Proliferation and Differentiation Potential of Bone Marrow–Derived Mesenchymal Stem Cells From Children With Polydactyly and Adults With Basal Joint Arthritis

Isolation and Expansion of Adult and Infant BMSCs

The samples of human adult BMSCs samples (n = 3) were obtained from the excised carpal bones following surgery for basal joint arthritis, with an age range of 37–81 years. In addition, human infant BMSCs (n = 3) samples, aged between 1 and 3 years, were collected from the bone of excised redundant thumb or finger after surgical reconstruction for polydactyly children. The protocol and procedure employed in this study were approved by the Institutional Review Board (IRB). To isolate the mononuclear BMSCs, the density gradient centrifugation method was utilized. The nucleated cells were subsequently plated at clonal density and cultured in a modified minimum essential medium (α-MEM), supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B. The growth medium was refreshed every 2 days, and the cells were passaged every 4 days at 1:5 split, prior to reaching 80% confluence in terms of cellular densities.

All subsequent experiments involving multiple differentiation were performed using three donors from both the infant and adult groups. Individual testing was conducted for each donor, and the results were then averaged.

Flow Cytometric Analysis

The BMSCs were harvested in 5 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS) and incubated with the following anti-human antibodies, including anti-CD29–phycoerythrin (PE), anti-CD73–allophycocyanin (APC), anti-CD90–fluorescein isothiocyanate (FITC, Becton Dickinson, San Jose, CA), anti-CD105–cyanine (Cy5.5), anti-CD44–PE, anti-CD14–PE, anti-CD79a–PE, anti-CD11b–PE, anti-CD19–PE, anti-CD34–PE, anti-CD45–PE, and anti-HLA-DR–PE. Mouse isotype antibodies (Becton Dickinson and Beckman Coulter) were used as controls. Ten thousand labeled cells were analyzed using a FACSCanto II Cytometer System running Diva software (Becton Dickinson, San Jose, CA, USA).

Proliferation

The adult and infant BMSCs, which were seeded at a density of 1 × 10⁵ cells in 10-cm dish, were passaged every 7 days. The seeding density and incubation times remained constant throughout the study. The increased cellular numbers at each passage were counted to calculate the fold changes of cell number and doubling time of BMSCs.

Real-Time Quantitative Polymerase Chain Reaction

Total RNA was extracted using Trizol reagent (Invitrogen). First-strand cDNA was synthesized by random sequence primers and SuperScript^TM III Reverse Transcriptase (Invitrogen) to prime reverse transcription reactions. The cDNA in a reaction mixture containing specific primer pairs (Table 1) and the fast SYBR® Green 189 Master Mix (Applied Biosystems, Foster City, CA, USA) to achieve Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was as an internal control. The results were analyzed using the software supplied with the StepOne^TM (Applied Biosystem) using the comparative CT (ΔΔCT) method ²⁰ .

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Table 1.

Primer Sequences Used for RT-qPCR Analysis.

Gene	Forward primer (5′–3′)	Reverse primer (5′–3′)
p16	ATCATCAGTCACCGAAGG	TCAAGAGAAGCCAGTAACC
p21	CATCTTCTGCCTTAGTCTCA	CACTCTTAGGAACCTCTCATT
p53	CGGACGATATTGAACAATGG	GGAAGGGACAGAAGATGAC
TERT	AAATGCGGCCCCTGTTTCT	CAGTGCGTCTTGAGGAGCA
SOD1	GTGATTGGGATTGCGCAGTA	TGGTTTGAGGGTAGCAGATGAGT
SOD2	TTAACGCGCAGATCATGCA	GGTGGCGTTGAGATTGTTCA
SOD3	CATGCAATCTGCAGGGTACAA	AGAACCAAGCCGGTGATCTG
SOX9	CCAGGGCACCGGCCTCTACT	TTCCCAGTGCTGGGGGCTGT
COL2	TTCAGCTATGGAGATGACAATC	AGAGTCCTAGAGTGACTGAG
COL10	CAAGGCACCATCTCCAGGAA	AAAGGGTATTTGTGGCAGCATATT
ALP	ACCATTCCCACGTCTTCACATTTG	AGACATTCTCTCGTTCACCGCC
OPN	GGACCTGCCAGCAACCGAAGT	GCACCATTCAACTCCTCGCTTTCC
DCN	CTCTGCTGTTGACAATGGCTCTCT	TGGATGGCTGTATCTCCCAGTACT
MMP3	CTGTTGATTCTGCTGTTGAG	AAGTCTCCATGTTCTCTAACTG
PPARγ	TCAGGTTTGGGCGGATGC	TCAGCGGGAAGGACTTTATGTATG
LPL	TGTAGATTCGCCCAGTTTCAGC	AAGTCAGAGCCAAAAGAAGCAGC
AFP	GGCGGTGAGTGTCAGGATAG	AGGTGCATACAGGAAGGGATG
ALB	TGCTTGAATGTGCTGATGACAGGG	AAGGCAAGTCAGCAGGCATCTCATC
MAP2	CAAACGTCATTACTTTACAACTTGA	CAGCTGCCTCTGTGAGTGGAG
NEFH	AGTGGTTCCGAGTGAGATTG	CTGCTGAATTGCATCCTGGT
GCLM	GGCGGTGAGTGTCAGGATAG	GGCGGTGAGTGTCAGGATAG
CAT	TTAATCCATTCGATCTCACC	GGCGGTGAGTGTCAGGATAG
GADPH	ATATTGTTGCCATCAATGACC	GATGGCATGGACTGTGGTCATG

