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Alterations in immunomodulatory potential of ADSCs under-going osteogenic differentiation in the context of future thera-peutic applications

ABSTRACT: For proteomic analysis, ADSCs from four independent donors were analyzed (n = 4 biological replicates per condition, OM vs. SM). Cell pellets were lysed in 5% sodium de-oxycholate buffer containing TCEP and CAA, supplemented with protease and phospha-tase inhibitors, followed by sonication and incubation at 60 °C. Protein concentration was determined by BCA assay, and 25 µg of protein was processed with a modified FASP pro-tocol employing 30 kDa filters. After trypsin/LysC digestion, peptides were purified on Waters HLB plates, eluted with 70% acetonitrile/0.1% TFA, dried, and stored at −80 °C. Background: Adipose-derived mesenchymal stem/stromal cells (ADSCs) are gaining recognition in regenerative medicine thanks to their potential for adipogenic, osteogenic, and chondrogenic differentiation, as well as their immunomodulatory properties. How-ever, ADSC-based therapies focus either on differentiation for tissue replacements or on counteracting the unrestrained inflammation to prevent tissue destruction and initiate re-generation. Here, we aim to examine the immunomodulatory potential of osteogenically differentiated ADSCs by analyzing their proteomic profile. Methods: using the LC-MS/MS technique, we created the proteomic profile of differentiated and undifferentiated ADSCs, and compared them with the Reactome database. The transcriptomic analysis was also performed and compared to the proteomic profile. Results: the comparison of proteomic (499 up-regulated; 355 down-regulated) and transcriptomic (212 up-regulated; 232 down-regulated) profiles showed 60,1% concordance – both proteins and transcripts showed the same trend. Significantly upregulated proteins in differentiating ADSCs (−log₁₀ p >5 and >10) were grouped into four categories: propensity for osteogenic differen-tiation; immunomodulation/immune/inflammatory response; cell senescence; cell cycle regulation. Among those proteins, thirteen were reported to play some role in processes such as immunomodulation, inflammatory signaling, transplant rejection, or graft-versus-host disease. Conclusions: we observed that differentiating ADSCs might still exert immunomodulatory effects, which could be used in the treatment of e.g., GvHD.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Dominik Cysewski

LAB HEAD: Dominik Cysewski

PROVIDER: PXD073297 | Pride | 2026-04-27

REPOSITORIES: Pride

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Alterations in Immunomodulatory Potential of ADSCs Undergoing Osteogenic Differentiation in the Context of Future Therapeutic Applications.

Szabłowska-Gadomska Ilona I Rudziński Stefan S Mroczko Agnieszka A Mrozikiewicz-Rakowska Beata B Cysewski Dominik D Gasperowicz Piotr P Bocian Katarzyna K

Cells 20260330 7

<h4>Background</h4>Adipose-derived mesenchymal stem/stromal cells (ADSCs) are gaining recognition in regenerative medicine for their potential for adipogenic, osteogenic, and chondrogenic differentiation, as well as their immunomodulatory properties. However, ADSC-based therapies focus either on differentiation for tissue replacement or on counteracting unrestrained inflammation to prevent tissue destruction and initiate regeneration. Here, we aim to examine the immunomodulatory potential of ost ...[more]

PMID: 41972704

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Project description:Bone tissue engineering is considered the optimal solution for the repair of large bone defects. Previous studies by our team have demonstrated the effectiveness of a "bottom-up" microtissue engineering strategy using bone marrow mesenchymal stem cells (BMSCs) to improve the utilization efficiency of seed cells. However, this strategy requires the extraction of bone marrow, which is associated with significant trauma and difficulties in obtaining the cells. Adipose-derived mesenchymal stem cells (ADSCs), obtained from aspirated adipose tissue, offer a less invasive and more accessible alternative, but their osteogenic capacity is noticeably weaker, severely limiting their application as seed cells in bone tissue engineering repair.Cell-free fat extract (CEFFE) is a cell-free liquid component extracted from adipose tissue, which exhibits beneficial properties such as promoting proliferation, antioxidation, anti-apoptosis, and angiogenesis. In this study, during the preparation of ADSC-based osteogenic microtissues, we first discovered that CEFFE significantly enhanced the osteogenic activity of ADSCs. Furthermore, we observed the formation of dense osteogenic membranes as early as day 7 of osteogenic induction. When the microtissues based on CEFFE-treated ADSCs were implanted subcutaneously in nude mice, we found a significant enhancement in their osteogenic activity. Finally, to further investigate the mechanism underlying the enhancement of ADSC osteogenic differentiation by CEFFE, we performed transcriptomic analysis of CEFFE-treated ADSCs and validated the activation of the key PI3K-Akt pathway using Western blotting.In conclusion, this study presents a novel approach for the preparation of ADSC-based osteogenic microtissues using CEFFE and provides insights into the mechanisms underlying the enhancement of osteogenic activity. These findings hold significant implications for the field of bone tissue engineering and offer potential advancements in the clinical translation of ADSC-based therapies.

Project description:Adipogenesis occurs through a specific gene program in undifferentiated fat progenitors. We hypothesized that the properties of the fat progenitors are regulated by hox genes, the developmental genes essential in different tissue stem cells. Their biased expression in white and brown fat implies roles in distinguishing the two fat types. Among 39 Hox genes, Hoxc8 is highly enriched in undifferentiated adipose tissue stem cells (ADSCs) and down-regulated in differentiated adipocytes. Forced expression of Hoxc8 suppressed adipocyte differentiation of ADSCs. Using microarrays, we investigated the effect of Hoxc8 overexpression on global transcripts in ADSCs. We compared among four groups: untreated ADSCs, adipogenic induction media (MDI)-treated ADSCs, MDI-treated ADSC-vector and MDI-treated ADSC-Hoxc8. A number of, but not all, adipogenesis-related genes are suppressed by Hoxc8. This dataset illustrates the global effect of Hoxc8, a developmental transcription factor, on the expression of adipogenesis-related genes. Gene expression was compared among untreated ADSCs (control), adipogenic induction media-treated ADSCs, adipogenic induction media-treated ADSC-vector (ADSCs transduced with control vector), and adipogenic induction media-treated ADSC-Hoxc8 (ADSCs transduced with human Hoxc8). Total RNA was isolated from ADSCs using the Qiagen RNeasy kit (Qiagen). At NimbleGen, quality and yield were verified before cDNA synthesis and Cy3-end labeling. The labeled cDNA samples were hybridized to Homo sapiens 4-Plex arrays (Roche NimbleGen, A4487001-00-01) that represent 24,000 human genes. Raw data files for each sample were normalized and background-corrected using a Robust Multi-Array Analysis as implemented by NimbleScan software. Students’ two-tail t-tests were conducted among the samples for each transcript and fold-change was determined. Transcripts whose abundance was significantly altered (P < 0.05) and an absolute fold change greater than 2 were defined as differentially regulated.

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