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Hydrogel supplemented with human platelet lysate enhances multi-lineage differentiation of mesenchymal stem cells.

ABSTRACT: Stem cells from human exfoliated deciduous teeth (SHED) can be used as a potential clinical material. But the use of xenogeneic ingredients will increase the risk of zoonotic disease transmission. Human platelet lysate (HPL) is a potential surrogate and used in human cell expansion with reliability in clinical applications. In this study, we synthesized chitosan/gelatin/gellan gum hydrogel supplemented with HPL and investigated the effect of 3D culture for SHED. TMT-tagged proteomics was used to decipher the secretome protein profiles of SHEDs and a total of 3209 proteins were identified, of which 23 were up-regulated and 192 were down-regulated. The results showed that hydrogel supplemented with HPL promoted SHED proliferation. After induction, the hydrogel coating contributed to osteogenic differentiation, adipogenic differentiation and differentiation into neural-like cells of SHED. SHED encapsulated in a hydrogel promotes migration and angiogenesis of HUVEC. In conclusion, our research found that hydrogel supplemented with HPL can be used as a method for SHED in standardized production and can contribute to the clinical application of SHED in cell therapy.

SUBMITTER: Lei T

PROVIDER: S-EPMC8976277 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Hydrogel supplemented with human platelet lysate enhances multi-lineage differentiation of mesenchymal stem cells.

Lei Tong T Liu Yanyan Y Deng Shiwen S Xiao Zhuangzhuang Z Yang Yanjie Y Zhang Xiaoshuang X Bi Wangyu W Du Hongwu H

Journal of nanobiotechnology 20220402 1

Stem cells from human exfoliated deciduous teeth (SHED) can be used as a potential clinical material. But the use of xenogeneic ingredients will increase the risk of zoonotic disease transmission. Human platelet lysate (HPL) is a potential surrogate and used in human cell expansion with reliability in clinical applications. In this study, we synthesized chitosan/gelatin/gellan gum hydrogel supplemented with HPL and investigated the effect of 3D culture for SHED. TMT-tagged proteomics was used to ...[more]

PMID: 35366889

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Project description:UnlabelledMesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration.Statement of significanceInnovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.

Project description:BackgroundThe use of distant autografts to restore maxillary bone defects is clinically challenging and has unpredictable outcomes. This variation may be explained by the embryonic origin of long bone donor sites, which are derived from mesoderm, whereas maxillary bones derive from neural crest. Gingival stem cells share the same embryonic origin as maxillary bones. Their stemness potential and ease of access have been repeatedly shown. One limitation in human cell therapy is the use of foetal calf serum during cell isolation and culture. To overcome this problem, a new serum-free medium enriched with an alternative to foetal calf serum, i.e., platelet lysate, needs to be adapted to clinical grade protocols.MethodsDifferent serum-free media enriched with platelet lysate at various concentrations and supplemented with different growth factors were developed and compared to media containing foetal calf serum. Phenotypic markers, spontaneous DNA damage, and stem cell properties of gingival stem cells isolated in platelet lysate or in foetal calf serum were also compared, as were the immunomodulatory properties of the cells by co-culturing them with activated peripheral blood monocellular cells. T-cell proliferation and phenotype were also assessed by flow cytometry using cell proliferation dye and specific surface markers. Data were analysed with t-test for two-group comparisons, one-way ANOVA for multigroup comparisons and two-way ANOVA for repeated measures and multigroup comparisons.ResultsSerum-free medium enriched with 10% platelet lysate and growth hormone yielded the highest expansion rate. Gingival stem cell isolation and thawing under these conditions were successful, and no significant DNA lesions were detected. Phenotypic markers of mesenchymal stem cells and differentiation capacities were conserved. Gingival stem cells isolated in this new serum-free medium showed higher osteogenic differentiation potential compared to cells isolated in foetal calf serum. The proportion of regulatory T cells obtained by co-culturing gingival stem cells with activated peripheral blood monocellular cells was similar between the two types of media.ConclusionsThis new serum-free medium is well suited for gingival stem cell isolation and proliferation, enhances osteogenic capacity and maintains immunomodulatory properties. It may allow the use of gingival stem cells in human cell therapy for bone regeneration in accordance with good manufacturing practice guidelines.

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.

Project description:Bone disorders are major health issues requiring specialized care; however, the traditional bone grafting method had several limitations. Thus, bone tissue engineering has become a potential alternative. In therapeutic treatments, using fetal bovine serum (FBS) as a culture supplement may result in the risk of contamination and host immunological response; therefore, human platelet lysate (hPL) has been considered a viable alternative source. This study attempted to compare the effectiveness and safety of different culture supplements, either FBS or hPL, on the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid (hAF-MSCs) under a three-dimensional gelatin scaffold. The results indicate that hAF-MSCs have the potential to be used in clinical applications as they meet the criteria for mesenchymal stem cells based on their morphology, the expression of a particular surface antigen, their proliferation ability, and their capacity for multipotent differentiation. After evaluation by MTT and Alamar blue proliferation assay, 10% of hPL was selected. The osteogenic differentiation of hAF-MSCs under three-dimensional gelatin scaffold using osteogenic-induced media supplemented with hPL was achievable and markedly stimulated osteoblast differentiation. Moreover, the expressions of osteoblastogenic related genes, including OCN, ALP, and COL1A1, exhibited the highest degree of expression under hPL-supplemented circumstances when compared with the control and the FBS-supplemented group. The induced cells under hPL-supplemented conditions also presented the highest ALP activity level and the greatest degree of calcium accumulation. These outcomes would indicate that hPL is a suitable substitute for animal derived serum. Importantly, osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells using hPL-supplemented media and three-dimensional scaffolds may open the door to developing an alternative construct for repairing bone defects.

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Hydrogel supplemented with human platelet lysate enhances multi-lineage differentiation of mesenchymal stem cells.

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Hydrogel supplemented with human platelet lysate enhances multi-lineage differentiation of mesenchymal stem cells.

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