#METABOLOMICS WORKBENCH jvaorodrigues1980_20250117_080129 DATATRACK_ID:5538 STUDY_ID:ST003765 ANALYSIS_ID:AN006180 PROJECT_ID:PR002348 VERSION 1 CREATED_ON February 25, 2025, 1:32 am #PROJECT PR:PROJECT_TITLE 1H NMR metabolomics applied to detect metabolic markers of early PR:PROJECT_TITLE ostedifferentiation of mesenchymal stem cells from multiple donors PR:PROJECT_TYPE 1H NMR metabolomics to detect metabolic markers of early ostedifferentiation of PR:PROJECT_TYPE human adipose-derived mesenchymal stem cells from multiple donors PR:PROJECT_SUMMARY Mesenchymal stem cells (MSC) are pivotal bioengineering tools that can be used PR:PROJECT_SUMMARY effectively in tissue regeneration. However, their inherent biological PR:PROJECT_SUMMARY variability due to inter-donor and tissue source heterogeneity often limits PR:PROJECT_SUMMARY therapeutic applications. In addition, the lack of standardized user-independent PR:PROJECT_SUMMARY protocols for MSC handling also contributes to this heterogeneity. Although PR:PROJECT_SUMMARY existing assays are invaluable for detecting well-known gene/protein markers, PR:PROJECT_SUMMARY they are often time-consuming, user-dependent and prone to reproducibility PR:PROJECT_SUMMARY issue. The resulting high variability in MSC behavior and differentiation PR:PROJECT_SUMMARY performance calls for improved characterization guidelines, ideally based on new PR:PROJECT_SUMMARY robust markers. Increasing interest has therefore arisen in the development of PR:PROJECT_SUMMARY new strategies to unambiguously monitor and predict MSC behavior, for an PR:PROJECT_SUMMARY effective selection of donors/cells with superior therapeutic quality. MSC PR:PROJECT_SUMMARY metabolomics appears as a valuable tool, mainly applied to: i) profile the PR:PROJECT_SUMMARY endometabolome of different cell types/origins, ii) identify the effects of PR:PROJECT_SUMMARY donor characteristics, and iii) measure metabolic adaptations to differentiation PR:PROJECT_SUMMARY or other culture conditions. The relevance of metabolomics in stem cell research PR:PROJECT_SUMMARY has been reviewed recently, and both intracellular and extracellular metabolomes PR:PROJECT_SUMMARY have been shown to respond to osteodifferentiation, their articulated PR:PROJECT_SUMMARY interpretation having been attempted. In this study, the intra- and PR:PROJECT_SUMMARY extracellular metabolic features accompanying the osteodifferentiation of hAMSC PR:PROJECT_SUMMARY were characterized for 3 independent donors, over a period of 21 days and PR:PROJECT_SUMMARY compared to proliferating cells. By employing untargeted nuclear magnetic PR:PROJECT_SUMMARY resonance (NMR) metabolomics, it was established a reliable donor-independent PR:PROJECT_SUMMARY metabolic signatures (sets of metabolite variations) that may serve as PR:PROJECT_SUMMARY predictive markers of osteodifferentiation, while elucidating particularly PR:PROJECT_SUMMARY responsive metabolic pathways accompanying the process. The integration of endo- PR:PROJECT_SUMMARY and exometabolomic adaptations should facilitate the development of rapid and PR:PROJECT_SUMMARY non-invasive strategies to detect and predict early MSC osteodifferentiation. PR:INSTITUTE University of Aveiro PR:DEPARTMENT CICECO – Aveiro Institute of Materials, Department of Chemistry PR:LABORATORY Metabolomics Group PR:LAST_NAME Gil PR:FIRST_NAME Ana PR:ADDRESS CICECO - Departamento de Química, Universidade de Aveiro, Campus de Santiago PR:EMAIL agil@ua.pt PR:PHONE +351234370707 #STUDY ST:STUDY_TITLE 1H NMR metabolomics applied to detect metabolic markers of early ST:STUDY_TITLE ostedifferentiation of mesenchymal stem cells from multiple donors ST:STUDY_TYPE 1H NMR metabolomics to detect metabolic markers of early ostedifferentiation of ST:STUDY_TYPE human adipose-derived mesenchymal stem cells from multiple donors ST:STUDY_SUMMARY In this study, the intra- and extracellular metabolic features accompanying the ST:STUDY_SUMMARY osteodifferentiation of hAMSC were characterized for 3 independent donors, over ST:STUDY_SUMMARY a period of 21 days and compared to proliferating cells. By employing untargeted ST:STUDY_SUMMARY nuclear magnetic resonance (NMR) metabolomics, it was established a reliable ST:STUDY_SUMMARY donor-independent metabolic signatures (sets of metabolite variations) that may ST:STUDY_SUMMARY serve as predictive markers of osteodifferentiation, while elucidating ST:STUDY_SUMMARY particularly responsive metabolic pathways accompanying the process. The ST:STUDY_SUMMARY integration of endo- and exometabolomic adaptations should facilitate the ST:STUDY_SUMMARY development of rapid and non-invasive strategies to detect and predict early MSC ST:STUDY_SUMMARY osteodifferentiation. ST:INSTITUTE University of Aveiro ST:DEPARTMENT CICECO – Aveiro Institute of Materials, Department of