Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) into Osteoclasts    

Materials and Reagents

Materials

1. Pipet tips (200 μl, 1 ml)

2. Cell strainer 70 μm (Corning, catalog number: 352350)

3. Nunclon Sphera 96-well plates, U-bottom (Thermo Fisher Scientific, catalog number: 174929)

4. Tissue culture 96-well plate, polystyrene (Greiner Bio-One, catalog number: 655180)

5. Tissue culture 6-well plate, polystyrene (BD, Falcon, catalog number: 08-772-1B)

6. Serological pipet, 5 ml (Fisher Scientific, catalog number: 13-678-11D)

7. Serological pipet, 10 ml (Fisher Scientific, catalog number: 13-678-11E)

8. 15 ml polypropylene conical tubes (Thermo Scientific, catalog number: 339650)

9. 50 ml polypropylene conical tubes (Thermo Scientific, catalog number: 339652)

10. Nalgene filter (0.2 μm PES membrane) 150 ml (Thermo Scientific, catalog number: 5650020)

11. Nalgene filter (0.2 μm PES membrane) 250 ml (Thermo Scientific, catalog number: 5680020)

12. Microcentrifuge tube (Fisher Scientific, catalog number: 05-408-129)

    
    

1. hiPSCs generated from healthy donors (Chen et al., 2013 and 2017)

2. Matrigel (Corning, catalog number: 354234)

3. PeproGrow embryonic stem cell (hESC) medium (Peprotech, catalog number: BM-hESC)

4. Accutase (Millipore Sigma, catalog number: SCR005)

5. Phosphate buffered saline, no calcium, no magnesium (Thermo Fisher Scientific, catalog number: 10010049)

6. DMEM/F12 medium (Thermo Fisher Scientific, catalog number: 11330032)

7. Stempro-34 medium (Thermo Fisher Scientific, catalog number:10639011)

8. L-Glutamine (200 mM) (Thermo Fisher Scientific, catalog number: 25030081)

9. 1-Thioglycerol (MTG) (Sigma-Aldrich, catalog number: M6145)

10. Ascorbic acid (Sigma-Aldrich, catalog number: A4034)

11. MEM Non-essential amino acids (NEAA 100x) (Thermo Fisher Scientific, catalog number: 11140-050)

12. Rock inhibitor Y-27632 (Selleck Chemicals, catalog number: S1049)

13. Recombinant human BMP4 (Peprotech, catalog number: 120-05ET)

14. Recombinant human VEGF (Peprotech, catalog number: 100-20)

15. Recombinant human TPO (Peprotech, catalog number: 300-18)

16. Recombinant human Flt3-ligand (Peprotech, catalog number: 300-19)

17. Recombinant human IL-3 (Peprotech, catalog number: 200-03)

18. Recombinant human MCSF (Peprotech, catalog number: 300-25)

19. Recombinant human RANKL (Peprotech, catalog number: 310-01)

20. Recombinant human TGF-β1 (Peprotech, catalog number: 100-21C)

21. Recombinant human SCF (Peprotech, catalog number: 300-07)

22. Recombinant human FGF basic (Peprotech, catalog number: 100-18B)

23. Gelatin 0.1% solution (EMD Millipore, catalog number: ES-006-B)

24. Advanced DMEM medium (Thermo Fisher Scientific, catalog number: 12491023)

25. Alpha MEM medium (Thermo Fisher Scientific, catalog number: 12571063)

26. Fetal bovine serum (Gibco, catalog number:10437020)

27. 1α, 25-dihydroxyvitamin D₃ (Sigma Aldrich, catalog number: D1530)