γH2AX Immunostaining

The adult and infant BMSCs at passage three to five were permeabilized by a permeabilization buffer (0.1% Triton X-100 in PBS) and treated with primary antibodies against γH2AX (tcba13051; Taiclone Biotech Corp., Taipei, Taiwan). The DyLight 488-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibodies (GTX213110-01; GeneTex Inc., Irvine, CA, USA) were used to show green fluorescence. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; F6057; Sigma-Aldrich, St Louis, MO, USA). Fluorescence intensity from 60 to 100 cells was determined by the Image-Pro Plus (v4.5.0.29, Media Cybernetics, Silver Spring, MD, USA). The intensity of adult BMSCs was considered as a standard to compare the relative folds of intensity of infant BMSCs.

Chondrogenic Differentiation In Vitro

At passages 3–5, 2.5 × 10⁵ adult or infant BMSCs were incubated in an induction medium contained the serum-free and high-glucose (4.5 g/l) Dulbecco’s modified Eagle’s medium (HG-DMEM) (Gibco, Grand Island, NY, USA) with 10^-7 M dexamethasone (Sigma-Aldrich), 50 μg/ml ascobate-2-phosphate (Sigma-Aldrich), 50 μg/ml L-proline, 10 ng/ml transforming growth factor-β1 (TGF-β1) (R&D Systems, Minneapolis, MN, USA) and Insulin-Transferrin-Selenium (ITS) plus Premix (BD Biosciences, San Jose, CA, USA). After centrifugation at 1500 rpm for 5 min to form a pellet, the sample was transferred into a culture incubator for cultivation and cultured in 5% CO₂ at 37°C. The medium changed every 3 days, and the chondrogenic pellets were observed and harvested at days 7, 14, and 21 for RT-qPCR detection during the induction ²¹ .

Immunohistochemistry Staining and Quantification

The equivalent diameter of the chondrogenic pellets formed by the adult and infant BMSCs was measured on day 21. The sections of the paraffin embedded pellets were stained with Alcian blue (ANC500; ScyTek Laboratories, UT, USA) to detect proteoglycan, and then stained with nuclear fast red staining (NFS125; ScyTek). For collagen type 2 (COL2) and collagen type 10 (COL10) detection, the sections were deparaffinized, hydrated, and treated with 0.4 mg/ml proteinase K for 15 min, and 3 % hydrogen peroxide (Sigma-Aldrich) was used to block the endogenous peroxidase activity. After washing and blocking, the sections were incubated with the primary antibodies against COL2 (MAB8887; Millipore Corporation, Burlington, MA, USA) or COL10 (ab58632; Abcam, Cambridge, MA, USA) overnight at 4°C, and incubated with the secondary anti-rabbit polymer–horseradish peroxidase (HRP) in the Super Sensitive™ Polymer-HRP Detection Systems (QD420-YIKE, Biogenex, San Ramon, CA, USA) for 30 min. The 3,3′-diaminobenzidine (DAB) substrate and hematoxylin were used to reveal the staining. The staining intensities in the areas of the adult and infant BMSC-differentiated pellets were quantified by the image analysis software (ImageJ, National Institute of Health, USA), and the values were compared to calculate the relative fold.

Osteogenic Differentiation

The adult and infant BMSCs at passages 3–5 were treated in an induction medium to induce osteogenic differentiation. The medium was an α-MEM supplemented with 10% FBS, 50 μg/ml ascorbic acid-2 phosphate (Nacalai, Kyoto, Japan), 0.01 μM dexamethasone (Sigma-Aldrich), and 1 mM b-glycerol phosphate (Sigma-Aldrich), and cultured at 37°C and 5% CO_2. The differentiation was monitored and harvested at days 7, 14, and 21 for RT-qPCR detection during the induction.

