Dataset Information

Influence of Human Platelet Lysate on Extracellular Matrix Deposition and Cellular Characteristics in Adipose-Derived Stem Cell Sheets.

ABSTRACT: Adipose-derived stem cell (ASC) is a valuable source of cell therapy. By stimulating extracellular matrix (ECM) secretion, ASC sheets can be fabricated with enhanced regenerative capabilities. In recent years, human platelet lysate (HPL) provides an attractive alternative to fetal bovine serum (FBS) for the ex vivo expansion of ASCs for clinical use. However, the effect of HPL on ASC sheet formation has not been previously determined. In this study, we compared ECM composition and cellular characteristics of ASC sheets cultured in growth medium supplemented with either FBS or HPL. HPL supplement significantly enhanced ASC proliferation without obvious change in the expression pattern of cell surface markers. We found that culturing ASCs with HPL rendered thicker cell sheets with significantly more ECM deposition, including collagen and fibronectin. Proteomic analysis of the FBS or HPL-cultured cell sheets showed diversity in ECM composition. HPL-cultured ASC sheets exhibited up-regulation of interleukin-6 and an anti-inflammatory cytokine, C1q/tumor necrosis factor-related protein-3. Conditioned medium of HPL-cultured ASC sheets significantly enhanced fibroblast migration and tube formation of endothelial cells in vitro, while it inhibited the migration of macrophages toward stimulated macrophages in vitro. TGF-?1-stimulated fibroblasts cultured in ASC sheet-conditioned medium showed down-regulation of ?-SMA and TGF-?1. By adding an anti-hepatocyte growth factor (HGF) neutralizing antibody in conditioned medium, we indicated that an anti-fibrosis effect of HPL-cultured ASC sheets is partially mediated through the increased secretion of HGF. Moreover, chick embryo chorioallantoic membrane (CAM) assay showed comparable capillary density after applying either FBS or HPL-cultured ASC sheets, both of which were significantly higher than the control. In conclusion, robust ECM formation with altered ECM composition was noted in ASC sheets cultured in HPL-supplemented medium. Their immunomodulatory and pro-angiogenesis capabilities were largely maintained. Our findings paved the way to elucidate the potential of HPL-cultured ASC sheets for clinical application in tissue regeneration.

SUBMITTER: Cheng NC

PROVIDER: S-EPMC7642065 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Influence of Human Platelet Lysate on Extracellular Matrix Deposition and Cellular Characteristics in Adipose-Derived Stem Cell Sheets.

Cheng Nai-Chen NC Tu Yuan-Kun YK Lee Ning-Hsu NH Young Tai-Horng TH

Frontiers in cell and developmental biology 20201022

Adipose-derived stem cell (ASC) is a valuable source of cell therapy. By stimulating extracellular matrix (ECM) secretion, ASC sheets can be fabricated with enhanced regenerative capabilities. In recent years, human platelet lysate (HPL) provides an attractive alternative to fetal bovine serum (FBS) for the <i>ex vivo</i> expansion of ASCs for clinical use. However, the effect of HPL on ASC sheet formation has not been previously determined. In this study, we compared ECM composition and cellula ...[more]

PMID: 33195191

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Project description:Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine adipose-derived (AD-) MSC culture so far, as it supported AD-MSC proliferation and basic characteristics. The aim of the current study was to further analyze the functional properties of equine AD-MSC cultured with the same ePL, focusing on cell fitness, genetic stability and pro-angiogenic potency. All experiments were performed with AD-MSC from n = 5 horses, which were cultured either in medium supplemented with 10% FBS, 10% ePL or 2.5% ePL. AD-MSC cultured with 2.5% ePL, which previously showed decreased proliferation potential, displayed higher apoptosis but lower senescence levels as compared to 10% ePL medium (p < 0.05). Non-clonal chromosomal aberrations occurred in 8% of equine AD-MSC cultivated with FBS and only in 4.8% of equine AD-MSC cultivated with 10% ePL. Clonal aberrations in the AD-MSC were neither observed in FBS nor in 10% ePL medium. Analysis of AD-MSC and endothelial cells in an indirect co-culture revealed that the ePL supported the pro-angiogenic effects of AD-MSC. In the 10% ePL group, more vascular endothelial growth factor (VEGF-A) was released and highest VEGF-A concentrations were reached in the presence of ePL and co-cultured cells (p < 0.05). Correspondingly, AD-MSC expressed the VEGF receptor-2 at higher levels in the presence of ePL (p < 0.05). Finally, AD-MSC and 10% ePL together promoted the growth of endothelial cells and induced the formation of vessel-like structures in two of the samples. These data further substantiate that buffy-coat-based ePL is a valuable supplement for equine AD-MSC culture media. The ePL does not only support stable equine AD-MSC characteristics as demonstrated before, but it also enhances their functional properties.

