Increased proliferation and differentiation capacity of placenta-derived mesenchymal stem cells from women of median maternal age correlates with telomere shortening

PDMSCs isolated from donors 22-35 years of age showed higher self-renewal properties and proliferative capacity

To analyze the effect of maternal age on the self-renewal and proliferative capacity of PDMSCs, we performed CGC, CPD and CFU-F assays. The results showed that cells derived from maternal age group A presented a slower growth rate than cells from the higher age groups (Figure 1A). Groups B, C, D and E presented a similar growth pattern until day 6, when group E showed a significant (p<0.05) decline in growth rate compared to those of groups B, C and D.

Figure 1. Proliferative capacity of placenta-derived mesenchymal stem cells from different maternal age group. (A) Cell growth curve showing 5 age groups. Cells were seeded at 1x10⁴ per cm² in 24-wells plates and counted every 48 hours for 10 days. (B) Cumulative Doubling Population curve showing 5 age groups. Cells were seeded at 1x10⁴ per cm² in 24-wells plates and subculture every 4 to 5 days. Population Doublings (PD) was calculated using the formula ΔPD= log(total cell number/initial cell number)log2. Each dot represents one passage. Bar graph represents the cumulative population doubling among the 5 age groups at day 46. (C) MTT cell proliferation assay performed using Thiazolyl Blue Tetrazolium Bromide in a 96-well ELISA plate. (D) Graph showing Colony Forming Unit-Fibroblast (CFU-F) assay of 5 age group. 1x10³ cells were seeded in 12-wells plates and allowed to grow for 8 days, before staining with Crystal Violet. Colonies with more than 40 cells were counted. (E) Graph showing the number of cells in colonies counted for the CFU-F of 5 age groups. (F) Representative image showing cellular morphology of 5 age groups at 4 days of culture. (G) Images represent crystal violet staining in cells from each maternal age group. Group A: 18-21; Group B: 22-25; Group C: 26-30; Group D: 31-35; Group E: 36 and over. *, p<0.05; **, p<0.01; ***, p<0.0005; ****, p<0.0001. Scale bar 200 μm.

CPD data showed that groups A and E presented significantly (p<0.0005) lower doubling capacity than cells from groups B, C and D (Figure 1B). Results from the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay confirmed our observations of PDMSCs isolated from donors 22-35 years of age showing higher self-renewal properties and proliferative capacity (Figure 1C).

Following cell expansion, adherent PDMSCs obtained from all 5 maternal age groups revealed a similar spindle-shaped, fibroblastic morphology, which corresponds to the typical appearance of MSCs in primary culture at passage 4 (Figure 1F).

CFU-F assays were carried out to quantify the colony formation capacity of cells from all 5 maternal age groups. Cells were seeded at 1x10³ and allowed to grow for 8 days; after staining with crystal violet, colonies with cell numbers <40 were counted. The results showed that Group C presented higher colony formation capacity (Figure 1D, 1E) than the other groups, whereas groups A and E showed the lowest number of colonies formed. Furthermore, when the number of cells per colony was counted (Figure 1G), Group C consistently showed more cells per colony than the other maternal age groups. Colony formation capacity correlated with the results obtained for CGC and CPD. Taken together, these results demonstrated that PDMSCs isolated from mothers aged 22-35 years have better self-renewal and proliferative capacity than cells isolated from mothers aged 18-21 or >36 years.

PDMSCs isolated from donors 22-35 years of age presented greater expression of pluripotency and multipotency markers

Since the expression of multipotency and pluripotency markers is a major characteristic of PDMSCs, we next decided to examine the effect of maternal age on these markers. Flow cytometry results revealed specific staining for each of the positive markers of MSC stemness (Figure 2A, 2B), while the expression of the negative markers CD45 and CD3 (Figure 2C) was undetectable. No expression was detected in isotype controls (Supplementary Figure 1). We then measured the difference in the mean fluorescence expression of pluripotency and multipotency markers among all 5 age groups (Figure 2D, 2E) and observed significantly (p<0.0001) reduced expression in age groups 18-21 and >36 compared to age group 22-35.