Alizarine Red S Staining

The osteogenic differentiated cells were fixed and stained with ARS (A5533; Sigma-Aldrich), an anthraquinone dye that was used to detect calcium deposits. The quantification assay was applied to determine the osteogenesis induction of the adult and infant BMSCs by extracting the ARS from the stained cells with a 10% cetylpyridinium chloride (CPC) buffer, and the optical density (OD) of the extract was measured at 550 nm by an enzyme-linked immunosorbent assay (ELISA) reader (Spectra MAX 250; Molecular Devices, Sunnyvale, CA, USA).

Tenogenic Differentiation

The adult and infant BMSCs at passages 3–5 were induced in a medium that contained Dulbecco’s modified Eagle medium with low glucose (LG-DMEM) with 10% FBS, 50 μg/ml ascorbic acid (AA; Sigma-Aldrich), and 100 ng/ml connective tissue growth factor (CTGF) ²² , and maintained at 37°C and 5% CO₂ and monitored at days 7, 14, and 21 for RT-qPCR detection during the induction.

Picro-Sirius Red Staining

The total collagen deposition of the differentiated cells was evaluated at the cell density of 3 × 10³ cells/cm². Samples were fixed in Bouin’s solution (Bouin’s Fixative, Electron Microscopy Sciences, USA) for 1 h and collagen fibers stained with 0.1% Picro-Sirius Red (ScyTek Laboratories) saturated in picric acid (Sigma-Aldrich). The collagen matrix deposition was finally visualized under polarized light microscopy ²³ . After staining, the Picro-Sirius Red from the stained cells were extracted with a 0.2 M NAOH and methanol (1:1) buffer to quantify the stained collagen, and the OD of the extract was measured at 550 nm using an ELISA reader (Spectra MAX 250; Molecular Devices, Sunnyvale, CA).

Adipogenic Differentiation

The adult and infant BMSCs at passages 3–5 were induced in an induction medium that contained an α-MEM supplemented with 10% FBS, 50 μg/ml ascorbic acid-2 phosphate, 0.1 μM dexamethasone, 50 μM indomethacin (Sigma-Aldrich), 45 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), and 1 μg/ml insulin (Sigma-Aldrich), which were applied to induce adipogenic differentiation at 37°C and 5% CO₂ and monitored at days 7, 14, and 21 for RT-qPCR detection during the induction.

Oil Red O Staining

The differentiated cells were fixed in 10% formalin for over 1 h at room temperature and stained with Oil Red-O (O9755; Sigma-Aldrich) for 2 h. The Oil Red-O working solution was prepared by mixing 15 ml of a stock (0.5% in isopropanol) and 10 ml of distilled water, and filtered through a polyvinylidene difluoride (PVDF) membrane (0.2 µm) filter. After staining, the Oil Red-O from the stained cells was extracted with isopropanol to quantify the lipid accumulation, and the OD of the extract was measured at 510 nm using an ELISA reader (Spectra MAX 250).

Hepatogenic Differentiation

The adult and infant BMSCs were induced in a step 1 induction medium that was an LG-DMEM containing the 20 ng/ml human epidermal growth factor (hEGF, Sigma-Aldrich) and a 10 ng/ml human fibroblast growth factor-basic (hBFGF, Sigma-Aldrich) at 37°C and 5% CO₂ for 24 h. Then, the differentiation was treated with a step 2 induction medium comprising the LG-DMEM supplemented with the 20 ng/ml human hepatocyte growth factor (hHGF, Sigma-Aldrich), 10 ng/ml hBFGF, and 0.61 mg/ml nicotinamide (Sigma-Aldrich) for 7 days. The step 3 induction medium, which contained the LG-DMEM supplemented with 20 ng/ml oncostatin (R&D System), 10⁻⁶ M dexamethasone (Sigma-Aldrich), and ITS-premix (Corning life science), was used for induction for 7 days ²⁴ . The total process of differentiation lasted 15 days. The differentiation was monitored at days 2, 9, and 16 for RT-qPCR detection during the induction.

Neurogenic Differentiation

The adult and infant BMSCs at passages 3–5 were induced by an LG-DMEM supplemented with 50 μM all-trans-retinoic acid (RA; Sigma-Aldrich) and 1 mM 2-mercaptoethanol (β-ME; Sigma-Aldrich) in a 37°C and 5% CO₂ incubator for 24 h ²⁵ . Then, the differentiated cells were maintained with the LG-DMEM containing 1% FBS and monitored from 1, 3, 5, and 7 days for RT-qPCR detection.

Immunofluorescence Staining and Quantification

Primary antibodies against albumin (Taiclone Biotech Corp., Taipei, Taiwan), microtubule-associated protein 2 (MAP2; GTX11267; GeneTex Inc.) and neurofilament heavy polypeptide (NEFH; BM4078S; OriGene Technologies, Medical Center Drive, MD, USA) at appropriate dilution were incubated with the differentiated cells on slides, then the cells were incubated with DyLight 488-conjugated goat anti-mouse IgG (A90-516D2; Bethyl Laboratories Inc., Montgomery, TX, USA) secondary antibodies. Each sample was counterstained with DAPI (Vector Labs) and detected by an Olympus BX43 microscope (Tokyo, Japan). Immunofluorescence intensities were quantified by the ImageJ.

