Identification of Senescence‐associated Genes in Rhesus Monkey Bone Marrow‐Derived Mesenchymal Stem Cells Cultured in A Defined Serum‐free Media

Abstract

BackgroundMesenchymal stem cells (MSCs) are multipotent cells that are currently being investigated in a wide variety of clinical trials for their anti‐inflammatory and immunomodulatory properties. The purity and properties for the resulting populations can be affected by the conditions under which MSCs are cultured. Serum‐free medium would greatly facilitate the development of robust, clinically acceptable bioprocesses for reproducibly generating quality‐assured cells. Clinical trials as well as animal studies have shown that the regeneration potential of bone and other tissues declines with age due to a decline in the number or frequency of stem cells present in adult organs; these factors may contribute to human aging and age‐related disease.MethodsOur in‐house serum free medium, consists of α‐MEM supplemented with PDGF, TGF‐β1, bFGF, IGF, Sodium Bicarbonate, L‐Glutamine, Hepes, Putrescine, progesterone, hydrocortisone, defined lipid concentrate, Fetuin, ascorbic acid, Insulin‐transferrin‐selenium, human albumin, serotonin and Folic acid. MSCs were first cultured under both our defined serum‐free medium and 10% FBS medium conditions. Immunophenotype analysis was performed with CD90, CD29, CD105, HL‐ADR, CD44 and CD34. And cells were treated with CCK‐8 for 24 h, 48 h, 96 h, 120 h,144 h,168 h and 192 h, respectively. Then, this study compared rBMSCs in three age groups: young (< 5 years), middle (8–10 years), and old (>15years). Using NOISeq method to filter 1909 differentially expressed genes (DEGs) out of 24,204 total genes analyzed, we identified 224 highly senescence‐related DEGs.ResultsThe expansion levels of MSCs in serum‐free culture exhibited better cell morphology and immunophenotype, positive for CD29 (99.9%) and CD105 (98.2%), and negative for CD34 (2.4%) and HLA‐DR (0.2%)(Figure. 1–2). And CCK‐8 assay showed that MSCs cultured in serum‐containing medium exhibited better cell proliferation activity. Also, the serum‐free culture cells showed better differentiation capacity. And then, GO term categorization revealed that the identified genes are strongly related to known biological process, such as angiogenesis and immune responses (Figure 3–5). Notably, many of the SASP components over‐secreted by senescent rBMSCs are to the immune system process. And we found a robust increase of several inflammatory cytokine genes in senescent rBMSCs, including IL‐6, IL‐8, CCL‐2, GM‐CSF, MMP3 and ICAM. Meanwhile, we found that the senescence of MSCs are strongly related to the angiogenesis genes, such as PDGFR, WISP1, SDF‐1 and TGF‐β1(Figure 6). And WNT‐induced secreted protein 1 (WISP1), also a downstream factor of the canonical WNT signaling pathway, is known to affect proliferation and differentiation of MSCs.ConclusionsOur results indicate that the use of SFM in MSC culture can give better cell quality than serum‐containing medium. And SASP increases the complexity of paracrine communication among rBMSCs and their physiological/pathological microenvironment. The genes identified using RNA‐seq will facilitate future studies of the mechanisms underlying the cellular senescence of MSCs.Support or Funding InformationFounding: Chengdu Giant Panda Breeding Research Foundation (2016cdspt001) Project Name: Rhesus Monkey Mesenchymal Stem Cells Biological Study and Resource Pool EstablishmentThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.