Impact of differentiation protocols on the functionality of mesenchymal stem cells derived from induced pluripotent stem cells

Abstract

Background: The discovery of induced pluripotent stem cells revolutionized regenerative medicine, providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs).

Aim: To evaluate and compare five iMSC differentiation protocols, assessing their efficiency, phenotypic characteristics, and functional properties relative to primary mesenchymal stem cells (MSCs).

Methods: Five iMSC differentiation protocols were assessed: SB431542-based differentiation (iMSC1, iMSC3), an iMatrix-free method (iMSC2), growth factor supplementation (iMSC4), and embryoid body formation with retinoic acid (EB-iMSC). iMSC identity was confirmed according to the International Society for Cell & Gene Therapy 2006 criteria, requiring expression of surface markers (CD105, CD73, CD90) and absence of pluripotency markers. Functional assays were conducted to evaluate differentiation potential (osteogenic and adipogenic), proliferation, mitochondrial function, reactive oxygen species, senescence, and migration.

Results: All iMSC types expressed MSC markers and lacked pluripotency markers. EB-iMSC and iMSC2 showed enhanced osteogenesis (runt-related transcription factor 2; P ≤ 0.01 and P ≤ 0.0001, respectively), while adipogenic potential was reduced in iMSC2 (Adipsin; P ≤ 0.01) and EB-iMSC (Adipsin and peroxisome proliferator-activated receptor gamma; P ≤ 0.0001 and P ≤ 0.01, respectively). Proliferation was comparable or superior to bone marrow MSCs, except in iMSC1, with iMSC4 showing the highest rate (MTT assay; P values ranged from 0.01 to 0.001). Despite reduced mitochondrial health in iMSC3 and iMSC4 (P ≤ 0.001), reactive oxygen species levels were lower in all iMSCs (P values ranged from 0.001 to 0.0001), and senescence was significantly reduced in all iMSCs with the exception of iMSC1 (P values ranged from 0.01 to 0.0001). Migration was most reduced in iMSC4 (P ≤ 0.001 at 24 hours and P ≤ 0.0001 at 48 hours).

Conclusion: While all protocols generated functional iMSCs, variations in differentiation, proliferation, and function emphasize the impact of protocol selection. These findings contribute to optimizing iMSC generation for research and clinical applications.

Keywords: Differentiation protocols; Induced mesenchymal stem cells; Induced pluripotent stem cells; Regenerative medicine; Tissue engineering.

Figure 1

Flow cytometry analysis of pluripotency cell surface markers in induced pluripotent stem cells-derived mesenchymal stem cell lines generated using different protocols. A: Representative microscopic images showing the morphology of induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cell (iMSC) lines compared to bone marrow mesenchymal stem cells (BM-MSCs), scale bar = 100 μm; B: Flow cytometry histograms of pluripotency markers NANOG and TRA-1-60 in iMSC lines compared to iPSCs (positive control) and BM-MSCs (negative control); C: Bar graph displaying the percentages of pluripotency markers in iMSC lines relative to control cells (BM-MSCs and iPSCs). Data represent mean ± SE from at least three independent technical replicates. BM-MSCs: Bone marrow mesenchymal stem cells; EB-iMSCs: Embryoid body-induced pluripotent stem cells-derived mesenchymal stem cells; iMSC: Induced pluripotent stem cells-derived mesenchymal stem cell; iPSCs: Induced pluripotent stem cells.

Figure 2

Flow cytometric analysis of human mesenchymal stem cell surface markers. A-D: Quantification of CD90 (A), CD105 (B), CD73 (C), and CD44 (D) expression levels in induced pluripotent stem cells-derived mesenchymal stem cell lines compared to bone marrow mesenchymal stem cells. Statistical analysis was performed using ordinary one-way ANOVA, with bone marrow mesenchymal stem cells as the control group. Data represent mean ± SE from at least three independent technical replicates. ^aP ≤ 0.05; ^bP ≤ 0.01; ^cP ≤ 0.001. BM-MSCs: Bone marrow mesenchymal stem cells; EB-iMSCs: Embryoid body-induced pluripotent stem cells-derived mesenchymal stem cells; iMSC: Induced pluripotent stem cells-derived mesenchymal stem cell.

