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.

Key Words: Induced pluripotent stem cells; Induced mesenchymal stem cells; Regenerative medicine; Differentiation protocols; Tissue engineering

Core Tip: Induced pluripotent stem cell-derived mesenchymal stem cells (MSCs) meet standard MSC criteria, yet their differentiation, proliferation, and functional properties vary considerably depending on the generation protocol. We compared five differentiation strategies, identifying distinct strengths and limitations in osteogenesis, adipogenesis, mitochondrial function, oxidative stress, senescence, and migration. Our findings highlight the need to optimize and standardize differentiation methods to produce reproducible, high-quality induced pluripotent stem cell-derived MSCs for research and therapeutic applications.

INTRODUCTION

Mesenchymal stem cells (MSCs) are multipotent cells that play essential roles in tissue recovery and regenerative medicine because of their capacity for self-renewal and differentiation into various cell types[1,2]. MSCs can be derived from several adult tissues including bone marrow, adipose tissue, amniotic fluid, dental pulp, placenta, umbilical cord blood, Wharton’s jelly, brain, kidney, liver, lung, spleen, pancreas, and thymus[3]. According to criteria established by the International Society for Cell & Gene Therapy (ISCT) in 2006, human MSCs must meet the following characteristics: (1) Plastic adherent under standard culture conditions; (2) Presence of CD105, CD73, CD90, and absence of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface molecules; and (3) Differentiation ability into osteoblasts, adipocytes, and chondrocytes in vitro[4]. While MSCs have shown promise in treating diseases lacking effective therapies, such as heart failure and osteoarthritis[5,6], their usage in regenerative medicine is hindered by several limitations. For example, the isolation of MSCs often involves invasive procedures, and their capacity for large-scale expansion is limited[7,8]. Furthermore, the exact characteristics of MSCs can vary highly depending on several factors that include tissue source and medium composition[9,10].

Recently, induced pluripotent stem cells (iPSCs) have emerged as a promising source for cell-based therapies[11]. iPSCs can be derived through reprogramming of cells from different sources, such as saliva and dermal fibroblasts, making them more accessible compared to other stem cell types[11,12]. Moreover, iPSCs possess the remarkable ability to differentiate into cell types from all three germ layers, exhibit high proliferative capacity, and can be generated in a patient-specific manner[13]. These features make iPSCs valuable resources for regenerative medicine and patient-specific therapy. Furthermore, induced MSCs derived from iPSCs (iMSCs) retain the defined characteristics of MSCs, including multipotent differentiation potential and the expression of key surface markers such as CD105, CD73, and CD90[8,14,15]. Several previous studies comparing iMSCs to traditional MSCs have shown iMSCs to be more promising, as they address several limitations of conventional MSCs[15-17]. Notably, iMSCs demonstrate enhanced accessibility and superior proliferative and expansion capacities[14,15]. Additionally, while the potential of conventional MSCs is limited by replicative senescence during extended culture, iMSCs can retain sustained growth capacity even after prolonged passaging[18,19]. Therefore, iMSCs are considered an attractive alternative to conventional MSCs, highlighting the need for further investigation into their characteristics and generation methods to facilitate their use in regenerative therapy.

Various protocols have been established to differentiate iPSCs into iMSCs, which can be broadly categorized into direct spontaneous induction and directed lineage-specific differentiation approaches. One widely used approach involves direct transition of iPSCs into MSC-specific culture media, which promotes mesodermal lineage specification over several weeks of passage[18,20,21]. This method reliably generates iMSCs that exhibit the characteristic surface markers and functional properties of traditional MSCs, while offering the added benefits of scalability and patient specificity. These qualities make iMSCs a powerful tool for regenerative medicine, disease modeling, and cell-based therapy development. Additionally, the differentiation of iPSCs into iMSCs can be directed using small molecule inhibitors and growth factors.

Although several studies have described methods for generating iMSCs from iPSCs, most have assessed individual protocols in isolation and under differing conditions[20,22]. In this study, we sought to investigate how different differentiation strategies influence iMSC characteristics by applying five previously reported protocols within the same culture system. One approach involved embryoid body (EB) formation, enhanced with retinoic acid, while the other four relied on combinations of small molecules and supplements to induce mesenchymal differentiation. Our study presents a comparative analysis of five distinct iMSC protocols under unified culture conditions. We also performed a multi-parameter functional evaluation, assessing MSC identity, differentiation, proliferation, reactive oxygen species (ROS) levels, mitochondrial health, senescence, and migration, providing a broader and more robust evaluation. The ultimate objective was to identify the most reliable and effective method for producing high-quality iMSCs suitable for applications in regenerative medicine and therapeutic interventions.

MATERIALS AND METHODS

The human bone marrow MSCs (BM-MSCs) used in this study were previously prepared at the Cell Therapy Center, University of Jordan, and reported in our previous studies[23,24]. This study was conducted in accordance with the Declaration of Helsinki, and Institutional Review Board approval was obtained (approval No. IRB-CTC/2-2021/09) at the Cell Therapy Center. All cell lines were confirmed to be free of mycoplasma contamination and verified for sterility.

