Retinoic Acid Mediates Visceral-specific Adipogenic Defects of Human Adipose-derived Stem Cells

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1 Retinoic Acid Mediates Visceral-specific Adipogenic Defects of Human Adipose-derived Stem Cells Kosuke Takeda, Sandhya Sriram, Xin Hui Derryn Chan, Wee Kiat Ong, Chia Rou Yeo, Betty Tan, Seung-Ah Lee, Kien Voon Kong, Shawn Hoon, Hongfeng Jiang, Jason J. Yuen, Jayakumar Perumal, Madhur Agrawal, Candida Vaz, Jimmy So, Asim Shabbir, William S. Blaner, Malini Olivo, Weiping Han, Vivek Tanavde, Sue-Anne Toh, and Shigeki Sugii Supplementary Experimental Procedures RNA-Seq experiment and analysis Total RNA was extracted from SC and VS fat depots using Qiagen RNeasy plus kit. Poly-A mrna was then enriched from ~5 µg of total RNA with oligo dt beads (Life Technologies). Approximately 100 ng of poly-a mrna recovered was used to construct multiplexed strand-specific RNA-seq libraries as per manufacturer s instruction (NEXTflexTM Rapid Directional RNA-SEQ Kit (dutp-based) v2). Individual library quality was assessed with an Agilent 2100 Bioanalyzer and quantified with a QuBit 2.0 fluorometer before pooling for sequencing on Illumina HiSeq 2000 (1x101 bp read). The pooled libraries were quantified using the KAPA quantification kit (KAPA Biosystems) prior to cluster formation. Fastq formatted reads were processed with Trimmomatic to remove adapter sequences and trim low quality bases (LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36). Reads were aligned to the human genome (hg19) using Tophat version 2 (settings --no-coverage-search --librarytype=fr-firststrand). Feature read counts were generated using htseq-count (Python package HTSeq default union-counting mode, strand=reverse). Differential Expression analysis was performed using the edger package in both classic and generalized linear model (glm) modes to contrast SC and VS adipose tissues from 8 non-diabetic patients. ipathwayguide (Advaita bioinformatics), which is a webbased pathway analysis tool, was further employed to identify the top biological pathways regulated. A false discovery rate cutoff of 0.05 and fold change of at least 2.0 was used to call significance. Microarray experiment and analysis RNA from ASCs was isolated using the TRIzol reagent (Invitrogen) and purified with the Column RNA easy kit (Qiagen) according to the manufacturer s instruction. Double-stranded cdna was synthesized from 500 ng of isolated mrna with an oligo (dt) primer by using SuperScript Choice (Invitrogen) containing a T7 RNA polymerase promoter site, and then purified with Phase Lock Gel (Eppendorf). Biotin-labeled crna was prepared by transcribing with an X transcript labeling kit (Enzo). After adjusting for possible carryover of residual RNA, a mixture containing 15 mg of biotinylated crna was prepared and hybridized to the Illumina Human HT-12 v4 array that has 47,000 probe sets representing more than 31,000 different annotated genes. After 16 hours of hybridization, the Beadchips were washed, Cy3-labeled, and scanned on Illumina BeadArray reader. The data files were uploaded to GenomeStudio (Illumina) for background subtraction and analyzed on Partek Genomic Suite. The data was normalized using quantile normalization and an Analysis of Variance (ANOVA) was performed. A list of differentially expressed genes was created using a fold-change cut-off larger than 2.0 and a False Discovery Rate (FDR) cut-off less than The data will be deposited in NCBI database.