28. CD14 antibody (FITC-conjugated; Biolegend, catalog number: 325604)

29. CD43 antibody (APC-conjugated, Miltenyi Biotec, catalog number: 560198)

30. CD45 antibody (APC-conjugated, Biolegend, catalog number:304012)

31. EDTA 0.5 M, pH 8.0 (Thermo Fisher Scientific, catalog number: 15575-038)

32. TRIzol (Thermo Fisher Scientific, catalog number: 10296028)

33. Direct-zol RNA (Zymo Research, catalog number: R2052)

34. DNase I (Invitrogen, catalog number: 18068015)

35. Superscript II reverse transcriptase (Invitrogen, catalog number: 18064071)

36. SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, catalog number: 1725271)

37. Glutaraldehyde 25% solution (Alfa Aesar, catalog number: A17876)

38. Acid phosphatase, leukocyte (TRAP) kit (Sigma-Aldrich, catalog number: 387A)

39. Primers for detection of mesoderm differentiation and osteoclast maturation by qPCR (Table 1)

40. Matrigel coating solution (see Recipes)

41. hiPSC culture medium (see Recipes)

42. EB mesoderm differentiation medium-1 (see Recipes)

43. EB mesoderm differentiation medium-2 (see Recipes)

44. Myelomonocytic expansion medium (see Recipes)

45. FACS buffer (see Recipes)

46. Osteoclast differentiation medium (see Recipes)

47. TRAP staining solution (see Recipes)

48. Reconstitution of hBMP4 (see Recipes)

49. Reconstitution of other cytokines (see Recipes)

    
    

    Table 1. Sequences of qPCR primers to detect marker gene expression for mesoderm differentiation and osteoclastogenesis
    

Equipment

1. Pipettes

2. Water bath

3. AirClean 600 PCR workstation (AirClean Systems, model: AC648A)

4. Stereomicroscope (Carl Zeiss, model: Stemi 508)

5. Cell culture incubator (Thermo Fisher Scientific, model: Heracell 240i, catalog number: 51026332)

6. Eppendorf refrigerated centrifuge (Eppendorf, model: 5810R)

7. CFX96 Touch Real-time PCR Detection System (Bio-Rad, catalog number: 1855196)

8. MACSQuant Analyzer 10 (Miltenyi Biotech, catalog number: 130-096-343)

9. Upright microscope (Carl Zeiss, model: Axio Imager.D2m)

10. Tabletop scanning electron microscope (Hitachi, model: TM1000)

Software

1. CFX manager software (Bio-Rad, 18450000)

2. FlowJo^TM software (BD Bioscience, https://www.flowjo.com/)

3. GraphPad Prism (GraphPad, https://www.graphpad.com/)

4. Fiji ImageJ (NIH, image.nih.gov)

5. ZEN microscope software (Zeiss, https://www.zeiss.com/microscopy/us/products/microscope-software/zen-lite.html)

Procedure

1. Maintenance of hiPSCs cultures

   1. Prepare Matrigel-coated 6-well plate by adding 1 ml of coating solution (see recipe Table 4) to each well and leave the plate at room temperature for 1 h before use.

   2. Maintain hiPSCs in PeproGrow hESC medium (see recipe Table 5) and change medium every other day.

   3. Passage undifferentiated hiPSCs every 4-5 days.

      1. Aspirate old medium and add 2 ml fresh PeproGrow hESC medium to each well.

      2. Scrape undifferentiated hiPSC colonies into small pieces using 200 μl pipet tips under a stereomicroscope within an AirClean PCR Workstation. Depending on the size, a hiPSC colony can be broken up into 15-40 pieces.

      3. Aspirate the Matrigel coating solution from the freshly prepared plates and transfer the lifted hiPSC fragments to new Matrigel-coated plates.

         
         

2. Mesoderm differentiation of hiPSC-derived embryoid bodies (EBs) (Stage 1)

   1. Examine hiPSC cultures and ensure hiPSCs are undifferentiated. Lift and remove the differentiated hiPSCs, if any, using a 200 μl pipet tip under a microscope in the PCR Workstation.

   2. Prewarm an aliquot of Accutase solution in 37 °C water bath for 10 min.

   3. Aspirate hiPSC culture medium. Wash hiPSCs with pre-warmed PBS (2 ml per well) twice. Add 1 ml of Accutase solution to each well and incubate culture plates in 37 °C incubator for 10 min.

