Comparative characterization of frozen-thawed CD146+ and CD146- subsets of CD73+CD90+CD105+CD34+ human ASCs

Abstract

Background: Mesenchymal stem cells are currently used to treat several diseases. Populations of putative stem cells found in the adipose tissue (ASCs) have been shown to possess particularly enhanced functionalities. Nonetheless, there is lack of evidence that evaluates the effects of cryopreservation techniques on well-defined functional ASC populations characterized by immunophenotypical repertoire.

Objective: We therefore embarked a study to compare the frozen-thawed ASC subsets: CD73+CD90+CD105+CD34+CD146-(CD34+CD146), CD73+CD90+CD105+CD34+CD146+(CD34+CD146+), and CD73+CD90+CD105+CD34+(CD34+). We assessed their characterization in different functional assays.

Method: The ASC immunophenotypical subsets-purified by a flow cytometry sorting technique-were frozen in liquid nitrogen. After a period, they were thawed to examine their differentiation ability, colony-forming units, viability, and growth rate.

Results: We confirmed that inside the primary cell culture system, the proportion of CD34+, CD34+CD146-, and CD34+CD146+ took up 80%, 62%, and 19% on average, respectively. All populations could be frozen and stored in liquid nitrogen with retention of more than 85% of cell viability and displayed comparable stemness characteristics. Most importantly, the CD34+CD146+ subpopulation displayed a higher proliferation rate than other groups.

Conclusion: Our data demonstrated that the frozen-thawed CD34+CD146+ cells might represent a promising source for autologous cellular-based therapy. These findings set the basis for ASC subpopulations-based application in future potential clinical settings.

Keywords: Adipose-derived mesenchymal stem cells; Heterogeneous; Proangiogenesis; Regenerative medicine; Subpopulation.

Figure 1:. Experimental scheme. A) ASCs were harvested from 5 donors and proceed to the primary culture. B) CD73+CD90+CD90+CD34+, CD73+CD90+CD90+CD34+CD146-, and CD73+CD90+CD90+CD34+CD146+ were isolated by FACS. C) After culturing the sorted cells for recovery from the mechanical stress, the resulting cell populations were frozen and stored in liquid nitrogen until use. D) The post-thawed cells were evaluated by an assay of potency tests. CD, cluster of differentiation; CFU, colony-formation unit; FACS, fluorescence-activating cell sorting.

Figure 2:. Gate strategies and the phenotype characterization of ASC. A) basic gate strategies were used to select the stable and live singlets. The analyzed cell sample was further separated by virtue of the expression of CD34, CD146 and CD34. Representative gating strategies were shown for the three cell populations, CD34+, CD34+CD146-, and CD34+CD146+. FMO controls and Boolean gates were employed to determine the boundary. B) The proportion of each subsets derived from 5 independent donors. Data were displayed as mean + SD. C) flow cytometric profiles of ASC single surface markers obtained from 5 donors.

Figure 3:. Stem cell properties and viability of the phenotypical selected ASC subpopulations. A) Histochemical analysis of the tri-lineage differentiation using Oil Red O, Alizarin Red, and Alcian blue for adipo-, osteo-, and chondrogenesis, respectively. Magnification: 200 X. Scale bar denotes 1mm.B) The colony-forming capacity was assessed from 5 independent subjects. C) The percent of live cells was conducted by the staining with viability dye and analyzed by flow cytometry. The data are presented as mean + SD. CFU: colony-formation unit assay.

Figure 4:. Growth rates of the selected cell populations. A) Cell confluency was visualized by the light microscope on day 8. Magnification: 100 X, Scale bar denotes 1mm. B) The quantitative measurement of cell growth rate(n=5) for each group. The results are shown as mean + SD. *, indicates a statistical significance.