Papel del óxido nítrico en la regulación de la respuesta a hipoxia y la función mitocondrial en células madre

  1. Estefanía Caballano Infantes
Supervised by:
  1. Francisco J. Bedoya Director
  2. J.R. Tejedo Director

Defence university: Universidad Pablo de Olavide

Year of defence: 2017

Committee:
  1. Bernat Soria Escoms Chair
  2. Ana Isabel Rojas González Secretary
  3. F. Javier Rivera-Guzmán Committee member

Type: Thesis

Teseo: 466553 DIALNET lock_openRIO editor

Abstract

Stem Cells (SCs) are non-specialized cells that present the capacity of self-renewal and differentiation into any cell type that are going to give place to the organism, which confers them the capacity to be used in cellular therapies for the treatment of numerous pathologies. Due to the vast number of applications that SCs could have in regenerative medicine and cell therapy, there is a growing interest in the knowledge of the mechanisms that regulate the self-renewal and plasticity of these cells. In addition, the control of pluripotency and cell differentiation in vitro culture, is of vital importance in the design of differentiation protocols towards specific cellular lineages for several clinical treatments. The expansion of pluripotent stem cells (PSCs) (embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)) under conditions that maintain their pluripotency is necessary to implement a cell therapy program. Previously, we have described that low nitric oxide (NO) donor diethylenetriamine/nitric oxide adduct (DETA-NO) added to the culture medium, promotes the expansion of these cell types. In addition, we have also found that low NO regulates the expression of genes involves in the control of survival, anaerobic metabolism, hypoxia response, etc. It has been reported that hypoxia prevents differentiation of human ESCs and is required to maintain a pluripotent state. Because of this, we proposed that low doses of NO in normoxia culture induce a similar hypoxia response in PSCs, thus improving the maintenance of pluripotency in culture. First, we have found that the treatment with low NO in normoxia displays stable levels of Hypoxia Inducible Factors (HIF-1α and HIF-2α) in PSCs. The expression of pluripotency markers as NANOG, SOX2 and OCT4 increases in cells cultured with low NO with respect to normoxia. The same pattern is observed in cells cultured fewer than 5% of oxygen. Because of the close relationship between hypoxia, metabolism, mitochondrial function and pluripotency regulation we have analyzed the role of NO on mitochondrial function and cell metabolism. We have observed that the expression of genes involved in glycolytic pathway as HK-2, LDHA, PDK1 and PKM2 is increased in cells cultured with low NO. In addition, VEGF-A expression, which is a transcriptional target of HIF-1α and HIF-2α, increases with low doses of NO. We further analyzed the role of NO on mitochondrial function and we have concluded that low NO reduces mitochondrial activity by a reduction in the mitochondrial potential membrane, low oxygen consumption and the increase in the expression of mitochondrial fusion regulators, as DRP1. Moreover, we observed that low NO does not increase oxygen reactive species levels. These results show that low NO mimic hypoxia response in PSCs. The knowledge of the role of NO in the Response to Hypoxia and the mechanisms that help to maintain self-renewal in pluripotent cells grown under normoxia, can help to the design of culture media where NO could be optimal for stem cell expansion in the performance of future cell therapies. Because of this, we have also focused on the validation of a serum-free medium previously formulated in our laboratory which is supplemented with low doses of NO for long-term culture of human mesenchymal stem cells (MSCs) derived from adipose tissue and derived from bone marrow. As a result of this study, a safer and more economical culture medium for MSCs is proposed