Generation of iMSCs

We utilized four iPSC lines (JUCTCi010-A, JUCTCi010-B, JUCTCi011-A, and JUCTCi011-B) that we have previously generated and characterized from human dermal fibroblasts of male and female donors[25,26]. This was followed by the application of five different protocols to produce iMSCs from the iPSC lines. iPSCs at passage 13 were cultured on Matrigel (Corning, NY, United States)-coated plates and maintained in mTeSR medium (StemCell Technologies, MA, United States) before differentiation into iMSCs. The differentiation protocols were named as: IMSC1, iMSC2, iMSC3, iMSC4, and EB-iMSC. Cells from both BM-MSCs and iMSC groups were assessed at passage 3 and maintained under standard culture conditions (37 °C, 21% O₂, and 5% CO₂).

iMSC1 generation protocol: In this protocol, iPSCs were cultured on Matrigel-coated six-well plates and maintained on mTeSR™ plus medium. Once cells reached approximately 50% confluence, the medium was switched to minimum essential medium Eagle-alpha modification (α-MEM; Gibco, NY, United States) supplemented with 10% fetal bovine serum (FBS; Hyclone, UT, United States), 1% GlutaMAX supplement (Gibco, NY, United States), and 1% antibiotic-antimycotic (Gibco, NY, United States). The medium was further enriched with 10 μM SB431542 with daily medium change for 14 days. Cells were then detached using 1 × TrypLE Express (Gibco, NY, United States) and cultured on uncoated plates in α-MEM complete culture medium (CCM) without SB431542[27].

iMSC2 generation protocol: Differentiation of iPSCs to iMSCs was performed by directly replacing mTeSR with α-MEM supplemented with 1% GlutaMAX, 1% MEM nonessential amino acids, 10% FBS, and 2% penicillin/streptomycin[28].

iMSC3 generation protocol: iPSCs were cultured in mTeSR1 medium supplemented with 10 μM SB431542 and daily medium exchange until exhibiting MSC-like morphology (within 4-5 weeks of SB431542 treatment)[29].

iMSC4 generation protocol: iMSCs were produced from iPSCs maintained in mTeSR before differentiation. Briefly, cells were digested into clumps with collagenase IV and cultured on fibronectin-coated surfaces in activin A (25 ng/mL) and CHIR99021 (3 μM) for one day in basal medium (DMEM: F12, 1% ITS, 2% B27, 2 mmol/L L-glutamine, 90 μM β-mercaptoethanol), followed by another day with activin A (25 ng/mL), CHIR99021 (3 μM), and fibroblast growth factor 2 (FGF2) (20 ng/mL) to promote differentiation into primitive streak cells. Mesoderm differentiation was induced with FGF2 (20 ng/mL), bone morphogenetic protein 4 (40 ng/mL), Y27632 (5 μM), and follistatin (100 ng/mL) for eight days, followed by MSC specification with FGF2 (50 ng/mL), platelet derived growth factor (50 ng/mL), epidermal growth factor (100 ng/mL), and ascorbic acid (500 μg/mL) for 11 days[30].

EB-iMSC generation protocol: iPSCs were cultured in MSC differentiation medium containing α-MEM with 15% FBS, 1% GlutaMAX, and 1% antibiotic-antimycotic. Retinoic acid was added on day 2 (10 μM) and day 4 (0.1 μM), followed by retinoic acid-free medium from day 6. On day 7, EBs were transferred to Matrigel-coated plates and maintained in MSC medium, refreshed every two days. From day 12, the medium was supplemented with 2.5 ng/mL basic FGF (bFGF)[24,31].

Flow cytometry analysis of pluripotency and MSC markers

Prior to iMSC generation from iPSCs, the four iPSC lines (JUCTCi010-A, JUCTCi010-B, JUCTCi011-A, and JUCTCi011-B) were assessed for the expression of pluripotency markers NANOG and TRA-1-60 using flow cytometry. To confirm successful differentiation into iMSCs, cells at passage 3 were evaluated for the loss of these markers. iPSC lines served as a positive control, while three different primary BM-MSC samples were used as a negative control. For flow cytometry staining, cells were detached, washed twice with 1 × phosphate-buffered saline (PBS), and resuspended in 400 μL of staining buffer (2% bovine serum albumin in 1 × PBS). Each sample was aliquoted into four test tubes containing 100 μL of the cell suspension, followed by the addition of 5 μL of the appropriate antibody. Samples were incubated in the dark for 40 minutes with gentle shaking (Supplementary Table 1). After incubation, cells were washed twice with 1 × PBS, centrifuged, and resuspended in 200 μL PBS. Flow cytometry was performed using a BD FACSCanto II, and data were analyzed with BD FACSDiva software.

Flow cytometry analysis of hMSC markers

For further characterization of the iMSC phenotype, cells were tested for the expression of human MSC surface markers using the Stemflow hMSC Analysis Kit (BD Biosciences, NJ, United States). A cocktail of conjugated antibodies targeting MSC-positive markers (CD105, CD73, CD90, and CD44) was applied to iMSC lines and BM-MSCs, alongside their respective isotype controls. Three BM-MSCs samples served as a positive control. Cells at passage 3 were detached, centrifuged, and resuspended in 800 μL of staining buffer. Each sample was aliquoted into four test tubes (200 μL per tube), followed by the addition of 100 μL of the appropriate antibody cocktail. Samples were incubated in the dark for 30 minutes, washed twice with 1 × PBS, centrifuged, and resuspended in 200 μL of 1 × PBS. Flow cytometry analysis was conducted using a BD FACSCanto II, and data were processed with BD FACSDiva software.

