Materials

Ascorbic acid-2-phosphate, ß-glycerophosphate, calf thymus DNA standard, dexamethasone, Dulbecco’s Modified Eagle’s Medium - high glucose (DMEM-hg), Dulbecco’s phosphate buffered saline (DPBS), gelatin, fetal bovine serum (FBS), 3-isobutyl-1-methylxanthine (IBMX), L-glutamine solution (L-glut), L-proline, phosphate buffered saline (PBS), penicillin-streptomycin (pen-strep), p-nitrophenol standard, trypsin-EDTA (10 x solution), tissue culture water, transforming growth factor-beta 3 human (TGF-β3) and TRI Reagent® were purchased from Sigma-Aldrich (St.Louis, USA). DNase I, GlutaMAX, HyClone™ fetal bovine serum (HyClone-FBS), insulin-transferrin-selenium-ethanolamine supplement mixture (ITS-X), KnockOut™ DMEM (KO-DMEM), β-mercaptoethanol, nonessential aminoacids and sodium pyruvate were purchased from Thermo Fisher Scientific (Waltham, USA). Basic fibroblast growth factor (bFGF) was purchased from PreproTech (Rocky Hill, USA) or Thermo Fisher Scientific.

Isolation and cultivation of human bone marrow-derived MSCs

Human bone marrow MSCs were isolated from the femoral bone marrow of 11 patients (6 women, 5 men, aged 43 to 90 years) undergoing hip replacement surgery at the Lorenz Bohler Unfallkrankenhaus, Vienna, Austria, with informed consent and full ethical approval (Ethics Committee for the AUVA Hospitals No. 1/2005, February 9th 2006). In addition, mononuclear cells from the bone marrow of two young adult donors (20-year-old woman and 22-year-old man) were acquired from Lonza (Basel, Switzerland). An overview of the characteristics of the donors is shown in Fig. 1. For the isolation of MSCs, remnants of the femoral tissue were transferred to a Petri dish, minced and washed with DMEM-hg supplemented with 1% pen-strep to obtain the bone marrow. The harvested bone marrow was centrifuged at 300 x g for 5 min. Commercial bone marrow-derived mononuclear cells were thawed by dilution in pre-warmed DMEM-hg supplemented with 10% FBS and 1% pen-strep, and centrifuged at 300 x g for 5 min. The resulting cell pellets from both sources were resuspended and cultured in MSC growth medium consisting of DMEM-hg supplemented with 10% FBS, 1% pen-strep, 2 mM L-glutamine and 1 ng/ml bFGF to establish primary cultures. The cultures were incubated at 37 °C and 5% CO2 with media changes twice a week. After reaching confluence, the plastic-adherent cells (i.e. MSCs) were trypsinized and subcultured at a seeding density of 5000 cells/cm2. The potential for proliferation in vitro was assessed over 6 consecutive passages. At the end of each passage, cells were counted and cumulative cell growth was determined as previously reported [33], taking into account the cells used for the characterization analyses. Average specific proliferation rate was determined for passages 1–6 for the analysis of correlation with donor age.

Fig. 1

Characterization of bone marrow MSCs. A) Cummulative proliferation of MSCs during 6 passages in vitro. B-C) Tri-lineage differentiation potential and proportion of senescence-associated beta-galactosidase positive cells. Individual MSC responses are summarized in the table (B). Donor responses were evaluated qualitatively as high (+++), positive (++), low (+) and negative (-). Representative images of three-lineage differentiation and positive beta-galactosidase staining are shown for donors F20 and M90 (C). Insets represent cultures in control media. Images for all donors are presented in Fig. S1A. D) Expression levels of surface antigens associated with MSCs. Individual flow cytometry charts for all donors are presented in Fig. S1B