2 LC/MS analyses of retinoids Samples were extracted using 100 μl out of 400 μl cell solution (2.5 x 10 5 ASCs) diluted in PBS. 500 µl of absolute ethanol containing 100 ng of retinyl acetate (Sigma-Aldrich) was then added as an internal standard. Subsequently, 4 ml of hexane was mixed with the samples and centrifuged for 10 min at 2000 g. The organic upper phase was then isolated and evaporated with N 2. The dried lipid was resuspended in 30 μl of methanol:acetonitrile (vol/vol, 1:1) and transferred to an autosampler vial (Waters Corp.) for analysis. For atra extraction, 0.5 ml of M KOH in ethanol was added to 300 μl cell solution (1.5 x 10 6 ASCs). 5 ng of atra-d5 dissolved in absolute ethanol was added to each extraction as internal standard. The aqueous phase was extracted with 4 ml of hexane. The organic phase containing nonpolar retinoids (retinol and retinyl ester) was removed. 30 μl of 8 M HCl was added to the aqueous phase, and polar retinoids (atra) were removed by extraction with 4 ml hexane. Organic phases were removed under a gentle stream of N 2. RA extracts were resuspended in 40 μl of acetonitrile and transferred to amber LC/MS vials. All LC/MS analyses were performed using a Waters Xevo TQ MS Acquity UPLC system (Waters Corp.), under the control of MassLynx software V4.1. Prior to injection, samples were maintained in the autosampler at 4 C. Samples were injected into an Acquity UPLC BEH Phenyl column ( mm; with 1.7 μm particles) for retinol and an ACQUITY UPLC BEH HSS column (3.0 X 100 mm; 1.7 µm particle size) for analysis of atra. Samples were separated in the presence of formic acid using water/acetonitrile gradients for atra. Positive ESI-MS was performed in the multiple reaction monitoring (MRM) mode. Penta-deuterated atra (atra-d5) was employed as an internal standard. Measurement of endogenous RA levels by Surface-enhanced Raman spectroscopy (SERS) ASC pellet samples were sonicated for 30 min, and centrifuged. 2 μl of the supernatant was diluted with 48 μl of sample diluents (Biosource). 50μl of the diluted samples were placed into the micro-well followed by 50μl of HRP conjugate, which had been pre-coated with anti-retinoic acid (Biosource). Samples were mixed well with the plate shaken gently for 60 seconds and incubated for 60 minutes at 37 C. They were then washed five times with 200μl Wash Buffer (Biosource), and let stood for 2 minutes. After any remaining liquid was decanted, 90μl of TMB (3,3,5,5 -Tetramethylbenzidine) Substrate (Sigma-Aldrich) was added to each well and incubated for 20 minutes at 37 C, protected from light. 50μl of 0.5 M H 2 SO 4 was then added to each well to stop the reaction. Before taking SERS measurement, 45 μl of 60 nm Au colloid (BBI Solutions) is added to the reaction mixture. 10 μl of the solution mixture was dropped onto the glass slide and protected with coverslip. SERS measurements were performed in reflection mode with a Raman microscope (Renishaw InVia) using a 633 nm excitation laser with 5% laser power, a 1800 line/mm grating and a cooled CCD (-70 C). A 20X objective lens (NA 0.4) delivered the laser beam and collected the back scattered light. Rayleigh scattering was blocked with a notch filter. The laser spot size was ~3 µm with a power of 0.28mW. Measurements were performed with a 10 second integration time and at multiple positions across each SERS region with data averaged. Background corrections and curve fittings were carried out using WiRE 3.2 (Renishaw software). Spectra were background subtracted by a 6-order polynomial fit before the curve-fitting procedure. The instrument is calibrated with signal from a silicon standard at 520 cm 1. Different atra concentrations were calculated from standard RA curve based on the intensity of 1605 cm -1 peak from TMB 2+ generated from TMB (Figure S2).

3 Supplementary Figure S1. RA induces majority of VS-ASC genes and suppresses a subset of SC- ASC genes SC-ASCs were treated with varying concentrations of RA, ranging from 0.01 µm to 10 µm RA for 48 hours and RT-qPCR was performed on the RNA collected. (A) Representative graphs showing significant increase in mrna expression of VS-ASC enriched genes from Figures 2 and 3, including CRBP1 (i), MGP (i), RARRES1 (ii), RARRES2 (ii), NR2F1 (iii), CDKN2B (iii), PLAT (iv), HOXA4 (iv), HOXA5 (iv), F3 (v) and CDH6 (v) and no change in BAPX1 (i), PTGS1 (iii) and MEIS1 (v) expression in SC-ASCs from S11, upon RA treatment. (B) Representative graphs showing significantly decreased mrna expression of SC-ASC enriched genes from Figure 3, such as MME (i), TNC (i), ENPP2 (ii) and CRYAB (ii), and no change in PITX2 (i) and CTSK (ii) expression in SC-ASCs from S11 upon RA treatment (n=2). *p<0.05 denotes significance.

4 Supplementary Figure S2. Ultrasensitive SERS measurement of RA (A) The scheme of TMB to TMB 2+ reaction (i) and SERS measurement using Au colloid (ii). (B) Comparison of the SERS spectra for both reactant TMB and product TMB 2+. (C) RA concentration dependent SERS spectra of TMB 2+. RA levels were calculated based on the intensity of 1605 cm -1 peak from TMB 2+. (D) Representative graph showing increased levels of RA in VS-ASCs compared to SC- ASCs as measured by SERS. The data shown are calculated from cells of five subjects (S2, S4, S6, S11 and S13). **p<0.01 denotes significant change (n=5). This figure is related to Figure 4A-B.

5 Supplementary Figure S3. RA inhibits the early stage of adipocyte differentiation of VS-ASCs Representative 10X images showing the lipid accumulation (AdipoRed-green) and the nuclei (Hoechst blue) in S11 VS-ASCs treated with 1 µm or 10 µm of RA at different time points as indicated. (i) No induction, (ii) No RA, (iii) D-2 to D0, (iv) D0 to D3, (v) D3 to D6, (vi) D6 to D9, or (vii) D9 to D12 treatment with RA. For the presentation purpose, the fluorescent intensities were enhanced to the same degree for all images. Scale bar represents 100 µm (n=2). This figure is related to Figure 5.