Chemistry ST:LABORATORY Metabolomics Group ST:LAST_NAME Gil ST:FIRST_NAME Ana ST:ADDRESS CICECO - Departamento de Química, Universidade de Aveiro, Campus de Santiago ST:EMAIL agil@ua.pt ST:PHONE +351234370707 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE donor 1: unknown; donor 2: 27 years-old; donor 3: 42 years-old; osteoblasts SU:AGE_OR_AGE_RANGE donor: 26 years-old. SU:GENDER Male and female SU:CELL_BIOSOURCE_OR_SUPPLIER donor 1: obtained via abdominoplasty, under an agreement between the University SU:CELL_BIOSOURCE_OR_SUPPLIER of Aveiro and “Hospital da Luz”, Aveiro, dated 17th February 2023; donor 2: SU:CELL_BIOSOURCE_OR_SUPPLIER American Type Culture Collection (Lot 70017032, Ref. ATCC PCS-500-011); donor 3: SU:CELL_BIOSOURCE_OR_SUPPLIER Lonza (Lot 22TL018258, Ref. LSLZPT-5006); osteoblasts donor: Lonza (Lot SU:CELL_BIOSOURCE_OR_SUPPLIER 19TL217387, Ref. CC-2538) SU:CELL_PASSAGE_NUMBER For all MSC donors, osteodifferentiation of hAMSC was induced at passages 5 or 6 SU:CELL_PASSAGE_NUMBER (for 21 days). #FACTORS #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 - PE_dn1_CTR_day0_01 Sample source:Cells | Factor:PE_dn1_CTR RAW_FILE_NAME(Raw_file_name)=PE_dn1_CTR_day0_s1.raw SUBJECT_SAMPLE_FACTORS - PE_dn1_CTR_day0_02 Sample source:Cells | Factor:PE_dn1_CTR RAW_FILE_NAME(Raw_file_name)=PE_dn1_CTR_day0_s2.raw SUBJECT_SAMPLE_FACTORS - PE_dn1_CTR_day01_01 Sample source:Cells | Factor:PE_dn1_CTR RAW_FILE_NAME(Raw_file_name)=PE_dn1_CTR_day01_s1.raw SUBJECT_SAMPLE_FACTORS - PE_dn1_CTR_day01_02 Sample source:Cells | Factor:PE_dn1_CTR RAW_FILE_NAME(Raw_file_name)=PE_dn1_CTR_day01_s2.raw SUBJECT_SAMPLE_FACTORS - PE_dn1_CTR_day01_03 Sample source:Cells | Factor:PE_dn1_CTR RAW_FILE_NAME(Raw_file_name)=PE_dn1_CTR_day01_s3.raw SUBJECT_SAMPLE_FACTORS - 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PE_dn3_OI_day01_02 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day01_s2.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day01_03 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day01_s3.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day04_01 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day04_s1.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day04_02 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day04_s2.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day04_03 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day04_s3.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day07_01 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day07_s1.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day07_02 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day07_s2.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day07_03 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day07_s3.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day14_01 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day14_s1.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day14_02 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day14_s2.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day14_03 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day14_s3.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day21_01 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day21_s1.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day21_02 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day21_s2.raw SUBJECT_SAMPLE_FACTORS - PE_dn3_OI_day21_03 Sample source:Cells | Factor:PE_dn3_OI RAW_FILE_NAME(Raw_file_name)=PE_dn3_OI_day21_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d01_01 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d01_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d01_02 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d01_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d01_03 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d01_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d04_01 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d04_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d04_02 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d04_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d04_03 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d04_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d11_01 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d11_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d11_02 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d11_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d11_03 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d11_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d14_01 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d14_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d14_02 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d14_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d14_03 