Cryopreserved H₂ O₂ -preconditioned human adipose-derived stem cells exhibit fast post-thaw recovery and enhanced bioactivity against oxidative stress

Abstract

Human adipose-derived stem cells (ASCs), despite being one of the most attractive cell populations for tissue engineering and regenerative medicine, currently have certain limitations that reduce their therapeutic efficacy. One of the most serious problems is the poor engraftment of cryopreserved ASCs at injured tissue, attributed to the diminished biological activity of ASCs immediately post-thaw and their poor survival under harsh conditions of oxidative stress. Seeking to address these issues, we have developed a hormetic strategy to preadapt human ASCs to oxidative stress based on a new hydrogen peroxide preconditioning procedure, resulting in cells we call HC016. These cells rapidly recover their biological activity and functionality after cryopreservation while maintaining their mesenchymal stem cell status. Compared with non-preconditioned ASCs, HC016 cells showed (a) faster in vitro adhesion capacity and cell cycle progression immediately post-thaw, (b) enhanced cell survival under oxidative stress in a serum-free environment, and (c) heightened chemotaxis towards damage signals of oxidized glial cells. In addition, compared with ASCconditioned medium, HC016-conditioned medium showed a greater cytoprotective and pro-recovery effect on oxidized fibroblasts under serum-free conditions. Consistent with these results, in HC016 cells exposed to oxidative stress, we observed markedly higher expression of insulin-like growth factor-1 (a key factor in cell survival and migration) and of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 and pyruvate dehydrogenase kinase isozyme 1 (essential enzymes to upregulate glycolysis and downregulate oxidative phosphorylation) along with lower basal mitochondrial activity. Taking into account all the aforementioned advantages, HC016 cells might be considered an important breakthrough in ASC-based cell therapies.

Cell Secretome: Basic Insights and Therapeutic Opportunities for CNS Disorders

“…Preconditioning the cells with other molecular stimuli also seems to modulate the secretome composition. As in the case of hydrogen peroxide (H 2 O 2 ), when it is provided to ASCs in culture, their secretome shows a better capability to promote cytoprotective and pro-recovery effects on fibroblasts under oxidative stress when compared to ASCs in regular culture [52]. In a case of skin flap, only the exosome fraction was used and it was observed that pre-stimulation with H 2 O 2 promoted survival and angiogenesis while reducing inflammation when compared with normal ASC secretome [53].…”

Efficacy of human HC016 cell transplants on neuroprotection and functional recovery in a rat model of acute spinal cord injury

“…Studies performed by Histocell S.L. showed that hHC016 cells, after being exposed to this condition of mild oxidative stress, exhibit enhanced bioactivity and increased gene expression of key factors on survival, migration, and enzymes involved in reactive oxygen species metabolism (Castro et al, ).…”

“…All donors signed a free informed consent, and all procedures were approved by the Ethics Committee of the Basque Country Public Health System. Cell isolation and cultured was performed as previously described (Castro et al, ).…”

“…For details, see the Supporting Information. Once GFP‐tagged and in order to guarantee that the benefits obtained after transplantation derive of the HC016 treatment, the same batch of cells was used to prepare both the hAMSC and the hHC016 transplants, which were transplanted after 3/4 passages, as also described in detail by Castro et al ().…”

“…In this context, hHC016 cells (hydrogen peroxide (H 2 O 2 )‐preconditioned human AMSCs [hAMSCs]) were specifically developed by Histocell S.L. to obtain cells with improved resistance to oxidative stress as described (Castro et al, ). Hence, hHC016 cells are expected for improved survival of transplanted cells in pathological conditions as those encompassing acute SCI, and as a result higher competence to exert their therapeutic effects.…”