6 Supplementary Figure S4. Retinol (ROL) and Retinaldehyde (RAL) inhibit adipocyte differentiation of ASCs (A) (i) Representative graph showing relative fluorescence units (RFU) of AdipoRed staining on S11 SC- and VS-ASCs. ASCs were treated with 50 µm of ROL at different time points as indicated. **p<0.01 denotes significant fold change against respective No ROL control SC-ASC samples and ^^^p<0.001 denotes significant fold change against No ROL control VS-ASC samples in RFU corresponding to the quantitation of lipid accumulation during adipocyte differentiation (n=2). (A) (ii) Representative 10X images showing the lipid accumulation (AdipoRed-green) in S11 SC-ASCs and VS- ASCs treated with 50 µm of ROL at different time points as indicated. Similar results were obtained from experiments using S12. For the presentation purpose, the fluorescent intensities were enhanced to the same degree for all original images. Scale bar represents 100 µm (n=2). (B) (i) Representative graph showing relative fluorescence units (RFU) of AdipoRed staining on S11 SC- and VS-ASCs. ASCs were treated with 10 µm of RAL at different time points as indicated. **p<0.01 and ***p<0.001 denotes significant fold change against respective No RAL control SC-ASC samples and ^^^p<0.001 denotes significant fold change against No RAL control VS-ASC samples in RFU corresponding to the quantitation of lipid accumulation during adipocyte differentiation (n=2). (B) (ii) Representative 10X images showing the lipid accumulation (AdipoRed-green) in S11 SC-ASCs and VS-ASCs treated with 10 µm of RAL at different time points as indicated. Similar results were obtained from experiments using S12. For the presentation purpose, the fluorescent intensities were enhanced to the same degree for all original images. Scale bar represents 100 µm (n=2).

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8 Supplementary Table S1. RNA-seq analysis of differentially expressed genes from VS and SC depots Gene Symbol logfc Fold Change (VS Vs SC) P Value SCEL E-59 LRP E-77 KLK E-56 WT E-40 KLK E-57 C21orf E-51 WT1-AS E-26 LRRN E-49 CFTR E-34 SYT E-51 ISL E-39 SGPP E-42 DNAJC E-37 CDH E-83 ANXA E-22 LOC E-39 PKHD1L E-28 MUC E-52 DSC E-45 TMEM151A E-49 ANXA8L E-22 KLK E-26 GATA E-23 MSLN E-27 UPK1B E-40 KLK E-22 KRT6A E-23 CGN E-25 ITLN E-26 ALOX E-35 GALNT E-13 CBLN E-14 ITLN E-30 BARX E-29 CKMT1B E-27 BNC E-26 KIRREL E-08 COX6B E-16 LOC E-34 CLDN E-18 FAM163A E-13 MYRF E-18 CKMT1A E-24 PSAPL E-08 KRT E-16 RBFOX E-14 ILDR E-19 KRT E-19 KCNH E-19 C1QL E-30 CXCL E-22 SIAH E-12 CACNG E-19 RIPK E-16 WIF E-09 MISP E-21 KRT E-20 LRRTM E-15 MGARP E-27 KRT E-15 SLITRK E-28 WNK E-35 HS3ST E-10 FAM101A E-12 EPS8L E-22 GREM E-19 TCF E-100 CA E-07 LRAT E-22 UPK3B E-16 IGSF E-15 HSD17B E-08 CSDC E-26 SERPINE E-18 DPYS E-27 TMPRSS E-35 IGFL E-16 ANGPTL E-19 KLK E-15 GRB E-14 LINC E-27 CAMSAP E-14 SLC26A E-05 ASTN E-15 RNF E-07 LHX E-14 HSD17B E-10 C4B E-65 HOOK E-18 FAM110C E-18 HNRNPA1P E-18 NPHS E-12 CRB E-10 SEC14L E-15 FLRT E-17 TCEAL E-12 RSPO E-05 PTN E-63 TNNT E-09 CARNS E-14 SMPD E-15 C10orf E-12 TCEAL E-15 SBK E-09 EPPK E-39 NELL E-15 KCTD E-14 FAM153B E-20 RPRM E-10 ISM E-13 FAM160A E-18 DPP E-12