Figure 3

Analysis of osteogenic and adipogenic differentiation potential of induced pluripotent stem cells-derived mesenchymal stem cells. A: Microscopy images showing red-stained calcium deposits in differentiated osteocytes (top row) and red-stained fat vacuoles in differentiated adipocytes (bottom row), scale bar = 50 μm; B and C: Quantitative reverse transcription polymerase chain reaction analysis of the two osteogenic-related genes runt-related transcription factor 2 (B) and OCN2 (C) in induced pluripotent stem cells-derived mesenchymal stem cells compared to bone marrow mesenchymal stem cells (control); D and E: Quantitative reverse transcription polymerase chain reaction analysis of the adipogenic-related genes Adipsin (D), and peroxisome proliferator-activated receptor gamma (E) in induced pluripotent stem cells-derived mesenchymal stem cells compared to bone marrow mesenchymal stem cells. Statistical analysis was performed using one-way ANOVA. All data are expressed as mean ± SE from at least three independent technical replicates. ^bP ≤ 0.01; ^cP ≤ 0.001; ^dP ≤ 0.0001. BM-MSCs: Bone marrow mesenchymal stem cells; EB-iMSCs: Embryoid body-induced pluripotent stem cells-derived mesenchymal stem cells; iMSC: Induced pluripotent stem cells-derived mesenchymal stem cell; RUNX2: Runt-related transcription factor 2; PPAR: Peroxisome proliferator-activated receptor.

Figure 4

Analysis of cell viability, mitochondrial membrane potential, and intracellular reactive oxygen species cellular for induced pluripotent stem cells-derived mesenchymal stem cell lines. A: Absorbance results of MTT assay showing viable cell numbers in induced pluripotent stem cells-derived mesenchymal stem cell (iMSC) lines compared to bone marrow mesenchymal stem cells at 24 hours and 48 hours after cell seeding; B: Percentage of colony-forming units among iMSC lines compared to bone marrow mesenchymal stem cells (control); C: Red/green fluorescence ratio indicating mitochondrial membrane potential in untreated and carbonyl cyanide 3-chlorophenylhydrazone-treated iMSC samples; D: Fluorescent intensities of total reactive oxygen species production in iMSC lines. Tert-butyl hydroperoxide was used as a positive control to induce reactive oxygen species production. ^bP ≤ 0.01; ^cP ≤ 0.001; ^dP ≤ 0.0001. BM-MSCs: Bone marrow mesenchymal stem cells; EB-iMSCs: Embryoid body-induced pluripotent stem cells-derived mesenchymal stem cells; iMSC: Induced pluripotent stem cells-derived mesenchymal stem cell; CFUs: Colony-forming units; MMP: Mitochondrial membrane potential; CCCP: Carbonyl cyanide 3-chlorophenylhydrazone; TBHP: Tert-butyl hydroperoxide; ROS: Reactive oxygen species.

Figure 5

Assessment of senescence and migration in induced pluripotent stem cells-derived mesenchymal stem cell lines. A: Representative micrographs of senescence in induced pluripotent stem cells-derived mesenchymal stem cell (iMSC) lines compared to bone marrow mesenchymal stem cells (BM-MSCs) (control), scale bar = 200 μm; B: Quantification of senescence-associated beta-galactosidase-positive cells in iMSCs; C: Representative microscopic images showing wound closure in iMSC lines compared to BM-MSCs at 0, 6, 24, and 48 hours post-scratch, scale bar = 200 μm; D: Quantification of migration area over time presented as bar graphs. Data are presented as mean ± SE from at least three independent biological replicates. ^bP ≤ 0.01; ^cP ≤ 0.001; ^dP ≤ 0.0001. BM-MSCs: Bone marrow mesenchymal stem cells; EB-iMSCs: Embryoid body-induced pluripotent stem cells-derived mesenchymal stem cells; iMSC: Induced pluripotent stem cells-derived mesenchymal stem cell; SA-β Gal: Senescence-associated beta-galactosidase.