Cell proliferation assays

MTT assay: The proliferation of iMSC lines generated using different protocols, compared to BM-MSCs, was assessed using the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ATCC, VA, United States]. Cells were seeded in three 96-well plates at a density of 7 × 10³ cells/well in 100 μL of CCM. At 24-hour and 48-hour post-seeding, 10 μL of MTT solution was added to each well, followed by incubation at 37 °C for 3 hours. Subsequently, 100 μL of solubilization stop solution was added, and plates were incubated for an additional 30 minutes at 37 °C. Absorbance was measured at 570 nm using a BioTek Cytation 5, and data were analyzed with BioTek Gen5 data analysis software (BioTek, VT, United States).

Colony-forming unit assay: Both iMSC lines and BM-MSCs were seeded in duplicate at densities of 100, 200, and 300 cells/well. Cells were cultured for nine days at 37 °C with 5% CO₂, with medium replacement every three days. Following incubation, cells were washed with 1 × PBS, fixed with methanol for 15 minutes, and stained with 5% crystal violet for 5 minutes. Excess stain was removed by washing twice with 1 × PBS, and plates were left at room temperature to dry. Colonies consisting of ≥ 50 cells were counted under a microscope. The colony formation rate was calculated by dividing the average colony count from duplicate wells by the initial seeding density, representing the percentage of cells that successfully formed colonies.

Mitochondrial membrane potential

The MitoProb™ JC-1 assay kit (Invitrogen, CA, United States) was used to assess mitochondrial membrane potential (MMP). Briefly, cell samples at a density of 7 × 10³ cells/well were seeded in black tissue culture-treated 96-well plates (Costar, Corning, NY, United States) and incubated for 48 hours. Afterward, samples were treated with 100 μL of freshly prepared 2 μM JC-1 (5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide) in serum-free media and incubated at 37 °C with 5% CO₂ for 60 minutes. For control cells, 50 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added after 30 minutes of incubation. For MMP assessment, cells were washed with PBS, and fluorescence intensities were measured using the BioTek Cytation 5. Green J-monomers were detected at an excitation of 485 nm and emission of 528 nm, while red J-aggregates were measured at an excitation of 535 nm and emission of 590 nm. Imaging and red/green ratio calculations were performed using BioTek Gen5 software. The red/green ratio was determined by calculating the proportion of the red fluorescent area to the green fluorescent area[24].

ROS level measurement

ROS levels were measured using the Total Reactive Oxygen Species Assay Kit (520 nm; Invitrogen, CA, United States). Each cell sample was seeded at 7 × 10³ cells/well in black tissue culture-treated 96-well plates (Costar, Corning, NY, United States). After 48 hours of incubation, a 1 × ROS stain was prepared by adding 10 μL of a 500 × stock reagent to 5 mL of prewarmed serum-free medium. After aspirating the CCM, 50 μL of the 1 × ROS stain solution was added to the wells. Cells treated with the stain were incubated at 37 °C for 60 minutes. Control wells were treated with 200 μmol/L tert-butyl hydroperoxide (TBHP) in serum-free medium after the first 30 minutes of incubation. Fluorescence was then measured at an excitation of 488 nm and an emission of 520 nm using the BioTek Cytation 5 and assessed with BioTek Gen5 software[24].

Osteogenic differentiation

iMSC lines were seeded in six-well tissue culture plates at a density of 200 × 10³ cells/well in CCM. Upon reaching at least 50% confluency, the medium was replaced with osteogenic differentiation medium, consisting of α-MEM supplemented with 15% FBS, 1% GlutaMAX (100 ×), 1% antibiotic-antimycotic (100 ×), 10 nM dexamethasone, 50 μg/mL ascorbic acid 2-phosphate, and 10 mmol/L β-glycerophosphate (Carbosynth, IL, United States). After 21 days, Alizarin Red-positive mineralization was assessed by staining with Alizarin Red, while osteogenic differentiation was also evaluated at the gene expression level using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stained crystals were visualized and imaged using the EVOS XL Core Imaging System (Thermo Fisher Scientific, MA, United States)[24].

Adipogenic differentiation

For adipogenic differentiation, iMSC lines were seeded in six-well plates at a density of 200 × 10³ cells/well in CCM. Once cells reached at least 50% confluency, the medium was replaced with adipogenic differentiation medium, which included α-MEM supplemented with 15% FBS, 1% GlutaMAX (100 ×), and 1% antibiotic-antimycotic (100 ×), 1 nM dexamethasone, 500 μM 3-isobutyl-1-methylxanthine, 200 μM indomethacin, and 10 μg/mL insulin. After 21 days, Oil Red O-stained lipid droplets were observed under a microscope and samples were stained with Oil Red-O to assess lipid accumulation or harvested for further analysis using qRT-PCR. Imaging was performed using the EVOS XL Core Imaging System[24].

Wound scratch assay

To perform the scratch assay, approximately 3 × 10⁵ cells per well from iMSC and BM-MSC lines were plated in α-MEM. Once the cells reached 80%-90% confluence, a 200 μm pipette tip was used to create a scratch across the cell monolayer. The cells were then washed twice with 1 × PBS to remove debris and incubated under standard culture conditions. Images of the scratched area were captured at 0, 6, 24, and 48 hours using an inverted microscope. Wound closure percentages were analyzed using the ImageJ software.

