#METABOLOMICS WORKBENCH vangrouwa_20230207_104744 DATATRACK_ID:3727 STUDY_ID:ST002469 ANALYSIS_ID:AN004027 PROJECT_ID:PR001595 VERSION 1 CREATED_ON February 9, 2023, 7:40 am #PROJECT PR:PROJECT_TITLE Development of a Robust Consensus Modeling Approach for Identifying Cellular and PR:PROJECT_TITLE Media Metabolites Predictive of Mesenchymal Stromal Cell Potency PR:PROJECT_TYPE MS Untargeted Analysis PR:PROJECT_SUMMARY Mesenchymal stromal cells (MSCs) have shown promise in regenerative medicine PR:PROJECT_SUMMARY applications due in part to their ability to modulate immune cells, such as T PR:PROJECT_SUMMARY cells. However, MSCs demonstrate significant functional heterogeneity in terms PR:PROJECT_SUMMARY of their immunomodulatory function because of differences in MSC donor/tissue PR:PROJECT_SUMMARY source, as well as non-standardized manufacturing approaches. As MSC metabolism PR:PROJECT_SUMMARY plays a critical role in their ability to expand to therapeutic numbers ex vivo, PR:PROJECT_SUMMARY we comprehensively profiled intracellular and extracellular metabolites PR:PROJECT_SUMMARY throughout the expansion process to identify predictors of MSC immunomodulatory PR:PROJECT_SUMMARY function (T cell modulation and indoleamine-2,3-dehydrogenase (IDO) activity). PR:PROJECT_SUMMARY Here, we profiled media metabolites in a non-destructive manner through daily PR:PROJECT_SUMMARY sampling and nuclear magnetic resonance (NMR), as well as MSC intracellular PR:PROJECT_SUMMARY metabolites at the end of expansion using mass spectrometry (MS). Using a robust PR:PROJECT_SUMMARY consensus machine learning approach, we were able to identify panels of PR:PROJECT_SUMMARY metabolites predictive of MSC immunomodulatory function for 10 independent MSC PR:PROJECT_SUMMARY lines. PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT Chemistry and Biochemistry PR:LABORATORY Fernandez Lab PR:LAST_NAME Van Grouw PR:FIRST_NAME Alexandria PR:ADDRESS 311 Ferst Dr. NW Atlanta, GA 30332 PR:EMAIL agrouw3@gatech.edu PR:PHONE 7072391412 PR:FUNDING_SOURCE NSF Center for Cell Manufacturing Technologies #STUDY ST:STUDY_TITLE Mesenchymal stromal cell (MSC) Metabolite MS study ST:STUDY_SUMMARY Metabolomics and lipidomics workflows were used to analyze Mesenchymal stromal ST:STUDY_SUMMARY cell (MSC) metabolites. Metabolite abundances were used to model MSC potency ST:STUDY_SUMMARY results in IDO and T-cell proliferation assays. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT Chemistry and Biochemistry ST:LABORATORY Fernandez Lab ST:LAST_NAME Van Grouw ST:FIRST_NAME Alexandria ST:ADDRESS 311 Ferst Dr. NW Atlanta, GA 30332 ST:EMAIL agrouw3@gatech.edu ST:STUDY_TYPE Untargeted Metabolite Study ST:PHONE 7072391412 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female SU:CELL_BIOSOURCE_OR_SUPPLIER RoosterBio SU:CELL_STRAIN_DETAILS Mesenchymal Stromal Cells #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS RB139 alphaMEM_RB139 Media:Gibco MEM alpha Functional_Composite_Score=0.96; Sex=Male; Age=25; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=8.10E+06; Initial_Population_Doubling_Level=12.2; Final_Population_Doubling_Level=16.6; Batch=1; Tcell_proliferation_01_CD4=81.58; Tcell_proliferation_01_CD8=88.55; Tcell_proliferation_02_CD4=50.59; Tcell_proliferation_02_CD8=35.42; IDO_Activity=49.31 SUBJECT_SAMPLE_FACTORS RB174 alphaMEM_RB174_01 Media:Gibco MEM alpha