Statistical Analysis

The quantitative analysis of results was performed by Prism (version 5.03, GraphPad, La Jolla, California, USA). Data were exhibited in mean ± standard error. Statistical significance of the data was determined by the Mann–Whitney U test, and a result was considered significant when the P value was less than 0.05.

Cell Proliferation of the Adult and Infant Human BMSC

According to the criteria established by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT), mesenchymal stem cells (MSCs) are required to adhere to plastic in culture conditions, display distinct cell surface markers, and demonstrate differentiation potential toward osteogenic, adipogenic, and chondrogenic lineages ²⁶ . The flow cytometry analysis confirmed the cell-surface protein profiles of both adult and infant BMSCs. Both cell types exhibited positive expression for CD29, CD44, CD73, CD90, and CD105, while they were negative for CD11b, CD14, CD19, CD34, CD45, CD79a, and HLA-DR (as shown in Fig. 1A). The fold increase in cell number of infant BMSCs was significantly higher than that of adult BMSCs from passages 2–6 and passages 8–9 (Fig. 1B). In addition, the cell doubling time of infant BMSCs remained consistent from passage 2–9 and was significantly shorter than adult BMSCs at passages 2–6 and passages 8–9 (Fig. 1C). To visualize the cellular morphologies and densities, images were captured at early stage (passage 3) and late stage (passage 7) (Fig. 1D). These images further confirmed the cells’ ability to adhere to plastic substrates.

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Figure 1.

Proliferation and replicative stress of adult and infant BMSCs. (A) Human adult and infant BMSCs were isolated and analyzed using antibodies against CD29, CD44, CD73, CD90, and CD105 showed positive expression. Antibodies against CD11b, CD19, CD34, CD45, CD14, CD79a, and (HLA-DR) showed negative expression. Flow cytometry analysis was performed using the FACSCanto II Cytometer System, and isotype control staining was used as a control. (B) The fold increase in cell number was calculated based on the consistent seeding density of 1 × 10⁵ cells per dish, with passages conducted every 7 days. (C) The cell doubling time was computed for both adult and infant BMSCs across passages 2 (P2) to 9 (P9). The data presented here represent the mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Statistical significance between adult and infant BMSCs was determined using the Mann–Whitney U test. Statistical significance is indicated by *P < 0.05. (D) Phase contrast microscopy images of adult and infant BMSCs at passages 3 and 7 were captured at 40× magnification. The scale bar represents 200 μm. BMSC: bone marrow–derived mesenchymal stem cell. HLA-DR: human leukocyte antigen-DR.

Senescence, Replicative Stress, and SOD Expression in the Adult and Infant BMSCs

Compared with adult BMSCs, markers of senescence, such as cyclin-dependent kinase inhibitor 2A (p16, Fig. 2A), cyclin-dependent kinase inhibitor 1A (p21, Fig. 2B), and p53 (Fig. 2C) were significantly downregulated in infant BMSCs at early stages (passages 3–5). Conversely, the telomerase reverse transcriptase (TERT), responsible for maintaining genomic integrity, was significantly upregulated in infant BMSCs at both early and late stages (Fig. 2D). The expression levels of p16, p21, and p53 in adult BMSCs during late stages were all significantly higher than those observed during early stages. Moreover, the expressions of p21 and p53 in infant BMSCs during late stages were significantly elevated compared with their levels during early stages. Cellular senescence was evaluated using SA-β-gal assays, which resulted in the appearance of a blue color (Fig. 2E). The percentage of SA-β-gal positive adult BMSCs at passage 8 (6%) was significantly higher than that of infant BMSCs (2%) (Fig. 2E). Replicative stress was assessed by analyzing the presence of γH2AX, a marker of replicative stress, in the nuclei of adult and infant BMSCs at passage 7, which appeared as green fluorescence (Fig. 2F). The quantified fluorescent intensities of infant BMSCs were significantly lower than those of adult BMSCs (Fig. 2F). Although the proliferation rate of adult and infant BMSCs at passage 7 did not show a significant difference, these results suggested that adult BMSCs experienced higher levels of replicative stress.

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Figure 2.

