Comparative analysis of mesenchymal stem/stromal cells derived from human induced pluripotent stem cells and the cognate umbilical cord mesenchymal stem/stromal cells

Abstract

Mesenchymal stem/stromal cells (MSCs) show tremendous potential for regenerative medicine due to their self-renewal, multi-differentiation and immunomodulatory capabilities. Largely studies had indicated conventional tissue-derived MSCs have considerable limited expandability and donor variability which hinders further application. Induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) have created exciting source for standardized cellular therapy. However, the cellular and molecular differences between iMSCs and the cognate tissue-derived MSCs remains poorly explored. In this study, we first successfully reprogrammed human umbilical cords-derived mesenchymal stem/stromal cells (UMSCs) into iPSCs by using the cocktails of mRNA. Subsequently, iPSCs were further differentiated into iMSCs in xeno-free induction medium. Then, iMSCs were compared with the donor matched UMSCs by assessing proliferative state, differentiation capability, immunomodulatory potential through immunohistochemical analysis, flow cytometric analysis, transcriptome sequencing analysis, and combine with coculture with immune cell population. The results showed that iMSCs exhibited high expression of MSCs positive-makers CD73, CD90, CD105 and lack expression of negative-maker cocktails CD34, CD45, CD11b, CD19, HLA-DR; also successfully differentiated into osteocytes, chondrocytes and adipocytes. Further, the iMSCs were similar with their parental UMSCs in cell proliferative state detected by the CCK-8 assay, and in cell rejuvenation state assessed by β-Galactosidase staining and telomerase activity related mRNA and protein analysis. However, iMSCs exhibited similarity to resident MSCs in Homeobox (Hox) genes expression profile and presented better neural differentiation potential by activation of NESTIN related pathway. Moreover, iMSCs owned enhanced immunosuppression capacity through downregulation pools of pro-inflammatory factors, including IL6, IL1B etc. and upregulation anti-inflammatory factors NOS1, TGFB etc. signals. In summary, our study provides an attractive cell source for basic research and offers fundamental biological insight of iMSCs-based therapy.

Keywords: Immunomodulatory; Induced pluripotent stem cells (iPSCs); Mesenchymal stem/stromal cells (MSCs); Transcriptomics; iPSC-derived MSCs (iMSCs).

Fig. 1

Generation and characterization of human iPSCs. a. Workflow of the generation of UMSCs-derived iPSCs through transfection with mRNA factor cocktails. b. Morphology of iPSCs during culture-expansion in vitro. Scale bar represents 100 μm. c. Immunofluorescence staining of OCT4, NANOG, SOX2, TRA160 and SSEA4. DAPI staining was done to visualize the nucleus. d. Barplot showing the relative expression level of representative pluripotent-, cell cycle- and telomere maintaining-related genes in iPSCs and UMSCs population using the RNA-sequencing data analysis.

Fig. 2

Differentiating the human iPSCs into iMSC. a. Schematic illustration of generation of iMSCs from human iPSCs. b. Morphology of iMSCs during culture-expansion in vitro. Scale bar represents 100 μm. c. The expression of MSC surface-positive marker (CD73, CD90, CD105) and lack expression of MSC surface-negative marker cocktails (CD34, CD45, CD11b, CD19, and HLA-DR) in culture-expanded iMSCs as assessed by FACS. d. Differentiation of adipocytes, osteocytes, and chondrocytes of iMSCs at passage 5. Scale bar 100 μm.

Fig. 3

No significant difference in proliferative capacity between iMSCs and UMSCs. a. The cell viability of iMSCs and UMSCs detected by the CCK-8 assay. b. The mRNA expression level of the representative cell cycle-related genes in iMSCs and UMSCs. c. Immunofluorescence staining of CD90 and MKI67 in iMSCs and UMSCs. DAPI staining was done to visualize the nucleus. Scale bar represents 100 μm. Column chart showing the percentage of MKI67-positive cells in CD90-positive cells of iMSCs and UMSCs. d. The mRNA expression level of the representative β-Galactosidase (β-GAL) related genes and telomere maintaining-related gene in iMSCs and UMSCs. e. β-galactosidase staining in iMSCs and UMSCs. Scale bar represents 100 μm. f. Column chart showing the percentage of β-GAL positive cells in total cells of iMSCs and UMSCs. g. The protein levels of telomerase activity determined by ELISA in the culture medium of iMSCs and UMSCs.

Fig. 4

iMSCs exhibited higher neural differentiation potential. a. The selected representative upregulation genes in DEGs of iMSCs and UMSCs respectively. b. Selected GO terms of upregulation genes in iMSCs compared to UMSCs. c. Barplot showing the relative expression level of selected genes in iMSCs and UMSCs. d. The regulatory network of the selected genes using the STRING databased. e. Immunofluorescence staining of NESTIN and HOXD11 in iMSCs, UMSCs and the umbilical cord tissue, respectively. DAPI staining was done to visualize the nucleus. Scale bar represents 100 μm. f. Column chart showing the percentage of NESTIN-positive cells in HOXD11-positive cells of iMSCs, UMSCs and the umbilical cord tissue, respectively.

Fig. 5

iMSCs showed higher immunosuppressive function compared to UMSCs in vitro. a. Selected representative GO terms of down-regulation genes in iMSCs compared to UMSCs. b. Heatmap showing the expression level of representative immunoregulation related genes in iMSCs and UMSCs. c. The expression levels of selected representative immunoregulation genes in iMSCs and UMSCs. d. Immunofluorescence staining of IL6 in iMSCs and UMSCs. DAPI staining was done to visualize the nucleus. Scale bar represents 100 μm. Column chart showing the percentage of IL6 positive cells in total cells of iMSCs and UMSCs. e. Column chart showing the proliferative activity of T cells detected by the CCK-8 assay. T cells cultured alone as the control group. f. T cell subpopulation analysis by flow cytometry using specific antibody including CD3, CD8, CD25, CD127, IFN-γ, IL4 and IL17A. Column chart showing the percentage of T cell subpopulations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.