In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells

Abstract

Human mesenchymal stem or stromal cells (hMSCs) are known for their potential in regenerative medicine due to their differentiation abilities, secretion of trophic factors, and regulation of immune responses in damaged tissues. Due to the limited quantity of hMSCs typically isolated from bone marrow, other tissue sources, such as adipose tissue-derived mesenchymal stem cells (hASCs), are considered a promising alternative. However, differences have been observed for hASCs in the context of metabolic characteristics and response to in vitro culture stress compared to bone marrow derived hMSCs (BM-hMSCs). In particular, the relationship between metabolic homeostasis and stem cell functions, especially the immune phenotype and immunomodulation of hASCs, remains unknown. This study thoroughly assessed the changes in metabolism, redox cycles, and immune phenotype of hASCs during in vitro expansion. In contrast to BM-hMSCs, hASCs did not respond to culture stress significantly during expansion as limited cellular senescence was observed. Notably, hASCs exhibited the increased secretion of pro-inflammatory cytokines and the decreased secretion of anti-inflammatory cytokines after extended culture expansion. The NAD+/NADH redox cycle and other metabolic characteristics associated with aging were relatively stable, indicating that hASC functional decline may be regulated through an alternative mechanism rather than NAD+/Sirtuin aging pathways as observed in BM-hMSCs. Furthermore, transcriptome analysis by mRNA-sequencing revealed the upregulation of genes for pro-inflammatory cytokines/chemokines and the downregulation of genes for anti-inflammatory cytokines for hASCs at high passage. Proteomics analysis indicated key pathways (e.g., tRNA charging, EIF2 signaling, protein ubiquitination pathway) that may be associated with the immune phenotype shift of hASCs. Together, this study advances our understanding of the metabolism and senescence of hASCs and may offer vital insights for the biomanufacturing of hASCs for clinical use.

Polychromatic Flow Cytometric Analysis of Stromal Vascular Fraction from Lipoaspirate and Microfragmented Counterparts Reveals Sex-Related Immunophenotype Differences

“…Although renowned as MSC signature markers, the expression of CD73, CD90, CD105 and CD146 are far from uniform and their function in MSC biology is still little understood. Special attention needs to be paid to the sparse literature data from human native as opposed to the more common cultured MSC, as the expression of signature and functional markers appear to be donor-dependent or change during the ex vivo-manipulation of MSC [ 16 , 26 , 27 ].…”

Adipose Tissue: Understanding the Heterogeneity of Stem Cells for Regenerative Medicine

“…Jeske et al found that ASCs were less susceptible to culture stress relative to BM-MSCs. An increased secretion of pro-inflammatory cytokines and decreased secretion of anti-inflammatory cytokines was observed after extended culture expansion of ASCs [68].…”

A Novel 3D Culture Model of Human ASCs Reduces Cell Death in Spheroid Cores and Maintains Inner Cell Proliferation Compared With a Nonadherent 3D Culture

“…However, ASC expansion on the flat layer of the plastic surface cannot accurately mimic the natural microenvironment for cell growth in vivo due to a lack of three-dimensional tissue structure information ( Yin et al, 2020 ). Furthermore, more evidence has shown that the extensive passage of ASCs in planar culture can cause cell morphology changes, leading to cell cycle arrest, induction of replication and senescence, and loss of differentiation potential ( Delben et al, 2021 ; Jeske et al, 2021 ). As a result, scientists are trying to find new cultivation methods that can overcome or partially overcome these problems, and 3D ASC culture is a novel approach.…”