   4. Add 2 ml of DMEM/F12 medium to each well. Pipette the cell suspension with 10 ml pipette gently up and down to dislodge the hiPSCs. Transfer the contents to 50 mL conical tubes.

   5. Centrifuge the cells at 340 x g for 7 min at 4 °C. Resuspend cells in EB basal medium-1 (see recipe Table 6). Filter the cells using a 70 μm strainer. Count cells.

   6. Plate 15,000 cells to each well in 150 μl EB mesoderm differentiation medium-1 (see recipe Table 6) on a Nunclon Sphera 96-well plate. Keep the plates at 37 °C in a 5% CO₂ and 5% O₂ incubator for 2 days. Each well forms one EB.

   7. Change half medium by pipetting out 75 μl of EB mesoderm differentiation medium-1 and adding 75 μl of EB mesoderm differentiation medium-2 (see recipe Table 7).

   8. Keep the plates at 37 °C in a 5% CO₂ and 5% O₂ incubator for 2 more days.

   9. Analyze mesoderm differentiation by examining the expression levels of mesoderm marker genes in EBs cultured for 1, 2, 3, and 4 days by qPCR (Figure 1).

      
      

      
      Figure 1. Expression levels of mesoderm marker genes hCDX2, hCD34, hSCL, hT, hHand, in EBs cultured for 1, 2, 3, and 4 days (d1, d2, d3 and d4). * P < 0.05 by one-way ANOVA compared to day 1 samples. Data was relative quantification and presented as mean ± SEM.

      
      

3. Expansion of myelomonocytic population released from EBs (Stage 2)

   1. Prepare gelatin-coated plate by adding 1 ml of 0.1% gelatin solution to each well of a 6-well plate and leave at room temperature for 1 h before use.

   2. Collect EBs from Nunclon Sphera 96-well plate by suctioning out the medium with EBs using a 1 ml pipet tip and transfer these EBs to a 50 ml conical tube.

   3. Let EBs sink down by gravity for 3-5 min.

   4. Aspirate medium with a glass Pasteur pipette carefully without removing EBs in the bottom.

   5. Resuspend EBs in myelomonocytic expansion medium (see recipe Table 8) according to numbers of EBs. Seed 40-50 EBs in one well of 6-well plate with 4 ml of myelomonocytic expansion medium. For example: EBs collected from a full 96-well plate can be resuspended in 8 ml medium and plated in 2 wells of a gelatin-coated 6-well plate.

   6. Keep the plates at 37 °C in a 5% CO₂ and 5% O₂ incubator.

   7. EBs attach to the plates. Some cells are released from EBs. The floating non-adherent cells are the myelomonocytic population.

   8. Change expansion medium after 4 days with the same medium but increase hMCSF concentration from 50 ng/ml to 100 ng/ml.

   9. Continue culture for another 13 days (medium changes at days 4, 8, 12, and 16).

   10. Floating cells released from EBs at days 10, 13, 17, 21 are collected and stained with primary antibodies against CD14 (1:20), CD43 (1:50), and CD45 (1:50) in FACS buffer (see recipe Table 9) for 30 min on ice. Protect from light. Table 2 shows FACS analysis data.

       
       

       Table 2. CD14, CD43, and CD45 expression in cells collected at day 10, 13, 17, and 21 days in Stage 2 analyzed by flow cytometry
       

       Data presented: mean ± SD. Data collected from 3 hiPSC lines. Each line had 3-4 technical repeats.

       
       

4. Promotion of osteoclast maturation (Stage 3)

   1. Collect floating cells (17 days of Stage 2) to a 15 ml or 50 ml conical tube. Typically, 0.2-0.45 million cells can be expected from EBs grown on one well of a gelatin-coated 6-well plate.

   2. Centrifuge the cells at 340 x g for 7 min at 4 °C. Count cells.

   3. Plate cells at a density of 10,000 cells per well in a 96-well plate and on bone chips.

   4. Culture osteoclast progenitors in osteoclast differentiation medium (see recipe Table 10). Change medium every 2-3 days.

