Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures

Abstract

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-γ and TNF-α 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-γ and TNF-α. 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.

Melatonin Synergizes With Mesenchymal Stromal Cells Attenuates Chronic Allograft Vasculopathy

“…After 96 h, the MSCs adhered to the surface of the culture dishes. Then the dishes were washed with RPMI medium and the suspended splenocytes were removed by aspiration with a pipette ( 46 ). The proliferation capacity of CD4 + and CD8 + T cells was evaluated by Ki67 staining through flow cytometry.…”

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

“…By comparing the ASC sheets cultured with FBS or HPL, this study revealed prominent differences in the abundance of ECM and the composition of the microenvironment deposited in vitro. Human platelet lysate prepared from human platelets has been increasingly suggested to replace FBS in clinically oriented MSC expansion applications, allowing efficient cell propagation under animal serum-free conditions for clinical use (Cowper et al, 2019;Pasztorek et al, 2019;Schallmoser et al, 2020). Although autologous blood is a reliable resource for producing HPL, requesting each patient to donate his/her blood during the long-term in vitro cell expansion process is certainly not cost effective.…”

“…ASCs release an array of cytokines that exert potent immunomodulatory effects capable of regulating multiple signaling pathways and cell types that contribute to the pathogenesis of inflammatory or immunerelated diseases (Zimmermann and Mcdevitt, 2014). Particularly, MSCs cultivated in 3D structure are sensitive to medium supplemented with HPL, resulting in changes in actin filament formation, mitochondrial dynamics and membrane elasticity that can have an impact on the immunomodulatory function of MSCs (Pasztorek et al, 2019). IL-6 is a regulator of inflammation, and previous reports have shown that IL-6 released from ASCs can increase proliferation of several types of cells (Lu et al, 2016;Pu et al, 2019).…”

The role and mechanism of mitochondrial functions and energy metabolism in the function regulation of the mesenchymal stem cells

“…Moreover, melatonin can promote the mitochondrial dynamics and metabolism of the BMSCs, enhance the functions of the mitochondria, and protect the BMSCs from excessive ageing in mice with chronic kidney disease [ 51 ]. Further, the mitochondrial dynamics of amniotic membrane-derived MSCs can affect the immune regulatory function of the MSCs [ 52 ]. In conclusion, the above studies suggest that the mitochondrial dynamics play a critical role in regulating the multi-directional differentiation, ageing, and immune regulation of MSCs via different mechanism (Fig.…”