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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.
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.
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 OCs
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
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 .......
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.......
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....
Name | Company | Catalog Number | Comments |
2-Mercaptoethanol | Sigma Aldrich | M6250-10ML | |
Antibody - Anti-Cathepsin K | Abcam | ab19027 | |
Antibody - APC-conjugated Anti-Human CD45 | BD | 555485 | |
Antibody - APC-conjugated Mouse IgG1, κ Isotype Control | BD | 555751 | |
Antibody - BV711-conjugated Anti-Human CD14 | BD | 563372 | |
Antibody - BV711-conjugates Mouse IgG2b, κ Isotype Control | BD | 563125 | |
Antibody - Goat Anti-Rabbit IgG H&L Alexa Fluor® 647 | Abcam | ab150079 | |
Antibody - PE-conjugated Anti-Human CD14 | R&D Systems | FAB3832P-025 | |
Antibody - PE-conjugated Anti-Human Integrin alpha M/CD11b | R&D Systems | FAB16991P-025 | |
Antibody - PE-Cy7-conjugated Anti-Human CD34 | BD | 560710 | |
Antibody - PE-Cy7-conjugated Mouse IgG1 κ Isotype Control | BD | 557872 | |
Antibody - PE/Cyanine5-conjugated Anti-Human CD11b | Biolegend | 301308 | |
Antibody - PE/Cyanine5-conjugated Mouse IgG1, κ Isotype Ctrl | Biolegend | 400118 | |
Antibody - PerCP-Cy5.5-conjugated Mouse IgG1 κ Isotype Control | BD | 550795 | |
Antibody - PerCpCy5.5-conjugated Anti-Human CD43 | BD | 563521 | |
Bone Resorption Assay Kit | CosmoBioUSA | CSR-BRA-24KIT | |
Countess 3 Automated Cell Counter | ThermoFisher | 16812556 | |
Cultrex Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract | R&D Sytems | 3434-010-02 | Basal membrane extract |
DAPI | R&D Systems | 5748/10 | |
Dispase (5 U/mL) | STEMCELL Technologies | 7913 | |
DMEM/F-12 with 15 mM HEPES | Stem Cell | 36254 | |
DMSO | Sigma Aldrich | D2650 | |
DPBS | Sigma Aldrich | D8537-500ML | |
Human Bone Morphogenetic Protein 4 (hBMP4) | STEMCELL Technologies | 78211 | |
Human IL-3 | STEMCELL Technologies | 78146.1 | |
Human Macrophage Colony-stimulating Factor (hM-CSF) | STEMCELL Technologies | 78150.1 | |
Human Soluble Receptor Activator of Nuclear Factor-κB Ligand (hsRANKL) | STEMCELL Technologies | 78214.1 | |
Human Stem Cell Factor (hSCF) | STEMCELL Technologies | 78155.1 | |
Human TruStain FcX (Fc Receptor Blocking Solution) | Biolegend | 422301 | |
Human Vascular Endothelial Growth Factor-165 (hVEGF165) | STEMCELL Technologies | 78073 | |
Invitrogen Rhodamine Phalloidin | Invitrogen | R415 | |
MEM α, nucleosides, no phenol red | ThermoFisher | 41061029 | |
mFreSR | STEMCELL Technologies | 05855 | Serum free cryopreservation medium |
mTeSR Plus medium | STEMCELL Technologies | 100-0276 | Human iPSC-serum free medium (hiPSC-SFM) |
Nunclon Sphera 96-Well, Nunclon Sphera-Treated, U-Shaped-Bottom Microplate | Thermo Scientific | 174925 | Round bottom ultra-low attachment 96-well plate |
P1000 Wide Bore Tips | ThermoFisher | 2079GPK | |
ROCK-Inhibitor Y-27632 | STEMCELL Technologies | 72304 | |
StemSpan SFEM | StemCell | 09650 | Hematopoietic cell culture medium |
TrypLE Select Enzyme (1X), no phenol red | Thermo Fisher | 12563011 | Single-cell dissociation reagent |
Ultraglutamine | Bioscience Lonza | BE17-605E/U1 | |
X-VIVO 15 Serum-free Hematopoietic Cell Medium | Bioscience Lonza | 04-418Q | Hematopoietic basal medium |
µ-Slide 8 Well High | Ibidi | 80806 |
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