Serum Deprivation Blunts the Angiogenic Response to Hypoxia in Human Adipose Stem Cells

Introduction:  

      Reconstruction of large soft tissue defects remains a challenge to surgeons. Tissue engineering of thick tissues is limited by the lack of inherent vascularity of the graft. Also chronic wound beds are ischemic and hypoxic further compromising the oxygenation of the graft. Human adipose stem cells (hASCs) are proving to be the ideal cells for soft tissue engineering because of their availability, viability and plasticity. A tissue engineering approach that uses implantable hASC-seeded natural or synthetic polymer scaffolds should result in a living soft tissue substitute, which could overcome current disadvantages of autograft and allogenic volume substitutes.

     However, despite the fact that hASCs have a lower oxygen consumption and higher tolerance for survival than mature fat cells the major disadvantage of cell-based strategies is that the pre-cultured tissue constructs, which must become vascularized once implanted within the recipient, may not be as successful as methods which foster a primary neovascularization of a biological construct. Previous studies have demonstrated that prolonged hypoxic preconditioning enhances the angiogenic potential of hASCs^{1, 2}. However since most wound beds are hypoxic and ischemic we propose to study gene expression in the setting of serum deprivation (SD) along with hypoxia.

Methods:

      Isolated hASCs were plated in 60mm petri dishes and cultured in conditions of normoxia, hypoxia (1% oxygen and 10% serum) and hypoxia and SD (1% oxygen and 0% serum).Total RNA isolation was performed at 0, 12 and 24 hrs. 5 samples were collected from each experiment including 0 hour normoxia, 12 hours hypoxia, 24 hours hypoxia and 24 hours of hypoxia and SD. The experiment was repeated three times. Gene expression profiling was conducted using the Human Angiogenesis RT˛ Profiler™ PCR Array from Sabiosciences. Relative gene expression was determined using the ΔΔCT method.

Results:

     CXCL6, ANGLT4, ANPEP and S1PR1 were found to be up regulated in the setting of hypoxia alone with a maximum response at 24 hours (Figure 1).This response was blunted in the setting of setting of SD along with hypoxia.

Conclusion:

     Serum Deprivation blunts the angiogenic response of hASCs in the setting of hypoxia. This could comprise cell viability and proliferation leading to loss of the graft. Hence further studies are needed to improve primary neovascularization of the biological scaffold to enhance cell survival.

Figure 1: