-
Pipet tips (200 μl, 1 ml)
-
Cell strainer 70 μm (Corning, catalog number: 352350)
-
Nunclon Sphera 96-well plates, U-bottom (Thermo Fisher Scientific, catalog number: 174929)
-
Tissue culture 96-well plate, polystyrene (Greiner Bio-One, catalog number: 655180)
-
Tissue culture 6-well plate, polystyrene (BD, Falcon, catalog number: 08-772-1B)
-
Serological pipet, 5 ml (Fisher Scientific, catalog number: 13-678-11D)
-
Serological pipet, 10 ml (Fisher Scientific, catalog number: 13-678-11E)
-
15 ml polypropylene conical tubes (Thermo Scientific, catalog number: 339650)
-
50 ml polypropylene conical tubes (Thermo Scientific, catalog number: 339652)
-
Nalgene filter (0.2 μm PES membrane) 150 ml (Thermo Scientific, catalog number: 5650020)
-
Nalgene filter (0.2 μm PES membrane) 250 ml (Thermo Scientific, catalog number: 5680020)
-
Microcentrifuge tube (Fisher Scientific, catalog number: 05-408-129)
-
hiPSCs generated from healthy donors (Chen et al., 2013 and 2017)
-
Matrigel (Corning, catalog number: 354234)
-
PeproGrow embryonic stem cell (hESC) medium (Peprotech, catalog number: BM-hESC)
-
Accutase (Millipore Sigma, catalog number: SCR005)
-
Phosphate buffered saline, no calcium, no magnesium (Thermo Fisher Scientific, catalog number: 10010049)
-
DMEM/F12 medium (Thermo Fisher Scientific, catalog number: 11330032)
-
Stempro-34 medium (Thermo Fisher Scientific, catalog number:10639011)
-
L-Glutamine (200 mM) (Thermo Fisher Scientific, catalog number: 25030081)
-
1-Thioglycerol (MTG) (Sigma-Aldrich, catalog number: M6145)
-
Ascorbic acid (Sigma-Aldrich, catalog number: A4034)
-
MEM Non-essential amino acids (NEAA 100x) (Thermo Fisher Scientific, catalog number: 11140-050)
-
Rock inhibitor Y-27632 (Selleck Chemicals, catalog number: S1049)
-
Recombinant human BMP4 (Peprotech, catalog number: 120-05ET)
-
Recombinant human VEGF (Peprotech, catalog number: 100-20)
-
Recombinant human TPO (Peprotech, catalog number: 300-18)
-
Recombinant human Flt3-ligand (Peprotech, catalog number: 300-19)
-
Recombinant human IL-3 (Peprotech, catalog number: 200-03)
-
Recombinant human MCSF (Peprotech, catalog number: 300-25)
-
Recombinant human RANKL (Peprotech, catalog number: 310-01)
-
Recombinant human TGF-β1 (Peprotech, catalog number: 100-21C)
-
Recombinant human SCF (Peprotech, catalog number: 300-07)
-
Recombinant human FGF basic (Peprotech, catalog number: 100-18B)
-
Gelatin 0.1% solution (EMD Millipore, catalog number: ES-006-B)
-
Advanced DMEM medium (Thermo Fisher Scientific, catalog number: 12491023)
-
Alpha MEM medium (Thermo Fisher Scientific, catalog number: 12571063)
-
Fetal bovine serum (Gibco, catalog number:10437020)
-
1α, 25-dihydroxyvitamin D3 (Sigma Aldrich, catalog number: D1530)
-
CD14 antibody (FITC-conjugated; Biolegend, catalog number: 325604)
-
CD43 antibody (APC-conjugated, Miltenyi Biotec, catalog number: 560198)
-
CD45 antibody (APC-conjugated, Biolegend, catalog number:304012)
-
EDTA 0.5 M, pH 8.0 (Thermo Fisher Scientific, catalog number: 15575-038)
-
TRIzol (Thermo Fisher Scientific, catalog number: 10296028)
-
Direct-zol RNA (Zymo Research, catalog number: R2052)
-
DNase I (Invitrogen, catalog number: 18068015)
-
Superscript II reverse transcriptase (Invitrogen, catalog number: 18064071)
-
SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, catalog number: 1725271)
-
Glutaraldehyde 25% solution (Alfa Aesar, catalog number: A17876)
-
Acid phosphatase, leukocyte (TRAP) kit (Sigma-Aldrich, catalog number: 387A)
-
Primers for detection of mesoderm differentiation and osteoclast maturation by qPCR (Table 1)
-
Matrigel coating solution (see Recipes)
-
hiPSC culture medium (see Recipes)
-
EB mesoderm differentiation medium-1 (see Recipes)
-
EB mesoderm differentiation medium-2 (see Recipes)
-
Myelomonocytic expansion medium (see Recipes)
-
FACS buffer (see Recipes)
-
Osteoclast differentiation medium (see Recipes)
-
TRAP staining solution (see Recipes)
-
Reconstitution of hBMP4 (see Recipes)
-
Reconstitution of other cytokines (see Recipes)
Table 1. Sequences of qPCR primers to detect marker gene expression for mesoderm differentiation and osteoclastogenesis
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).
The author declares no conflict of interest.
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).