Functional_Composite_Score=0.57; Sex=Male; Age=25; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=9.80E+06; Initial_Population_Doubling_Level=13.06; Final_Population_Doubling_Level=17.8; Batch=1; Tcell_proliferation_01_CD4=75.15; Tcell_proliferation_01_CD8=78.96; Tcell_proliferation_02_CD4=50.49; Tcell_proliferation_02_CD8=38.16; IDO_Activity=38.90 SUBJECT_SAMPLE_FACTORS RB177 alphaMEM_RB177 Media:Gibco MEM alpha Functional_Composite_Score=-1.62; Sex=Male; Age=22; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=4.23E+06; Initial_Population_Doubling_Level=15.1; Final_Population_Doubling_Level=18.6; Batch=1; Tcell_proliferation_01_CD4=73.11; Tcell_proliferation_01_CD8=70.64; Tcell_proliferation_02_CD4=35.57; Tcell_proliferation_02_CD8=29.35; IDO_Activity=70.43 SUBJECT_SAMPLE_FACTORS RB179 alphaMEM_RB179 Media:Gibco MEM alpha Functional_Composite_Score=1.27; Sex=Male; Age=21; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=8.20E+06; Initial_Population_Doubling_Level=12.6; Final_Population_Doubling_Level=17.1; Batch=1; Tcell_proliferation_01_CD4=85.35; Tcell_proliferation_01_CD8=84.80; Tcell_proliferation_02_CD4=45.71; Tcell_proliferation_02_CD8=34.36; IDO_Activity=22.49 SUBJECT_SAMPLE_FACTORS RB183 alphaMEM_RB183 Media:Gibco MEM alpha Functional_Composite_Score=0.50; Sex=Female; Age=26; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=5.30E+06; Initial_Population_Doubling_Level=12; Final_Population_Doubling_Level=15.8; Batch=1; Tcell_proliferation_01_CD4=83.28; Tcell_proliferation_01_CD8=80.65; Tcell_proliferation_02_CD4=48.26; Tcell_proliferation_02_CD8=35.15; IDO_Activity=53.76 SUBJECT_SAMPLE_FACTORS RB71 alphaMEM_RB71 Media:Gibco MEM alpha Functional_Composite_Score=2.13; Sex=Female; Age=18-30; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=7.50E+06; Initial_Population_Doubling_Level=12.72; Final_Population_Doubling_Level=17; Batch=2; Tcell_proliferation_01_CD4=86.95; Tcell_proliferation_01_CD8=89.51; Tcell_proliferation_02_CD4=55.34; Tcell_proliferation_02_CD8=45.80; IDO_Activity=46.26 SUBJECT_SAMPLE_FACTORS RB174 alphaMEM_RB174_02 Media:Gibco MEM alpha Functional_Composite_Score=0.92; Sex=Male; Age=25; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=8.43E+06; Initial_Population_Doubling_Level=13.06; Final_Population_Doubling_Level=17.6; Batch=2; Tcell_proliferation_01_CD4=73.37; Tcell_proliferation_01_CD8=79.08; Tcell_proliferation_02_CD4=49.34; Tcell_proliferation_02_CD8=40.72; IDO_Activity=21.98 SUBJECT_SAMPLE_FACTORS RB175 alphaMEM_RB175 Media:Gibco MEM alpha Functional_Composite_Score=0.96; Sex=Male; Age=25; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=6.24E+06; Initial_Population_Doubling_Level=12; Final_Population_Doubling_Level=16.1; Batch=2; Tcell_proliferation_01_CD4=74.21; Tcell_proliferation_01_CD8=75.19; Tcell_proliferation_02_CD4=50.93; Tcell_proliferation_02_CD8=42.01; IDO_Activity=20.45 SUBJECT_SAMPLE_FACTORS RB182 alphaMEM_RB182 Media:Gibco MEM alpha Functional_Composite_Score=-1.21; Sex=Female; Age=26; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=3.65E+06; Initial_Population_Doubling_Level=11.48; Final_Population_Doubling_Level=14.8; Batch=2; Tcell_proliferation_01_CD4=65.76; Tcell_proliferation_01_CD8=61.28; Tcell_proliferation_02_CD4=48.56; Tcell_proliferation_02_CD8=36.16; IDO_Activity=54.69 SUBJECT_SAMPLE_FACTORS iMSC alphaMEM_iMSC Media:Gibco MEM alpha Functional_Composite_Score=-4.47; Sex=not available; Age=not available; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=1.86E+06; Initial_Population_Doubling_Level=not