A Comparison of Freshly-Isolated vs. Cryopreserved Adipose Stromal Cells with Respect to their Vascular Smooth Muscle Cell Differentiation Potential

Zhang, Xiaoqing 1, 4 ;  Simmons, Craig A 1, 2, 3, 4 ;  Santerre, J. Paul  1, 3, 4

1. Institute of Biomaterials and Biomedical Engineering; 2. Department of Mechanical and Industrial Engineering; 3. Faculty of Dentistry; 4. Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research,  University of Toronto, Toronto, ON, Canada

Introduction: Regeneration of functional vascular smooth muscle tissue requires access to a VSMC source that is effective, robust and safe. Previous studies have shown that multipotent adipose derived stromal cells (ASCs) are easy to harvest by liposuction and would be able to differentiate into VSMCs. However, there is a lack of direct knowledge related to cryopreserved ASCs vs. freshly isolated on their potential to differentiate into functional VSMCs. Such a direct comparison study will provide insight into the possibility of ASC banking for vascular tissue regeneration. 

Objective and Hypothesis: The objective of the current study was to investigate the effect of cryopreservation for ASCs on their differentiation into VSMCs. It was hypothesized that short-term cryopreservation will not have a negative effect on the ASCs' ability to differentiate towards a VSMC cell fate. 

Materials and Methods: ASCs were isolated from fat tissues donated by patients undergoing liposuction surgeries using collagenase digest solution (protocol #33176, UHN, Toronto) . Freshly isolated passage 5 ASCs were analyzed for ASC surface markers CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD14, CD31 and CD45 using flow cytometry. Both freshly isolated and cryopreserved passage 5 ASCs were differentiated towards VSMC-like cells with supplementation of VSMC differentiation factors: 5 ng/ml transforming grow factor ß1 (TGF-ß1) and 1 µM retinoic acid for 1 week. The expression of early VSMC markers (a-SMA and SM22a), middle VSMC markers (calponin and caldesmon) and late VSMC-specific markers (SM-MHC and smoothelin) were analyzed at the gene and protein levels with q-PCR and western blots respectively. Data were analyzed by independent samples t-test (SPSS 17.0). Statistical significance was reported for p < 0.05. 

Results: Based on flow cytometry data analysis, passage 5 ASCs expressed positive markersCD13 (99.83±0.29), CD29 (99.55±0.07), CD44 (99.68±0.45), CD73 (99.97±0.06), CD90 (98.97±0.50), CD105 (81.10±0.57) and CD166 (87.47±1.27), while lacked of negative markers CD14 (4.33±1.05), CD31 (0.26±0.06) and CD45 (0.99±0.18). The q-PCR and western blot data demonstrated similar expression of a-SMA, SM22a, calponin, caldesmon, and  SM-MHC genes and proteins of the VSMCs derived from fresh and cryopreserved ASCs. However, VSMCs derived from the cryopreserved ASCs (vs. fresh ASCs) showed decreased expression of late VSMC-specific marker smoothelin at gene and protein levels (p < 0.05). 

Significance: This direct comparison study of VSMC differentiation from fresh vs. cryopreserved ASCs will provide insight into the possibility of ASC banking for vascular tissue regeneration, and provide direction towards improved ASC cryopreservation methods required  in order to achieve ASC banking for vascular tissue engineering.