iPSC culture
The OC proliferation and differentiation of two iPSC lines derived from distinct tissue origins were compared. MCND-TENS2, a peripheral blood mononuclear CD34+/CD38− cell-derived iPSC line from a healthy donor (37) (received from NIH National Heart Lung Blood Institute, Bethesda, MD, USA; registered at https://hpscreg.eu/cell-line/RTIBDi001-A) was compared to GM28404*B, a fibroblast-derived iPSC line documented to originate from “apparently healthy individuals” (received from Coriell Institute Cell Repository, Camden, NJ, USA; registered at https://www.cellosaurus.org/CVCL_C0M4). Both cell lines were created using a Sendai virus reprogramming kit (37, 43). Upon defrosting, cells were cultured with mTeSR Plus (StemCell Technologies, Vancouver, Canada) on Cultrex basement membrane extract (R&D Systems, Minneapolis, USA) coated 6-well plates and incubated at 37°C and supplied with 5% CO2. 10 µM of Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor Y-276432 was used when defrosting to improve cell survival. Media changes were performed every second day. iPSCs were passaged at 70–80% confluency using 5 U/mL Dispase (StemCell Technologies, Vancouver, Canada).
Mesodermal induction, hematopoietic differentiation, and osteoclast differentiation
In this study a cytokine defined differentiation protocol published by Rössler et al.(31) was compared to a commercially available differentiation kit (STEMdiff hematopoietic kit, StemCell Technologies, Vancouver, Canada), using the above-mentioned iPSC lines from different tissue origins. Both cell lines underwent mesodermal induction at passage 27.
EB differentiation according to Rössler et al. (31) was performed by creating a single cell suspension which was seeded in a round bottom ultra-low attachment 96-well plate at a cell density of 1.25x104 cells in 100 µL of mTeSR Plus supplemented with 50 ng/mL human bone morphogenetic protein 4 (BMP4) (StemCell Technologies, Vancouver, Canada), 50 ng/mL human vascular endothelial growth factor-165 (VEGF165) (StemCell Technologies, Vancouver, Canada), 20 ng/mL human stem cell factor (SCF) (StemCell Technologies, Vancouver, Canada), and 10 µM ROCK inhibitor Y-27632 (StemCell Technologies, Vancouver, Canada). Subsequently, the 96-well plate was centrifuged for 3 min at 100 x g and half medium changes performed after one and two days. Four days after mesodermal induction, embryoid bodies were transferred to a 6-well plate and further differentiated in X-VIVO 15 Medium (Bioscience Lonza, Basel, Switzerland) supplemented with 2 mM Ultraglutamine (Bioscience Lonza, Basel, Switzerland), 55 µM 2-mercaptoethanol (ThermoFisher, Waltham, USA), 25 ng/mL human interleukin 3 (IL-3) (StemCell Technologies, Vancouver, Canada), 100 ng/mL human macrophage colony-stimulating factor (M-CSF) (StemCell Technologies, Vancouver, Canada). A full medium change of 3 mL was performed after 5 days. After 10 days of differentiation, floating suspension cells were harvested and used for further OC differentiation.
MB differentiation using STEMdiff hematopoietic kit (37) (StemCell Technologies, Vancouver, Canada) was performed according to manufacturer’s instruction. In short, 16–40 cell aggregates were seeded onto each 12-well plate coated with Cultrex basement membrane extract and treated for 3 days with medium A (containing bFGF, BMP4, VEGFA) (37) and consecutively for 10 days with medium B (containing bFGF, BMP4, VEGFA, SCF, Flt3L, TPO) (37). The floating suspension cells were harvested at the end of the period and used for further OC differentiation.
Following hematopoietic differentiation cells were transferred to 6-well plates at 2 x 105 cells/cm2 and incubated in alpha-MEM (Bioscience Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 50 ng/mL human M-CSF for 3 days after which cells were incubated in alpha-MEM supplemented with 10% heat-inactivated FBS, 50 ng/mL human M-CSF and 70 ng/mL human RANKL (StemCell Technologies, Vancouver, Canada) for 7 days. Medium changes were performed every 2–3 days. OC differentiation was finished by a final treatment using alpha-MEM supplemented with 10% heat-inactivated FBS and 80 ng/mL human RANKL for 2 days.
Enzymatic staining
To assess the degree of spontaneous differentiation during cell expansion, both iPSC lines were stained for alkaline phosphatase (ALP) (Abcam, Cambridge, UK) as a marker for pluripotency each time the cell lines were passaged. Additionally, ALP expression was assessed throughout differentiation. For this, cells were seeded onto 8-chamber slides, fixed at 70–80% confluency and stained according to manufacturer’s protocol. Tiled full well images were taken using an inverted widefield microscope (DMI6000, Leica, Wetzlar, Germany).
Tartrate-resistant acid phosphatase (TRAP) staining was performed after M-CSF matured hematopoietic cells were seeded onto a calcium-phosphate 24-well bone resorption assay plate (Cosmo Bio, Tokyo, Japan) in order to assess osteoclastogensis using a TRAP staining kit (Sigma-Aldrich, St. Louis, MO, USA). Staining was performed according to manufacturer’s instructions. In short, cells were fixed using 4% PFA, washed, staining mix added to the plate and incubated for 20 min at 37°C. OCs were then counterstained with methyl green nuclear stain (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes at room temperature. Image acquisition was performed using a Leica DMI6000 inverted microscope in phase-contrast mode and tiled full well images were analyzed for cell size and multi-nucleation using ImageJ.
