have provided significant insights into role of hypoxia in MSCs behavior, which may in turn reveal a reliable tool to enhance the clinical safety and effectiveness of MSC-based therapy. This work leaves us with an important question: are we ready to perform hypoxia preconditioning in the standardized protocol of MSCs preparation for clinical applications? Improved experimental outcomes have been observed in the treatment of some diseases in animals by hypoxia-cultured MSCs compared with those cultured in normoxia [4, 7]; but according to the database of clinicaltrials.gov, this procedure has been considered only in a handful of clinical studies, such as pulmonary emphysema (NCT01849159) and ischemic limb disease (NCT02336646), in the attempt to (re-)educate cells to survive in an oxygen-deprived environment/tissue. There are a number of possible reasons that may explain this phenomenon. First, hypoxia is good for MSCs expansion, but to some degree it also negatively affects differentiation ability, which is a potential mechanism for tissue regeneration. Second, the precise oxygen tension that enables/promotes effectiveness and safety of MSCs in the targeted tissue microenvironment needs further investigation. The third concern is identifying the suitable culture time in hypoxia as prolonged hypoxia has been reported to alter the MSCs secretome and oxidative metabolism [13, 14], therefore, the effective culture time in hypoxia needs to be optimized; cryopreservation has been demonstrated not to alter the main characteristics of Good Manufacturing Practice (GMP)-grade MSCs [15], but the question remains as to which stage during the expansion of allogenic MSCs is best for hypoxic preconditioning, that is, prior to cryopreservation or after thawing. Furthermore, the culture system used for MSCs should also be considered, taking into account the level of culture medium, the coating methods and the scaffolds, all of which may affect the hypoxia benefit. Last but not least, the influence of hypoxia in the paracrine action of MSCs should be investigated, as the paracrine function, especially that performed via microvesicles or exosomes, has been found to play a very promising role in MSC-based regeneration [16, 17], whilst hypoxia, combined with serum deprivation, has been shown to enhance the release of extracellular vesicles when compared to standard culture conditions (10% FBS and normoxia), hypoxia, or serum-free conditions alone, and importantly is able to promote angiogenesis in an ischemic heart [18]. In conclusion, the study of Bigot and colleagues [10] is relevant not only for showing the genomic stability of BM-MSCs under hypoxia, but also raises an essential issue for further discussion and investigations to consider the possibility of adding hypoxia preconditioning to the standardized protocol of MSCs expansion for clinical use. Hypoxia preconditioning is beneficial to some extent, but further investigations are required to standardize the protocol by basic scientific researchers and clinicians; importantly, a gathering of experts led by regulatory agencies, academic societies or scientific journals in the field of MSC-based therapy is encouraged to optimize the use of hypoxia preconditioning for MSCs expansion, as MSC-based therapy in clinics is increasing significantly worldwide. This work was financially supported by National Natural Science Foundation of China (No.:31271049 & 31570970) and by Shenzhen Science and Technology Innovation Committee Projects (No.:JCYJ20130402113615042 & JCYJ20150403091443318). The authors are very grateful to the five reviewers for their excellent suggestions to improve this manuscript. Y.C.H. and O.P.: conception, design and manuscript writing; L.D. and B.S.Y.: conception, design and final approval of manuscript. The authors indicate no potential conflicts of interest.