Time- and cell-specific activation of BMP signaling restrains chondrocyte hypertrophy

. 2024 Jul 18;27(8):110537. doi: 10.1016/j.isci.2024.110537

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Rabbit anti-MGP	Abcam	Cat. No. ab224367
Rabbit anti-COMP	Abcam	Cat. No. ab231977
Rabbit anti-DPT	ThermoFisher	Cat. No. PA514396; RRID: AB_2094317
Rabbit anti-COL1A1	Abcam	Cat. No. ab34710; RRID: AB_731684
Mouse anti-COL2A1	Dev. Studies Hybridoma Bank	Cat. No. II-II6B3; RRID: AB_528165
Rabbit anti-COL10A1	Kuznetsov et al., 2019²⁹	Made by SBS/NIDCR/NIH
Rabbit anti-ACAN	Millipore Sigma	Cat. No. AB1031; RRID: AB_90460
Goat anti-ALPL	ThermoFisher	Cat. No. PA547419; RRID: AB_2609590
Rabbit anti-PRG4	Abcam	Cat. No. ab28484; RRID: AB_776089
Alexa 488 Goat anti-hVIMENTIN	R&D Systems	Cat. No. IC8104G; RRID: AB_2827831
Alexa Flour 488 Donkey anti-Goat	ThermoFisher	Cat. No. A11055; RRID: AB_2534102
Alexa 488 Donkey anti-Mouse	ThermoFisher	Cat. No. R37114; RRID: AB_2556542
Alexa Flour 546 Donkey anti-Rabbit	ThermoFisher	Cat. No. A10040; RRID: AB_2534016

Biological samples

Human bone marrow stromal cells/skeletal stem cells	Human bone specimens	see Table S1
NCRM NL5 human induced pluripotent stem cell line	Baghbaderani et al., 2015⁷²	NIH-Center for Regenerative Medicine
MIXL1-GFP human induced pluripotent stem cell line	This paper	N/A
SOX9-mCherry human induced pluripotent stem cell line	This paper	N/A

Chemicals, peptides, and recombinant proteins

Fetal Bovine Serum	GeminiBio	Cat. No. 100-50
Dexamethasone	Sigma	Cat. No. D4902
L-ascorbic acid phosphate magnesium salt n-hydrate	Wako	Cat. No. 013-19641
Vitronectin XF	Stemcell Technologies	Cat. No. 07180
Insulin-transferrin-selenium	ThermoFisher	Cat. No. 51500056
Recombinant TGFβ1	Peprotech	Cat. No. 100-21
Collagenase II	ThermoFisher	Cat. No. 17101015
Hyaluronidase	Merck	Cat. No. H3506
Dispase	Sigma	Cat. No. D4693
Bovine serum albumin	Miltenyi Biotec	Cat. No. 130-091-376
Pepsin	Millipore Sigma	Cat. No. R2283
Citrate buffer	Millipore Sigma	Cat. No. C9999
Matrigel	Corning	Cat. No. 356237
Polyvinyl alcohol	Sigma	Cat. No. P8136
Lipid concentrate	ThermoFisher	Cat. No. 11905031
Monothioglycerol	Sigma	Cat. No. M6145
Recombinant Activin A	R&D systems	Cat. No. 338-AC-050/CF
CHIR99021 (Wnt agonist)	R&D Systems	Cat. No. 4423
Recombinant FGF-2	R&D Systems	Cat. No. 233-FB
PIK90 (PI3K inhibitor)	Millipore Sigma	Cat. No. 528117
SB-431542 (TGFβ inhibitor)	Millipore Sigma	Cat. No. S4317
LDN-193189 (BMP inhibitor)	Sigma	Cat. No. SML0559
C59 (Wnt inhibitor)	Cellagen Technology	Cat. No. C7641-2s
PD173074 (FGF inhibitor)	R&D Systems	Cat. No. 3044
Purmorphamine (Hedgehog agonist)	ReproCell	Cat. No. 04-0009
Recombinant BMP-4	R&D systems	Cat. No. 314-BP-050
Recombinant BMP-2	Peprotech	Cat. No. AF-120
Recombinant GDF-5	Peprotech	Cat. No. 120-01

Critical commercial assays

EndoFree Plasmid Maxi Kit	Qiagen	Cat. No/12362
RNeasy Micro Kit with DNase treatment	Qiagen	Cat. No. 74004
SuperScript III First-Strand Synthesis SuperMix	ThermoFisher	Cat. No. 11752050

Deposited data

hiPSC RNA sequencing data	GEO	NCBI GEO accession number: GSE242526
Code	Github	GitHub: https://github.com/gadomskisj/Gadomski_et_al_iScience_2024

Experimental models: organisms/strains

Mouse: NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ	The Jackson Laboratory	Strain No. 005557
Rat: Sprague Dawley-Rag2^em2heraIl2rg^em1hera/HblCrl	Charles River	Strain No. 400

Oligonucleotides

Primers for qRT-PCR	U.S. Food and Drug Administration	see Table S2

Recombinant DNA

Plasmid: pUC57	Genscript Biotech Corp.	Cat. No. SD1176
Plasmid: LentiCRISPR v2	Addgene	Plasmid #52961

Software and algorithms

Cell Ranger version 7.0	10x Genomics	RRID:SCR_017344
SoupX (ambient RNA removal)	Young and Behjati, 2020⁷³	RRID:SCR_019193
scDblFinder (doublet removal)	Germain et al., 2022⁷⁴	RRID:SCR_022700
Seurat (integration and cluster analysis)	Stuart et al., 2019⁴⁵	RRID:SCR_016341
Clustree (determining cluster resolution)	Zappia & Oshlack, 2018⁷⁵	RRID:SCR_016293
clusterProfiler (pathway analysis)	Wu et al., 2021⁷⁶	RRID:SCR_016884
Bioconductor’s edgeR package (RNA sequencing analysis)	Robinson et al., 2009⁷⁷	RRID:SCR_012802
gProfiler (gene ontology)	Raudvere et al., 2019⁷⁸	version e107_e.g.,54_p17_bf42210
ComBat-Seq (adjusting batch effects with cross-comparison of published datasets)	Zhang et al., 2020⁷⁹	RRID:SCR_010974
Incucyte Image Analysis Software	Sartorius, UK	RRID:SCR_017316
ImageJ/Fiji Software	National Institutes of Health	RRID:SCR_002285
Flowjo 10.6 Software	FLOWJO, LLC	RRID:SCR_008520
GraphPad Prism 8 Software	GraphPad Software	RRID:SCR_002798

Other

HyA-FMBs	Gorodetsky et al., 1999²⁶; Kuznetsov et al., 2019²⁹	N/A
0.9 mm and 2.1 mm drill bits	FineScienceTools	Cat. No. 19007