Characterization of human bone marrow-derived MSCs

The profile of surface antigen expression was determined in passage 5 cells. Briefly, the cells were trypsinized, washed and resuspended in a blocking buffer (PBS with 1% FBS). Aliquots of 100,000 cells were incubated for 30 min on ice in the dark with fluorescence-conjugated primary antibodies against CD44 (Cat. No. 555478), CD73 (Cat. No. 550257), CD90 (Cat. No. 555596), CD146 (Cat. No. 561013), CD31 (Cat. No. 555446) and the isotype controls IgG-PE (Cat. No. 555749) and IgG-FITC (Cat. No. 555786), all from BD Biosciences (San Jose, USA). In addition, antibodies against CD105 (Cat. No. ab53321, Abcam, Cambridge, United Kingdom), CD14 and CD45 (Cat. Nos. 21279143 and 21270453, ImmunoTools, Friesoythe, Germany), CD34 (Cat. No. IM1870, Beckman Coulter, Brea, USA) and HLA-DR (Cat. No. 12-9952-4, eBioscience, San Diego, USA) were used. After staining, cells were washed, resuspended in blocking buffer and immediately analyzed using the Cytoflex flow cytometer and CytExpert software (Beckman Coulter). Positive expression of surface markers was determined using a combination of fluorescence minus one and isotype controls. For the negative controls, the gates were set to 1%.

Tri-lineage differentiation potential was determined in monolayer and pellet cultures, as previously reported [30, 34], using cells from passages 3 to 5. For osteogenic differentiation, cells were seeded in 24-well plates at a density of 5000 cells/cm2 and cultured in osteogenic medium consisting of DMEM-hg supplemented with 10% FBS, 1% pen-strep, 2 mM L-glutamine, and an osteogenic supplements 10 nM dexamethasone, 50 µM ascorbic acid-2-phosphate and 10 mM ß-glycerophosphate. For adipogenic differentiation, the cells were seeded in 24-well plates at a density of 7400 cells/cm2 (a higher seeding density provided an additional adipogenic stimulus) and cultured in an adipogenic medium, consisting of DMEM-hg supplemented with 10% FBS, 1% pen-strep, 2 mM L-glutamine and adipogenic supplements 0.5 mM IBMX, 60 µM indomethacin, 0.5 µM hydrocortisone and 1 µM dexamethasone. Control cultures for osteogenic and adipogenic differentiation were seeded in 24-well plates at a density of 5000 cells/cm2 and cultured in MSC growth medium without bFGF. The cells were inspected regularly and harvested after 25–28 days of culture before the monolayers detached due to cell overgrowth. Osteogenic and adipogenic differentiation was assessed according to standard procedures for Alizarin Red staining of calcium deposits and Oil Red O staining of lipid accumulations. The differentiation potential was qualitatively assessed as high (+++), positive (++), low (+) and negative (-). For correlation analysis, high differentiation potential was assigned value 3, positive potential value 2, low potential value 1 and negative potential value 0. For combined differentiation rank, the three differentiation ranks were summed-up.

For chondrogenic differentiation, 250,000 cells were transferred to microcentrifuge tubes and centrifuged at 300 x g for 5 min. The resulting cell pellets were either cultured in chondrogenic medium consisting of DMEM-hg supplemented with 1% pen-strep, 1% ITS-X, 100 µg/ml sodium pyruvate, 40 µg/ml L-proline, 100 nm dexamethasone, 50 µg/ml ascorbic acid-2-phosphate and 10 ng/ml TGF-β3, or in MSC growth medium without bFGF as a control. After 4 weeks of culture, the pellets were harvested and processed for histologic analysis as described in the following section.

Senescence was determined in cells of passage 5. The cells were seeded in three replicates in 12-well plates at a density of 5000 cells/cm2. After 2–3 days, when the cultures had reached 30–50% confluence, senescence was determined using senescence β-galactosidase staining kit (Cell Signaling Technology, Danvers, USA), according to the manufacturer’s instructions. The stained cultures were imaged using an inverted microscope (Primovert) mounted with a digital camera (AxioCam ICc5, Zeiss, Oberkochen, Germany). Six non-overlapping fields were imaged for each well, and the number of senescent cells and the total number of cells were counted independently by two investigators. Senescence was expressed as the percentage of beta-galactosidase-positive cells out of the total number of cells.

