In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells

Abstract

Human mesenchymal stem or stromal cells (hMSCs) are known for their potential in regenerative medicine due to their differentiation abilities, secretion of trophic factors, and regulation of immune responses in damaged tissues. Due to the limited quantity of hMSCs typically isolated from bone marrow, other tissue sources, such as adipose tissue-derived mesenchymal stem cells (hASCs), are considered a promising alternative. However, differences have been observed for hASCs in the context of metabolic characteristics and response to in vitro culture stress compared to bone marrow derived hMSCs (BM-hMSCs). In particular, the relationship between metabolic homeostasis and stem cell functions, especially the immune phenotype and immunomodulation of hASCs, remains unknown. This study thoroughly assessed the changes in metabolism, redox cycles, and immune phenotype of hASCs during in vitro expansion. In contrast to BM-hMSCs, hASCs did not respond to culture stress significantly during expansion as limited cellular senescence was observed. Notably, hASCs exhibited the increased secretion of pro-inflammatory cytokines and the decreased secretion of anti-inflammatory cytokines after extended culture expansion. The NAD+/NADH redox cycle and other metabolic characteristics associated with aging were relatively stable, indicating that hASC functional decline may be regulated through an alternative mechanism rather than NAD+/Sirtuin aging pathways as observed in BM-hMSCs. Furthermore, transcriptome analysis by mRNA-sequencing revealed the upregulation of genes for pro-inflammatory cytokines/chemokines and the downregulation of genes for anti-inflammatory cytokines for hASCs at high passage. Proteomics analysis indicated key pathways (e.g., tRNA charging, EIF2 signaling, protein ubiquitination pathway) that may be associated with the immune phenotype shift of hASCs. Together, this study advances our understanding of the metabolism and senescence of hASCs and may offer vital insights for the biomanufacturing of hASCs for clinical use.

Keywords: NAD redox cycle; adipose-derived mesenchymal stem cells; immune phenotype; proteomics; replicative senescence; transcriptomics.

Figure 1

Stem cell properties of hASCs during in vitro culture expansion. (A) Representative images of hASC morphology during culture expansion. Scale bar: 250 µm. (B) The population doubling time of hASCs at different passages. (C) mRNA levels of cell cycle markers of hASCs at different passages. mRNA levels of (D) stemness genes and genes for (E) osteogenic differentiation and (F) adipogenic differentiation of hASCs at different passages. (G) Colony-forming ability (CFU) and (H) β-gal activity of hASCs at P5 and P12. *indicates p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Metabolic characteristics of hASCs during in vitro culture expansion. Metabolic activity and mitochondrial fitness is relatively stable during in vitro culture expansion of hASCs. (A) Glucose consumption and lactate production is stable during culture expansion of hASCs. (B) Total ATP and (C) the ratio of glycolytic ATP to total ATP is well maintained during culture expansion of hASCs. The ratio of glycolytic ATP was calculated by the delta value of total ATP and 2-DG treated ATP normalized to total ATP. (D) Gene expression of critical enzymes involved in glycolysis and pentose phosphate pathway (PPP) of hASCs at different passages. (E) Mitochondrial fitness determined by flow cytometry: mass, Mitochondrial Membrane Potential (MMP), total reactive oxygen species (ROS), and mitochondrial ROS for P5 and P12 cells. (F) Electron transport complex-I (ETC-I) activity of hASCs at P4 and P12. (G) mRNA levels of genes involved in mitochondrial fusion and fission dynamics. (H) mRNA levels of genes related to autophagy. (I) Autophagic flux of hASCs at P5 and P12 via flow cytometry. Statistical analysis was performed and no statistical significance was observed.

Figure 3

NAD+/NADH redox cycle and Sirt-1 activity in hASCs during in vitro culture expansion. (A) The levels of NAD+ and NADH, (B) the ratio of NAD+/NADH, and (C) mRNA expression of CD38, CD73, NAMPT, Sirt-1, and Sirt-3 for P4 and P12 hASCs. (D) Flow cytometry histograms depicting little change in protein expression of Sirt1, Sirt3, CD38, CD73, and NAMPT. (E) Western Blot images and quantifications for the Sirt-1 and Sirt-3 enzymes normalized to α-tubulin. Data are shown in mean and standard deviation. *indicates p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Immunomodulation ability of hASCs during in vitro culture expansion. Immunomodulatory potentials of hASCs was significantly changed during culture expansion. (A) mRNA levels for the genes involved in M1 macrophage polarization and (B) M2 macrophage polarization of macrophage co-cultured with hASCs from different passages. (C) (i) Indoleamine 2,3-dioxygenase (IDO) activity from basal level and interferon-gamma (IFN-γ) priming of hASCs at P4 and P12. (ii) IDO activity with reactive oxygen species (ROS) depletion. Rapamycin (Rapa, 100 nM) or mitoquinone (MitoQ, 1 µM) were used to treat hASCs together with IFN-γ stimulation. Secretion of (D) Pro-inflammatory cytokines (CXCL10, IL-1β, and IL-6); and (E) anti-inflammatory cytokines (PGE2, IL-10, and HGF) of hASCs at different passages (determined by ELISA assay normalized to cell number). *indicates p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Global alterations of transcriptome in hASCs during in vitro culture expansion. (A) Venn diagram depicting variation and overlap of differentially expressed genes (DEGs) in P4, P8, and P12 hASCs from mRNA-sequencing transcriptome analysis. (B) Interleukin mRNA expression (shown in fold change) for P12 vs. P4 hASCs, showing upregulation at P12. (C) Chemokine mRNA expression (shown in fold change) for P12 vs. P4 hASCs, showing upregulation at P12. (D) Growth factor mRNA expression (TGF, PDGF, and VEGF) for P12 vs. P4 hASCs. (E) Integrin and matrix metalloproteinase (MMP) mRNA expression for P12 vs. P4 hASCs. The numbers are the Log2 values of ratios of P12 cells to P4 cells. Negative values indicate that the genes are present in higher amounts in the P4 group, while positive values indicate that the genes are present in higher amounts in the P12 group. The number 1 indicates two-fold increase.

Figure 6

Global alterations of proteome in hASCs during in vitro culture expansion. (A) Venn diagram depicting variation and overlap of differentially expressed proteins (DEPs) in P4, P8, and P12 hASCs from proteomics analysis. (B) Principle component analysis (PCA) showing P4, P8, and P12 protein expression as three distinct groups. (C) Volcano plots depicting greater variation in protein fold change between the P4 vs. P12 group than the P4 vs. P8 group. (D) Gene ontology (GO) analysis for DEPs between P4 and P12 hASCs for biological processes, cellular components, and molecular functions. (E) Ingenuity Pathway Analysis (IPA) depicts signaling pathways with most significant evidence for alterations by in vitro passaging.