9 CCDC E-21 PRR15L KCTD E-21 TFF E-10 CXADR E-09 CYP2J E-19 IL E-11 CST E-17 IGFBP E-17 CLUL E-09 SLC27A E-11 RAET1E E-11 SERTM E-07 IL CLEC4M E-06 KNDC E-12 WFDC E-08 THSD E-20 ESRP E-15 ANXA E-12 MTMR E-20 SLC28A E-10 MMP E-09 PCSK E-08 VSNL E-13 MSI E-13 BCHE E-22 FAM153C E-23 CNTNAP E-19 CACNB E-11 DMKN E-24 VIPR E-36 LINC E-29 C4A E-22 SCG E-13 EEF1A E-08 FGF E-14 SBSPON E-11 SLC6A E-11 B4GALNT E-14 ARMC E-06 FRAS E-08 NTNG E-18 CCL E-10 ROR E-16 STAB E-12 UNC5B-AS E-07 CHAC E-09 ITGB E-09 VAT1L E-07 NSG E-10 UCP E-08 LSAMP E-13 MAPK E-15 CDH E-05 ASPHD E-10 TRIM E-08 GRIK E-13 LOC E-06 TGM E-08 DPCR RORB E-13 FAM167A E-12 GPM6A E-19 ATRNL E-08 SYN E-12 LOC E-14 PEX5L E-16 PCP E-05 SPEG E-10 LYPD E-12 FMN E-13 IQCA E-08 POF1B E-07 FAXC E-05 REEP E-11 NKX E-17 NGEF E-07 SMTNL E-06 DKFZp434J E-06 ERBB E-17 TFPI E-23 NR2F E-33 WNT10A E-20 BEX E-09 BMP E-05 CHGB E-07 PCDHA E-09 LINC E-05 DLGAP E-16 IL1A NPNT E-16 DOC2A E-16 RARRES E-23 FBXO E-11 GPR E-09 TMEM255A E-12 PCDH E-08 RHPN E-11 WISP E-05 DSP E-09 HPR E-10 PLEKHG4B E-09 SMPDL3B E-05 NRG E-07 FAM153A E-11 CYP21A1P E-08 GJB E-11 CBLN E-06 GAL3ST CPA E-05 WNT5A E-19 DAW E-06 LOC E-10 NGF E-10 FAM189A E-11 HS6ST E-05 ST6GAL E-06 SLPI E-10 TRPM E-07

10 CACNA1G E-13 TNFSF E-13 ZDHHC8P E-06 RAET1G E-07 SULF E-11 LOC E-12 NPAS E-10 PARD6B E-07 RGS E-06 ART E-08 LRRN E-19 FBXL E-06 RASEF E-05 CLIC E-13 NTRK E-09 C E-26 MMRN E-06 HHIP E-06 KCNA E-15 KIAA E-06 NELL E-11 PRPH E-09 INMT E-16 BRINP E-06 RIMBP E-13 TRNP E-10 GAL3ST E-09 IGSF9B E-14 STK E-07 LRRN E-09 DPY19L2P E-11 SCUBE E-06 RAMP E-08 CHST E-07 ALDH1A E-08 LINC E-05 MTUS E-07 PLLP E-11 SHROOM E-11 PRR E-05 CYP21A E-07 MYO5B E-05 MST1R E-12 SLC44A E-09 EFNB E-13 GADL PTPRZ NFASC E-28 MKX E-14 SSTR E-05 PGM5P3-AS E-09 GATA E-15 AMN E-07 TVP23A E-11 COBL E-06 PDPN E-10 NWD E-10 HOXB-AS E-14 GPR E-12 TIMD CFB E-05 DNER E-07 PAPPA E-13 SIX E-11 KRT GATA6-AS E-11 OSR E-12 MAP3K E-11 ASPG E-06 SCN3A E-10 KCNN E-07 ALDH1L1-AS E-13 TUBB4A E-08 SAMD E-11 BCAS BDKRB SEZ6L E-08 BEGAIN E-08 SDR42E E-09 AADACL ADAMTSL E-23 MCTP E-06 BMPR1B E-06 KBTBD E-05 KCNK E-09 OBSCN E-12 SPTBN E-12 PCSK1N E-08 RASSF RAB TSPAN E-12 LIF E-05 DCT E-08 LAMP AMHR KLK FOXF E-12 PCSK E-11 RSPO E-16 LOC GALNT E-09 CDH E-08 CLIC E-13 HS3ST E-06 GNG E-05 ARNT E-07 TNFAIP8L E-06 C1orf E-06 CTXN E-05 APLP E-10 CDCP E-05 PTPRQ E-18 VWA E-07 AP1M BCO E-09 PPAPDC1A FERMT E-05 SLAIN E-11 GOLGA2P E-09 TNNT E-05