Preparation of conditioned media from hiPSC-MPs and bone marrow-derived MSCs

hiPSC-MPs were derived in our previous study (line 1013 A) and showed a strong growth and trilineage differentiation potential, an expression profile of surface antigens similar to that of bone marrow-derived MSCs, and a normal karyotype [30]. The cells were thawed, seeded in tissue culture flasks pre-coated with 0.1% gelatin in tissue culture water and cultured in a medium consisting of KO-DMEM supplemented with 10% Hyclone-FBS, 1% pen-strep, 2 mM GlutaMAX, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol and 1 ng/ml bFGF (Thermo Fisher Scientific). For CM preparation, hiPSC-MPs of passages 7–9 were seeded at a density of 10,000 cells/cm2 and cultured up to 80% confluent. Cultures were washed twice with PBS and incubated in DMEM-hg with 1% pen-strep for 48 h. CM from different flasks was then collected, pooled, filtered and stored in aliquots at -80 °C. After CM collection, the cells in the different flasks were counted to determine comparable cell numbers. Control medium (CTRL) was prepared according to the same protocol in the absence of cells. Two large pools of hiPSC-MP-CM and CTRL medium were merged and used in all experiments except for evaluating the effects of prolonged hiPSC-MP cultivation and predifferentiation, in which separate CTRL and CM were prepared from subconfluent hiPSC-MP cultures. CM was also prepared from bone marrow-derived MSCs from three donors (M22, F71, F85) using the same conditioning protocol as described for hiPSC-MPs.

To investigate the effects of prolonged cultivation and pre-differentiation, CM was prepared from hiPSC-MPs cultured in either growth medium (d14-CM) or osteogenic medium (d14OST-CM) for 14 days. Osteogenic and growth medium were composed as described above for MSC characterization studies, except that the dexamethasone concentration in the osteogenic medium was increased to 1 µM, as previously reported for osteogenic differentiation of hiPSC-MPs [30], and the growth medium did not contain bFGF. Proliferation (DNA content) and alkaline phosphatase (ALP) activity were assessed at the beginning of conditioning in parallel hiPSC-MP cultures seeded in 24-well plates to confirm early osteogenic differentiation (see following sections). Osteogenic differentiation was further verified by staining for ALP activity (Leukocyte Alkaline Phosphatase Kit, Sigma-Aldrich) according to the manufacturer’s instructions.

Effect of conditioned media on adult/aged bone marrow-derived MSC osteogenesis

Different types of CM and treatments have been used to investigate the effects of CM on osteogenic differentiation of bone marrow-derived MSCs (see below).

Duration of hiPSC-MP-conditioned medium supplementation to enhance osteogenic differentiation

The effect of hiPSC-MP-CM on early osteogenic differentiation was tested with passage 1 MSCs in monolayer cultures. MSCs were seeded in 96-well plates at a density of 8000 cells/cm2 in DMEM-hg containing 10% FBS, 0.1% pen-strep and 2 mM L-glutamine to ensure uniform seeding. After 24 h, the medium was replaced with CTRL and CM media freshly supplemented with 10% FBS, 0.1% pen-strep and 2 mM L-glutamine. In addition, the CTRL + O and CM + O groups were formed by adding osteogenic supplements: 10 nM dexamethasone, 50 µM ascorbic acid-2-phosphate and 10 mM ß-glycerophosphate.

To assess the duration of CM supplementation, cultures were either maintained in CM media and controls for 14 days, with complete media changes every 2–3 days (tested with MSCs from all 13 donors). Alternatively, short-term CM supplementation was tested (using MSCs from donors F71 and M89), in which MSC cultures were incubated in CTRL, CM, CTRL + O and CM + O media for three days, and then switched to regular control and osteogenic media (without CM or CTRL) for the remaining 14 days of culture. In parallel groups, the three-day supplementation was repeated in the second week (days 7–10). Cultures were harvested after 7 and 14 days to determine proliferation (DNA content) and ALP activity.

