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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

Abstract

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

Introduction

Osteoclasts (OCs) are hematopoietic-derived1,2, versatile cell types that are commonly used by researchers in areas such as bone disease research3,4, cancer research5,6, tissue engineering7,8, and endoprosthesis research9,10. Nevertheless, OC differentiation can be challenging as fusion of mononuclear precursors into multinucleated OCs is necessary to create functional OCs11. Several biological factors, such as receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), are necessary for OC differentiation. M-CSF has been reported to have a positive effect on cell proliferation, cell survival, and RANK expression12,13,14. On the other hand, RANKL binds to RANK, which activates downstream signaling cascades that induce osteoclastogenesis. Activation is mediated via TNF receptor-associated factor 6 (TRAF6), which leads to the degradation of nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκB-α), a binding protein that binds NF-kB dimers16,17. Hence, IκB-α degradation releases NF-kB dimers, which then translocate into the nucleus and induce the expression of the transcription factors c-Fos and Nuclear Factor of Activated T-Cells 1 (NFATc1). This, in turn, triggers the transcription of a multitude of OC differentiation-related proteins15,18. Upregulated proteins such as DC-Stamp and Atp6v0d2 mediate cell-cell fusion of OC precursors, leading to syncytium formation19,20,21.

With respect to human primary cells, CD34+ and CD14+ PBMCs are currently the most widely used cell types for differentiation into OCs22. However, this approach is limited by the heterogeneity within the CD34+ population of harvested cells from donors23 and their limited expandability. Human iPSCs present an alternative source for OCs. As they can be propagated indefinitely24, they allow for expandability and upscaling of OC production. This allows for the differentiation of large numbers of OCs, which facilitates OC research.

Several protocols for the differentiation of iPSCs into OCs have been published25,26,27. The entire differentiation process can be divided into an iPSC propagation part, a mesodermal and hematopoietic differentiation part, and OC differentiation. Propagation of iPSCs before the differentiation process allows for the upscaling of OC production prior to differentiation. Several approaches exist regarding mesodermal and hematopoietic differentiation. Traditionally, embryoid body (EB) formation has been used to differentiate hematopoietic cells, but monolayer-based approaches represent another hematopoietic differentiation strategy that does not require EB induction. Nevertheless, monolayer-based systems appear to require further optimization, as we and others have found EB-based approaches to be more robust for the differentiation of OCs.

Here, we describe the differentiation of OCs from human iPSCs using an EB-based protocol. This protocol was adapted from Rössler et al.26 and modified to increase robustness and allow for cryopreservation during the differentiation process. First, we harvested hematopoietic cells only once after 10 days of differentiation. Hematopoietic cells were then cryopreserved to allow for more flexibility during the differentiation process. Additionally, we increased the hematopoietic cell seeding density from 1 x 105 to 2 x 105 cells/cm2 for OC differentiation. A more recent human iPSC serum-free medium (hiPSC-SFM, see Table of Materials) was used, and coating of wells was performed with 200-300 µg/mL of a basal membrane extract (see Table of Materials) instead of 0.1% gelatin. Penicillin/streptomycin was not added to the media.

The protocol by Rössler et al.26 was originally adapted from an iPSC to a macrophage differentiation protocol28 that uses EB formation for hematopoietic differentiation. While EB formation has been used for an extended time by researchers for hematopoietic differentiation29,30, several methods of EB induction have been described in the literature, such as spontaneous aggregation, centrifugation in a round-bottom well plate, hanging drop culture, bioreactor culture, conical tube culture, slow turning lateral vessel, and micromold gel culture31. This protocol uses centrifugation of dissociated iPSCs in a round-bottom well plate to bring single iPSC cells into proximity to each other and to allow for sphere (EB) formation, as described hereafter.

Protocol

NOTE: All reagents used in this protocol can be found in the Table of Materials. Unless otherwise specified, all media were pre-equilibrated to 37 °C before use. All centrifugation steps are performed at 37 °C and by using the slowest acceleration/deceleration mode. Unless otherwise specified, supernatant is always removed using disposable Pasteur glass pipettes.