11 LOC E-08 FAM155A E-09 EZR E-07 LY6H OGN E-08 TNK E-07 CGNL E-09 STK E-06 SUSD E-06 ARSJ E-08 ARHGAP E-12 HAND E-07 RBM SLC4A E-17 SHISA E-06 WWC E-06 MKRN9P WNT2B E-11 SLC39A E-05 CLDN E-09 SCN7A E-08 PRIMA E-16 COLGALT E-06 SLC2A E-12 PLEK LOC CHODL E-05 CXCR E-09 MFSD2A FGFR E-08 SYT E-05 RASSF E-09 GDF E-06 ALDH1A E-07 NEK CCDC E-08 SGOL E-07 GPR E-06 BCAS PDZK1IP E-07 PNMA E-09 NUDT E-06 CA E-08 PROX E-13 NEBL E-07 SRSF E-06 PRKCZ E-07 CFI E-12 PRSS E-05 COL9A E-06 WDR86-AS E-08 HAND2-AS E-10 GLS E-05 BEAN E-05 FOLH E-08 BZRAP E-12 RDH E-08 GREM PTGS E-10 SLC12A E-07 SLC16A E-14 RGS E-07 EVPL SEMA3B E-20 METTL E-06 HHIP-AS E-05 FAR2P E-07 NKD IL2RA E-09 LOC E-05 KIAA E-07 TFAP2C E-07 KCTD E-07 TRIM KLF WDR E-12 LAYN E-06 CABP KLHL PLEKHB E-07 MUM1L E-13 DIRAS E-05 SLC14A E-07 KLHL E-05 CD E-10 CACNA1H E-07 CRMP MET E-08 CMYA E-09 FAIM E-06 TTC E-06 CFAP NRK E-15 NCR3LG E-05 PTPRH E-06 SPATA E-06 LOC E-08 IL E-05 HOXA E-09 DIRAS DIAPH TIMP E-06 EGFEM1P E-08 PPFIA RARB E-08 KCNK CPNE E-07 HTR2A B3GALT SLC38A E-07 PRG HIGD1B E-06 PLEKHA E-08 LAMB E-13 LMO E-05 COL4A E-08 SDC E-05 LXN E-08 WNT TMEM

12 DLG E-09 SCN2A E-07 GPR E-08 LRRC4B E-08 HIST1H2BD E-06 SUSD E-05 KIAA SEMA4G E-11 KIAA E-07 CLDN SMOC E-10 IDO E-06 PLAT E-10 DSCAML E-06 GFPT COL21A ABHD12B SLC16A E-06 SNX E-05 PKP FHDC E-07 LONRF E-14 HOXA-AS E-10 FLNC GRIP E-07 ESYT PTHLH ACSM E-07 PROCR E-05 MST E-07 RIMS PTGIS E-05 RORC NAP1L E-07 VSIG RALGPS E-09 SEMA5A E-05 AGT E-05 LOC E-07 MAMSTR E-07 IGFBP E-12 MT1L E-11 COL4A MROH E-05 POU6F GLB1L E-05 MEIS E-09 NPAS E-05 CYP1B1-AS SHROOM DAPK E-09 NAPEPLD E-11 CIDEA E-07 TTC RGS7BP SLC16A E-07 CNTN PITPNM E-05 MUC SPTB E-06 SFRP CFAP TMEM E-06 MICAL E-09 TMEM132B E-05 PAMR LRFN E-08 SOX E-13 TNFRSF E-06 JPH GLIS E-08 KIAA SAMD E-05 DLGAP DNAAF RELN SSX2IP E-05 MT1G TMEM E-16 PTGDS E-07 IL20RA E-06 PCDHAC RXRG E-05 SYCE NLRP SLCO2A E-09 GRIA E-05 ZC3H12B E-08 FAM46B PTK E-08 MARVELD SLC7A E-07 IL1RL GFI E-05 ALDH1L E-07 PCDHGA E-06 PM20D E-11 SPOCK E-06 SLC1A CDC42BPG CD CCBE E-05 RIC E-07 ZBTB7C E-06 BAI E-06 CPT1C E-08 SORBS E-06 ENOX E-08 CCDC85C SMOX E-05 KCNAB E-05 ANO E-06 LOC PROSER2-AS ROBO E-13 RAB E-06 RBP E-09 ZNF E-08 ARHGEF E-17 GAS E-06