For gene expression analysis, MSCs (donors M22, F71, M73, F87, M89 and M90) were seeded in 24-well plates at a density of 5000 cells/cm2, cultured in CTRL, CM, CTRL + O and CM + O media for 14 days, harvested in TRI Reagent® and stored at -80 °C until analysis.

Due to frequent overgrowth and detachment of monolayer cultures between the second and third week of culture in preliminary experiments, the effects on late markers of osteogenesis were tested in pellet cultures, as previously reported [30, 34], using passage 3 MSCs. Pellets were prepared from ~ 450,000 MSCs (donors F71 and M89) as described above for chondrogenic differentiation and incubated in CTRL, CTRL + O CM and CM + O media for 6 weeks, with complete media changes every 2–3 days. At the end of the culture, the pellets were harvested and processed for histological analysis as described in the following section.

Effect of hiPSC-MP-conditioned medium dose

For the dose-response experiments, CM and CTRL media were successively concentrated using VivaSpin 20 ultrafiltration devices with 30 kDa and 3 kDa MWCO (Sartorius, Göttingen, Germany) to obtain a 20-fold concentrated CM fraction. Dilution series of the concentrated CM and CTRL media (1:1, 1:2, 1:4 and 1:8 dilutions) were prepared using fresh KO-DMEM supplemented with 10% FBS, 0.1% pen-strep and 2 mM L-glutamine. In parallel, CM and CTRL dilution series were prepared supplemented with osteogenic supplements. CM and CTRL dilution series with/without osteogenic supplements were tested in 96-well plates with monolayer cultures of MSCs (donors F71, F85 and M89). Cultures were continuously supplemented for 14 days as described above for CM and harvested at week 1 and 2 to determine proliferation (DNA content) and ALP activity.

Effects of cell source and hiPSC-MP prolonged cultivation/osteogenic differentiation on conditioned medium stimulatory activity

The effects of hiPSC-MP-CM were compared with the effects of CM derived from MSCs from three donors (M22, F71 and M85) using responding MSCs from two donors (F71 and F85).

To assess the effects of prolonged cultivation or pre-differentiation of hiPSC-MPs on CM functionality, CTRL, CM, d14-CM and d14OST-CM media with or without osteogenic supplements were tested with MSCs from donor F85. MSCs of passage 1 were seeded in monolayer cultures and continuously supplemented with different CM types for 14 days as described above, after which proliferation (DNA content) and ALP activity were determined.

DNA content determination

Proliferation of bone marrow-derived MSC in response to different CM and control media was analysed using the Molecular Probes™ CyQUANT™ Cell Proliferation Assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cultures were harvested in 96-well plates, washed once with PBS and cell monolayers were stored at -80 °C until analysis. The plates were thawed to room temperature, then 200 µl of freshly prepared fluorescent reagent was added to the wells and incubated for 5 min in the dark. The fluorescence was measured at 485/520 nm. The DNA concentration of the samples was determined using a standard curve prepared in parallel with the DNA standard provided with the assay kit.

To assess the proliferation of hiPSC-MPs after 14 days of culture in 24-well plates in growth and osteogenic medium (before the start of conditioning), DNA content was determined as previously described [35]. Briefly, cultures were harvested in the well plates, washed once with PBS and digested overnight at 60 °C in a buffer containing 150 mM NaCl, 55 mM Na citrate * 2H2O, 20 mM EDTA * 2 H2O, 0.2 M NaH2PO4 * 1 H2O, 10 mM EDTA * 2 H2O, 6 U/mL papain, and 10 mM cysteine in ddH2O (with a pH of 6.0). The digested samples were collected, centrifuged at 300 × g for 5 min, and the DNA content of the supernatant was determined using Hoechst 33,342 dye in assay buffer (2 M NaCl, 50 mM NaH2PO4, pH 7.4). Samples were incubated for 5 min at 37 °C in the dark with slow shaking, and fluorescence was measured at 355/460 nm. The DNA concentration of the samples was determined using a standard curve generated with calf thymus DNA solutions of known concentrations.