available; Final_Population_Doubling_Level=not available; Batch=2; Tcell_proliferation_01_CD4=60.06; Tcell_proliferation_01_CD8=62.65; Tcell_proliferation_02_CD4=11.82; Tcell_proliferation_02_CD8=17.11; IDO_Activity=80.56 SUBJECT_SAMPLE_FACTORS RB139 RBio_RB139 Media:RoosterBasal-MSC media Functional_Composite_Score=1.05; Sex=Male; Age=25; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=1.51E+07; Initial_Population_Doubling_Level=12.2; Final_Population_Doubling_Level=17.5; Batch=1; Tcell_proliferation_01_CD4=55.84; Tcell_proliferation_01_CD8=64.27; Tcell_proliferation_02_CD4=68.63; Tcell_proliferation_02_CD8=66.47; IDO_Activity=8.87 SUBJECT_SAMPLE_FACTORS RB174 RBio_RB174_01 Media:RoosterBasal-MSC media Functional_Composite_Score=0.53; Sex=Male; Age=25; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=2.55E+07; Initial_Population_Doubling_Level=13.06; Final_Population_Doubling_Level=19.1; Batch=1; Tcell_proliferation_01_CD4=56.11; Tcell_proliferation_01_CD8=60.94; Tcell_proliferation_02_CD4=58.77; Tcell_proliferation_02_CD8=53.87; IDO_Activity=2.48 SUBJECT_SAMPLE_FACTORS RB177 RBio_RB177 Media:RoosterBasal-MSC media Functional_Composite_Score=-1.35; Sex=Male; Age=22; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=2.12E+07; Initial_Population_Doubling_Level=15.1; Final_Population_Doubling_Level=20.9; Batch=1; Tcell_proliferation_01_CD4=51.37; Tcell_proliferation_01_CD8=45.73; Tcell_proliferation_02_CD4=54.93; Tcell_proliferation_02_CD8=46.49; IDO_Activity=14.93 SUBJECT_SAMPLE_FACTORS RB179 RBio_RB179 Media:RoosterBasal-MSC media Functional_Composite_Score=-0.10; Sex=Male; Age=21; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=2.66E+07; Initial_Population_Doubling_Level=12.6; Final_Population_Doubling_Level=18.7; Batch=1; Tcell_proliferation_01_CD4=55.07; Tcell_proliferation_01_CD8=61.90; Tcell_proliferation_02_CD4=49.48; Tcell_proliferation_02_CD8=42.74; IDO_Activity=0.16 SUBJECT_SAMPLE_FACTORS RB183 RBio_RB183 Media:RoosterBasal-MSC media Functional_Composite_Score=0.69; Sex=Female; Age=26; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=7.80E+06; Initial_Population_Doubling_Level=12; Final_Population_Doubling_Level=16.4; Batch=1; Tcell_proliferation_01_CD4=63.36; Tcell_proliferation_01_CD8=64.09; Tcell_proliferation_02_CD4=61.39; Tcell_proliferation_02_CD8=54.84; IDO_Activity=10.14 SUBJECT_SAMPLE_FACTORS RB71 RBio_RB71 Media:RoosterBasal-MSC media Functional_Composite_Score=1.50; Sex=Female; Age=18-30; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=1.50E+07; Initial_Population_Doubling_Level=12.72; Final_Population_Doubling_Level=12.72; Batch=2; Tcell_proliferation_01_CD4=63.09; Tcell_proliferation_01_CD8=65.10; Tcell_proliferation_02_CD4=67.70; Tcell_proliferation_02_CD8=64.11; IDO_Activity=6.33 SUBJECT_SAMPLE_FACTORS RB174 RBio_RB174_02 Media:RoosterBasal-MSC media Functional_Composite_Score=1.72; Sex=Male; Age=25; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=1.29E+07; Initial_Population_Doubling_Level=13.06; Final_Population_Doubling_Level=13.06; Batch=2; Tcell_proliferation_01_CD4=66.93; Tcell_proliferation_01_CD8=77.71; Tcell_proliferation_02_CD4=60.63; Tcell_proliferation_02_CD8=56.73; IDO_Activity=3.97 SUBJECT_SAMPLE_FACTORS RB175 RBio_RB175 Media:RoosterBasal-MSC media Functional_Composite_Score=1.44; Sex=Male; Age=25; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=1.92E+07; Initial_Population_Doubling_Level=12; Final_Population_Doubling_Level=12; Batch=2; Tcell_proliferation_01_CD4=62.30; Tcell_proliferation_01_CD8=70.30; Tcell_proliferation_02_CD4=61.80; Tcell_proliferation_02_CD8=58.35; IDO_Activity=2.21 