13 HEG E-07 HR E-06 C3orf SLC23A E-05 FER1L SQLE AOX ADH1C E-09 AKAP IGF E-09 EML SLC8A E-07 ABCB E-05 GCNT EBF E-08 ZNF E-06 PTX FAAH SNAP GDF CYP39A E-07 LOC E-05 FZD E-06 FZD E-05 RASL10B E-06 PHYHIP E-05 AZGP E-05 STAP E-06 DTX E-08 ANXA E-11 NRIP ZC3HAV1L E-06 CLEC4F LOC E-05 AMMECR E-05 ALDH1A E-07 ARL4D PIEZO P4HA CLUHP E-09 DFNA E-05 PPP1R13L E-06 MPZL RTKN E-07 SDK AXIN F2RL EMB E-05 SCN2B SALL CCDC E-06 ALDH8A SNHG KCNJ E-06 RAP1GAP DISP CAPN TRO E-05 PLCB E-05 TMEM E-05 CELSR E-08 KCNH PROSER E-07 HOMER LETM MIAT E-07 PAPLN E-11 LPAR DISP FGF BAI PRKG E-07 PODXL E-05 TMOD E-09 PRODH ARAP HOXB E-06 ACOT E-05 FAM150B FAM83H ATP7B E-07 ARHGEF E-09 H E-06 ATAD3C E-05 MMP23B VEGFC E-09 GPD1L E-09 RAB RTN4R MEIS E-08 SAMD HPSE E-05 NR2F1-AS E-07 BACH MOV10L SULT1C E-07 DDR E-08 ABO PPP1R3C E-05 TEAD E-17 NRXN E2F INTU E-11 ATP1A C10orf E-05 EPHB E-07 LSR E-05 BOC E-08 IGSF E-05 PITX FZD E-05 CASC ZNF PITX PTPN E-10 SLC25A E-10 PIP5K1B GPR CDCA7L E-05 PYGM E-07

14 LINC GRB ENPP SLC44A E-07 NKD CMAHP E-05 CFAP HOXB E-06 TAS2R LOC E-05 SLC4A E-06 SCHIP HYDIN MIR E-05 RGS TMEM9B-AS E-05 LOC PDE10A PEG E-07 SIX ZNF E-05 CCR E-05 MAMDC E-06 EBF E-05 CNKSR PON BNC E-07 REC C GPR EYA E-09 SH2D1A TAF1A E-07 SHC GRAMD1C AKAP C2orf E-06 ELFN TMEM30B E-06 VEGFA E-07 IGSF FAM188B GJC LOC CYP1B E-05 TPD52L E-07 NGFR E-07 CDK E-06 POMC PLAG SLC26A SCN3B E-06 PCSK MIR100HG E-06 LMNTD CCDC102B E-05 HSD17B HCG MAPK E-06 PDE1A E-08 SLC9A7P CDH E-07 LTBP E-05 KLF3-AS CNKSR ZNF LEF GNG E-07 LOC SLC7A LOC GXYLT E-07 CPE PPARGC1B BBS MRGPRF-AS FAM227B ATP1B SUCO E-06 ZNF JAG VENTX E-05 ADAMTS E-09 HACL E-12 MXRA RCAN E-06 HOXD PLXNB POSTN E-05 NINJ E-06 DUSP ASS E-08 CCDC E-06 CXXC E-09 NMUR KLHL E-06 AFF E-06 FAM72A FAM69B E-05 SCNN1B E-05 LRRC HDC TPRG E-05 ANGPTL LINC E-09 OLFML2A FABP VIPR E-05 EOGT E-09 MELK ALCAM RHOXF ITGAX VIM-AS CLCA MIR TMEM E-08 PHKG C4orf E-06 SREBF E-05

15 PPP2R1B E-07 PNMAL MYADM NRIP FBLN E-05 SULT1C KIAA E-05 GLDN E-06 GNG E-09 ACE E-05 CSMD RGCC E-05 MGLL E-06 THRSP E-05 EYS SLC5A CSPG E-06 SLC35G E-05 MEGF E-07 LIPE-AS DUSP P2RY E-05 COL6A E-05 SATB E-06 ANPEP E-07 DCLK E-06 AKR1B FAT E-08 FGF E-06 CYP26B E-05 PRRX E-10 FILIP1L ANK E-06 COL5A E-09 PLBD E-11 MSX TNN TLE E-09 MGAT E-08 DOCK E-07 ROBO PTGES E-05 LINC MMP E-05 LOC RBM E-09 P2RX CILP CES ALPL CSF2RA E-07 CD DNASE1L SERINC E-07 RAB E-06 CCR SNCG E-06 ASAH E-08 DHRS E-10 LAMP CD COL4A HTRA FGF E-05 NR5A E-06 MYH CEP TCTEX1D SPARC E-06 LOC E-06 NRXN APOE E-05 FAM26E E-07 CYP2B7P NLRP AKR1C E-08 ENDOU VIT E-05 LINC E-06 UBE2QL C16orf E-06 TMEM E-09 CACNA2D E-07 ZFAT E-06 COL5A E-08 REEP E-05 GYPE FADS E-07 ELANE PNPLA LPHN SHC OASL ADAMTS E-10 AKR1C E-11 NDNF NOVA E-10 NTRK E-09 TRDN CD E-05 ATP10A E-09 RANBP SEMA3E FAM171A E-06 MYO1H KCNA ADRA2A E-06 FOXF FMOD E-09 TREM SIGLEC17P EPHB E-05 MAP7D SEL1L E-07 SPP LOC IL20RB E-06 CDH E-05 HTR2B E-05 MEOX E-06