Alkaline phosphatase activity determination

To determine ALP activity, cell monolayers were washed in 96-well plates and stored as described above for DNA content. After thawing to room temperature, ALP activity was determined using Alkaline Phosphatase Yellow (pNPP) Liquid ELISA substrate (P7998, Sigma-Aldrich), as previously reported [36]. Briefly, 100 µl of the substrate was added to the wells, and the plates were incubated at 37 °C until yellow color development. The reaction was stopped by adding 100 µl of 0.1 M NaOH, the reaction time was recorded, and the absorbance was determined at 405 nm. ALP activity was determined using a standard curve generated with p-nitrophenol solutions of known concentrations.

ALP activity of hiPSC-MPs after 14 days of culture in 24-well plates in growth and osteogenic medium (at the beginning of conditioning) was determined as previously described [35]. Cultures were harvested, washed and lysed in a solution containing 0.5% Triton X-100 in 0.5 M 2-amino-2-methyl-1-propanol buffer with 2 mM MgCl2 (pH 10.3). The lysed samples were centrifuged at 300 x g for 5 min and the ALP activity of the supernatant was determined by adding the 0.02 M p-nitrophenyl phosphate substrate solution to the extracted supernatant and incubating at 37 °C until yellow color development. The reaction was stopped by adding 0.2 M NaOH stop solution and the reaction time was recorded. The absorbance was measured at 405 nm and the ALP activity was determined using a standard curve generated with p-nitrophenol solutions of known concentrations.

Gene expression analyses

MSCs grown for 14 days in 24-well plates in CM and control media were harvested in TRI Reagent® and total RNA was isolated according to the manufacturer’s instructions. The isolated RNA was treated with amplification-grade DNase I, and 300–800 ng of RNA was reverse transcribed into cDNA with the GoScript™ Reverse Transcription System 100 (Promega, Wisconsin, USA) using random hexamer primers. Real-time PCR was performed using the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, USA). 2 µL of cDNA was added to a 25 µL reaction containing the TaqMan® Universal PCR Master Mix and one of the TaqMan® gene expression assays (Thermo Fisher Scientific) for alkaline phosphatase (ALP, Hs01029144_m1), ostepontin (OPN, Hs00959010_m1), bone sialoprotein (BSP, Hs00173720_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs02786624_g1) (URL links to assay information are provided in Suppl. Data File 1). Standard cycling conditions were used: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s (denaturation) and 60 °C for 60 s (annealing and extension). Results were exported with CFX Manager 3.1 (Bio-Rad Laboratories, California, USA) and analyzed in Excel (Microsoft, Redmond, USA) using the ΔΔCt method. The expression levels of osteogenic target genes were normalized to the expression level of the housekeeping gene GAPDH as in our previous studies.

Histological and immunohistochemical analyses

The collected pellets were washed with PBS and fixed in 4% phosphate-buffered formaldehyde (Roti®-Histofix 4%, P087.3, Carl Roth, Karlsruhe, Germany) for 24 h. Samples were then rinsed, dehydrated in an increasing series of EtOH concentrations and embedded in paraffin using a vacuum infiltration device (Tissue Tek, Sakura Finetek, Nagano, Japan). 4 μm thin sections were cut with a microtome (Microm HM 355 S, Thermo Fisher Scientific), placed on glass slides and stored overnight at 37 °C to ensure optimal adhesion of the sections to the glass slides.

Immunohistochemical staining was performed to determine the presence of collagen type II. Sections were rehydrated in a descending EtOH series. Antigen retrieval was performed by pepsin treatment (Pepsin Reagent Antigen Retriever, R2283, Merck KGaA, Darmstadt, Germany) for 10 min at 37 °C in a humidified chamber. The sections were then incubated with a primary antibody against collagen type II (MS 306-P1; Collagen II Ab-3 (clone 6B3), mouse monoclonal antibody, Thermo Fisher Scientific) for one hour at RT. After washing, the samples were incubated with a secondary HRP anti-mouse antibody (Bright Vision poly HRP-Anti-Mouse IgG, VWRKDPVM110HRP, ImmunoLogic, Duiven, The Netherlands) for 30 min at RT. The signal was detected using the NovaRed® HRP peroxidase substrate kit (ImmPACTTMNova RedTM, SK4805, Vector Laboratories, Burlingame, USA) according to the manufacturer’s instructions, and cell nuclei were stained with Haemalaun.

