Pluripotency and immunomodulatory signatures of canine induced pluripotent stem cell-derived mesenchymal stromal cells are similar to harvested mesenchymal stromal cells

Abstract

With a view towards harnessing the therapeutic potential of canine mesenchymal stromal cells (cMSCs) as modulators of inflammation and the immune response, and to avoid the issues of the variable quality and quantity of harvested cMSCs, we examined the immunomodulatory properties of cMSCs derived from canine induced pluripotent stem cells (ciMSCs), and compared them to cMSCs harvested from adipose tissue (cAT-MSC) and bone marrow (cBM-MSC). A combination of deep sequencing and quantitative RT-PCR of the ciMSC transcriptome confirmed that ciMSCs express more genes in common with cBM-MSCs and cAT-MSCs than with the ciPSCs from which they were derived. Both ciMSCs and harvested cMSCs express a range of pluripotency factors in common with the ciPSCs including NANOG, POU5F1 (OCT-4), SOX-2, KLF-4, LIN-28A, MYC, LIF, LIFR, and TERT. However, ESRRB and PRDM-14, both factors associated with naïve, rather than primed, pluripotency were expressed only in the ciPSCs. CXCR-4, which is essential for the homing of MSCs to sites of inflammation, is also detectable in ciMSCs, cAT- and cBM-MSCs, but not ciPSCs. ciMSCs constitutively express the immunomodulatory factors iNOS, GAL-9, TGF-β1, PTGER-2α and VEGF, and the pro-inflammatory mediators COX-2, IL-1β and IL-8. When stimulated with the canine pro-inflammatory cytokines tumor necrosis factor-α (cTNF-α), interferon-γ (cIFN-γ), or a combination of both, ciMSCs upregulated their expression of IDO, iNOS, GAL-9, HGF, TGF-β1, PTGER-2α, VEGF, COX-2, IL-1β and IL-8. When co-cultured with mitogen-stimulated lymphocytes, ciMSCs downregulated their expression of iNOS, HGF, TGF-β1 and PTGER-2α, while increasing their expression of COX-2, IDO and IL-1β. Taken together, these findings suggest that ciMSCs possess similar immunomodulatory capabilities as harvested cMSCs and support further investigation into their potential use for the management of canine immune-mediated and inflammatory disorders.

Figure 1

Canine iPSC-derived MSCs more closely resemble harvested bone marrow-derived MSCs than the iPSCs from which they were generated. (A) Venn analysis of expressed genes (CPM ≥ 1) identified 83% of the 14,765 genes analysed as being co-expressed by canine bone marrow-derived mesenchymal stem cells (cBM-MSCs), canine induced pluripotent stem cell-derived mesenchymal stem cells (ciPSC-MSCs) and canine induced pluripotent stem cells (ciPSCs). 135 genes, representing 0.9%, are shared by ciPSC-MSCs and the ciPSCs, while the ciPSC-MSCs and cBM-MSCs share 818 (5.5%) genes in common. (B) Venn analysis using the transcriptome data for 224 pluripotency factors showed that ciPSC-MSCs, cBM-MSCs and ciPSCs co-express 213 (94.2%) of these pluripotency factors. (C) Multidimensional scaling (MDS) plots using both raw gene count and (D) normalised gene count data placed the ciPSC-MSCs and cBM-MSCs as a superimposed cluster while the ciPSCs formed a separate cluster. (E) Quantitative RT-PCR analysis of the expression of key genes identified from the RNAseq as being differentially expressed between the cMSCs and ciPSCs. Both the RNAseq and qRT-PCR data point to the ciMSCs as being more similar in their transcriptional profiles to AT- and BM-MSCs than to the ciPSCs from which they were derived.

Figure 1

Canine iPSC-derived MSCs more closely resemble harvested bone marrow-derived MSCs than the iPSCs from which they were generated. (A) Venn analysis of expressed genes (CPM ≥ 1) identified 83% of the 14,765 genes analysed as being co-expressed by canine bone marrow-derived mesenchymal stem cells (cBM-MSCs), canine induced pluripotent stem cell-derived mesenchymal stem cells (ciPSC-MSCs) and canine induced pluripotent stem cells (ciPSCs). 135 genes, representing 0.9%, are shared by ciPSC-MSCs and the ciPSCs, while the ciPSC-MSCs and cBM-MSCs share 818 (5.5%) genes in common. (B) Venn analysis using the transcriptome data for 224 pluripotency factors showed that ciPSC-MSCs, cBM-MSCs and ciPSCs co-express 213 (94.2%) of these pluripotency factors. (C) Multidimensional scaling (MDS) plots using both raw gene count and (D) normalised gene count data placed the ciPSC-MSCs and cBM-MSCs as a superimposed cluster while the ciPSCs formed a separate cluster. (E) Quantitative RT-PCR analysis of the expression of key genes identified from the RNAseq as being differentially expressed between the cMSCs and ciPSCs. Both the RNAseq and qRT-PCR data point to the ciMSCs as being more similar in their transcriptional profiles to AT- and BM-MSCs than to the ciPSCs from which they were derived.