   5. Multinucleated mature osteoclasts form in 7-10 days in osteoclast differentiation medium (Figure 2A).

   6. Fix hiPSC-OCs at day 10-12 for TRAP staining (Figures 2B-2C).

      1. Remove culture medium. Wash gently with PBS once.

      2. Fix cells in 2.5% glutaraldehyde (150 μl per well) at room temperature for 10 min.

      3. Prepare TRAP staining solution (see recipe Table 11).

      4. Add 100 μl of TRAP solution in each well of 96-well plate.

      5. Incubate culture plate at 37 °C for 45 min to 1 h.

      6. Remove the TRAP solution. Rinse plates with distilled water.

      7. Dry the plate before taking images.

   7. Fix TRAP stained cells on bone chips at day 14. Take images of resorption pits with TM1000 tabletop scanning electron microscope (Figure 2D).

   8. Confirm differentiation status of osteoclasts by examining expression levels of osteoclast marker genes by qPCR (Table 3).

      
      

      
      Figure 2. hiPSC-OC cultures. A. Bright field of hiPSC-OC culture at day 10 in OC differentiation medium. Scale bar = 100 μm; B. TRAP staining of one well of 96-well plate. Purplish large sized cells are TRAP positive multinucleated osteoclasts; C. High magnification of TRAP-positve multinucleated cells. Scale bar = 100 μm; D. Resorption pits (indicated by yellow asterisks) on bone chip. Large black spots are TRAP positive osteoclasts. Scale bar = 200 μm.

      
      

      Table 3. qPCR data of expression levels of osteoclast marker genes in hiPSC-osteoclasts
      

      Data presented: mean ± SD. Data collected from 3 hiPSC lines. Each line had 3 technical repeats. *P < 0.05 by Student’s t-test. OC progenitors are the floating cells collected at day 17 of stage 2 and cultured in osteoclast differentiation medium without 1α,25-dihydroxyvitamin D₃, hTGFβ-1, and hRANKL.

Data analysis

1. qPCR data were analyzed using CFX manager software. The significant difference was determined by Student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test using GraphPad Prism software.

2. Flow cytometry data were analyzed using FlowJo software.

Notes

1. Differentiation efficiency of stem cells can vary based on the size of EBs (Moon et al., 2014, Ng et al., 2015). The step of forming uniform-sized EBs is important to achieve consistant efficiency of hiPSC-OCs differentiation.

2. We determined the duration of Stage 2 as 17 days based on flow cytometry data for the expression levels of CD14, CD43, and CD45, which are expressed highest during these 17 days with little variability in comparison to other time points.

3. The non-specific/spontaneous differentiation of hiPSCs can be identified by their heterogeneous morphology within a hiPSC colony. Differentiation usually starts in the center of a large hiPSC colony and appears as a dark field under a brightfield microscope.

4. Isogenic hiPSCs have identical genetic background except for a genetic variant that has been introduced. Thus isogenic hiPSCs are the best controls to use when researchers aim to determine the impact of a specific disease mutation.

Recipes

1. Matrigel coating solution (Table 4)

   1. Thaw frozen Matrigel in refrigerator overnight and aliquot Matrigel in ice-cold microcentrifuge tubes (60 μl for one 6-well plate)

   2. Thaw Matrigel aliquots on ice and transfer Matrigel to ice-cold DMEM/F12 medium. For one 6-well plate, add 60 μl Matrigel to 6 ml DMEM/F12 medium

      
      

      Table 4. Matrigel coating solution
      

      
      

2. hiPSC culture medium (Table 5)

   1. Centrifuge the vial with lyophilized Peprotech growth factor prior to opening and reconstitute with sterile water. Use 500 μl water for each 500 ml PeproGrow hESC basal medium kit

   2. Add the reconstituted growth factor to the basal medium aseptically and mix well by swirling or pipetting. Store at 2-8 °C and use within 2 weeks

