title: High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells.
abstract: Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.
authors.affiliation: Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.
authors.forename: Amel
authors.initials: A
authors.lastname: Dudakovic
authors.forename: Emily
authors.initials: E
authors.lastname: Camilleri
authors.forename: Scott M
authors.initials: SM
authors.lastname: Riester
authors.forename: Eric A
authors.initials: EA
authors.lastname: Lewallen
authors.forename: Sergiy
authors.initials: S
authors.lastname: Kvasha
authors.forename: Xiaoyue
authors.initials: X
authors.lastname: Chen
authors.forename: Darcie J
authors.initials: DJ
authors.lastname: Radel
authors.forename: Jarett M
authors.initials: JM
authors.lastname: Anderson
authors.forename: Asha A
authors.initials: AA
authors.lastname: Nair
authors.forename: Jared M
authors.initials: JM
authors.lastname: Evans
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Krych
authors.forename: Jay
authors.initials: J
authors.lastname: Smith
authors.forename: David R
authors.initials: DR
authors.lastname: Deyle
authors.forename: Janet L
authors.initials: JL
authors.lastname: Stein
authors.forename: Gary S
authors.initials: GS
authors.lastname: Stein
authors.forename: Hee-Jeong
authors.initials: HJ
authors.lastname: Im
authors.forename: Simon M
authors.initials: SM
authors.lastname: Cool
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Westendorf
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Kakar
authors.forename: Allan B
authors.initials: AB
authors.lastname: Dietz
authors.forename: Andre J
authors.initials: AJ
authors.lastname: van Wijnen
mesh_terms: D050156:Adipogenesis / Q000235:genetics; D000273:Adipose Tissue / Q000166:cytology*; D001483:Base Sequence; D002450:Cell Communication / Q000235:genetics; D059447:Cell Cycle Checkpoints / Q000235:genetics; D002454:Cell Differentiation; D049109:Cell Proliferation / Q000235:genetics; D064987:Cell- and Tissue-Based Therapy; D020219:Chondrogenesis / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D005109:Extracellular Matrix / Q000235:genetics; D005434:Flow Cytometry; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016130:Immunophenotyping; D000090062:Kruppel-Like Factor 4; D008565:Membrane Proteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008938:Mitosis / Q000235:genetics; D010012:Osteogenesis / Q000235:genetics*; D017423:Sequence Analysis, RNA; D018800:Thy-1 Antigens / Q000096:biosynthesis
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D008565:Membrane Proteins; D018800:Thy-1 Antigens
keywords: ACTA2; ADH1B; ADIPOGENESIS; ADIPOSE-TISSUE DERIVED STROMAL CELLS; ASPN; CCNB2; CCND1; CD105; CD44; CD73; CD90; CELL CYCLE; CHI3L1; CHONDROGENESIS; CYCLIN; E2F1; E2F7; E2F8; ECM2; ENG; EXTRACELLULAR MATRIX; FIBROBLAST; FMOD; H19; HINFP; HIST1H3H; HIST1H4A; HIST2H4A; HIST2H4B; HISTONE; KLF4; LINEAGE-COMMITMENT; MESENCHYMAL STEM CELL; MULTIPOTENT; NANOG; NES; NPAT; NT5E; OCT4; OGN; OSTEOGENESIS; PLURIPOTENT; PODN; POU5F1; RARRES2; SFRP2; SFRP4; THY1; TNNT3; WISP2; WNT2; WNT2A; WNT5A; WNT5B; WNT7B