1. Thawing and propagation of human iPSCs

  1. One day prior to thawing iPSCs, coat a well of a 6-well plate with 1 mL of a basal membrane extract at a concentration of 200-300 µg/mL. Place the well plate at 4 °C overnight.
  2. The next day, thaw and transfer the cells into a 15 mL tube using a P1000 pipette. Dropwise, add 5-7 mL of DMEM/F-12 with 15 mM HEPES.
  3. Centrifuge the cells at 300 x g for 5 min. Gently remove the cells from the centrifuge, and be sure not to disturb the cell pellet.
  4. Carefully remove the supernatant with a Pasteur glass pipette and resuspend the cells in 1 mL of hiPSC-serum free medium (hiPSC-SFM containing 10 µM of Rho kinase (ROCK) inhibitor Y-27632) using P1000 with wide bore tips.
  5. Aspirate the basal membrane extract from the well coated the previous day (step 1.1) and transfer 1 mL of hiPSC-SFM with 10 µM Y-27632 into the well. Add resuspended iPSCs to the well to reach a final volume of 2 mL per well.
  6. Swirl the plate to evenly distribute iPSC aggregates before placing in an incubator at 37 °C and 5% CO2.
  7. Perform full medium changes every other day using hiPSC-SFM (without the addition of ROCK inhibitor Y-27632). iPSCs usually reach 70-80% confluency after 3-4 days of propagation.

2. Passaging iPSCs

  1. One day prior to passaging iPSCs, coat wells of a 6-well plate with a basal membrane extract at a concentration of 200-300 µg/mL. Place the well plate at 4 °C for overnight.
    NOTE: Confirm that cells have reached approximately 70-80% confluency. Ensure iPSCs do not get overconfluent (more than 80% confluency), as this will promote spontaneous differentiation.
  2. Begin passaging iPSCs by removing differentiated regions or regions with many dead iPSC aggregates under the stereomicroscope by using 10 µL or 20 µL pipette tips or a cell scraper. Differentiated regions will appear denser or whiter in color.
    NOTE: Scraped-off cell aggregates may still partially attach to the well bottom.
  3. Wash the well bottom several times with a wide bore P1000 pipette to remove any aggregates that may still be partially attached to the well's bottom.
  4. Discard the spent medium in which the iPSCs have been cultivated that contains the detached iPSC aggregates. Repeat the washing step two more times with phosphate buffered saline (PBS).
  5. Next, add 1 mL of 5 U/mL Dispase per well. Edges of the iPSC colonies will lift off from the well plate, which can be observed under the stereomicroscope after 3-5 min incubation at room temperature.
  6. Carefully remove Dispase to avoid dislodging slightly detached aggregates and add 1 mL of DMEM/F-12 with 15 mM HEPES. Use a disposable cell lifter to slice the aggregates into small sizes. The consistency of iPSC aggregate sizes can be improved with a stem cell passaging tool.
  7. Wash off the sliced iPSC aggregates with the medium in the well and transfer the medium with aggregates into a 15 mL conical tube using a 5 mL serological pipette or P1000 with a wide bore tip.
  8. Rinse the well with DMEM/F-12 and transfer the medium to the 15 mL tube with the iPSC aggregates.
  9. Centrifuge the sliced iPSC aggregates at 200 x g for 3 min. Remove the supernatant with a Pasteur glass pipette and add 2 mL of hiPSC-SFM to dislodge and resuspend the iPSCs using a 5 mL serological pipette or P1000 with wide bore tips.
  10. Aspirate the leftover basal membrane extract from the pre-coated well plate(s) and add 1 mL of hiPSC-SFM to each well of the 6-well plate.
  11. Transfer iPSCs into new wells of a 6-well plate so that the final volume is 2 mL of hiPSC-SFM per well using a P1000 with wide bore tips. Depending on the iPSC line, split ratios need to be optimized. Here, a 1:6 split ratio was used.
  12. Check the well for floating aggregates and aggregate size under the stereomicroscope. Ideally, the aggregate size should be between 50-200 µm.
  13. Swirl the well plate to distribute the cells evenly across the plate after the aggregates have been transferred and incubate at 37 °C at 5% CO2 until 70-80% confluency.