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d14_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d18_01 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d18_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d18_02 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d18_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d18_03 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d18_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d21_01 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d21_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d21_02 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d21_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_d21_03 Sample source:Media | Factor:EXO_dn1_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_d21_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d01_01 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d01_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d01_02 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d01_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d01_03 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d01_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d04_01 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d04_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d04_02 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d04_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d04_03 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d04_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d11_01 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d11_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d11_02 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d11_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d11_03 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d11_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d14_01 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d14_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d14_02 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d14_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d14_03 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d14_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d18_01 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d18_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d18_02 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d18_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d18_03 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d18_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d21_01 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d21_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d21_02 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d21_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_d21_03 Sample source:Media | Factor:EXO_dn1_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_d21_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_day0_Br Sample source:Media | Factor:EXO_dn1_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_day0_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_day04_Br Sample source:Media | Factor:EXO_dn1_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_day04_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_day14_Br Sample source:Media | Factor:EXO_dn1_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_day14_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_day18_Br Sample source:Media | Factor:EXO_dn1_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_day18_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_CTR_day21_Br Sample source:Media | Factor:EXO_dn1_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn1_CTR_day21_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_day0_Br Sample source:Media | Factor:EXO_dn1_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_day0_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_day14_Br Sample source:Media | Factor:EXO_dn1_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_day14_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn1_OI_day18_Br Sample source:Media | Factor:EXO_dn1_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn1_OI_day18_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d01_01 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d01_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d01_02 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d01_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d01_03 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d01_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d04_01 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d04_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d04_02 