SUBJECT_SAMPLE_FACTORS RB182 RBio_RB182 Media:RoosterBasal-MSC media Functional_Composite_Score=-0.32; Sex=Female; Age=26; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=1.47E+07; Initial_Population_Doubling_Level=11.48; Final_Population_Doubling_Level=11.48; Batch=2; Tcell_proliferation_01_CD4=60.47; Tcell_proliferation_01_CD8=60.44; Tcell_proliferation_02_CD4=53.46; Tcell_proliferation_02_CD8=45.15; IDO_Activity=11.97 SUBJECT_SAMPLE_FACTORS iMSC RBio_iMSC Media:RoosterBasal-MSC media Functional_Composite_Score=-5.16; Sex=not available; Age=not available; Total_Cells_Seeded=3.75E+05; Final_Cell_Yield=1.90E+07; Initial_Population_Doubling_Level=not available; Final_Population_Doubling_Level=not available; Batch=2; Tcell_proliferation_01_CD4=37.12; Tcell_proliferation_01_CD8=23.57; Tcell_proliferation_02_CD4=25.68; Tcell_proliferation_02_CD8=21.86; IDO_Activity=21.54 #COLLECTION CO:COLLECTION_SUMMARY For intracellular lipidomic/metabolomic analysis, cell pellets were washed twice CO:COLLECTION_SUMMARY by resuspending in PBS and centrifuged at 10,000 rpm. All supernatant was CO:COLLECTION_SUMMARY removed and cell pellets were then stored at –80° C. CO:SAMPLE_TYPE Bone marrow #TREATMENT TR:TREATMENT_SUMMARY Bone marrow-derived MSCs (BMMSCs) were purchased from RoosterBio, Inc. TR:TREATMENT_SUMMARY (Frederick, MD), and iMSCs were purchased from Fujifilm Cellular Dynamics Inc TR:TREATMENT_SUMMARY (Madison, WI). Prior to this study’s expansion, MSCs were previously expanded TR:TREATMENT_SUMMARY to an initial population doubling level (PDL0). Cryopreserved vials from each TR:TREATMENT_SUMMARY donor were thawed, and 106 MSCs were seeded into an initial T-150 tissue culture TR:TREATMENT_SUMMARY flask in complete media containing Gibco™ Minimum Essential Media α with TR:TREATMENT_SUMMARY nucleosides (Thermo Fisher Scientific, Waltham, MA), 10% fetal bovine serum TR:TREATMENT_SUMMARY (FBS; HyClone Laboratories, Logan, UT), and 1% penicillin-streptomycin solution TR:TREATMENT_SUMMARY (10,000 U/mL; Sigma-Aldrich, St. Louis, MO) for a culture rescue period of 48 TR:TREATMENT_SUMMARY hr. The same lot of FBS was used throughout the study. MSCs were washed with TR:TREATMENT_SUMMARY endotoxin-free Dulbecco’s phosphate buffered saline (PBS) without calcium and TR:TREATMENT_SUMMARY magnesium (Millipore Sigma), harvested using 1X TrypLE™ Express Enzyme (Thermo TR:TREATMENT_SUMMARY Fisher Scientific), neutralized with complete media, and centrifuged 300g to TR:TREATMENT_SUMMARY create a cell pellet. MSCs were then resuspended in complete media and counted. TR:TREATMENT_SUMMARY Next, MSCs from each donor were seeded at 500 cells/cm2 into 10 T-75 flasks TR:TREATMENT_SUMMARY containing 10 mL complete media. Control flasks containing 10 mL complete media TR:TREATMENT_SUMMARY only were also prepared. All flasks were then transferred to a humidified TR:TREATMENT_SUMMARY incubator set to 37° C and 5% CO2. MSC conditioned medium (CM) sample TR:TREATMENT_SUMMARY collection of 300 µl was performed for each flask at approximately the same TR:TREATMENT_SUMMARY time each day (±1 hr) and total complete media exchange was performed every 3 TR:TREATMENT_SUMMARY days until MSCs achieved 70-80% confluence. All media samples were placed TR:TREATMENT_SUMMARY directly into –80° C storage until