Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry

Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, K.; Knöbel, Sebastian; Dickinson, AM; Filby, Andrew; Wang, Xiao-Nong

doi:10.1038/s41598-018-22326-5

Cited by 14 publications

(10 citation statements)

References 69 publications

(75 reference statements)

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“…In addition, a recent publication has reported a global phenotypic analysis of surface markers for MSCs cultured in FBS and HPL. Importantly, it was reported that differential expression on multiple markers in HPL‐expanded cells was specifically related to pathways involved in proliferation, osteogenic differentiation, and inflammatory responses . This further supports our observation of increased proliferation and higher in vivo bone formatting capacity of hPDCs expanded in spinner flasks using HPL supplemented medium.…”

Section: Discussionsupporting

confidence: 90%

Human Platelet Lysate Improves Bone Forming Potential of Human Progenitor Cells Expanded in Microcarrier-Based Dynamic Culture

Gupta

Hall

Geris

et al. 2019

Stem Cells Translational Medicine

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Xenogeneic‐free media are required for translating advanced therapeutic medicinal products to the clinics. In addition, process efficiency is crucial for ensuring cost efficiency, especially when considering large‐scale production of mesenchymal stem cells (MSCs). Human platelet lysate (HPL) has been increasingly adopted as an alternative for fetal bovine serum (FBS) for MSCs. However, its therapeutic and regenerative potential in vivo is largely unexplored. Herein, we compare the effects of FBS and HPL supplementation for a scalable, microcarrier‐based dynamic expansion of human periosteum‐derived cells (hPDCs) while assessing their bone forming capacity by subcutaneous implantation in small animal model. We observed that HPL resulted in faster cell proliferation with a total fold increase of 5.2 ± 0.61 in comparison to 2.7 ± 02.22‐fold in FBS. Cell viability and trilineage differentiation capability were maintained by HPL, although a suppression of adipogenic differentiation potential was observed. Differences in mRNA expression profiles were also observed between the two on several markers. When implanted, we observed a significant difference between the bone forming capacity of cells expanded in FBS and HPL, with HPL supplementation resulting in almost three times more mineralized tissue within calcium phosphate scaffolds. FBS‐expanded cells resulted in a fibrous tissue structure, whereas HPL resulted in mineralized tissue formation, which can be classified as newly formed bone, verified by μCT and histological analysis. We also observed the presence of blood vessels in our explants. In conclusion, we suggest that replacing FBS with HPL in bioreactor‐based expansion of hPDCs is an optimal solution that increases expansion efficiency along with promoting bone forming capacity of these cells. Stem Cells Translational Medicine 2019;8:810&821

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Section: Discussionsupporting

confidence: 90%

Human Platelet Lysate Improves Bone Forming Potential of Human Progenitor Cells Expanded in Microcarrier-Based Dynamic Culture

Gupta

Hall

Geris

et al. 2019

Stem Cells Translational Medicine

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“…Most of these studies evaluated the immunophenotype of the cells after expansion by analyzing cell surface markers with fluorescence-activated cell sorting (FACS): stem cell defining surface markers [46] were analyzed in almost any study; the consistent results were that expansion of MSC in hPL-supplemented ESM does not alter the expression of stem cell defining surface markers in comparison to MSC expanded in FCS-supplemented ESM [16][17][18]22,23,26,28,29,43,44,47]. Viau et al [18] and Reis et al [47] assessed the expression of further surface markers, revealing that the expression of some surface markers differs significantly between MSC expanded in hPL or FCS, but the majority of the markers are only mildly influenced by the source of protein supplement in ESM. Some studies additionally performed gene expression analyses of expanded MSC and revealed that, along with the results of protein expression of the investigated markers, the ESM's protein supplement only mildly influences the gene expression pattern of MSC [29,43,44].…”

Section: Discussionmentioning

confidence: 99%

Human Platelet Lysate Can Replace Fetal Calf Serum as a Protein Source to Promote Expansion and Osteogenic Differentiation of Human Bone-Marrow-Derived Mesenchymal Stromal Cells

Karadjian

Senger

Essers

et al. 2020

Cells

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Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates and happens to be a stable and reliable protein source. In this study, we investigated the influence of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed population doublings during cell expansion, performed alizarin red staining to evaluate the calcium content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a protein supplier in cell culture media and does not negatively affect the osteogenic differentiation capacity of BMSC.

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“…In brief, bone marrow mononuclear cells (MNCs) were isolated by density gradient centrifugation using Lymphoprep™ (Axis-Shield). MNCs were then plated at a density of 2 × 10 7 cells/flask in T-25 tissue culture flasks in basal medium containing Dulbecco's modified eagle medium, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 IU/ml heparin and 2 mM L-glutamine (all from Sigma-Aldrich), supplemented with 5% human platelet lysate (hPL; PLTMax, Mill Creek Lifesciences) ( 23 ). The cells were cultured for 3 days at 37°C in a 5% CO 2 incubator.…”

Section: Methodsmentioning

confidence: 99%

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function

et al. 2018

Self Cite

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Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro. MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro. Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro. MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.

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Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry

Cited by 14 publications

References 69 publications

Human Platelet Lysate Improves Bone Forming Potential of Human Progenitor Cells Expanded in Microcarrier-Based Dynamic Culture

Human Platelet Lysate Improves Bone Forming Potential of Human Progenitor Cells Expanded in Microcarrier-Based Dynamic Culture

Human Platelet Lysate Can Replace Fetal Calf Serum as a Protein Source to Promote Expansion and Osteogenic Differentiation of Human Bone-Marrow-Derived Mesenchymal Stromal Cells

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function

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