To visualize pellet morphology and growth in CM and control media with/without osteogenic supplements, sections were stained with hematoxylin and eosin, and pellet mineralization was assessed using the standard von Kossa staining procedure.

Determination of TGFβ−1 content

The concentrations of TGFβ−1 in CTRL, CM, d14-CM and d14OST-CM media samples were determined with the Quantikine® ELISA kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions using a standard curve generated with standards of known concentrations included in the kit.

Proteomic analysis

Samples of CTRL, CM and d14-CM from three independent preparations were collected and stored at -80 °C until mass spectrometry (MS) proteomic analysis. Detailed MS analysis procedures are described in Suppl. Data File 1. Briefly, proteins were precipitated from the CM with a mixture of dichloromethane and methanol, followed by a series of centrifugation steps to remove the supernatant, leaving a pellet that was then air-dried. The pellets were dissolved in 50 µl of 50 mM triethylammonium bicarbonate (Sigma Aldrich) containing 0.1% RapiGest SF (Waters, Milford, USA) for protein solubilization. Protein concentrations were measured using a nano spectrophotometer (DS-11, DeNovix, Wilmington, USA). The proteins were then reduced, alkylated, and digested with MS-grade trypsin (Thermo Fisher Scientific) before being stored at -80 °C until further analysis. For nanochromatographic separation, a Nano-RSLC UltiMate 3000 system (Thermo Fischer Scientific) was used, employing a PepMap C18 trap-column (Thermo Fischer Scientific) for sample loading and desalting and a 200 cm C18 µPAC column (PharmaFluidics, Ghent, Belgium) for separation of the digested proteins. The separation used a cooled aqueous loading phase and a gradient elution in which two mobile phases were mixed to improve the capture of hydrophilic analytes and optimize peptide separation. Peptides were then analyzed using both UV at 214 nm (3 nl UV cell) and a Q-Exactive Orbitrap Plus mass spectrometer (both Thermo Fisher Scientific). The collected raw MS data were searched against the human UniProt protein database (February 2023 version) using FragPipe 21.1 (Nesvilab, Michigan, USA; https://fragpipe.nesvilab.org/) [37]. Statistical analysis and visualization was performed using Perseus version 1.6.5.0 (Cox lab, Martinsried, Germany; https://cox-labs.github.io/coxdocs/perseus_instructions.html) [38]. The program String (https://string-db.org/) [39] was used to investigate the biological relationship between the differentially expressed proteins and functional enrichments. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [40] via the PRIDE partner repository with the dataset identifier PXD052766 [41].

Statistical analyses

Data are presented either as individual values, or as a combination of mean ± SD with individual values. Normal distribution of the data was first tested using the Shapiro-Wilk test. Data showing a normal distribution were tested using t-test, one-way ANOVA or two-way ANOVA, followed by Tukey’s multiple comparison test. Data that did not show a normal distribution were analysed using the Friedman test followed by Dunn’s test for multiple comparisons. Correlations between specific proliferation rate, senescence-associated beta-galactosidase expression (middle) and MSC donor age were evaluated using Pearson’s correlation coefficient. Correlations between osteogenesis, chondrogenesis, adipogenesis, trilineage potential and MSC donor age were evaluated using Spearman’s rank correlation coefficient. The significance level was set at 0.05 for all analyses. The analyses were performed using Microsoft Excel (Redmond, USA) for the t-test and GraphPad Prism 10 software (GraphPad Software, San Diego, USA). The type of analysis, number of samples and significant differences between groups are indicated in the legends of each figure. Detailed statistical analyses of the proteomic data are provided in Suppl. Data File 1.