Senescence-related genes expression in adult and infant BMSC. The expression levels of (A) p16, (B) p21, (C) p53, and (D) TERT genes in adult and infant BMSCs at early passages (P3–P5) and late passages (P7–P9) were analyzed using RT-qPCR. The expression values were normalized to the reference gene GAPDH. The data are presented as mean ± SEM from three experimental replicates. Using the data from adult BMSCs in the early stage as the baseline, we calculated the relative fold change of adult BMSCs in the late stage and infant BMSCs in both early and late stages. The statistical comparisons of differences were individually analyzed using Mann–Whitney U analysis for the following groups: differences between adult and infant BMSCs in the early passage, differences between adult and infant BMSCs in the late passages, differences between adult BMSCs in the early and late stages, and statistical comparisons between infant BMSCs in the early and late stages. (E) SA-β-gal staining was performed using a Senescence Detection Kit (BioVision, USA) to detect cell senescence in adult and infant BMSCs at passage 8. SA-β-gal positive BMSCs exhibited a blue color upon staining. The cells were visualized using an Olympus BX43 microscope at 200× magnification, and the scale bar represents 150 μm. The percentage of SA-β-gal positive cells was quantified. (F) Immunofluorescence staining was performed on cells at passage 7 using an anti-γH2AX antibody (green) and DAPI (blue). The stained cells were visualized using an Olympus BX43 microscope at 400× magnification, and the scale bar represents 50 μm. The immunofluorescence intensity of γH2AX in the nuclei of adult and infant BMSCs was quantified using Image-Pro Plus v4.5.0.29, normalized to the cell numbers, and presented as fluorescence intensity (arbitrary unit, a.u.). The mean ± SEM calculated from three experimental replicates within both the infant and adult groups. BMSC: bone marrow–derived mesenchymal stem cell; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SEM: standard error of the mean; SA-β-gal: Senescence-associated β-galactosidase; DAPI: 4′,6-diamidino-2-phenylindole; RT-qPCR: real-time quantitative polymerase chain reaction; TERT: telomerase reverse transcriptase. Statistical significance is indicated by *P < 0.05; ***P < 0.001.

The expression of superoxide dismutase (SOD) genes, specifically SOD1, SOD2, and SOD3, was examined in both adult and infant BMSCs at early and late stages. The expression levels of all three SODs were significantly higher in infant BMSCs compared with adult BMSCs at early and late stages (Fig. 3A–C). Furthermore, in both infant and adult BMSCs, the expression levels of SOD1, SOD2, and SOD3 were significantly downregulated during the late stages when compared with the early stages (Fig. 3A–C). The gene expressions of glutathione synthesis gene (GCLM) and catalase (CAT), along with other antioxidation-related genes, were also detected (Fig. S1 in the supplemental material). However, the expression levels of both genes in infant BMSCs were notably lower than those in adult BMSCs during the early stages. Furthermore, CAT expression in infant BMSCs showed a significant decrease during the late stages. In comparison to the early stages, GCLM expression in adult BMSCs during the late stages exhibited a significant downregulation. In addition, GCLM and CAT gene expressions in infant BMSCs during the late stages were significantly lower than those in adult ones observed during the early stages.

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Figure 3.

Superoxide dismutase (SODs) expressions in adult and infant BMSC. The expression levels of (A) SOD1, (B) SOD2, and (C) SOD3 genes in adult and infant BMSCs at early passages (P3–P5) and late passages (P7–P9) were analyzed using RT-qPCR. The expression values were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Using the data from adult BMSCs in the early stage as the baseline, the relative fold change of adult BMSCs in the late stage and infant BMSCs in both early and late stages were calculated. The statistical comparisons of differences were individually analyzed using Mann–Whitney U analysis for the following groups: differences between adult and infant BMSCs in the early passage, differences between adult and infant BMSCs in the late passages, differences between adult BMSCs in the early and late stages, and statistical comparisons between infant BMSCs in the early and late stages. BMSC: bone marrow–derived mesenchymal stem cell; RT-qPCR: real-time quantitative polymerase chain reaction. The significance levels are indicated by *P < 0.05.

Chondrogenic Differentiation in Adult and Infant BMSCs

After 21 days of chondrogenic differentiation, infant BMSCs formed larger chondrogenic pellets compared with adult BMSCs (Fig. 4A). The mean diameter of the chondrogenic pellets derived from infant BMSCs was approximately 0.18 cm, which was significantly larger than the pellets derived from adult BMSCs, measuring about 0.15 cm (Fig. 4B). The expression levels of chondrogenic genes, including SRY-box transcription factor 9 (SOX9), COL2, and COL10, were monitored in the chondrogenic pellets derived from adult and infant BMSCs from days 7 to 21. The fold increase in the expression of SOX9, COL2, and COL10 in the chondrogenic pellets derived from infant BMSCs was significantly higher than those derived from adult BMSCs on day 21 (Fig. 4C–E). In addition, on day 14, the expression of COL2 in the chondrogenic pellets derived from infant BMSCs was also significantly higher than that in adult BMSC-derived pellets (Fig. 4E). To evaluate the content of glycosaminoglycans (GAGs), the 21-day differentiated chondrogenic pellets derived from adult and infant BMSCs were stained with Alcian blue (Fig. 5A, B). Immunohistochemistry staining was performed to assess the protein expressions of COL2 (Fig. 5C, D) and COL10 (Fig. 5E, F) in the pellets. The staining intensities were quantified using ImageJ for relative expression analysis. The Alcian blue staining intensities of the chondrogenic pellets derived from infant BMSCs were significantly higher than those derived from adult BMSCs (Fig. 5A, B). Moreover, both the relative expressions of COL2 and COL10 in the pellets derived from infant BMSCs were significantly higher than those in the pellets derived from adult BMSCs (Fig. 5H, I).

