3.1. Cell Culture Alters the Transcriptional Profiles of Human Adipose-Derived Stem Cells
Utilizing a microfluidic-based single-cell gene expression platform previously described by our laboratory [20], the transcriptional profiles of 60–100 individual cells from fresh, P0, and P1 adipose-derived stem cells (ASCs) were simultaneously evaluated for 48 surface-marker coding gene (Table S1). In this analysis, ASCs isolated from all passages displayed significant heterogeneity at the single-cell level, both within and across groups (Figure 1).
Differences in the transcriptional profiles of genes related to cell stemness, proliferation, and tumor biology, such as the CD47, CD151, DPPA3, and IGF2R were also observed at the single cell level (Figure 2), suggesting a potential evolution of these properties throughout expansion in cell culture. These differences are embodied in both the fraction of cells expressing these genes, as well as the characteristics of their distributions across cell types.
Figure 1  Single cell transcriptional analysis of fresh, passage 0, and passage 1 adipose-derived stem cells. Hierarchical clustering of simultaneous gene expression for single cells from fresh (left; black), passage 0 (middle; dark grey), and passage 1 (right; light grey) human adipose-derived stem cells (ASCs). Gene expression is presented as fold change vs. median on a color scale from yellow (high expression, 32-fold above median) to blue (low expression, 32-fold below median). Cell/gene qPCR reactions failing to amplify after 40 cycles are designated as non-expressers and represented in grey.
Figure 2  Cell culture alters the transcriptional profile of adipose-derived stem cells. Differentially-expressed genes among primary, passage 0, and passage 1 cells identified using non-parametric two sample Kolmogorov–Smirnov testing. Twelve genes exhibit significantly different (p < 0.01 following Bonferroni correction for multiple comparisons) distributions of single cell expression between fresh→P0 or P0→P1 populations, illustrated here using median-centered Gaussian curve fits. The left bar for each panel represents the fraction of qPCR reactions that failed to amplify in each group. The curves above the dashed line represent transcriptional comparisons between primarily isolated cells versus cells at passage 0, whereas the curves below the dashed line represent comparisons between cells at passage 1 versus passage 0.   Furthermore, the top molecular networks associated with each set of significantly-altered genes, generated using the Ingenuity Knowledge Base, appear to link key mediators of cell proliferation, embryonic development, and organogenesis (Figure 3).
Collectively, these data support the transcriptional evolution of cultured stem cells toward gene expression profiles characterized by more robust, proliferative, and stem-like properties.
Figure 3  Network analysis of gene expression changes in of fresh, passage 0, and passage 1 adipose-derived stem cells. Top scoring Ingenuity Pathway Analysis (IPA)-constructed transcriptome networks based genes that were significantly up-regulated in fresh→P0 (A) and P0→P1 (B) human adipose-derived stem cells (ASCs). Direct relationships are indicated by solid lines, and dashed lines represent indirect relationships.

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