Project description:BackgroundHuman platelet lysate (HPL) is emerging as the preferred xeno-free supplement for the expansion of mesenchymal stromal cells (MSCs) for bone tissue engineering (BTE) applications. Due to a growing demand, the need for standardization and scaling-up of HPL has been highlighted. However, the optimal storage time of the source material, i.e., outdated platelet concentrates (PCs), remains to be determined. The present study aimed to determine the optimal storage time of PCs in terms of the cytokine content and biological efficacy of HPL.MethodsDonor-matched bone marrow (BMSCs) and adipose-derived MSCs (ASCs) expanded in HPL or fetal bovine serum (FBS) were characterized based on in vitro proliferation, immunophenotype, and multi-lineage differentiation. Osteogenic differentiation was assessed at early (gene expression), intermediate [alkaline phosphatase (ALP) activity], and terminal stages (mineralization). Using a multiplex immunoassay, the cytokine contents of HPLs produced from PCs stored for 1-9 months were screened and a preliminary threshold of 4 months was identified. Next, HPLs were produced from PCs stored for controlled durations of 0, 1, 2, 3, and 4 months, and their efficacy was compared in terms of cytokine content and BMSCs' proliferation and osteogenic differentiation.ResultsBMSCs and ASCs in both HPL and FBS demonstrated a characteristic immunophenotype and multi-lineage differentiation; osteogenic differentiation of BMSCs and ASCs was significantly enhanced in HPL vs. FBS. Multiplex network analysis of HPL revealed several interacting growth factors, chemokines, and inflammatory cytokines. Notably, stem cell growth factor (SCGF) was detected in high concentrations. A majority of cytokines were elevated in HPLs produced from PCs stored for ≤ 4 months vs. > 4 months. However, no further differences in PC storage times between 0 and 4 months were identified in terms of HPLs' cytokine content or their effects on the proliferation, ALP activity, and mineralization of BMSCs from multiple donors.ConclusionsMSCs expanded in HPL demonstrate enhanced osteogenic differentiation, albeit with considerable donor variation. HPLs produced from outdated PCs stored for up to 4 months efficiently supported the proliferation and osteogenic differentiation of MSCs. These findings may facilitate the standardization and scaling-up of HPL from outdated PCs for BTE applications.

Project description:The mechanobiological behavior of mesenchymal stem cells (MSCs) in two- (2D) or three-dimensional (3D) cultures relies on the formation of actin filaments which occur as stress fibers and depends on mitochondrial dynamics involving vimentin intermediate filaments. Here we investigate whether human platelet lysate (HPL), that can potentially replace fetal bovine serum for clinical-scale expansion of functional cells, can modulate the stress fiber formation, alter mitochondrial morphology, change membrane elasticity and modulate immune regulatory molecules IDO and GARP in amnion derived MSCs. We can provide evidence that culture supplementation with HPL led to a reduction of stress fiber formation in 2D cultured MSCs compared to a conventional growth medium (MSCGM). 3D MSC cultures, in contrast, showed decreased actin concentrations independent of HPL supplementation. When stress fibers were further segregated by their binding to focal adhesions, a reduction in ventral stress fibers was observed in response to HPL in 2D cultured MSCs, while the length of the individual ventral stress fibers increased. Dorsal stress fibers or transverse arcs were not affected. Interestingly, ventral stress fiber formation did not correlate with membrane elasticity. 2D cultured MSCs did not show differences in the Young's modulus when propagated in the presence of HPL and further cultivation to passage 3 also had no effect on membrane elasticity. In addition, HPL reduced the mitochondrial mass of 2D cultured MSCs while the mitochondrial mass in 3D cultured MSCs was low initially. When mitochondria were segregated into punctuate, rods and networks, a cultivation-induced increase in punctuate and network mitochondria was observed in 2D cultured MSCs of passage 3. Finally, mRNA and protein expression of the immunomodulatory molecule IDO relied on stimulation of 2D culture MSCs with pro-inflammatory cytokines IFN-? and TNF-? with no effect upon HPL supplementation. GARP mRNA and surface expression was constitutively expressed and did not respond to HPL supplementation or stimulation with IFN-? and TNF-?. In conclusion, we can say that MSCs cultivated in 2D and 3D are sensitive to medium supplementation with HPL with changes in actin filament formation, mitochondrial dynamics and membrane elasticity that can have an impact on the immunomodulatory function of MSCs.

Dataset Information

Influence of Human Platelet Lysate on Extracellular Matrix Deposition and Cellular Characteristics in Adipose-Derived Stem Cell Sheets.

Publications

Influence of Human Platelet Lysate on Extracellular Matrix Deposition and Cellular Characteristics in Adipose-Derived Stem Cell Sheets.

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