Figure 2. Characterization of placenta-derived mesenchymal stem cells based on surface and nuclear markers expression from different maternal age group. (A) Representative histograms of detection by fluorescence-activated cell sorting (FACS) demonstrates positive expression of three MSC pluripotency intracellular markers. Populations are more than 95% positive for SSEA4 and NANOG. (B) Representative histograms of detection by FACS demonstrates positive expression of three MSC multipotency surface markers. Populations are more than 99% positive for CD90, CD73 and CD105. (C) Representative histograms showing no detection by FACS demonstrates no expression of the immunologic markers CD45 and CD3. Populations are more than 99% negative for CD45 and CD3. (D) Graphs of difference in mean fluorescence of MSC pluripotency intracellular markers of all 5 age groups. (E) Graphs of difference in mean fluorescence of MSC multipotency surface markers of all 5 age groups. (F) Graphs showing difference in expression of MSC pluripotency intracellular transcripts NANOG, OCT4 and SOX2 gene expression measure by Real Time PCR of all 5 age groups. Group A: 18-21; Group B: 22-25; Group C: 26-30; Group D: 31-35; Group E: 36 and over. *, p<0.05; **, p<0.01; ***, p<0.0005; ****, p<0.0001.

To ensure that the observed changes in intracellular pluripotency marker expression were consistent at the transcriptional level, we validated our findings by RT-qPCR. Gene expression analysis of OCT4, NANOG and Sox2 showed significantly (p<0.05) reduced expression in the 18-21 and >36 age groups compared to the 22-35 age group, consistent with our flow cytometry observations (Figure 2F).

Overall, while all age groups of PDMSCs maintained their multipotency under standard culture conditions, cells from groups A and E presented reduced plasticity given their decreased marker expression compared to that of groups B, C, and D.

PDMSCs isolated from donors 26-35 years of age have higher mesenchymal lineage differentiation potential

Based on the results obtained for the expression of pluri- and multipotency markers as an indicator of cell plasticity in PDMSCs among all 5 groups, we then induced mesenchymal multilineage differentiation to understand the effect of maternal age. All groups included in this study were differentiated into chondrogenic, adipogenic and osteogenic lineages. Chondrocytes stained positive for Alcian Blue, in contrast to undifferentiated controls (Figure 3A), indicating the synthesis of proteoglycans by chondrocytes. We then confirmed chondrogenic differentiation by RT-qPCR analysis of the gene expression of the chondrogenic markers Aggrecan (ACAN) and the transcription factor SOX9. ACAN showed significantly (p<0.0001) higher expression (~2.5- to ~3.5-fold change) (Figure 3B) along with a >20-fold increase in the transcription factor SOX9 expression (Figure 3C), in groups C and D compared to the other groups, consistent with the Alcian blue staining. Oil Red O, a dye that stains triglyceride and lipid deposits, showed positive staining for adipocyte-differentiated cells, in contrast to the undifferentiated controls (Figure 4A). Adipocyte lipid-binding protein (ALBP) gene expression confirmed adipogenic differentiation, with significantly (p<0.0.0001) higher expression (~7-fold change) in cells from group C than in cells from the other groups (Figure 4B), as well a 6-fold increase of the fatty acid-binding protein 4 (FABP4) and >3.8-fold of CCAAT/enhancer-binding protein beta (CEBPB) in group B, C, or D (Figure 4C, 4D). Osteocytes stained positive for Alizarin Red (Figure 5A), a stain that identifies calcium-containing osteocytes in differentiated culture. Osteocalcin gene expression confirmed osteocyte differentiation (Figure 5B) and showed significantly (p<0.0.001) higher expression in groups D (~4-fold change), A (~3-fold change), and group C (~2-fold change) than in the other groups. The expression of other osteogenic differentiation markers, such as collagen type 1 (COL1) and Runt-related transcription factor 2 (RUNX2), were highly upregulated in groups B, C, and D (Figure 5C, 5D), with more than 400-times increase of osteopontin (OPN) gene expression in group D (Figure 5E). Taken together, the results showed that although groups B, C and D presented higher expression of pluri- and multipotency markers, after induction of differentiation, groups B, C and D showed the highest differentiation potential, and group A also showed an elevated capacity for osteogenic differentiation compared to that of the undifferentiated controls.

