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 Bl&#252;mke 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.

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 &#176;C before use. All centrifugation steps are performed at 37 &#176;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
<ol>
	<li>One day prior to thawing iPSCs, co...

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 &#34;3. Freezing back iPSCs&#34;), one well at the point of passaging was frozen back into one cryovial. When thawing (see protocol step &#34;1...

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-&#954;B 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 (I&#954;B-&#945;), a binding protein that binds NF-kB dimers16,17. Hence, I&#954;B-&#945; 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 R&#246;ssler 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 &#181;g/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 R&#246;ssler 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M6250-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - Anti-Cathepsin K&#160;</td>
			<td>Abcam</td>
			<td>ab19027</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - APC-conjugated Anti-Human CD45</td>
			<td>BD</td>
			<td>555485</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - APC-conjugated Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555751</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - BV711-conjugated Anti-Human CD14</td>
			<td>BD</td>
			<td>563372</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - BV711-conjugates Mouse IgG2b, &#954; Isotype Control</td>
			<td>BD</td>
			<td>563125</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - Goat Anti-Rabbit IgG H&#38;L Alexa Fluor&#174; 647</td>
			<td>Abcam</td>
			<td>ab150079</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - PE-conjugated Anti-Human CD14</td>
			<td>R&#38;D Systems</td>
			<td>FAB3832P-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - PE-conjugated Anti-Human Integrin alpha M/CD11b</td>
			<td>R&#38;D Systems</td>
			<td>FAB16991P-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - PE-Cy7-conjugated Anti-Human CD34</td>
			<td>BD</td>
			<td>560710</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - PE-Cy7-conjugated Mouse IgG1 &#954; Isotype Control</td>
			<td>BD</td>
			<td>557872</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - PE/Cyanine5-conjugated Anti-Human CD11b</td>
			<td>Biolegend</td>
			<td>301308</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - PE/Cyanine5-conjugated Mouse IgG1, &#954; Isotype Ctrl</td>
			<td>Biolegend</td>
			<td>400118</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - PerCP-Cy5.5-conjugated Mouse IgG1 &#954; Isotype Control</td>
			<td>BD</td>
			<td>550795</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody - PerCpCy5.5-conjugated Anti-Human CD43</td>
			<td>BD</td>
			<td>563521</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Resorption Assay Kit</td>
			<td>CosmoBioUSA</td>
			<td>CSR-BRA-24KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess 3 Automated Cell Counter</td>
			<td>ThermoFisher</td>
			<td>16812556</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract</td>
			<td>R&#38;D Sytems</td>
			<td>3434-010-02</td>
			<td>Basal membrane extract</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>R&#38;D Systems</td>
			<td>5748/10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase (5 U/mL)</td>
			<td>STEMCELL Technologies</td>
			<td>7913</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 with 15 mM HEPES</td>
			<td>Stem Cell</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Bone Morphogenetic Protein 4 (hBMP4)</td>
			<td>STEMCELL Technologies</td>
			<td>78211</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-3</td>
			<td>STEMCELL Technologies</td>
			<td>78146.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Macrophage Colony-stimulating Factor (hM-CSF)</td>
			<td>STEMCELL Technologies</td>
			<td>78150.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Soluble Receptor Activator of Nuclear Factor-&#954;B Ligand (hsRANKL)</td>
			<td>STEMCELL Technologies</td>
			<td>78214.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Stem Cell Factor (hSCF)</td>
			<td>STEMCELL Technologies</td>
			<td>78155.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TruStain FcX (Fc Receptor Blocking Solution)</td>
			<td>Biolegend</td>
			<td>422301</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Vascular Endothelial Growth Factor-165 (hVEGF165)</td>
			<td>STEMCELL Technologies</td>
			<td>78073</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Rhodamine Phalloidin</td>
			<td>Invitrogen</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM &#945;, nucleosides, no phenol red</td>
			<td>ThermoFisher</td>
			<td>41061029</td>
			<td></td>
		</tr>
		<tr>
			<td>mFreSR</td>
			<td>STEMCELL Technologies</td>
			<td>05855</td>
			<td>Serum free cryopreservation medium</td>
		</tr>
		<tr>
			<td>mTeSR Plus medium</td>
			<td>STEMCELL Technologies</td>
			<td>100-0276</td>
			<td>Human iPSC-serum free medium (hiPSC-SFM)</td>
		</tr>
		<tr>
			<td>Nunclon Sphera 96-Well, Nunclon Sphera-Treated, U-Shaped-Bottom Microplate</td>
			<td>Thermo Scientific</td>
			<td>174925</td>
			<td>Round bottom ultra-low attachment 96-well plate</td>
		</tr>
		<tr>
			<td>P1000 Wide Bore Tips</td>
			<td>ThermoFisher</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK-Inhibitor Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>StemSpan SFEM</td>
			<td>StemCell</td>
			<td>09650</td>
			<td>Hematopoietic cell culture medium</td>
		</tr>
		<tr>
			<td>TrypLE Select Enzyme (1X), no phenol red</td>
			<td>Thermo Fisher</td>
			<td>12563011</td>
			<td>Single-cell dissociation reagent</td>
		</tr>
		<tr>
			<td>Ultraglutamine</td>
			<td>Bioscience Lonza</td>
			<td>BE17-605E/U1</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Bioscience Lonza</td>
			<td>04-418Q</td>
			<td>Hematopoietic basal medium</td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 Well High</td>
			<td>Ibidi</td>
			<td>80806</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation and characterization of osteoclasts from human induced pluripotent stem cells

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.

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 ...

Watch this Scientific Journal Video about Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells at JoVE.com

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

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.

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

Abstract