Senescence-associated beta-galactosidase staining

Senescence in treated MSC and iMSC cells was assessed using the Senescence Detection Kit (cat. No. ab65351; Abcam, MA, United States) following the manufacturer’s guidelines. Briefly, 3 × 10⁵ cells were cultured in the appropriate growth medium for each cell type until reaching 80%-90% confluence. Afterward, the cells were washed once with 1 × PBS and fixed with a fixative solution for 15 minutes at room temperature. Subsequently, a staining solution was applied, and the cells were incubated overnight at 37 °C. Senescent cells were identified by their blue color, visualized under a microscope, and counted. Additionally, nuclei were stained with 4’,6-diamidino-2-phenylindole to determine the total cell number using BioTek Cytation 5. The percentage of senescent cells [senescence-associated beta-galactosidase (SA-β-Gal)-positive] was calculated by dividing the number of positive cells by the total cell count, as described by Debacq-Chainiaux et al[32].

qRT-PCR

Total RNA was isolated from treated cells using the RNeasy Mini Kit (Qiagen, Germany), following the manufacturer’s protocol, with RNA concentration being measured on a NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific, MA, United States). For the analysis of gene expression, qRT-PCR was performed. In brief, cDNA was synthesized using 1 μg RNA in total reaction volume of 20 μL using the PrimeScript RT Master Mix kit (Takara, Japan). Real-time PCR was carried out in triplicates with a 20 μL reaction volume containing 10 ng/μL of cDNA, specific primers amplify OCN2, runt-related transcription factor 2 (RUNX2), peroxisome proliferator-activated receptor gamma (PPARG) and Adipsin (Supplementary Table 2), and the TB Green Premix EX Taq II (Tli RNase H Plus) kit (Takara, Japan), using the CFX96 real-time PCR cycler (Bio-Rad, CA, United States). The PCR cycling protocol included an initial denaturation at 95 °C for 30 seconds, followed by 40 cycles of denaturation at 95 °C for 5 seconds and annealing/extension at 60 °C for 1 minute. The annealing temperature varied depending on the specific primer set used (Supplementary Table 2). Gene expression levels were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and analyzed using the 2^-ΔΔCt method.

Statistical analysis

All data were analyzed using GraphPad Prism version 9.1.0 (GraphPad Software, La Jolla, CA, United States). An ordinary one-way analysis of variance (ANOVA) was used for the colony-forming unit and human MSC surface markers, while two-way ANOVA was used for the rest of the assays, followed by the Bonferroni post hoc test when indicated. BM-MSCs served as the control group, and for each test, the mean ± SE of each group was calculated relative to the control mean. Ninety-five percent confidence intervals (95%CIs) were calculated for key comparisons to assess the magnitude of observed differences. At least three independent technical replicates were performed for all assays, with each experiment repeated at least three times. P ≤ 0.05 was considered statistically significant.

RESULTS

iPSCs successfully differentiated into iMSCs

The iPSC lines used for iMSC generation exhibited compact colonies with well-defined edges and distinct borders (Supplementary Figure 1A). Before differentiation, the expression of iPSC surface markers NANOG and TRA-1-60 was assessed in all four iPSC lines, confirming high expression levels (≥ 95%) across all lines (Supplementary Figure 1B). The generated iMSCs exhibited a fibroblast-like morphology (Figure 1A) and were tested for the absence of iPSC markers (NANOG and TRA-1-60). All iMSC lines were negative for these markers, indicating efficient and successful differentiation from iPSCs to iMSCs (Figure 1B and C).

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.

Successful expression of hMSC surface markers in iMSCs and BM-MSCs

All iMSC lines and BM-MSCs used in this study were analyzed for MSC characteristics according to the ISCT minimum criteria[4], including the presence of human MSC surface markers. Flow cytometric analysis was performed to assess the expression of MSC surface markers CD90, CD105, CD73, and CD44 and the absence of negative human MSC marker cocktail (CD34, CD45, CD14, CD11b, CD79a, CD19, and HLA-DR). Both iMSCs and their corresponding primary BM-MSCs expressed high levels of surface markers as shown in the histograms (Supplementary Figure 2) and bar graphs (Figure 2A-D). However, some iMSCs significantly varied in their expression of certain markers compared to primary BM-MSCs, specifically cells from iMSC4 protocol, which had lower expression levels CD90, CD105, and CD73 compared to BM-MSCs (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001, respectively) (Figure 2A-D). The magnitude of these differences was substantial, with iMSC4 showing mean reductions of 3 ± 0.92 for CD90 (95%CI: 0.1808-5.819) (Figure 2A), 25.13 ± 4.56 for CD105 (95%CI: 11.19-39.08) (Figure 2B), and 8.63 ± 1.7 for CD73 (95%CI: 3.441-13.83) (Figure 2C) compared to BM-MSCs.

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.

Investigation of differentiation ability

Staining all iMSC lines with Alizarin Red demonstrated strong differentiation potential into osteocytes; however, the adipogenic differentiation level was clearly lower in all five iMSCs compared to BM-MSCs (Figure 3A). The qRT-PCR analysis of the osteogenic gene RUNX2 revealed that both EB-iMSC and iMSC2 expressed significantly higher levels than the control BM-MSCs (-593.3 ± 127.1, 95%CI: -981.7 to -205.0, P ≤ 0.01; -1109 ± 127.1, 95%CI: -1498.0 to -720.9, P ≤ 0.0001, respectively) (Figure 3B). Additionally, all iMSC samples showed higher expression for OCN2 than BM-MSCs, with mean differences of -8.08 ± 2.01, 95%CI: -4.20 to -1.96 (iMSC2; P ≤ 0.01); -8.20 ± 2.01, 95%CI: -14.33 to -2.08 (iMSC4; P ≤ 0.01); -11. 42 ± 2.01, 95%CI: -17.54 to -5.30 (iMSC1; P ≤ 0.001); -11.25 ± 2.01, 95%CI: -17.37 to -5.12 (iMSC3; P ≤ 0.001); and -26.59 ± 2.01, 95%CI: -32.71 to -20.46 (EB-iMSC; P ≤ 0.0001) (Figure 3C). Consistent with imaging results, the qRT-PCR analysis revealed that all iMSCs had lower expression of the adipogenic genes compared to BM-MSCs, except for iMSC1, which showed higher, but non-significant, levels of Adipsin and PPARG. EB-iMSC was the only protocol incapable of expressing either Adipsin or PPARG compared to BM-MSCs (30.2 ± 4.4, 95%CI: 16.74-43.67, P ≤ 0.0001; 18.86 ± 4.1, 95%CI: 6.28-31.44, P ≤ 0.01, respectively), with iMSC2 showing lower adipogenic differentiation potential as reflected by downregulation of Adipsin compared to BM-MSCs (17.44 ± 4.4, 95%CI: 3.97-30.90, P ≤ 0.01) (Figure 3D and E).

