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 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.
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
2. Passaging iPSCs
3. Freezing back iPSCs
4. Embryoid body induction
5. Hematopoietic differentiation
6. M-CSF maturation and OC differentiation
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 of iPSCs throughout the differentiation process to OCs (Figure 2). iPSC colonies (Figure 2A) were dissociated into a single-cell suspension, which appears as individual cells throughout the round bottom well plate before centrifugation (Figure 2B). Following centrifugation (step 4.9 in the protocol), cells will collect in the center of the round bottom ultra-low attachment well plate and subsequently form spheres (embryoid bodies, EBs, Figure 2C). EBs will increase two to three times in size throughout the mesodermal differentiation process (Figure 2D) and become easily visible to the eye at the end of the 4-day differentiation period (Figure 2E). EBs can be seen adhering and fusing with the well plate bottom following the transfer into wells of a 6-well plate for hematopoietic differentiation (step 5.3 in the protocol, Figure 2F). After 7-8 days, large quantities of floating hematopoietic cells become visible in the culture medium (Figure 2G). After harvesting and replating hematopoietic cells, an M-CSF maturation phase follows, and OC differentiation is initiated (step 6.4 in the protocol). Within 5-6 days, multinucleated cells with a large transparent cell body first become visible (Figure 2H). A large number of mononuclear cells are still visible at this stage. After 2-3 more days of OC differentiation, OCs will further fuse with adjacent cells to form large polykarions with even more nuclei (Figure 2I).
Assessing the EB-derived hematopoietic population
Hematopoietic differentiation can be performed for a variable length of time. Time periods starting with 7 days up to 9 weeks have been described in the literature. In this protocol, hematopoietic differentiation is performed for a period of 10 days. We found that 10 days of hematopoietic differentiation yielded low numbers of early CD34+ (0.53%, Figure 3A) and a larger number of midterm stage CD43+ (48.5%, Figure 3B) hematopoietic progenitors. More critically, sufficient quantities of CD45+ (96.2%, Figure 3C), CD14+ (33%, Figure 3D), and CD11b+ (35.9%, Figure 3E) HPCs were generated after a treatment period of 10 days to successfully differentiate them further into OCs. However, the cytokine treatment period for hematopoietic differentiation (step 5.4 in the protocol) may need to be adjusted and optimized based on the iPSC line to generate adequate amounts of CD45+, CD14+, and CD11b+ cells.
On average, 6 million hematopoietic cells were harvested after 10 days of differentiation from each well with 8 EBs.
Assessing OC morphology and activity
Following OC differentiation, OCs can be assessed morphologically and functionally. OC precursors are reseeded onto chamber slides or coverslip slides following the M-CSF maturation step to improve image quality when staining for TRAP or Cathepsin K. Enzymatic TRAP staining following OC differentiation shows large, multinucleated, TRAP-positive OCs (Figure 4A). Additionally, slightly TRAP-positive mononuclear cells can be seen interspersed in between multinuclear OCs. Negative controls without the addition of RANKL do not display fused multinuclear OC. Nevertheless, a small number of slightly TRAP-positive mononuclear cells can be depicted (Figure 4B).
Confocal laser scanning microscopy (CLSM) images show OCs stained for Cathepsin K (turquoise) and F-actin (red) in conjunction with DAPI nuclear stain (blue) (Figure 4C, 4D). Large multinuclear OCs are visible when treated with RANKL according to the protocol, which depicts an extensive F-actin cytoskeletal structure and stain positive for Cathepsin K (Figure 4C). Negative controls without the addition of RANKL, on the other hand, do not show fused multinuclear cells.