Figure 3. Chondrogenic differentiation ability of PDMSCs from different maternal age group. (A) Representative image of chondrogenic differentiation of all 5 age groups. Upper panel showed that undifferentiated cells did not retain Alcian blue staining. Lower panel showed that differentiated cells were positive to Alcian blue staining. Real Time PCR performed to measure chondrocyte markers gene expression of (B) Aggrecan (ACAN) and (C) SOX9 of all 5 age groups. Group A: 18-21; Group B: 22-25; Group C: 26-30; Group D: 31-35; Group E: 36 and over. *, p<0.05; **, p<0.01; ***, p<0.0005; ****, p<0.0001. Scale bar 200 μm.

Figure 4. Adipogenic differentiation ability of PDMSCs from different maternal age group. (A) Representative image of adipogenic differentiation of all 5 age groups. Upper panel showed that undifferentiated cells did not retain Oil-O Red staining. Lower panel showed that differentiated cells were positive to Oil-O Red staining. Real Time PCR performed to measure the gene expression of (B) Adipocyte lipid binding protein (ALBP) (C) Fatty acid-binding protein 4 (FABP4), and (D) CCAAT/enhancer-binding protein beta (CEBPB) of all 5 age groups. Group A: 18-21; Group B: 22-25; Group C: 26-30; Group D: 31-35; Group E: 36 and over. *, p<0.05; **, p<0.01; ***, p<0.0005; ****, p<0.0001. Scale bar 200 μm.

Figure 5. Osteogenic differentiation ability of PDMSCs from different maternal age group. (A) Representative image of Osteogenic differentiation of all 5 age groups. Upper panel showed that undifferentiated cells did not retain Alizarin staining. Lower panel showed that differentiated cells were positive to Alizarin staining. Real Time PCR performed to measure the gene expression of (B) Osteocalcin, (C) Collagen type 1 (COL1), (D) Runt-related transcription factor 2 (RUNX2), and (E) Osteopontin (OPN) of all 5 age groups. Group A: 18-21; Group B: 22-25; Group C: 26-30; Group D: 31-35; Group E: 36 and over. *, p<0.05; **, p<0.01; ***, p<0.0005; ****, p<0.0001. Scale bar 200 μm.

Telomere length was negatively correlated with PDMSC self-renewal and proliferative capacity in all 5 maternal age groups

The age-related decline in the regenerative potential of MSCs has been correlated with telomerase-related telomere elongation loss of function [19]. Based on this, we decided to investigate whether there was a correlation between increased maternal age and decreasing telomere length in PDMSCs. Telomere length was assessed through RT-qPCR. Surprisingly, the results showed that groups A and E presented longer telomeres than groups B, C and D (Figure 6A). Interestingly, when correlating telomere length with self-renewal and proliferative capacity (Figure 1), groups with reduced proliferative capacity (A and E) had longer telomere length. On the other hand, PDMSCs from mothers aged 22-35 year had higher cell proliferation and shorter telomeres, probably due to their increased cell division (Figure 6B). Measurement of the expression of p21 by RT-qPCR, revealed that no cellular senescence was present among the different maternal age groups (Supplementary Figure 2). These data suggest that the reduced telomere length in PDMSCs could be due to increased cellular proliferation rather than age-related effects. Taken together, our data demonstrated correlations of self-renewal properties, multipotency capacity and differentiation ability with maternal age (Figure 6C), showing that PDMSCs from mothers aged 22-35 years have better regenerative potential for biomedical research and clinical applications.