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.

MSC4 shows the highest viability, while all iMSCs, except iMSC1, exhibit enhanced colony formation, and increased ROS resistance

MTT assay was performed to measure cell viability at 24 hours and 48 hours, with absorbance values reflecting mitochondrial metabolic activity and indirectly indicating cell proliferation. After 48 hours, all iMSC lines were either similar to or showed slightly higher viability compared to BM-MSCs, except for the iMSC4 line, which showed significantly higher viability rate compared to BM-MSCs (-0.30 ± 0.78, 95%CI: -0.84 to 0.47, P ≤ 0.01), iMSC3 (-0.15 ± 0.78, 95%CI: -0.76 to 0.55, P ≤ 0.01), iMSC1 (-0.38 ± 0.78, 95%CI: -0.61 to 0.70, P ≤ 0.001) and EB-iMSC (-0.38 ± 0.78, 95%CI: -0.61 to 0.70, P ≤ 0.001) (Figure 4A). Although most iMSC lines showed similar viability scores to BM-MSCs in the MTT assay, all iMSC groups, except for iMSC1, exceeded the colony-forming ability of BM-MSCs (Figure 4B). EB-iMSC showed a mean difference of -3.87 ± 1.24, 95%CI: -7.451 to -0.2827, compared to BM-MSCs, which was statistically significant (P ≤ 0.05). Similarly, both iMSC2 and iMSC4 exhibited mean differences of -6.83 ± 1.24, 95%CI: -10.42 to -3.249, and -6.6 ± 1.24, 95%CI: -10.18 to -3.02, respectively, each showing highly significant differences (P ≤ 0.001). Notably, iMSC3 demonstrated the strongest effect with a mean difference of -11.27 ± 1.24, 95%CI: -14.85 to -7.68, reaching P ≤ 0.0001.

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.

To investigate cellular health under physiological and stress conditions, we assessed the mitochondrial function of iMSCs through MMP and ROS assays. JC-1 red/green fluorescence ratio indirectly reflects the health and function of mitochondria. At baseline conditions, iMSC1 and iMSC2 exhibited similar or slightly higher red/green ratios compared to BM-MSCs, while iMSC3 and iMSC4 showed significantly lower red/green ratios (1.35 ± 0.27, 95%CI: 0.60-2.10, P ≤ 0.001 and 1.18 ± 0.27, 95%CI: 0.43-1.92, P ≤ 0.001, respectively) (Figure 4C). After CCCP treatment, BM-MSCs experienced a sharp decline in red/green ratio, similar to all iMSC lines except EB-iMSC, which had a significantly lower red/green ratio compared to BM-MSCs (1.32 ± 0.27, 95%CI: 0.57-2.06, P ≤ 0.001). On the other hand, the total ROS production testing revealed an interesting finding: While there was no significant difference in ROS production at baseline conditions, after ROS induction with TBHP treatment, BM-MSCs showed a significantly higher ROS production compared to all iMSC lines. Specifically, EB-iMSC, iMSC2, iMSC3, and iMSC4 had mean differences of 2578984 ± 277748, 95%CI: 1802141-3355828, 1483776 ± 277748, 95%CI: 706933-2260620, 1763609 ± 277748, 95%CI: 986765-2540453, and 1501889 ± 277748, 95%CI: 725045-2278732, respectively (all P ≤ 0.0001), while iMSC1 showed a mean difference of 110134 ± 277749, 95%CI: 530064-2083751 (P ≤ 0.001) (Figure 4D). This finding that iMSC lines may be superior in resisting oxidative stress.

iMSCs revealed decreased senescence and migration rate compared to BM-MSCs

Cellular senescence was investigated by quantifying SA-β-Gal-positive cells in microscopic images of iMSCs, with BM-MSCs serving as control[32]. The results showed that the percentage of baseline SA-β-Gal-positive cells was significantly higher in BM-MSCs compared to all iMSC lines (66.97 ± 3.57, 95%CI: 7.12-29.29, P ≤ 0.01 for iMSC4; 10.24 ± 3.57, 95%CI: -0.84 to 21.33 for iMSC1; 37.86 ± 3.57, 95%CI: 26.78-48.94 for iMSC2; 38.13 ± 3.99, 95%CI: 25.73-50.52 for iMSC3; and 48.75 ± 3.57, 95%CI: 37.66-59.83 for EB-iMSC, all P ≤ 0.0001) (Figure 5A and B). These results indicate decreased number of senescenct iMSCs compared to BM-MSCs; the later were used as a model for conventional MSCs.

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.