3. Freezing back iPSCs

  1. To freeze iPSC cells back, passage cells as described above (see protocol step "2. Passaging of iPSCs"). After cells have been sliced into colonies and washed off the well bottom (step 2.10), spin down the sliced aggregates at 200 x g for 3 min. Aspirate the supernatant.
  2. Add 1 mL of serum-free cryopreservation medium per well of a 6-well plate to the 15 mL tube and resuspend the sliced aggregates using a P1000 with a wide bore tip.
  3. Transfer cells into prelabeled cryotubes. Close tubes and transfer tubes into a prechilled cryopreservation container at 4 °C. Store cells at -80 °C for 24-48 h.
  4. Transfer cryovials into liquid nitrogen for long-term storage.
    NOTE: A schematic summary of the OC differentiation process is illustrated in Figure 1.

4. Embryoid body induction

  1. Cultivate and expand sufficient iPSCs as described above for EB induction.
    NOTE: OC production can be upscaled by increasing the number of embryoid bodies, which in turn produce an overall higher yield of hematopoietic cells. One well of 70-80% confluent iPSCs yields approximately 8.4 x 105 cells per well of a 6-well plate well. 12,500 single-cell iPSCs are needed to form one embryoid body.
  2. Aspirate the spent medium from the iPSC cultures and rinse the iPSC colonies with D-PBS.
  3. Add 0.5 mL of prewarmed to room temperature single-cell dissociation reagent to each well of a 6-well plate and swirl the culture vessel to coat the entire well surface. Incubate the culture vessel at 37 °C for 5-8 min.
  4. Remove the vessel from the incubator, aspirate the single cell dissociation reagent, and add 1 mL of the Stage 1 differentiation medium to the wells, consisting of hiPSC-serum free medium with 50 ng/mL human bone morphogenetic protein 4 (hBMP4), 50 ng/mL human vascular endothelial growth factor-165 (hVEGF), 20 ng/mL human stem cell factor (hSCF), and 10 µM Y-27632.
  5. Gently detach cells by rinsing the well with the Stage 1 medium. Pool dissociated iPSCs into a 15 mL conical tube.
  6. Add 1 mL of Stage 1 differentiation medium to the wells and wash any remaining cells off or use a cell scraper for colonies that do not wash off easily.
  7. After transferring all cells to the tube, centrifuge at 200 × g for 5 min at room temperature to form a cell pellet. Aspirate and resuspend the cells in a total of 2 mL of pre-equilibrated Stage 1 differentiation medium using a 5 mL serological pipette or a P1000 with a wide bore tip.
  8. Count cells using a hemocytometer or automated cell counting device. Add the medium to the 15 mL tube containing the single cell suspension to plate 12,500 cells per well in a round bottom ultra-low attachment 96-well plate in 100 µL of the Stage 1 differentiation medium.
    NOTE: Under the microscope, cells will appear as a single-cell suspension with cells dispersed throughout the well.
  9. Centrifuge the 96-well plates for 3 min at 100 x g. After centrifuging, cells should begin to resemble spheroids when viewed under the microscope. Place the plate in a 37 °C incubator for 24 h.
  10. Change half of the medium on Day 1 and Day 2 with the Stage 1 differentiation medium. To improve efficiency, use a multichannel pipette to dispose of 50 µL of the spent Stage 1 differentiation medium into a Petri dish.
  11. After disposing of the medium from the 96-well plate, check for EBs under the stereomicroscope that might have accidentally been removed. Transfer accidentally removed EBs to the 96-well plate using a P1000 pipette with wide bore tips.
  12. Add 50 µL of fresh Stage 1 differentiation medium to each well of a round bottom ultra-low attachment 96-well plate using a multichannel pipette.