Figure 2

ciMSCs and cAT-MSCs constitutively express immunomodulatory and anti-inflammatory factors. ciMSCs and cAT-MSCs have similar constitutive expression profiles, although ciMSCs express higher levels of iNOS and HGF, and lower levels of VEGF, than the cAT-MSCs. Inducible nitric oxide (iNOS); Indoleamine 2,3 dioxygenase (IDO); Galectin-9 (GAL-9); Cyclooxygenase-2 (COX-2); Transforming growth factor-β1 (TGF-β1); Prostaglandin receptor-2α (PTGER-2α); Hepatocyte growth factor (HGF); Vascular endothelial growth factor (VEGF) ; Interleukin-8 (IL-8) and Interleukin-1β (IL-1β). Significance is defined as: ns = not significant p > 0.05; *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0002; ****p ≤ 0.0001.

Figure 3

Response of ciMSCs, cAT-MSCs and cBM-MSCs to priming with pro-inflammatory cytokines canine tumor necrosis factor-α (cTNF-α), canine interferon-γ (cIFN-γ) and a combination of both (cTNF-α + cIFN-γ). When stimulated with cTNF-α, cIFN-γ, or a combination of both, ciMSCs upregulated their expression of : (A) iNOS; (B) GAL-9; (C) COX-2; (D) TGF-β; (E) PTGER-2α; (F) IDO; (G) HGF; (H) VEGF; (I) IL-8 and (J) IL-1β. Significance is defined as: ns = not significant p > 0.05; *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0002; ****p ≤ 0.0001.

Figure 3

Response of ciMSCs, cAT-MSCs and cBM-MSCs to priming with pro-inflammatory cytokines canine tumor necrosis factor-α (cTNF-α), canine interferon-γ (cIFN-γ) and a combination of both (cTNF-α + cIFN-γ). When stimulated with cTNF-α, cIFN-γ, or a combination of both, ciMSCs upregulated their expression of : (A) iNOS; (B) GAL-9; (C) COX-2; (D) TGF-β; (E) PTGER-2α; (F) IDO; (G) HGF; (H) VEGF; (I) IL-8 and (J) IL-1β. Significance is defined as: ns = not significant p > 0.05; *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0002; ****p ≤ 0.0001.

Figure 3

Response of ciMSCs, cAT-MSCs and cBM-MSCs to priming with pro-inflammatory cytokines canine tumor necrosis factor-α (cTNF-α), canine interferon-γ (cIFN-γ) and a combination of both (cTNF-α + cIFN-γ). When stimulated with cTNF-α, cIFN-γ, or a combination of both, ciMSCs upregulated their expression of : (A) iNOS; (B) GAL-9; (C) COX-2; (D) TGF-β; (E) PTGER-2α; (F) IDO; (G) HGF; (H) VEGF; (I) IL-8 and (J) IL-1β. Significance is defined as: ns = not significant p > 0.05; *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0002; ****p ≤ 0.0001.

Figure 4

Effect of mitogen-stimulated canine lymphocytes on inflammatory cytokine expression of ciMSCs and cAT-MSCs. When co-cultured with mitogen-stimulated lymphocytes, ciMSCs downregulated their expression of iNOS, HGF, TGF-β1and PTGER-2α, while increasing their expression of COX-2, IDO and IL-1β. Significance is defined as: ns = not significant p > 0.05; *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0002; ****p ≤ 0.0001.

Figure 5

Effect of ciMSCs and cAT-MSCs on inflammatory cytokine expression of mitogen-stimulated canine lymphocytes. Lymphocytes cultured with ciMSCs and cAT-MSCs downregulated their expression of GAL-9, PTGER-2α and VEGF, while the expression of iNOS, IDO, IL-8 and IL-1β were unchanged. Lymphocytes co-cultured with cAT-MSCs increased their expression of COX-2 and TGF-β1. Significance is defined as: ns = not significant; p > 0.05; *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0002; ****p ≤ 0.0001.

Figure 6

Effects of co-culture on the secretion of factors by lymphocytes, ciMSCs and cAT-MSCs. Lymphocytes, ciMSCs and cAT-MSCs produce IL-1β, IL-8, TGF-β1 and VEGF. Based on mRNA levels (see Figs. 5 and 6), the increase in IL-1β measured in the medium from co-cultures of cMSCs and lymphocytes is likely produced by the ciMSCs and cAT-MSCs rather than the lymphocytes. When similarly referenced to mRNA levels, cAT-MSCs and lymphocytes in co-culture upregulate their expression of IL-8 while ciMSCs do not. Significance is defined as: *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0002; ****p ≤ 0.0001.