Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

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-derived^1,2, versatile cell types that are commonly used by researchers in areas such as bone disease research^3,4, cancer research^5,6, tissue engineering^7,8, and endoprosthesis research^9,10. Nevertheless, OC differentiation can be challenging as fusion of mononuclear precursors into multinucleated OCs is necessary to create functional OCs

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, co.......

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.......

Materials

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