16 C10orf E-08 NANOS E-11 HOXC E-06 TRIM E-08 SLC18A HRCT E-08 RND VCAN E-05 COL3A E-06 TRIM ADORA E-09 ITGA NOV E-06 RBP E-09 KLHL E-06 ODF3L SOX APOC ECM E-07 TBX E-08 TACR E-05 HPGD ABCC E-08 MAP1A E-10 HOXC E-06 PLA2G E-05 GRIA E-05 IFITM E-10 ACP E-06 TPSAB E-05 TPPP E-05 QPCT E-06 NTRK DHRS E-07 AOC4P E-06 MMP E-07 NNAT E-08 CSTA E-16 CXCL E-06 CTSG ENPP E-16 PLP E-07 ITIH E-09 PNMA E-05 FAM180B ADAM E-15 CD E-05 TPSB E-06 MME E-10 CD300LB E-05 DRD PTCH E-09 CETP S100A E-10 FAT E-06 GOLGA7B E-06 IL1RN ANKRD30A E-06 CTHRC E-07 CRIP E-05 PITX ST7-AS E-07 TENM E-11 DGAT E-08 LOXL E-13 COL15A E-10 ACR LOC LINC E-06 TUBB2A E-07 IRF E-05 KLF TF E-09 LRRC4C E-07 TTC39A E-05 FGFR E-09 PPP1R1B E-12 ADAMTS E-05 KRT E-05 AR E-21 TTYH CYP2A E-07 ZFHX4-AS E-05 WISP E-18 GLIPR E-13 CMA ZNF E-08 OSR E-14 HSD17B E-05 LOC E-11 HOXD E-09 BHMT E-07 NCAPH E-05 CSN1S IRF E-21 CCL E-05 OLFM E-18 MMP MAB21L E-05 PROK E-05 CCL E-06 C5orf E-07 SLC2A E-05 LOC E-05 PTGFR E-08 SMOC E-09 NRN E-16 P2RX6P E-05 PDE11A E-08 GAS2L E-10 ITGBL E-05 LINC E-16 TMEM E-07 SLC6A E-06 KCNQ E-05 ANGPTL E-06 AADAC HS3ST E-07 PHACTR CNTN E-05

17 ABCB E-10 ESR E-09 DIO E-07 LOC E-07 SLAMF SEMA3G E-12 XPNPEP E-09 PRR E-06 LBP E-10 DNASE2B FOXP HPSE E-07 CCL E-06 LINC E-07 TENM E-29 HORMAD AS1 1.15E-06 NOTCH E-10 CCL E-05 SPOCD E-09 MFAP E-07 CHIT CD E-16 STAC E-09 CCND E-23 PEMT E-24 CDC E-08 DEFB E-10 TM4SF E-05 KLHDC7A E-06 HMCN E-07 GLI E-11 MMP E-08 MMP DMRT E-11 LINC E-12 MS4A6E ANO E-17 FGF13-AS E-11 C14orf E-11 TWIST E-27 ATP6V0D ALDOC E-28 GALNT E-26 SIX E-12 LOC E-08 HOXD-AS E-10 LAMB E-22 ALPK E-06 HOXC E-33 LINC E-06 HOXA E-11 IGFN E-10 GABRA E-10 COL11A E-07 GYS E-11 OXT E-11 BBOX E-14 SHOX E-11 CNTNAP E-09 P2RX E-14 LOC E-06 MMP E-06 PRKG E-11 GDF E-15 KIAA1549L E-12 EN E-10 PCDH E-35 EMX2OS E-12 IRX E-27 SELE URAD E-08 FNDC E-06 HOXA E-23 XG E-14 LUZP E-13 LINC E-10 BMP E-08 C19orf E-12 SPAG E-16 MMP E-08 CLIC E-18 UNC13C E-07 DKK E-17 KRT E-18 EMX E-14 TBX5-AS E-12 TBX E-22 KRT E-10 AQP E-06 CSMD E-08 HOXC E-61 NRCAM E-24 CRNDE E-49 IRX E-31 HOXC-AS E-24 SERPINA E-31 TBX E-11 HOXA E-11 GDA E-07 LINC E-10 IRX E-32 C5orf E-40 EGFL E-22 HOXA11-AS E-12 HOXA10-AS E-17 HOXC E-72 DMRT E-54 MYEOV E-52 IRX E-39 STMN E-52 PAX E-20 HOXC E-49 SIM E-53