OCs can be further assessed functionally by measuring the resorptive activity. Bone or mineral resorption assays can be used to determine the resorptive activity. Here, OC precursors were seeded onto calcium phosphate-coated wells and terminally differentiated. Large areas where the mineral coating was resorbed are visible in a bluish-gray color (Figure 4E). Resorption pits of different sizes can be identified. Not resorbed, the remaining calcium phosphate-coating is visible in brown. The presence of resorption pits confirms the identity of the differentiated multinucleated cells as OCs. Additionally, resorption pits can further be quantified in order to assess and compare the resorptive activity. The total area of resorbed mineral over the total well surface area can be quantified as a percentage measure. Additionally, the size and number of resorption pits can further be quantified. Untreated negative controls did not display resorption pits (Figure 4F).
Figure 1: Schematic illustration of the osteoclast differentiation process from human iPSCs. Illustration drawn by Hannah Blümke using Affinity Designer 2.1.1. The illustration utilizes previously used drawings33. Please click here to view a larger version of this figure.
Figure 2: Microscopy images throughout the differentiation process of human iPSCs toward osteoclasts. (A) Undifferentiated iPSC colonies throughout propagation. (B) iPSCs in round bottom wells after dissociation into a single cell suspension prior to centrifugation. (C) Centrally collected single cell iPSCs following centrifugation. (D) EBs grow in size throughout the 4-day mesodermal differentiation period. (E) Visible embryoid bodies following mesodermal differentiation. (F) Following the transfer of embryoid bodies onto basal membrane extract coated 6-well plates, embryoid bodies can be seen adhering and fusing with the well bottom. (G) After 5-7 days of hematopoietic differentiation, a large number of floating hematopoietic cells can be observed in the medium. (H) Following M-CSF maturation, the first osteoclasts with 3-4 nuclei appear after 5-7 days of differentiation with RANKL. (I) At the end of osteoclast differentiation, large multinucleated cells can be observed. Scale bars: A, D, F, G = 200 µm, B, C, H, I = 50 µm, E = 1 mm. Please click here to view a larger version of this figure.
Figure 3: Surface marker analysis of embryoid body-derived hematopoietic cells using flow cytometry. Marker expression allows for the analysis of hematopoietic cells and identification of sub-populations after gating for singlets and live cells. (A) Ontogenetically early CD34+ hematopoietic progenitor cell population is very small to absent in conjunction with this protocol. (B) CD43+ cells make up approximately 50% of the entire population. (C) Later stage CD45+ hematopoietic progenitor cells make up the largest part of the hematopoietic population with 96.2%. (D, E) More direct CD14+ and CD11b+ OC precursors make up 33% and 36%, respectively. In red: unstained negative control, in blue: isotype controls, in yellow: cells stained with the respective marker antibody. Plots use previously published data33. Please click here to view a larger version of this figure.
Figure 4: Morphological and functional assessment of iPSC-differentiated human osteoclasts. (A) TRAP staining following osteoclast differentiation shows large, multinucleated, TRAP positive osteoclasts. Slightly TRAP positive mononuclear cells can be seen interspersed in-between multinuclear osteoclasts. (B) Negative controls without the addition of RANKL stained for TRAP do not display fused multinuclear osteoclasts. Nevertheless, a small number of slightly TRAP positive, mononuclear cells can be depicted. (C) Confocal laser scanning microscopy images of osteoclast differentiated hematopoietic cells on a coverslip slide stained for F-actin (red) and Cathepsin K (turquoise) in conjunction with DAPI nuclear stain (blue) show large multinucleated Cathepsin K positive osteoclasts. (D) Negative controls without the addition of RANKL show similar to (B) mononuclear cells at a lower cell density. (E) Functional assessment can be performed by assessing the resorption activity of osteoclasts. For this, osteoclast differentiation is performed on a bone or mineral resorption assay. Tiled well images acquired with an inverted widefield microscope in phase contrast mode depict large resorption areas of the calcium phosphate mineral layer. Resorptive activity can be further quantified by measuring the resorption area over the total area. (F) Negative controls without the addition of RANKL do not show resorption areas. Scale bars: A, B = 100 µm, C, D = 50 µm, E, F = 1 mm. Images have been edited from previously published data33. Please click here to view a larger version of this figure.