Figure 6. Maternal age influences PDMSCs telomere length and correlates with MSCs characteristics. (A) Graph showing the telomere length for all 5 age groups (B) Schematic representation of hypothesis for reduced telomere shortening. (C) Graph Showing correlation between age factor and cellular self-renewal capacity, cellular multipotency capacity and cellular differentiation capacity. Group A: 18-21; Group B: 22-25; Group C: 26-30; Group D: 31-35; Group E: 36 and over. *, p<0.05; **, p<0.01; ***, p<0.0005; ****, p<0.0001.

PDMSC isolation, expansion, and maternal age grouping

Human placentas were obtained from full-term pregnancies (38~40 weeks of gestation) with no evidence of prior chronic disease or obstetrical and surgical complications. Informed consent forms were signed by all of the participants after an explanation of the study was provided. This study was approved by the National Committee on Research Bioethics and the Institutional Review Boards of Hospital Santo Tomas and Hospital Nacional. All procedures were in compliance with the Declaration of Helsinki.

MSCs were isolated from the chorionic villi of freshly delivered placentas, as previously published [40]. Briefly, full-term human placentas were obtained from mothers with informed consent, and cell isolation initiated within 40 minutes after placental collection. Chorionic villi tissues were harvested and minced for digestion with Collagenase Type 2 at 37° C for 40min. The digested tissues were filtered and used for density gradient centrifugation to isolate mononuclear cells. Flow cytometry results revealed specific staining for each of the positive markers of MSC stemness, while the expression of the negative markers CD45 and CD3 (Figure 2C) was undetectable, validating the quality of our isolated cells. Isolated cells were seeded at 1 × 10⁴ per cm² in Dulbecco’s modified Eagle’s medium (DMEM)-low glucose with 10% fetal bovine serum (Gibco BRL, Life Technologies, NY, USA) at 37° C with 5% CO₂. PDMSCs (P4, n=3) from three different donors were grouped for pooling according to their respective maternal age (Table 1). After the cells reached 80% confluence, the PDMSCs were seeded on a polystyrene surface for further culture [41, 42].

Proliferation assays, generation time studies, and cell growth curves

To determine the cumulative population doubling (CPD) of PDMSCs, cells were seeded at a cell density of 1 × 10⁴ per cm² and subcultured approximately every 4 to 5 days. The CPD was plotted against time and calculated using the equation ΔPD=log (final total cell number/initial cell number seeded)/log2 [42].

A cell growth curve was generated by seeding the cells at a cell density of 1 × 10⁴ per cm² and then plotting cell number against time. Cell counts were performed using a hematocytometer every 48 hours during a 10-day culture period [42].

PDMSCs derived from the 5 donors age groups were seeded in a 96-well ELISA plate in triplicates. After cells reached 80% confluence MTT assay was performed using Thiazolyl Blue Tetrazolium Bromide (#M5655, SIGMA) diluted in PBS and filtered before used. 50μl of MTT working solution was added to each well and incubated for 4 hours at 37° C with 5% CO2. After incubation, 100μl of detergent reagent was added. Plate was gently shaken to dissolve crystals and absorbance was measure at 570 nm in a microplate reader.

Colony-forming unit-fibroblast (CFU-F) assay

Cells were seeded at a density of 1000 cells/per well in 12-well plates in DMEM supplemented with 10% FBS, and each group was seeded in triplicate. After 8 days of culture, the cells were washed with DPBS, fixed with formaldehyde at 4% and stained with crystal violet for 5 minutes followed by a ddH₂O wash. Colonies with < 40 cells were counted using a hemocytometer [43].