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Figure 4.

Chondrogenic differentiation of adult and infant BMSC. (A) Chondrogenic pellets were generated from adult and infant BMSCs at passages 3–5, with 3 × 10⁵ cells per pellet, and cultured for 21 days. The image shows representative chondrogenic pellets, with a scale bar indicating 1 mm. (B) The diameters of the chondrogenic pellets were measured on day 21. (C) The gene expression of SOX9, (D) COL2, and (E) COL10 in the chondrogenic pellets differentiated from adult and infant BMSCs was analyzed by RT-qPCR from days 7 to 21. The expression values were normalized to the reference gene (GAPDH). The gene expression levels in undifferentiated cells were used as controls for comparison with adult and infant BMSC-differentiated chondrogenic cells, and the results were presented as relative fold changes. The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Statistical significance between infant and adult BMSC-differentiated pellets was determined using the Mann–Whitney U test. BMSC: bone marrow–derived mesenchymal stem cell; GAPDH: glyceraldehyde-3-phosphate dehydrogenase RT-qPCR: real-time quantitative polymerase chain reaction. The significance levels are indicated by *P < 0.05; **P < 0.01.

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Figure 5.

Immunohistochemistry staining of adult and infant BMSC-differentiated pellets. The chondrogenic pellets differentiated from adult and infant BMSCs for 21 days were sectioned and stained with Alcian blue (A and B). Immunostaining was performed to detect the protein expressions of collagen type 2 (COL2) (C and D) and collagen type 10 (COL10) (E and F) in the pellets. Representative images were captured at a magnification of ×40, and a scale bar indicating 2 mm was included. The densities of each staining were calculated and normalized to the background of the entire pellet sections (G–I). The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Statistical significance between adult and infant BMSCs was determined using the Mann–Whitney U test. BMSC: bone marrow–derived mesenchymal stem cell. The significance levels are indicated by *P < 0.05; **P < 0.01.

Osteogenic Differentiation in Adult and Infant BMSCs

To assess the osteogenic differentiation of adult and infant BMSCs, the relative fold changes in the expression of osteocyte-related genes, including alkaline phosphatase (ALP) (Fig. 6A) and osteopontin (OPN) (Fig. 6B), were measured from days 7 to 21. The expression of ALP on day 14 and OPN on day 21 in the differentiated osteogenic cells derived from infant BMSCs was significantly higher than that in adult BMSCs (Fig. 6A, B). Calcium deposits in the osteogenic cells differentiated from adult and infant BMSCs on day 21 were stained with Alizarin Red S (ARS) (Fig. 6C). The ARS staining was dissolved in a buffer, and the absorbance at 550 nm was measured to calculate the relative fold change in staining intensity. The results indicated a significantly higher intensity of ARS staining in the osteogenic cells derived from infant BMSCs compared with those derived from adult BMSCs (Fig. 6D).

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Figure 6.

Detection of osteocyte-related gene expressions of adult and infant BMSCs during differentiation using OIM. (A) The expression of ALP and (B) OPN genes in adult and infant BMSCs during osteogenic differentiation from day 7 to day 21 was detected using RT-qPCR. The expression values of each gene were normalized to the expression of GAPDH. Undifferentiated cells were used as a control to compare the gene expression of adult and infant BMSCs after differentiation, showing the relative fold change. (C) Undifferentiated adult and infant BMSCs were stained with ARS as a control. The 21-day osteogenic differentiated adult and infant BMSCs were confirmed using ARS staining and observed under a Nikon eclipse TS100 microscope (magnification × 200, scale bar = 25 μm). (D) The ARS stain was extracted from the stained cells, and the optical density (OD) was measured at a wavelength of 550 nm. The OD values of the differentiated cells were normalized to the control values set as 1 (not shown in the figure), demonstrating the relative fold changes in comparison to the control. The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Statistical significance of comparing adult and infant BMSCs in osteogenic differentiation was determined using the Mann–Whitney U test. BMSC: bone marrow–derived mesenchymal stem cell; ALP: alkaline phosphatase; OPN: osteopontin; OIM: osteogenic induction medium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ARS: Alizarin Red S; RT-qPCR: real-time quantitative polymerase chain reaction. The significance levels are indicated by *P < 0.05; ***P < 0.001.