The migration ability of iMSC lines was assessed by calculating the area of migration at 0, 6, 24, and 48 hours post-scratch, performed using a 200 μL tip in all iMSC lines. BM-MSCs served as the control cells (Figure 5C and D). Wound-healing observations under the microscope showed slower migration across all cell types at 6 hours post-scratch. After 24 hours, an increase in migration was observed in all cell lines, with BM-MSCs exhibiting a significantly higher migration rate compared to iMSC3 (21.88 ± 5.07, 95%CI: 8.27-35.49, P ≤ 0.0001) and iMSC4 (38.83 ± 5.07, 95%CI: 24.77-51.99, P ≤ 0.001), but a significantly lower rate compared to EB-iMSC (-27.34 ± 5.07, 95%CI: -40.95 to -13.73, P ≤ 0.0001). At 48 hours, BM-MSCs maintained a significantly higher migration rate than both iMSC3 (41.4 ± 5.07, 95%CI: 27.79-55.01, P ≤ 0.0001) and iMSC4 (57.9 ± 5.07, 95%CI: 44.29-71.51, P ≤ 0.0001). Moreover, iMSC3 and iMSC4 exhibited reduced migration compared to other iMSC lines; however, this decrease was not statistically significant. Overall, these findings suggest that iMSC3 and iMSC4 showed lower migration potential, particularly in comparison to BM-MSCs.

DISCUSSION

In this study, we compared five iMSC differentiation protocols to evaluate their efficiency and characteristics (Table 1). The protocols were selected based on previous studies demonstrating their effectiveness in promoting iMSC differentiation. For instance, inhibition of the SMAD signaling pathway using SB431542, in combination with bFGF, has been shown to enhance differentiation by downregulating pluripotency-associated genes through transforming growth factor-beta pathway suppression[20]. Moreover, the use of L-ascorbic acid-2-phosphate and sodium pyruvate further supports the differentiation process[8,17,33]. Liu et al[30] demonstrated that ascorbic acid facilitates MSC specification by boosting self-renewal and osteochondrogenic potential in an iron-dependent manner. Each of these studies utilized distinct methods for generating iMSCs, which may contribute to the observed variations in differentiation outcomes. Additionally, Kang et al[34] used MSC differentiation media to direct iMSC differentiation, while Hynes et al[8] supplemented their media with sodium pyruvate and L-ascorbate-2-phosphate to enhance the process[20]. Similarly, Lian et al[18] used MSC differentiation medium supplemented with growth factors, including bFGF and FGF2, to further promote the differentiation process.

Table 1 Summary of key functional assessment results of induced pluripotent stem cell-derived mesenchymal stem cell differentiation protocols compared with bone marrow-derived mesenchymal stem cells.

Protocol	Mechanism of action	Viability	Osteogenesis	Adipogenesis	ROS level	Migration	Mitochondrial potential
BM-MSCs	Primary BM-MSCs	High	High	High	Low	High	Low
EB-iMSCs[31]	EB formation + retinoic acid enhance 3D signaling and mesenchymal lineage commitment	Moderate-high	Moderate-high	Moderate	Moderate	Moderate	High
iMSC1[27]	SB431542 inhibits TGF-β/SMAD signaling to promote mesenchymal transition	Moderate	Low	Low	Low	Moderate	Moderate
iMSC2[28]	Spontaneous mesenchymal induction following direct medium replacement with α-MEM	Low	Very low	Very low	High	Low	High
iMSC3[29]	Prolonged SB431542 exposure downregulates pluripotency genes, driving MSC fate	Highest	Highest	High	Low	Highest	Low
iMSC4[30]	Stepwise mesoderm induction (activin A/CHIR99021 → FGF2/BMP4/follistatin → PDGF/EGF/ascorbate) promotes MSC specification	Low-moderate	Moderate	Low-moderate	Moderate	Low	Low

ROS: Reactive oxygen species; 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; TGF-β: Transforming growth factor β; α-MEM: Alpha minimal essential medium; MSC: Mesenchymal stem cell; FGF2: Fibroblast growth factor 2; BMP4: Bone morphogenetic protein 4; PDGF: Platelet-derived growth factor; EGF: Epidermal growth factor.

Another successful approach involves the generation of EBs, which are three-dimensional cell aggregates that represent all three germ layers[35,36]. EBs facilitate improved intercellular signaling and substance exchange, enhancing the differentiation process and producing better-quality iMSCs[37,38]. This method has shown promise in producing iMSCs with functional properties similar to those of traditional MSCs, further expanding their potential for therapeutic applications and disease modeling[39,40]. EB formation is a common method used for the differentiation of iPSCs into various cell lineages[36,41]. Culturing of embryonic stem cells in suspension without anti-differentiation factors leads to spontaneous differentiation and formation of three-dimensional multicellular aggregates (EBs)[35,36]. However, researchers have introduced innovative strategies to enhance EB formation. Notably, Karam et al[42] used retinoic acid to promote EB formation, which in turn improved the ability of iMSCs to differentiate into adipocytes. This approach emphasizes the importance of refining differentiation protocols to maximize the potential of iMSCs for specific therapeutic applications, such as adipogenesis.

This study included five different protocols of iMSC generation: (1) SB431542 treatment in α-MEM without bFGF (iMSC1)[27]; (2) SB431542 treatment in α-MEM without iMatrix-511 (iMSC2)[28]; (3) Prolonged SB431542 supplementation in mTeSR1 (iMSC3)[29]; (4) Stepwise mesodermal induction with FGF2, platelet derived growth factor, and epidermal growth factor, together with ascorbic acid (iMSC4)[30]; and (5) EB formation enhanced with retinoic acid (EB-iMSC)[31]. We aimed to evaluate the differentiation potential and functional properties of iMSCs generated using different protocols in comparison to primary MSCs through a comprehensive analysis.