5. Hematopoietic differentiation

  1. One day prior to starting hematopoietic differentiation, coat a well of a 6-well plate with 1 mL of a basal membrane extract at a concentration of 200-300 µg/mL. Place the well plate at 4 °C overnight.
    NOTE: Each well will receive 8 EBs at a later point of this protocol. Depending on the number of EBs prepared, coat wells correspondingly.
  2. Aspirate the excess basal membrane extract and prefill wells of the 6-well plate with 3 mL of the Stage 2 differentiation medium, consisting of a hematopoietic basal medium to which 2 mM Ultraglutamine, 55 µM 2-mercaptoethanol, 25 ng/mL human interleukin 3 (hIL-3), and 100 ng/mL human macrophage colony-stimulating factor (hM-CSF) is added.
  3. Using a P1000 with wide bore tips, transfer 8 EBs into each well of the 6-well plate. After transferring, confirm by eye or under the stereomicroscope that 8 EBs are in each well.
    NOTE: After 1 day, the floating EBs will adhere to the well bottom. In the following 5-7 days, a floating cell population comprising hematopoietic cells should become visible. The hematopoietic differentiation period can be varied, starting with 7 days. Differentiation for 10 days showed a hematopoietic population comprised of large CD45+, CD14+ and CD11b+ subpopulations.
  4. After 5 days of treatment with the Stage 2 differentiation medium, perform a medium change by removing the spent medium and dispensing it into a 50 mL conical tube. Pipette slowly to try to remove as little floating cells as possible and keep the shear stress as low as possible. Pipetting under a stereomicroscope can help avoid accidental removal of cells.
  5. Immediately add 1 mL of fresh Stage 2 differentiation medium to the wells. In order to recover any floating hematopoietic cells that may already be present at this point in the differentiation process, do not discard the dispensed medium. Rather, spin down the tube with the spent medium at 300 x g for 5 min, and aspirate the supernatant.
  6. Add fresh Stage 2 differentiation medium to the tube and resuspend in order to detach cells that might have been transferred with the spent medium.
  7. Add 2 mL of the fresh Stage 2 differentiation medium with the recovered cells into each well, which was previously filled with 1 mL of the Stage 2 differentiation medium.
  8. On Day 10 of hematopoietic differentiation, check for the presence of large numbers of floating hematopoietic cells. Harvest by collecting them in a 50 mL tube. They can either be frozen back in 10% DMSO, 50% FBS, and 40% medium or used immediately to differentiate the cells into OCs.

6. M-CSF maturation and OC differentiation

  1. Seed cells at a concentration of 200,000 cells/cm2 onto tissue culture treated well plates and treat with alpha-MEM, supplemented with 10% FBS and 50 ng/mL hM-CSF.
  2. To perform functional assays or imaging, detach the M-CSF matured cells 3 days after seeding by washing with pre-warmed PBS to 37 °C using a P1000 with a wide bore tip. Repeated washing steps might be needed to fully detach cells.
  3. Transfer the detached cells into a 15 mL tube using a P1000 with a wide bore tip and centrifuge at 300 x g for 5 min. Discard the supernatant.
  4. Resuspend and dissolve the cell pellet with the OC differentiation medium, consisting of alpha-MEM supplemented with 10% FBS, 50 ng/mL hM-CSF, and 80 ng/mL hRANKL. Count the cells using a hemocytometer or automated cell counting device and reseed M-CSF matured cells at a density of 200,00-250,000 cells/cm2 in an appropriate culture ware for functional assays or imaging (i.e., bone resorption assays, coverslip slides for imaging, etc.).
  5. Differentiate with the OC differentiation medium for 7-9 days. Perform a complete well medium change with the fresh OC differentiation medium every 2-3 days.
    NOTE: Multinucleated OCs typically appear after 5-7 days.

Results

Monitoring cell morphology throughout the differentiation process
All results described below were generated using the MCND-TENS2 iPSC line for OC differentiation. This iPSC line has previously been used in several studies32,33. Nevertheless, other iPSC lines have also been successfully used with this differentiation protocol.

Regular visual assessment reveals differing and distinct morphological characteristics ...

Discussion

This protocol offers a reliable and robust method to differentiate iPSCs into OCs. Nevertheless, there are several pitfalls that can be encountered throughout the differentiation process. Human iPSC lines generated from cells of different tissue origins have successfully been differentiated using this protocol33. When freezing back iPSCs (see protocol step "3. Freezing back iPSCs"), one well at the point of passaging was frozen back into one cryovial. When thawing (see protocol step "1...

Disclosures

The authors declare no competing interests.