18 Supplementary Table S2. Microarray analysis of differentially expressed genes in the two depots of ASCs Supplementary Table S2A. Genes predominantly expressed in SC-ASCs Column # Gene Gene Name Fold Change Location 8766 HOXA9 Homeobox A Nucleus MME Membrane Metallo-Endopeptidase Cell membrane MOXD1 Monooxygenase, DBH-Like Endoplasmic reticulum membrane XG Xg Blood Group Cell membrane 4362 COLEC12 Collectin Sub-Family Member Membrane Cytoplasm. Cell junction, focal LPXN Leupaxin adhesion. Nucleus. Cytoplasm, perinuclear region 4772 CXCL5 Chemokine (C-X-C Motif) Ligand Secreted IRX5 Iroquois Homeobox Nucleus IRX2 Iroquois Homeobox Nucleus 8756 HOXA10 Homeobox A Nucleus IRX3 Iroquois Homeobox Nucleus LOC HSPB7 Heat Shock 27kDa Protein Family, Member Cytoplasm. Nucleus. Nucleus, Cajal body 7346 FOXQ1 Forkhead Box Q Nucleus 4374 COMP Cartilage Oligomeric Matrix Protein Secreted, extracellular space, extracellular matrix 5935 EMX2 Empty Spiracles Homeobox Nucleus 2774 C4orf31 Neuron-Derived Neurotrophic Factor Secreted 5955 ENPP1 Ectonucleotide Pyrophosphatase/Phosphodiesterase Cell membrane, Secreted 8152 GREM2 Gremlin 2, DAN Family BMP Antagonist Secreted RASD2 RASD Family, Member Cell membrane TMEM119 Transmembrane Protein Membrane NCALD Neurocalcin Delta Cytoplasm 7279 FNDC1 Fibronectin Type III Domain Containing Secreted PITX2 Paired-Like Homeodomain Nucleus RNF144 Ring Finger Protein 144A Membrane

19 6617 FBN2 Fibrillin Secreted, extracellular space, extracellular matrix LOC DDIT4L DNA-Damage-Inducible Transcript 4-Like Cytoplasm S100A4 S100 Calcium Binding Protein A Nucleus 4529 CRIP1 Cysteine-Rich Protein Cytoplasm LOC HAS1 Hyaluronan Synthase Membrane IL13RA2 Interleukin 13 Receptor, Alpha Membrane 7601 GAS1 Growth Arrest-Specific Cell membrane Mannan-Binding Lectin Serine Peptidase MASP SLC1A3 (C4/C2 Activating Component Of Ra-Reactive Factor) Solute Carrier Family 1 (Glial High Affinity Glutamate Transporter), Member Secreted Cell membrane 1462 BAALC Brain And Acute Leukemia, Cytoplasmic Cytoplasm. Membrane TNC Tenascin C Secreted, extracellular space, extracellular matrix LOC LIMCH1 LIM And Calponin Homology Domains MMP12 Matrix Metallopeptidase Secreted, extracellular space, extracellular matrix HS3ST3A CRISPLD CYP26B1 Heparan Sulfate (Glucosamine) 3-O- Sulfotransferase 3A1 Cysteine-Rich Secretory Protein LCCL Domain Containing 2 Cytochrome P450, Family 26, Subfamily B, Polypeptide Golgi apparatus membrane Secreted Endoplasmic reticulum membrane IFI44 Interferon-Induced Protein Cytoplasm RNF144A Ring Finger Protein 144A Membrane KRT81 Keratin ANGPT1 Angiopoietin Secreted 8785 HOXC9 Homeobox C Nucleus 4130 CLDN23 Claudin Cell junction, tight junction

20 8784 HOXC8 Homeobox C Nucleus 8003 GPNMB Glycoprotein (Transmembrane) Nmb Cell membrane LOC TBX15 T-Box Nucleus PCDH18 Protocadherin Cell membrane PODXL podocalyxin-like Apical cell membrane. Cell projection, lamellipodium 5956 ENPP2 Ectonucleotide Pyrophosphatase/Phosphodiesterase Secreted OLFML2B Olfactomedin-Like 2B Secreted UDP-N-Acetyl-Alpha-D GALNT5 Galactosamine:Polypeptide N-Acetylgalactosaminyltransferase Golgi apparatus membrane 8783 HOXC6 Homeobox C Nucleus CTSK Cathepsin K Lysosome 7259 FMN2 Formin Cytoplasm, cytoskeleton NTNG1 Netrin G Cell membrane TWIST1 Twist Basic Helix-Loop-Helix Transcription Factor Nucleus PRRX1 Paired Related Homeobox Nucleus PSG9 Pregnancy Specific Beta-1-Glycoprotein Secreted PSG5 Pregnancy Specific Beta-1-Glycoprotein Secreted 4568 CRYAB Crystallin, Alpha B Cytoplasm. Nucleus LOC Prostaglandin E Receptor 2 (Subtype EP2), PTGER2 53kDa Cell membrane 3577 CCRL Cytoplasm. Nucleus C13orf15 Regulator Of Cell Cycle cytoskeleton,,microtubule organizing center, centrosome SCARA3 Scavenger Receptor Class A, Member Endoplasmic reticulum membrane