We evaluated the efficiency of the generated iMSCs based on 2006 ISCT minimum standards for MSCs. These standards require the absence of pluripotent surface markers (TRA-1-60 and NANOG), the elevated expression of human MSC surface markers (CD90, CD105, CD73, and CD44), the ability to differentiate into osteogenic and adipogenic lineages, fibroblast-like morphology, and plastic adherence[4]. Flow cytometric analysis confirmed that all MSC types lacked pluripotent markers and displayed high levels of human MSC surface markers. However, while iMSC4 showed slightly lower expression of CD73 and CD90, and a more substantial reduction in CD105 compared to primary BM-MSCs, all other markers and functional assays still supported its MSC identity. This reduction in CD105 expression may warrant further validation in future studies.

While this study relied on the 2006 ISCT criteria, which were the accepted standards at the time this work was conducted, the updated 2025 guidelines[43] introduce important changes. Notably, the requirements for trilineage differentiation and plastic adherence are no longer mandatory, as these traditional stemness assays do not reliably distinguish true stem cells from specialized stromal cells. Instead, the 2025 criteria focus on defined thresholds for surface marker expression, mandatory inclusion of CD45 as a negative marker, and identification of tissue origin, which affects MSC characteristics. The guidelines also emphasize functional potency assays and critical quality attributes to better capture clinical relevance. Although our study meets many foundational criteria, future work should incorporate these potency assessments to align with current standards.

For further characterization of iMSCs, osteocyte and adipocyte development were visually confirmed in all MSC types by observing red-stained calcium deposits and Oil Red O-stained lipid droplets under the microscope. Differential gene expression analysis showed significant variation in osteogenic (RUNX2 and OCN2) and adipogenic (PPARG and ADIPSIN) markers compared to BM-MSCs. Our results demonstrated an overall enhancement of osteogenic differentiation across all iMSC lines, with particularly distinct ability in EB-iMSC and iMSC2, where elevated osteogenic expression was associated with reduced adipogenesis. While iMSCs are recognized for their trilineage differentiation potential, the effectiveness of their differentiation can vary considerably depending on the protocols used. For instance, Lian et al[18] reported that more than 80% of their iMSC population differentiated into adipocytes and osteocytes, with over 90% achieving chondrogenic differentiation. In contrast, other studies by Hynes et al[8], Kang et al[34], Sheyn et al[37], and Xu et al[44], in alignment with our findings, demonstrated strong osteogenic and chondrogenic differentiation in their iMSCs but found limited adipogenic potential in the same cell lines. Moreover, previous studies have shown that retinoic acid plays a direct inhibitory role in adipogenic differentiation by downregulating key transcription factors such as PPARγ and CCAAT enhancer-binding protein alpha, which are essential for adipocyte commitment and maturation[45,46]. This may partly explain the marked reduction in adipocyte formation observed in the EB-iMSC group, where retinoic acid was used during EB formation. Similarly, the reduced adipogenic potential observed in iMSC2 may be attributed to the absence of iMatrix-511 coating during spontaneous differentiation. iMatrix, an extracellular matrix-based substrate rich in laminin-511, has been shown to support trilineage differentiation in MSCs, including its ability to influence adipogenic specification. Its absence may therefore limit the adipogenic commitment of iMSCs, as extracellular matrix components like laminin and fibronectin have been reported to affect MSC lineage decisions[47]. These discrepancies demonstrate the influence of differentiation protocols on the outcomes of iMSC differentiation and highlight the need for optimized methods to harness their full therapeutic potential.

Assessment of proliferation and self-renewal ability of iMSCs through the MTT and colony-forming unit assays revealed that most iMSC lines exhibited similar or slightly higher viability compared to BM-MSCs, with iMSC4 showing significantly higher viability after 48 hours of seeding. Interestingly, while the MTT results showed comparable performance among iMSCs (with the exception of iMSC4) and BM-MSCs, the colony-forming ability of iMSC lines (excluding iMSC1) consistently surpassed that of BM-MSCs. These findings suggest a superior clonogenic potential for iMSCs, consistent with previous reports showing that iMSCs exhibit enhanced proliferative and self-renewal capacities compared to primary MSCs[23,38,48].

MMP assessment, as an indirect indicator of mitochondrial health, revealed a significant decrease in mitochondrial health in untreated iMSC3 and iMSC4 compared to primary MSCs. However, while CCCP treatment led to a reduction in MMP across various MSC groups, including the control and particularly EB-iMSC, it did not noticeably affect MMP in iMSC3 and iMSC4. This suggests that, unless due to experimental conditions, iMSC3 and iMSC4 may exhibit unique resilience or reduced sensitivity to CCCP-induced mitochondrial depolarization. This may be due to differences in mitochondrial properties or protective mechanisms that reduce responsiveness to CCCP treatment, resulting in a consistently lower baseline MMP. Although the precise mechanism remains unclear, this resistance could reflect differences in mitochondrial properties or stress responses intrinsic to these cell lines. While possibilities such as elevated expression of mitochondrial uncoupling proteins, which could moderate the impact of CCCP by allowing controlled proton leakage[49], enhanced mitophagy[50], or metabolic adaptation (such as a shift toward glycolysis)[51] could potentially explain this observation, our current data do not directly support these hypotheses. Future investigations are necessary to clarify whether the proposed mechanisms contribute to this apparent CCCP resistance.