further analysis by NMR. MSCs were then TR:TREATMENT_SUMMARY harvested using the same procedure described above. Cell pellets were split for TR:TREATMENT_SUMMARY cryopreservation (and functional analysis, see below) or preparation for TR:TREATMENT_SUMMARY intracellular lipidomic/metabolic analysis. Cell pellets designated for TR:TREATMENT_SUMMARY cryopreservation were prepared into cryovials containing 106 MSCs in 1 mL TR:TREATMENT_SUMMARY CryoStor® CS 10 (Sigma-Aldrich) and stored at –80° C for 24 hr using TR:TREATMENT_SUMMARY controlled rate freezing containers. Vials were then transferred to the vapor TR:TREATMENT_SUMMARY phase in a liquid nitrogen cryogenic freezer until further analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Approximately one million MSCs were analyzed for each sample. Frozen cell SP:SAMPLEPREP_SUMMARY pellets were thawed and washed prior to undergoing a modified Bligh-Dyer SP:SAMPLEPREP_SUMMARY extraction to yield two phases. Extraction solvent (2:2:1 SP:SAMPLEPREP_SUMMARY chloroform:methanol:water) and glass beads (400-600 µm) were added to cell SP:SAMPLEPREP_SUMMARY pellets for extraction and homogenization in a TissueLyser II to 30 Hz for 6 SP:SAMPLEPREP_SUMMARY minutes. Samples were then sonicated and centrifuged. Following extraction, 300 SP:SAMPLEPREP_SUMMARY µL aliquots from each layer were transferred to new microcentrifuge tubes and SP:SAMPLEPREP_SUMMARY solvent was dried using vacuum centrifugation. Dried organic phase samples were SP:SAMPLEPREP_SUMMARY re-constituted in isopropyl alcohol, while dried aqueous phase samples were SP:SAMPLEPREP_SUMMARY re-constituted in 80% methanol. Re-constitution was followed by sonication, SP:SAMPLEPREP_SUMMARY centrifugation, and transfer to liquid chromatography (LC) vials for ultrahigh SP:SAMPLEPREP_SUMMARY performance liquid chromatography mass spectrometry (UHPLC-MS) analysis. Media SP:SAMPLEPREP_SUMMARY samples without cells were also analyzed as blanks to remove any features SP:SAMPLEPREP_SUMMARY corresponding to remaining media components on the cells. Ten µL of media was SP:SAMPLEPREP_SUMMARY subject to the same Bligh-Dyer extraction as above and extracts were run SP:SAMPLEPREP_SUMMARY according to the instrumental methods listed above. A quality control (QC) SP:SAMPLEPREP_SUMMARY sample for hydrophilic interaction chromatography (HILIC) and reverse phase SP:SAMPLEPREP_SUMMARY datasets was created by pooling 20 µL from each experimental sample. The pooled SP:SAMPLEPREP_SUMMARY QC injections were used for drift correction of peak areas. Sample queue was SP:SAMPLEPREP_SUMMARY randomized with a mix of samples, QCs, and blanks. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Orbitrap ID-X Tribrid mass spectrometer CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) CH:SOLVENT_A 80% water/20% acetonitrile; 10 mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 5% A 0-8 min; 60% A 8-10.4 min; 5% A 10.5-14min CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mobile Phase A used for HILIC chromatography was 80:20 water:MeCN with 10 mM MS:MS_COMMENTS ammonium formate and 0.1% formic acid. Mobile phase B for HILIC chromatography MS:MS_COMMENTS was acetonitrile with 0.1% formic acid. The flow rate was set at 0.4 mL/min. MS:MS_COMMENTS The column temperature was set to 40 °C, the injection volume was 2 µL. MS:MS_RESULTS_FILE ST002469_AN004027_Results.txt UNITS:Peak Area Has m/z:Neutral masses Has RT:No RT units:No RT data #END