      
      

      Table 5. hiPSC medium
      

      
      

3. EB mesoderm differentiation medium-1 (Table 6)

   1. Prepare 100 ml EB mesoderm differentiation medium as shown in Table 6

   2. Sterilize EB basal medium (including Stempro-34, L-glutamine, MTG) by filtering through a 0.2 μm PES membrane filter and keep at 2-8 °C for up to 1 month

   3. Add hBMP4, hVEGF, Y-27632, and ascorbic acid in EB basal medium just before use

      
      

      Table 6. EB mesoderm differentiation medium-1
      

      
      

4. EB mesoderm differentiation medium-2 (Table 7)

   1. Prepare 100 ml EB mesoderm differentiation medium as shown in Table 7

   2. Sterilize EB basal medium (including Stempro-34, L-glutamine, MTG) by filtering through a 0.2 μm PES membrane filter and keep at 2-8 °C for up to 1 month

   3. Add 2x concentration of hBMP4, hVEGF, Y-27632, and ascorbic acid in EB basal medium just before use

      
      

      Table 7. EB mesoderm differentiation medium-2
      

      
      

5. Myelomonocytic expansion medium (Table 8)

   1. Prepare 100 ml myelomonocytic expansion medium as shown in Table 8

   2. Sterilize myelomonocytic expansion medium (including advanced DMEM, FBS, L-glutamine, β-mercaptoethanol) by filtering through a 0.2 μm PES membrane filter and keep at 2-8 °C for up to 1 month

   3. Add hIL-3 and hM-CSF aseptically only before use. Increase hM-CSF concentration from 50 to 100 ng/ml starting from day 4 medium change

      
      

      Table 8. Myelomonocytic expansion medium
      

      
      

6. FACS buffer (Table 9)

   1. Prepare 500 ml of FACS buffer as shown in Table 9

   2. Store at 2-8 °C for up to 6 months

      
      

      Table 9. FACS buffer
      

      
      

7. Osteoclast differentiation medium (Table 10)

   1. Prepare 100 ml osteoclast differentiation medium as shown in Table 10

   2. Sterilize osteoclast differentiation basal medium (including alpha-MEM, FBS) by filtering through a 0.2 μm PES membrane filter and keep at 2-8 °C for up to 1 month

   3. Add vitamin D, hTGFβ, hMCSF, and hRANKL aseptically only before use

      
      

      Table 10. Osteoclast differentiation medium
      

      
      

8. TRAP staining solution (Table 11)

   1. All reagents are included in the acid phosphatase, leukocyte (TRAP) kit

   2. Premix Fast Garnet GBC and sodium nitrate

   3. Add the rest of reagents after 2 min

      
      

      Table 11. TRAP staining solution
      

      
      

9. Reconstitution of hBMP4

   1. Centrifuge the vial prior to opening

   2. Reconstitute in 5 mM HCl, pH 3.0 to a concentration of 0.1-1.0 mg/ml

   3. Do not vortex

   4. Further dilute in a buffer containing 0.1% BSA as carrier protein and store in working aliquots at -80 °C

10. Reconstitution of other cytokines

    1. Centrifuge the vial prior to opening

    2. Reconstitute in water to a concentration of 0.1-1.0 mg/ml

    3. Do not vortex

    4. Further dilute in a buffer containing 0.1% BSA as carrier protein and store in working aliquots at -80 °C

Acknowledgments

The work was supported by funds from National Institute of Health (K99/R00 DE021442 and R01 DE025664). We adapted published protocols using M-CSF and IL-3 for myeloid differentiation (Panicker et al., 2012; Jeon et al., 2016).

Competing interests

The author declares no conflict of interest.

Ethics

The hiPSC lines from peripheral blood were generated as previous described (Chen et al., 2013; Chen et al., 2017). This work was in accordance with guidelines of the Institutional Review Board of the University of Connecticut Health (IRB protocol 09-199).

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