Acknowledgements

The authors would like to thank the members of the Giachelli lab for their technical help and support. We thank the W. M. Keck Microscopy Center and the Keck Center manager, Dr. Nathanial Peters, for assistance in obtaining the confocal microscopy and widefield microscopy images. We also thank the UW Flow Core Facility and the Flow Core Facility manager, Aurelio Silvestroni, for technical support and assistance. Finally, we thank Hannah Blümke for the support with illustration and graphic design.

Funding was provided through the National Institutes of Health grant R35 HL139602-01. We also acknowledge NIH S10 grant S10 OD016240 for instrument funding at the W. M. Keck Center as well as NIH grant 1S10OD024979-01A1 for instrument funding at the UW Flow Core Facility.

Materials

NameCompanyCatalog NumberComments
2-MercaptoethanolSigma AldrichM6250-10ML
Antibody - Anti-Cathepsin K Abcamab19027
Antibody - APC-conjugated Anti-Human CD45BD555485
Antibody - APC-conjugated Mouse IgG1, κ Isotype ControlBD555751
Antibody - BV711-conjugated Anti-Human CD14BD563372
Antibody - BV711-conjugates Mouse IgG2b, κ Isotype ControlBD563125
Antibody - Goat Anti-Rabbit IgG H&L Alexa Fluor® 647Abcamab150079
Antibody - PE-conjugated Anti-Human CD14R&D SystemsFAB3832P-025
Antibody - PE-conjugated Anti-Human Integrin alpha M/CD11bR&D SystemsFAB16991P-025
Antibody - PE-Cy7-conjugated Anti-Human CD34BD560710
Antibody - PE-Cy7-conjugated Mouse IgG1 κ Isotype ControlBD557872
Antibody - PE/Cyanine5-conjugated Anti-Human CD11bBiolegend301308
Antibody - PE/Cyanine5-conjugated Mouse IgG1, κ Isotype CtrlBiolegend400118
Antibody - PerCP-Cy5.5-conjugated Mouse IgG1 κ Isotype ControlBD550795
Antibody - PerCpCy5.5-conjugated Anti-Human CD43BD563521
Bone Resorption Assay KitCosmoBioUSACSR-BRA-24KIT
Countess 3 Automated Cell CounterThermoFisher16812556
Cultrex Stem Cell Qualified Reduced Growth Factor Basement Membrane ExtractR&D Sytems3434-010-02Basal membrane extract
DAPIR&D Systems5748/10
Dispase (5 U/mL)STEMCELL Technologies7913
DMEM/F-12 with 15 mM HEPESStem Cell36254
DMSOSigma AldrichD2650
DPBSSigma AldrichD8537-500ML
Human Bone Morphogenetic Protein 4 (hBMP4)STEMCELL Technologies78211
Human IL-3STEMCELL Technologies78146.1
Human Macrophage Colony-stimulating Factor (hM-CSF)STEMCELL Technologies78150.1
Human Soluble Receptor Activator of Nuclear Factor-κB Ligand (hsRANKL)STEMCELL Technologies78214.1
Human Stem Cell Factor (hSCF)STEMCELL Technologies78155.1
Human TruStain FcX (Fc Receptor Blocking Solution)Biolegend422301
Human Vascular Endothelial Growth Factor-165 (hVEGF165)STEMCELL Technologies78073
Invitrogen Rhodamine PhalloidinInvitrogenR415
MEM α, nucleosides, no phenol redThermoFisher41061029
mFreSRSTEMCELL Technologies05855Serum free cryopreservation medium
mTeSR Plus mediumSTEMCELL Technologies100-0276Human iPSC-serum free medium (hiPSC-SFM)
Nunclon Sphera 96-Well, Nunclon Sphera-Treated, U-Shaped-Bottom MicroplateThermo Scientific174925Round bottom ultra-low attachment 96-well plate
P1000 Wide Bore TipsThermoFisher2079GPK
ROCK-Inhibitor Y-27632STEMCELL Technologies72304
StemSpan SFEMStemCell09650Hematopoietic cell culture medium
TrypLE Select Enzyme (1X), no phenol redThermo Fisher12563011Single-cell dissociation reagent
UltraglutamineBioscience LonzaBE17-605E/U1
X-VIVO 15 Serum-free Hematopoietic Cell MediumBioscience Lonza04-418QHematopoietic basal medium
µ-Slide 8 Well HighIbidi80806

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