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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • 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 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.......

Representative 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.......

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....

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

References

  1. Bar-Shavit, Z. The osteoclast: A multinucleated, hematopoietic-origin, bone-resorbing osteoimmune cell. Journal of Cellular Biochemistry. 102 (5), 1130-1139 (2007).
  2. Boyce, B. F. Advances in the regu....

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