Interestingly, measurement of ROS production demonstrated decreased ROS levels in all iMSC types compared to primary MSCs. Low ROS production after TBHP treatment suggests that the iMSCs’ antioxidant defense system effectively neutralized ROS, indicating healthy cells with improved protective mechanisms, leading to better viability and function as shown by the MTT assay. Moreover, the senescence assay revealed a decrease in senescence in all iMSC types, except iMSC2. However, although iMSCs exhibited lower senescence levels compared to BM-MSCs, iMSCs (excluding EB-iMSCs) still displayed a relatively high baseline senescence based on microscopic observations. When quantified, however, iMSCs showed statistically lower number of senescent cells compared to BM-MSCs. In line with these findings, previous studies have shown that iMSCs exhibit improved cellular vitality. This includes enhanced survival, proliferation, and differentiation capabilities, compared to conventional MSCs. This is attributed to the rejuvenation process that occurs during reprogramming[14,15]. Consistently, their parental iPSCs have demonstrated remarkable self-renewal capacity without exhibiting notable signs of replicative senescence[14,52].

Cell migration assessment through monitoring scratch closure in vitro revealed a noticeable decrease in migration in iMSC3 and iMSC4 compared to primary MSCs. In contrast, other iMSC types showed comparable migration rates to the control, particularly at 48 hours after the scratch was made. Rajasingh et al[53] performed a comparative analysis between iMSCs and umbilical cord-derived MSC and found through wound-healing assay that iMSC showed superior migration compared to umbilical cord-derived MSCs. These findings, along with ours, suggest heterogeneous migration capacities of iMSCs, likely influenced by the protocol used for iMSC generation or the source of cells. The reduced migration observed in iMSC4 may be partly attributed to lower expression of specific MSC surface markers. In particular, CD105 expression was significantly lower compared to BM-MSCs, while CD73 and CD90 were also reduced, though to a lesser extent. Previous studies have reported that diminished CD105 expression can impair MSC migration, supporting this explanation for iMSC4[54-56]. However, this does not fully explain the migration deficit in iMSC3, where surface marker expression was largely preserved. This suggests that factors beyond canonical MSC surface markers may contribute to the observed differences. Migration is a fundamental property of MSCs, underpinning their role in tissue repair and regeneration[2]. The reduced migration in certain iMSC lines may be linked to reprogramming-associated changes, such as alterations in cytoskeletal organization, adhesion dynamics, or chemokine signaling, all of which are known to influence MSC motility[57]. These potential mechanisms were not examined in the present study which is one of its limitations. Furthermore, as the scratch assay reflects both cell proliferation and migration, it may not fully capture migratory capacity[58]. Future investigations should incorporate more direct methods, such as transwell migration assays, alongside targeted gene expression analyses of pathways regulating MSC motility.

While all five protocols successfully generated functional iMSCs meeting the 2006 ISCT minimal criteria, their comparative analysis revealed distinct strengths and limitations. Each protocol demonstrated unique characteristics in terms of differentiation potential, cellular vitality, and functional properties, indicating that the choice of protocol depends on the specific experimental needs or clinical applications being pursued. However, among the five protocols investigated, iMSC4 showed the highest MTT activity, indicating increased metabolic activity, which may suggest enhanced viability and/or proliferation under the tested conditions. This protocol also exhibited robust functional properties, with significant advantages over BM-MSCs in most assays, including ROS production, osteogenic differentiation, and senescence. However, other protocols showed distinct strengths in specific areas. iMSC2 and EB-iMSC excelled in osteogenic differentiation, as indicated by higher RUNX2 gene expression, making them ideal for applications requiring enhanced bone regeneration. Additionally, all iMSC types outperformed BM-MSCs by producing less ROS, indicating enhanced antioxidant defenses. While iMSC4 may be the best choice for overall viability and function, it was not the most effective in terms of migration. iMSC2 and EB-iMSC may be preferable for studies focused on osteogenesis and migration, respectively, depending on the specific needs of the experiment or clinical application.

These findings pave the way for understanding iMSC generation methods and their effects on cellular properties, providing a foundation for optimizing and standardizing iMSC production. This supports ongoing efforts to establish consistent MSC protocols[59], which are essential for improving reproducibility and clinical translation. Therefore, selecting and standardizing iMSC generation methods is crucial to advancing these objectives.

Several methodological considerations should be acknowledged in our study. The present study was designed as a proof-of-principle using two donor-derived iPSC lines (one male and one female), which limits biological diversity, as most replicates reflect technical rather than donor-independent variation. While statistical assumptions were assessed and data distribution verified, future studies could benefit from more rigorous, formal testing approaches. Nonetheless, future investigations should incorporate multiple unrelated donor-derived iPSC lines and employ single-cell analyses to better capture inter-donor and intra-clonal variability, addressing the inherent heterogeneity of iMSCs and improving the generalizability of protocol-dependent outcomes.

CONCLUSION

In conclusion, this study compared five differentiation protocols for generating iMSCs and assessed their efficiency and functional properties relative to primary MSCs. All protocols successfully produced functional iMSCs meeting the ISCT standards, but significant variations were observed in differentiation potential, proliferation, and cellular vitality. These differences underscore the impact of protocol choice on iMSC characteristics, indicating that selection should be tailored to the intended experimental or clinical application. These findings provide valuable insights into iMSC generation methods and pave the way for future optimization and standardization of iMSC production.

ACKNOWLEDGEMENTS

We would like to express our sincere gratitude to the staff of the Cell Therapy Center for their invaluable assistance and support throughout the study.