Tenogenic Differentiation in Adult and Infant BMSCs

During tenogenic differentiation, the expression levels of matrix metallopeptidase 3 (MMP3) (Fig. 7A) and decorin (DCN) (Fig. 7B) genes in adult and infant BMSCs were monitored from days 7 to 21. The relative fold changes in gene expression in the tenogenic cells differentiated from infant BMSCs were significantly higher than those in adult BMSCs throughout the entire differentiation period (days 7–21) (Fig. 7A, B). To visualize the presence of collagens in the 21-day differentiated tenogenic cells derived from adult and infant BMSCs, Picro-Sirius Red staining was performed, and collagen fibers appeared in red (Fig. 7C). The Picro-Sirius Red staining was dissolved using a buffer, and the absorbance at 550 nm was measured using an ELISA reader. The observed fold changes in collagen content of the differentiated cells from infant BMSCs were comparable with those derived from adult BMSCs (Fig. 7D).

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Figure 7.

Detection of tendon-related gene expressions of adult and infant BMSCs during differentiation using TIM. (A) The expression of DCN and (B) MMP3 genes in adult and infant BMSCs during tenogenic differentiation from days 7 to 21 was detected using RT-qPCR. The expression values of each gene were normalized to the expression of GAPDH. Undifferentiated cells were used as a control to compare the gene expression of adult and infant BMSCs after differentiation, showing the relative fold change. (C) Undifferentiated adult and infant BMSCs were stained with Picro-Sirius Red as a control. The 21-day tenogenic differentiated adult and infant BMSCs were confirmed using Picro-Sirius Red staining and observed under a Nikon eclipse TS100 microscope (magnification × 40, scale bar = 50 μm). (D) The Picro-Sirius Red stain was extracted from the stained cells, and the optical density (OD) was measured at a wavelength of 550 nm. Subsequently, the OD values of the differentiated cells were normalized against the control values set as 1 (not shown in the figure), presenting the relative fold changes compared with the control. The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Statistical significance in the comparison of tenogenic differentiation between adult and infant BMSCs was determined using the Mann–Whitney U test. BMSC: bone marrow–derived mesenchymal stem cell; DCN: decorin; MMP3: matrix metallopeptidase 3; TIM: tenogenic induction medium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR: real-time quantitative polymerase chain reaction. Significance levels are indicated by *P < 0.05; **P < 0.01.

Adipogenic Differentiation in Adult and Infant BMSCs

To assess adipogenic differentiation, the expression of adipogenic genes, including peroxisome proliferator-activated receptor γ (PPARγ) (Fig. 8A) and lipoprotein lipase (LPL) (Fig. 8B), was examined from days 7 to 21. The fold changes in PPARγ expression in the cells differentiated from adult BMSCs were significantly higher than those in infant BMSCs throughout the differentiation period (days 7–21). Similarly, the expression of LPL at days 14 and 21 in the differentiated cells derived from adult BMSCs was significantly higher than that in cells derived from infant BMSCs. To visualize the accumulation of lipids, Oil Red O staining was performed on both differentiated and undifferentiated cells from adult and infant BMSCs on day 21 (Fig. 8C). The relative fold change in the intensity of Oil Red O staining in the adipogenic cells derived from adult BMSCs on day 21 showed a significant increase compared with those in cells derived from infant BMSCs (Fig. 8D).

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Figure 8.

Detection of adipose-related gene expressions of adult and infant BMSCs during differentiation using AIM. (A) The expression of PPARγ and (B) LPL genes in adult and infant BMSCs after 7–21 days of factor-induced adipogenic differentiation was detected using RT-qPCR. The expression values of each gene were normalized to the expression of GAPDH. Undifferentiated cells were used as a control to compare the gene expression of adult and infant BMSCs after differentiation, showing the relative fold change. (C) Undifferentiated adult and infant BMSCs were stained with Oil Red O as a control. The presence of lipid droplets in adult and infant BMSCs after 21 days of adipogenic differentiation was confirmed using Oil Red O staining and observed under a Nikon Eclipse TS100 microscope. (Magnification × 200, scale bar = 25 μm). (D) The Oil Red O stain was extracted, and the optical density (OD) was measured at a wavelength of 510 nm. Subsequently, the OD values of the differentiated cells were adjusted against the OD values of undifferentiated cells, which were set as 1 (not shown in the figure), illustrating the relative fold changes compared with the control. The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Statistical significance of comparing adult and infant BMSCs in adipogenic differentiation was determined using the Mann–Whitney U test. BMSC: bone marrow–derived mesenchymal stem cell; PPARγ: peroxisome proliferator-activated receptor-γ; LPL: lipoprotein lipase; AIM: adipogenic induction medium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR: real-time quantitative polymerase chain reaction. The significance levels are indicated by *P < 0.05; ***P < 0.001.