Placenta-derived mesenchymal stem cell marker analysis

The effect of maternal age on the expression of multipotency and pluripotency markers in PDMSCs was analyzed by flow cytometry. PDMSCs were washed with Dulbecco’s phosphate-buffered saline (DPBS, Gibco) and resuspended in fluorescence activated cell sorting (FACS) buffer (Thermo Fisher Scientific) with FITC-conjugated anti-human CD90, PE-conjugated anti-human CD73, and APC-conjugated anti-human CD105 (BioLegend, CA, USA) for 20 minutes at room temperature in the dark. The cells were then washed with FACS buffer and resuspended in fixation buffer (Thermo Fisher Scientific). For intracellular staining, the cell membrane was permeabilized with 1x Perm buffer (BioLegend) for 20 minutes at room temperature. The cells were resuspended in 1x Perm buffer with PE-conjugated anti-human OCT4, FITC-conjugated anti-human SSEA4 and Alexa Fluor® 647-conjugated anti-NANOG antibodies for 2 hours in the dark. The cells were then washed and resuspended in fixation buffer. The fluorescence intensity of 20,000 cells was recorded on a flow cytometer (Guava, Merck) and analyzed using FlowJo software with excitation/emission wavelengths 494/520 for FITC, 496/578 for PE, 650/660 for APC and 650/660 for Alexa Fluor® 647.

Reverse transcription-quantitative polymerase chain reaction

Total RNA was extracted from cell cultures according to the guidelines of the RNeasy Mini Kit manual (Qiagen). Two micrograms of RNA were used to generate cDNA by reverse transcription with SuperScript™ III Reverse Transcriptase according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed using SYBR® Green RT-qPCR master mix (Applied BioSystems, MA, USA). Gene expression levels in the cells were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results are expressed as fold changes relative to the controls. Primer details are listed in Table 2.

In vitro induction of differentiation

Adipogenic, osteogenic and chondrogenic induction of PDMSCs was performed according to manufacturer’s instructions in the StemPro® Adipogenesis, Osteogenesis, and Chondrogenesis Differentiation Kit (Gibco). PDMSCs at passage 2 were seeded on polystyrene surfaces (control) and cultured in low-glucose DMEM-10% FBS for 3 days prior to induction of differentiation. For adipogenic induction, PDMSCs were cultured on StemPro® Adipocyte Differentiation Basal Medium supplemented with StemPro® adipogenesis supplement and gentamycin (10 mg/mL, Gibco) for 7 days. Adipogenesis was assessed by Oil Red O staining (Sigma-Aldrich, MO, USA). For induction of chondrogenic differentiation, PDMSCs were cultured in StemPro® Chondrocyte Differentiation Basal Medium with StemPro® chondrogenesis supplement and gentamycin reagent (10 mg/mL) for 12 days. Chondrogenesis was assessed with 1% Alcian blue stain (Sigma-Aldrich) prepared in 0.1 N HCL. For induction of osteogenic differentiation, PDMSCs were cultured in StemPro® Osteocyte Differentiation Basal Medium with StemPro® osteogenesis supplement and gentamycin reagent (10 mg/mL) for 13 days. Osteogenesis was assessed by Alizarin Red S staining (Sigma-Aldrich). Cells were fixed prior to staining. Images were captured with an inverted light microscope (Leica Microsystems, IL, USA).

Genomic DNA extraction and evaluation of telomere length by RT-qPCR

Genomic DNA was isolated using the Qiagen DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. Telomere length was evaluated by adding 5 ng of DNA into the Absolute Human Telomere Length Quantification PCR Assay Kit (ScienceCells, CA, USA) with SYBR® Green RT-qPCR master mix (Applied BioSystems) according to the manufacturer’s protocol. The 2^-∆∆Cq method was used to calculate the average telomere length. The change in telomere length was derived from the reference sample telomere length (695 ± 16 kb) x 2^-∆∆Cq.

Statistical analysis

All experiments were performed at least in triplicate (n=3). Experimental results are expressed as the mean SD of the samples. Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparison test. Homogeneity of variance was assumed with a 95% confidence interval level. The results for comparisons with at least n=3 and p<0.05 were considered significant. All statistical analyses were performed using GraphPad Prism software Version 6.0.