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d04_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d04_03 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d04_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d11_01 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d11_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d11_02 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d11_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d11_03 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d11_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d14_01 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d14_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d14_02 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d14_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d14_03 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d14_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d18_02 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d18_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d18_03 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d18_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d21_01 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d21_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d21_02 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d21_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_d21_03 Sample source:Media | Factor:EXO_dn2_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_d21_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d01_01 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d01_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d01_02 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d01_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d01_03 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d01_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d04_01 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d04_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d04_02 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d04_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d04_03 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d04_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d11_02 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d11_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d11_03 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d11_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d14_01 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d14_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d14_02 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d14_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d14_03 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d14_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d18_01 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d18_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d18_02 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d18_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d18_03 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d18_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_d21_01 Sample source:Media | Factor:EXO_dn2_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_d21_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_day0_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_day0_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_day04_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_day04_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_day11_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_day11_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_day14_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_day14_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_day18_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_day18_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_CTR_day21_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_CTR_day21_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_day0_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_day0_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_day04_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_day04_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn2_OI_day11_Br Sample source:Media | Factor:EXO_dn2_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn2_OI_day11_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d01_01 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d01_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d01_02 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d01_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d01_03 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d01_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d04_01 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d04_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d04_02 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d04_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d04_03 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d04_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d11_01 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d11_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d11_02 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d11_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d11_03 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d11_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d14_01 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d14_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d14_02 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d14_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d14_03 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d14_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d18_01 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d18_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d18_02 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d18_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d18_03 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d18_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d21_01 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d21_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d21_02 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d21_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_d21_03 Sample source:Media | Factor:EXO_dn3_CTR RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_d21_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d01_01 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d01_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d01_02 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d01_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d01_03 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d01_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d04_01 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d04_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d04_02 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d04_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d04_03 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d04_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d11_01 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d11_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d11_02 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d11_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d11_03 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d11_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d14_01 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d14_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d14_02 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d14_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d14_03 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d14_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d18_01 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d18_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d18_02 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d18_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d18_03 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d18_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d21_01 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d21_s1.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d21_02 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d21_s2.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_d21_03 Sample source:Media | Factor:EXO_dn3_OI RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_d21_s3.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_day0_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_day0_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_day04_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_day04_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_day11_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_day11_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_day14_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_day14_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_day18_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_day18_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_CTR_day21_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_CTR_day21_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_day0_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_day0_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_day04_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_day04_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_day11_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_day11_Br.raw SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_day14_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_day14_Br SUBJECT_SAMPLE_FACTORS - EXO_dn3_OI_day18_Br Sample source:Media | Factor:EXO_dn3_Br RAW_FILE_NAME(Raw_file_name)=EXO_dn3_OI_day18_Br.raw SUBJECT_SAMPLE_FACTORS - osteoblast_day00_01 Sample source:Osteoblasts | Factor:OST RAW_FILE_NAME(Raw_file_name)=osteoblast_day00_s1.raw SUBJECT_SAMPLE_FACTORS - osteoblast_day00_02 Sample source:Osteoblasts | Factor:OST RAW_FILE_NAME(Raw_file_name)=osteoblast_day00_s2.raw SUBJECT_SAMPLE_FACTORS - osteoblast_day00_03 Sample source:Osteoblasts | Factor:OST RAW_FILE_NAME(Raw_file_name)=osteoblast_day00_s3.raw SUBJECT_SAMPLE_FACTORS - osteoblast_day07_01 Sample source:Osteoblasts | Factor:OST RAW_FILE_NAME(Raw_file_name)=osteoblast_day07_s1.raw SUBJECT_SAMPLE_FACTORS - osteoblast_day07_02 Sample source:Osteoblasts | Factor:OST RAW_FILE_NAME(Raw_file_name)=osteoblast_day07_s2.raw SUBJECT_SAMPLE_FACTORS - osteoblast_day07_03 Sample source:Osteoblasts | Factor:OST RAW_FILE_NAME(Raw_file_name)=osteoblast_day07_s3.raw #COLLECTION CO:COLLECTION_SUMMARY For all donors, cryopreserved hAMSC were thawed, plated in culture flasks CO:COLLECTION_SUMMARY (T175), expanded in minimum essential alpha medium (α-MEM, Gibco™ 12000063, CO:COLLECTION_SUMMARY Waltham, MA, USA) supplemented with 10% v/v heat-inactivated fetal bovine serum CO:COLLECTION_SUMMARY (FBS, Gibco 10270106) and 1% v/v antibiotics (penicillin−streptomycin, Gibco CO:COLLECTION_SUMMARY 15240062) at 37°C in a humidified 5% CO2 incubator and passaged as described CO:COLLECTION_SUMMARY previously (Bispo et al. 2022a, https://doi.org/10.3390/cells11081257). CO:COLLECTION_SUMMARY Osteodifferentiation of hAMSC was induced at passages 5 or 6, for 21 days, in CO:COLLECTION_SUMMARY T175 culture flasks (for metabolomics) and in 48-well plates (for biochemical CO:COLLECTION_SUMMARY assays). An independent experiment was conducted for each of the donors. For CO:COLLECTION_SUMMARY metabolomics, cells were seeded at a 0.5 × 106 cells/flask density. After CO:COLLECTION_SUMMARY reaching 100% confluence, day 0 (D0) cell samples were collected in triplicate CO:COLLECTION_SUMMARY from independent flasks, and media were replaced in the remaining flasks as CO:COLLECTION_SUMMARY follows: 15 control (CTR) flasks in standard growth medium and 15 osteoinduced CO:COLLECTION_SUMMARY (OI) flasks in medium supplemented with 10 mM β-glycerophosphate (β-GP, CO:COLLECTION_SUMMARY Sigma-Aldrich G9422), 50 μg/mL L-ascorbic acid (Sigma A0278) and 10 nM CO:COLLECTION_SUMMARY dexamethasone (ACROS Organics 230300010). Media were replaced 2× per week, on CO:COLLECTION_SUMMARY days (Di) 0, 4, 7, 11, 14, 18 and 21. Cells were trypsinized and collected in CO:COLLECTION_SUMMARY triplicate on D0, D1, D4, D7, D14 and D21. Cell suspensions were filtered CO:COLLECTION_SUMMARY through 100 μm pore strainers, centrifuged (300 g, 4 °C, 5 min) and rinsed CO:COLLECTION_SUMMARY twice in phosphate-buffered saline (PBS) solution. For intracellular CO:COLLECTION_SUMMARY metabolomics (endometabolomics), cell numbers/sample were 3.8-21.1 × 106, CO:COLLECTION_SUMMARY 2.3-8.6 × 106 and 1.4-9.1 × 106 for donors 1, 2 and 3, respectively. For CO:COLLECTION_SUMMARY extracellular metabolomics (exometabolomics), media samples were collected on CO:COLLECTION_SUMMARY D1, D4, D7, D11, D14, D18 and D21 (days when cell collection and/or medium CO:COLLECTION_SUMMARY exchange was carried out) and filtered through 40 μm pore strainers to remove CO:COLLECTION_SUMMARY cellular debris. Due to contamination and/or technical issues, triplicate CO:COLLECTION_SUMMARY samples could not always be retrieved (donor 1: CTR D0 cells n = 2, OI D7 media CO:COLLECTION_SUMMARY n = 0; donor 2: CTR D7,D18 media n = 2, OI D11,D21 media n = 1-2, OI D21 cells n CO:COLLECTION_SUMMARY = 1). For biochemical assays, cell samples were rinsed 2× with PBS and lysed by CO:COLLECTION_SUMMARY osmotic/thermal shock. Collected media and cell samples were stored at − 80 CO:COLLECTION_SUMMARY °C. For osteoblast samples (OST), Cryopreserved Clonetics™ normal human CO:COLLECTION_SUMMARY osteoblasts (NHOst) from a healthy 26-year-old Caucasian male (Lonza, CC-2538, CO:COLLECTION_SUMMARY Lot 19TL217387) were subcultured using ReagentPack™ subculture reagents CO:COLLECTION_SUMMARY (Lonza, CC-5034) up to passage 5. Cells were maintained in Clonetics™ OGM™ CO:COLLECTION_SUMMARY Osteoblast media (Lonza, CC-3208) with OGM SingleQuot Kit supplements and growth CO:COLLECTION_SUMMARY factors (Lonza, CC-4193) for either 6 days (100% confluence) or 13 days, CO:COLLECTION_SUMMARY followed by cell collection. CO:COLLECTION_PROTOCOL_FILENAME hASCs_Experimental_Procedure.pdf CO:SAMPLE_TYPE Cell and cell media CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For all donors, cryopreserved hAMSC were thawed, plated in culture flasks TR:TREATMENT_SUMMARY (T175), expanded in minimum essential alpha medium (α-MEM, Gibco™ 12000063, TR:TREATMENT_SUMMARY Waltham, MA, USA) supplemented with 10% v/v heat-inactivated fetal bovine serum TR:TREATMENT_SUMMARY (FBS, Gibco 10270106) and 1% v/v antibiotics (penicillin−streptomycin, Gibco TR:TREATMENT_SUMMARY 15240062) at 37°C in a humidified 5% CO2 incubator and passaged as described TR:TREATMENT_SUMMARY previously (Bispo et al. 2022, https://doi.org/10.3390/cells11081257). TR:TREATMENT_SUMMARY Osteodifferentiation of hAMSC was induced at passages 5 or 6, for 21 days, in TR:TREATMENT_SUMMARY T175 culture flasks (for metabolomics) and in 48-well plates (for biochemical TR:TREATMENT_SUMMARY assays). An independent experiment was conducted for each of the donors. For TR:TREATMENT_SUMMARY metabolomics, cells were seeded at a 0.5 × 106 cells/flask density. After TR:TREATMENT_SUMMARY reaching 100% confluence, day 0 (D0) cell samples were collected in triplicate TR:TREATMENT_SUMMARY from independent flasks, and media were replaced in the remaining flasks as TR:TREATMENT_SUMMARY follows: 15 control (CTR) flasks in standard growth medium and 15 osteoinduced TR:TREATMENT_SUMMARY (OI) flasks in medium supplemented with 10 mM β-glycerophosphate (β-GP, TR:TREATMENT_SUMMARY Sigma-Aldrich G9422), 50 μg/mL L-ascorbic acid (Sigma A0278) and 10 nM TR:TREATMENT_SUMMARY dexamethasone (ACROS Organics 230300010). Media were replaced 2× per week, on TR:TREATMENT_SUMMARY days (Di) 0, 4, 7, 11, 14, 18 and 21. Cells were trypsinized and collected in TR:TREATMENT_SUMMARY triplicate on D0, D1, D4, D7, D14 and D21. Cell suspensions were filtered TR:TREATMENT_SUMMARY through 100 μm pore strainers, centrifuged (300 g, 4 °C, 5 min) and rinsed TR:TREATMENT_SUMMARY twice in phosphate-buffered saline (PBS) solution. For intracellular TR:TREATMENT_SUMMARY metabolomics (endometabolomics), cell numbers/sample were 3.8-21.1 × 106, TR:TREATMENT_SUMMARY 2.3-8.6 × 106 and 1.4-9.1 × 106 for donors 1, 2 and 3, respectively. For TR:TREATMENT_SUMMARY extracellular metabolomics (exometabolomics), media samples were collected on TR:TREATMENT_SUMMARY D1, D4, D7, D11, D14, D18 and D21 (days when cell collection and/or medium TR:TREATMENT_SUMMARY exchange was carried out) and filtered through 40 μm pore strainers to remove TR:TREATMENT_SUMMARY cellular debris. Due to contamination and/or technical issues, triplicate TR:TREATMENT_SUMMARY samples could not always be retrieved (donor 1: CTR D0 cells n = 2, OI D7 media TR:TREATMENT_SUMMARY n = 0; donor 2: CTR D7,D18 media n = 2, OI D11,D21 media n = 1-2, OI D21 cells n TR:TREATMENT_SUMMARY = 1). For biochemical assays, cell samples were rinsed 2× with PBS and lysed by TR:TREATMENT_SUMMARY osmotic/thermal shock. Collected media and cell samples were stored at − 80 TR:TREATMENT_SUMMARY °C. TR:TREATMENT_PROTOCOL_FILENAME hASCs_Experimental_Procedure.pdf TR:TREATMENT_PROTOCOL_COMMENTS After reaching 100% confluence, day 0 (D0) cell samples were collected in TR:TREATMENT_PROTOCOL_COMMENTS triplicate from independent flasks, and media were replaced in the remaining TR:TREATMENT_PROTOCOL_COMMENTS flasks as follows: 15 control (CTR) flasks in standard growth medium and 15 TR:TREATMENT_PROTOCOL_COMMENTS osteoinduced (OI) flasks in medium supplemented with 10 mM β-glycerophosphate TR:TREATMENT_PROTOCOL_COMMENTS (β-GP, Sigma-Aldrich G9422), 50 μg/mL L-ascorbic acid (Sigma A0278) and 10 nM TR:TREATMENT_PROTOCOL_COMMENTS dexamethasone (ACROS Organics 230300010). TR:CELL_MEDIA 15 control (CTR) flasks in standard growth medium; and 15 osteoinduced (OI) TR:CELL_MEDIA flasks in medium supplemented with 10 mM β-glycerophosphate (β-GP, TR:CELL_MEDIA Sigma-Aldrich G9422), 50 μg/mL L-ascorbic acid (Sigma A0278) and 10 nM TR:CELL_MEDIA dexamethasone (ACROS Organics 230300010). TR:CELL_MEDIA_LASTCHANGED Media were replaced 2× per week, on days (Di) 0, 4, 7, 11, 14, 18 and 21. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To prepare samples for NMR analysis, intracellular metabolites (endometabolites) SP:SAMPLEPREP_SUMMARY were extracted using a methanol-chloroform-water method, as described in Bispo SP:SAMPLEPREP_SUMMARY et al. 2022b (https://doi.org/10.3390/cells11233745). Briefly, cell pellets were SP:SAMPLEPREP_SUMMARY re-suspended in 1 mL of a cold solution of methanol (Honeywell Riedel-de-Haen SP:SAMPLEPREP_SUMMARY 14262) and Milli-Q water (in a 4:1 ratio), transferred to glass tubes containing SP:SAMPLEPREP_SUMMARY 150 mg of glass beads (ø = 0.5 mm), and vortexed for 2 min at room temperature SP:SAMPLEPREP_SUMMARY (RT 25 °C). Cold chloroform (400 μL, Honeywell Riedel-de-Haen 650471) was then SP:SAMPLEPREP_SUMMARY added and vortexed for 2 min at RT, followed by 