Hepatogenic Differentiation in Adult and Infant BMSCs

The expression fold changes of alpha-fetoprotein (AFP) (Fig. 9A) and albumin (ALB) (Fig. 9B), which serve as hepatogenic markers, were monitored in cells differentiated from adult and infant BMSCs from days 2 to 16. The AFP expression on day 9 and ALB expression on day 2 in the differentiated cells derived from infant BMSCs were significantly higher than those derived from adult BMSCs. To visualize the expression of the albumin protein, immunofluorescence staining was performed (Fig. 9C). The quantified expression levels in the hepatogenic cells differentiated from infant BMSCs were higher than those in adult BMSCs, although the difference was not statistically significant (Fig. 9D).

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Figure 9.

Detection of hepatocyte-related gene expressions of adult and infant BMSCs during differentiation using HIM. (A) The expression of AFP and (B) matrix ALB genes in adult and infant BMSCs after 13-15 days of factor-induced hepatogenic differentiation was detected using RT-qPCR. The expression values of each gene were normalized to the expression of GAPDH. Undifferentiated cells were used as a control to compare the gene expression of adult and infant BMSCs after differentiation, showing the relative fold change. (C) The presence of albumin protein in adult and infant BMSCs was detected using immunofluorescence staining, represented by green fluorescence, and evaluated using an Olympus BX43 microscope. Nuclear DNA stained by DAPI is shown in blue fluorescence. (Magnification × 400, scale bar = 100 μm). (D) Immunofluorescence intensity was quantified using ImageJ. The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Undifferentiated adult and infant BMSCs were stained with DAPI and antibody, establishing the control set at 1 (not shown in the figure), demonstrating the relative fold changes of differentiated cells compared with the control. Statistical significance of comparing adult and infant BMSCs in hepatogenic differentiation was determined using the Mann–Whitney U test. BMSC: bone marrow–derived mesenchymal stem cell; AFP: alpha-fetoprotein; ALB: albumin; HIM: hepatogenic induction medium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4′,6-diamidino-2-phenylindole; RT-qPCR: real-time quantitative polymerase chain reaction. Significance levels are indicated by *P < 0.05.

Neurogenic Differentiation in Adult and Infant BMSCs

To confirm neurogenic differentiation, the expression of MAP2 and NEFH genes was examined in adult and infant BMSCs from days 7 to 21. The fold changes in MAP2 expression in the differentiated cells derived from infant BMSCs were significantly higher on days 1 and 5 than those from adult BMSCs (Fig. 10A). Similarly, the fold changes in NEFH expression in the neurogenic cells differentiated from infant BMSCs were significantly higher on days 3 and 5 than those from adult BMSCs (Fig. 10B). To visualize the protein expression levels of MAP2 and NEFH, immunofluorescence staining was performed (Fig. 10C). The intensities of immunofluorescence staining for MAP2 and NEFH in the differentiated cells were quantified and normalized with the cell numbers. The intensities of both proteins in the neurogenic cells derived from infant BMSCs were significantly higher than those derived from adult BMSCs (Fig. 10D, E).

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Figure 10.

Detection of neuron-related gene expressions of adult and infant BMSC during differentiation using NIM. (A) The expression of MAP2 and (B) NEFH genes in adult and infant BMSCs after 1-7 days of factor-induced neurogenic differentiation was detected using RT-qPCR. The expression values of each gene were normalized to the expression of GAPDH. Undifferentiated cells served as a control to compare the gene expression of adult and infant BMSCs after differentiation, showing the relative fold change. (C) Undifferentiated adult and infant BMSCs were stained with DAPI and antibodies as a control. The presence of MAP2 and NEFH proteins in adult and infant BMSCs after neurogenic differentiation was detected using immunofluorescence staining, which appeared as green fluorescence, and observed under an Olympus BX43 microscope. Nuclear DNA stained with DAPI was shown in blue fluorescence. (Magnification × 200, scale bar = 100 μm). (D and E) Immunofluorescence intensity was quantified using Image-Pro Plus v4.5.0.29. The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. The undifferentiated adult and infant BMSCs were designated as the control set at 1 (not shown in the figure), illustrating the relative fold changes of differentiated cells in comparison to the control. Statistical significance of comparing adult and infant BMSC-differentiated cells was determined using the Mann–Whitney U test. BMSC: bone marrow–derived mesenchymal stem cell; MAP2: microtubule-associated protein 2; NEFH: neurofilament heavy polypeptide; NIM: neurogenic induction medium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4′,6-diamidino-2-phenylindole; RT-qPCR: real-time quantitative polymerase chain reaction. The significance levels are indicated by *P < 0.05; ***P < 0.001.