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Comprehensive RNA expression profile of therapeutic adipose?derived mesenchymal stem cells co?cultured with degenerative nucleus pulposus cells.

ABSTRACT: Stem cell?based therapy is a promising alternative to conventional approaches to treating intervertebral disc degeneration (IDD). However, comprehensive understanding of stem cell?based therapy at the gene level is still lacking. In the present study, we identified the expression profiles of messenger RNAs (mRNAs) and long non?coding RNAs (lncRNAs) expressed within a co?culture system of adipose?derived mesenchymal stem cells (ASCs) and degenerative nucleus pulposus cells (NPCs) and explored the signaling pathways involved and their regulatory networks. Microarray analysis was used to compare ASCs co?cultured with degenerative NPCs to ASCs cultured alone, and the underlying regulatory pattern, including the signaling pathways and competing endogenous RNA (ceRNA) network, was analyzed with robust bioinformatics methods. The results showed that 360 lncRNAs and 1757 mRNAs were differentially expressed by ASCs, and the microarray results were confirmed by quantitative PCR. Moreover, 589 Gene Ontology terms were upregulated, whereas 661 terms were downregulated. A total of 299 signaling pathways were significantly altered. A Path?net and a Signal?net were built to show interactions among differentially expressed genes. An mRNA?lncRNA co?expression network was constructed to reveal the interplay among differentially expressed mRNAs and lncRNAs, whereas a ceRNA network was built to investigate their connections with microRNAs involved in IDD. To the best of our knowledge, this original and comprehensive exploration reveals differentially expressed lncRNAs and mRNAs of ASCs stimulated by degenerative NPCs, underscoring the regulation pattern within the co?culture system at the gene level. These data may further understanding of NPC?directed differentiation of ASCs and facilitate the application of ASCs in future treatments for IDD.

SUBMITTER: Han Z

PROVIDER: S-EPMC7809910 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Comprehensive RNA expression profile of therapeutic adipose‑derived mesenchymal stem cells co‑cultured with degenerative nucleus pulposus cells.

Han Zhihua Z Wang Qiugen Q Wu Xiaoming X Wang Jiandong J Gao Liang L Guo Ruipeng R Wu Jianhong J

Molecular medicine reports 20210105 3

Stem cell‑based therapy is a promising alternative to conventional approaches to treating intervertebral disc degeneration (IDD). However, comprehensive understanding of stem cell‑based therapy at the gene level is still lacking. In the present study, we identified the expression profiles of messenger RNAs (mRNAs) and long non‑coding RNAs (lncRNAs) expressed within a co‑culture system of adipose‑derived mesenchymal stem cells (ASCs) and degenerative nucleus pulposus cells (NPCs) and explored the ...[more]

PMID: 33398382

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Project description:BACKGROUND:Stem cell therapy is considered as a promising alternative to treat intervertebral disc degeneration (IDD). Extensive work had been done on identifying and comparing different types of candidate stem cells, both in vivo and in vitro. However, few studies have shed light on degenerative nucleus pulposus cells (NPCs), especially their biological behavior under the influence of exogenous stem cells, specifically the gene expression and regulation pattern. In the present study, we aimed to determine messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs), which are differentially expressed during the co-culturing process with adipose-derived mesenchymal stem cells (ASCs) and to explore the involved signaling pathways and the regulatory networks. METHODS:We compared degenerative NPCs co-cultured with ASCs with those cultured solely using lncRNA-mRNA microarray analysis. Based on these data, we investigated the significantly regulated signaling pathways based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Moreover, 23 micro RNAs (miRNAs), which were demonstrated to be involved in IDD were chosen; we investigated their theoretic regulatory importance associated with our microarray data. RESULTS:We found 632 lncRNAs and 1682 mRNAs were differentially expressed out of a total of 40,716 probes. We then confirmed the microarray data by real-time PCR. Furthermore, we demonstrated 197 upregulated, and 373 downregulated Gene Ontology terms and 176 significantly enriched pathways, such as the mitogen-activated protein kinase (MAPK) pathway. Also, a signal-net was constructed to reveal the interplay among differentially expressed genes. Meanwhile, a mRNA-lncRNA co-expression network was constructed for the significantly changed mRNAs and lncRNAs. Also, the competing endogenous RNA (ceRNA) network was built. CONCLUSION:Our results present the first comprehensive identification of differentially expressed lncRNAs and mRNAs of degenerative NPCs, altered by co-culturing with ASCs, and outline the gene expression regulation pattern. These may provide valuable information for better understanding of stem cell therapy and potential candidate biomarkers for IDD treatment.

Project description:Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

Dataset Information

Comprehensive RNA expression profile of therapeutic adipose?derived mesenchymal stem cells co?cultured with degenerative nucleus pulposus cells.

Publications

Comprehensive RNA expression profile of therapeutic adipose‑derived mesenchymal stem cells co‑cultured with degenerative nucleus pulposus cells.

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