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. Thawing and propagation of human iPSCs"), one cryovial was thawed into a single well of a 6-well plate. Different iPSC lines will behave slightly differently, and proliferation rates will vary. The split rate will need to be adjusted correspondingly.
When passaging iPSCs or dissociating iPSCs into a single-cell suspension for EB induction, it is important to remove any spontaneously differentiated colonies or aggregates of dead cells to improve the effectiveness and efficiency of mesodermal and hematopoietic differentiation. This can be done under a stereomicroscope by using a cell scraper, pipette tips from a P10 or P20 pipette, or a scraping tool built from a Pasteur pipette35.
As mentioned above, this protocol involves the dissociation of iPSC colonies into a single-cell suspension for EB induction, while with other protocols, iPSCs are left as aggregates to be centrifuged for EB induction36. EB size, cell number, shape, and morphology have all been reported to influence differentiation37,38,39. Thus, we hypothesize that the dissociation into a single-cell suspension allows for better EB-to-EB uniformity in size and shape after centrifugation and, hence, more consistent results of hematopoietic cell production31.
Other methods for EB induction have been described. Such methods that use a single-cell suspension in conjunction with ROCK inhibitor to improve cell survival have been reported to be advantageous in controlling EB size and differentiation outcome31.
The round-bottom 96-well plate EB induction method described in this protocol is suitable for large-scale production of EBs and allows for upscaling of OC production. More novel methods for hematopoietic differentiation without an embryoid body induction step with the potential to facilitate the differentiation process have recently been described32. Nonetheless, these protocols have not yet been established for OC differentiation33.
In the above-mentioned protocol, we describe the medium change on Day 5 of hematopoietic differentiation. A small number of floating cells may already appear around Day 4-5 of differentiation. In order to avoid discarding any floating cells, the medium should be collected in a tube and centrifuged before being discarded. The cell pellet at the bottom of the tube must then be dislodged with the fresh medium and should be transferred back to wells of a 6-well plate. The significance of the early floating cell population in OC differentiation still needs to be determined, however.
The production of hematopoietic cells can be assessed using flow cytometry. High yields of CD45+, CD14+ and CD11b+ cells are desirable for osteoclast differentiation33. Cryopreservation of harvested floating hematopoietic cells has been reported to be challenging with generally limited recovery rates and low cell viability40,41. By cryopreserving hematopoietic cells in a cryopreservation medium consisting of 50% of a serum-free hematopoietic cell expansion medium (see Table of Materials), 40% FBS and 10% DMSO, we were able to recover cells with a cell viability of approximately 90.3% ± 2.62 SD (n = 7) post thawing.
Osteoclastogenesis requires the fusion of multiple mononucleated OC precursors to form multinucleated osteoclasts that are capable of mineral and bone resorption. While murine OC-precursor cell lines simply need the addition of RANKL to induce OC formation42, human precursors require additional M-CSF for cell survival and proliferation43. OSCAR has recently been discovered as an additional receptor involved in OC differentiation, even though only type I collagen has thus far been identified as a ligand. While research of OSCAR with iPSC-derived OCs is still limited, supraphysiological in vitro RANKL and M-CSF concentrations in a murine cell line seem to bypass the necessity for OSCAR activation44, activation of OSCAR in vivo appears to be a necessary costimulatory signal for osteoclastogenesis45. An additional factor that needs to be considered is the well plate surface. Chemical46 and physical47 surface properties are known to influence osteoclast differentiation and can either promote or hinder successful differentiation. A certain heterogeneity within the precursor population also appears critical for successful fusion of OCs, as different fusion-related factors such as CD47 and DC-STAMP act at different stages of osteoclast fusion48.
In conclusion, this protocol enables the differentiation of human OC from iPSCs to facilitate and accelerate OC research.
The authors have nothing to disclose.
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.
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 |