Immunocytochemistry
Immunocytochemistry (ICC) was performed after EB or MB induction in order to assess mesodermal differentiation and after termination of RANKL treatment in order to assess OCs.
After EB induction, cells were fixed with 4% paraformaldehyde (PFA) and blocked with PBS containing 10% normal goat serum (Abcam, Cambridge, UK), 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), and 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA). After blocking, embryoid bodies were incubated overnight with antibodies against SOX1 (NL 493-conjugated) and Otx-2 (NL557-conjugated) to assess ectodermal differentiation, Brachyury (NL557-conjugated) and HAND1 (NL637-conjugated) to assess mesodermal differentiation or GATA-4 (NL493-conjugated) and SOX17 (NL637-conjugated) to assess endodermal differentiation (all germ-layer antibodies from R&D Systems, Minneapolis, USA) (Supplementary information, Table 1). All cells were counterstained for 15 min with 4′,6-diamidino-2-phenylindole (DAPI) (R&D Systems, Minneapolis, USA). Images were acquired using a Leica SP8X confocal laser scanning microscope (CLSM) and analyzed using ImageJ.
Following OC differentiation, cells were fixed with 4% PFA, permeabilized with Triton X-100 (ThermoFisher, Waltham, USA) for 30 min and blocked with normal goat serum for 60 min. Following permeabilization and blocking, OCs were stained with a primary anti-Cathepsin K antibody (Abcam, Cambridge, UK) which was succeeded by staining with an Alexa 647-conjugated secondary antibody (Abcam, Cambridge, UK) (Supplementary information, Table 1). Cells were then additionally stained with TRITC-conjugated Phalloidin and DAPI nuclear stain. Images were acquired and analyzed as mentioned above.
Cell number and viability of hematopoietic cells
Following hematopoietic differentiation, floating suspension cells were collected and analyzed using a Countess 3 cell counter (ThermoFisher, Waltham, USA). Cell viability and cell number was measured after Trypan blue was added to testing samples.
Flow cytometry
Hematopoietic differentiation was assessed by harvesting monocyte-like suspension cells, staining with LIVE/DEAD Fixable Violet stain (ThermoFisher, Waltham, USA) and blocking Fc receptors using TruStain FcX (Biolegend, San Diego, USA). This was followed by fixing cells with 4% PFA (Electron Microscopy Sciences, Hatfield, USA) and subsequent staining with primary antibodies (Supplementary information, Table 1) against CD34 (PE-Cy7-conjugated) (BD, Franklin Lakes, USA), CD43 (PerCp-Cy5.5-conjugated) (BD, Franklin Lakes, USA), CD45 (APC-conjugated) (BD, Franklin Lakes, USA), CD14 (BV711-conjugated) (BD, Franklin Lakes, USA), CD11b (PE-Cy5-conjugated) (Biolegend, San Diego, USA) and CD265/RANK (PE-conjugated) (R&D Systems, Minneapolis, USA), at 4°C for 1h in PBS supplemented with 0.09% (w/v) sodium azide (ThermoFisher, Waltham, USA) and 1% heat-inactivated FBS. Data acquisition and analysis was performed as mentioned above. Positive control and fluorescence compensation was performed with UltraComp eBeads (ThermoFisher, Waltham, USA). Gating was performed using isotype controls following singlets and live/dead gating. Data was acquired using a BD FACSymphony A3 (BD, Franklin Lakes, USA) and analyzed with FlowJo 10 (BD, Franklin Lakes, USA).
Mineral resorption assay
In order to determine the mineral resorptive capacity of differentiated OCs, HPCs were plated onto a calcium-phosphate 24-well resorption assay plate and treated as mentioned above. After terminating OC differentiation, OCs were removed using 5% bleach and resorptive area was analyzed by taking tiled full well images as described above. OCs derived from human CD34+ PBMCs (healthy donor, received from Fred Hutch, Seattle) were used as positive control. In addition to image acquisition using the phase-contrast mode, images for resorption area quantification were taken using the yellow channel in fluorescence mode in order to facilitate differentiation between resorbed and un-resorbed areas in ImageJ.
Analysis of gene expression
RNA extraction and isolation throughout the differentiation process for both iPSC lines using either the EB or the MB method was performed using a RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. In short, a lysis buffer was added to the cells and lysed mechanically using a 20G needle and syringe. Following RNA extraction RNA was isolated and purified using spin-columns. RNA concentration was measured using a NanoDrop (ThermoFisher, Waltham, USA). A total of 250 ng of RNA was used to generate complementary DNA (cDNA) using Omniscript (Qiagen, Hilden, Germany) at 37°C for 1 hour. The cDNA was then used to determine the expression of POU5F1, CSF1R, TNFRSF11A, NFATC1, CA2, MMP9 using a TaqMan quantitative reverse transcription PCR (qRT-PCR) (Supplementary information, Table 2). Gene expression levels were normalized to 18s ribosomal RNA levels and calculated using the ∆∆Ct method to determine fold gene expression throughout the differentiation process.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 9. Data are shown as means ± SD. Statistical significance was assessed using Tukey’s multiple comparison post-hoc test unless indicated otherwise in individual experiments. An adjusted p-value of p < 0.05 was considered to be statistically significant.