400 μL of cold chloroform plus SP:SAMPLEPREP_SUMMARY 360 μL of cold Milli-Q water with further vortexing for 2 min at RT. After SP:SAMPLEPREP_SUMMARY incubating at −20 °C for 10 min, samples were centrifuged (2000 g, 20 min, SP:SAMPLEPREP_SUMMARY RT) and the polar extracts (PE) were collected, dried, and stored at − 80 °C SP:SAMPLEPREP_SUMMARY until analysis. For NMR analysis, PE samples were re-suspended in 625 μL of 100 SP:SAMPLEPREP_SUMMARY mM phosphate buffer (pH 7.4), in D2O (99.9% deuterium, Eurisotop D216) SP:SAMPLEPREP_SUMMARY containing 0.1 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, in D2O, SP:SAMPLEPREP_SUMMARY Sigma-Aldrich 293040) for chemical shift referencing. Extracellular metabolites SP:SAMPLEPREP_SUMMARY (exometabolites) were measured in cell media, upon protein-precipitation of SP:SAMPLEPREP_SUMMARY blank (Br) and conditioned media as described in Bispo et al. 2022a SP:SAMPLEPREP_SUMMARY (https://doi.org/10.3390/cells11081257). Briefly, 600 µL of 100% methanol at SP:SAMPLEPREP_SUMMARY – 80 °C were added to microcentrifuge tubes containing 300 µL of medium SP:SAMPLEPREP_SUMMARY sample. After incubating at – 20 °C for 30 min, samples were centrifuged SP:SAMPLEPREP_SUMMARY (13000 g, 20 min, RT), the supernatant was collected, dried under vacuum and SP:SAMPLEPREP_SUMMARY stored at – 80 °C. To prepare samples for NMR analysis, cellular polar SP:SAMPLEPREP_SUMMARY extracts were re-suspended in 625 μL of 100 mM phosphate buffer (pH 7.4), in SP:SAMPLEPREP_SUMMARY D2O (99.9% deuterium, Eurisotop D216) containing 0.1 mM SP:SAMPLEPREP_SUMMARY 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, in D2O, Sigma-Aldrich 293040) SP:SAMPLEPREP_SUMMARY for chemical shift referencing. For exometabolome analysis, dry media samples SP:SAMPLEPREP_SUMMARY were resuspended in 700 µL of the same phosphate buffer, centrifuged (13000 g, SP:SAMPLEPREP_SUMMARY 5 min, RT) and the supernatant was collected. A volume of 550 μL from each SP:SAMPLEPREP_SUMMARY sample (pH adjusted to 7.4) was transferred into a 5 mm NMR tube. SP:SAMPLEPREP_PROTOCOL_FILENAME hASCs_Experimental_Procedure.pdf SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Intracellular metabolites (endometabolites) were extracted using a SP:EXTRACTION_METHOD water/methanol/chloroform method, as described in (Bispo et al., 2022, SP:EXTRACTION_METHOD https://doi.org/10.3390/cells11233745). Extracellular metabolites SP:EXTRACTION_METHOD (exometabolites) were measured in cell media, upon protein-precipitation of SP:EXTRACTION_METHOD blank and conditioned media SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Cellular polar extracts were re-suspended in 625 μL of 100 mM phosphate buffer SP:SAMPLE_RESUSPENSION (pH 7.4), in D2O (99.9% deuterium, Eurisotop D216) containing 0.1 mM SP:SAMPLE_RESUSPENSION 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, in D2O, Sigma-Aldrich 293040) SP:SAMPLE_RESUSPENSION for chemical shift referencing. For exometabolome analysis, dry media samples SP:SAMPLE_RESUSPENSION were resuspended in 700 µL of the same phosphate buffer, centrifuged (13000 g, SP:SAMPLE_RESUSPENSION 5 min, RT) and the supernatant was collected. A volume of 550 μL from each SP:SAMPLE_RESUSPENSION sample (pH adjusted to 7.4) was transferred into a 5 mm NMR tube. SP:SAMPLE_SPIKING 0.1 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, in D2O, Sigma-Aldrich SP:SAMPLE_SPIKING 293040) , as a chemical shift reference. #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Metabolomics Group AN:OPERATOR_NAME Daniela Bispo AN:DETECTOR_TYPE NMR AN:SOFTWARE_VERSION TopSpin3.2 and Amix3.9.14 AN:ANALYSIS_PROTOCOL_FILE hASCs_Experimental_Procedure.pdf AN:ACQUISITION_PARAMETERS_FILE hASCs_Experimental_Procedure.pdf AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME NMR 500 Bruker Avance III NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 500.13 MHz NM:NMR_PROBE TXI probe NM:NMR_SOLVENT 100 mM phosphate buffer (pH 7.4), in D2O (99.9% deuterium, Eurisotop D216) NM:NMR_SOLVENT containing 0.1 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, in D2O, NM:NMR_SOLVENT Sigma-Aldrich 293040), for chemical shift referencing. NM:NMR_TUBE_SIZE 5 mm NMR tubes NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 90-degree NM:CHEMICAL_SHIFT_REF_CPD 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4) NM:TEMPERATURE 25 NM:NUMBER_OF_SCANS 256 scans (exometabolome) and 512 (endometabolome) NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 2.33 s NM:RELAXATION_DELAY 4 s NM:SPECTRAL_WIDTH 7,002.8 Hz NM:NUM_DATA_POINTS_ACQUIRED 32 k points NM:LINE_BROADENING 0.3 Hz NM:ZERO_FILLING 128k (exometabolome) or 64 k points (endometabolome) NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Manual NM:CHEMICAL_SHIFT_REF_STD 0 ppm for TSP-d4 NM:NMR_RESULTS_FILE ST003765_AN006180_Results.txt UNITS:ppm #END