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Decellularized ECM effects on human mesenchymal stem cell stemness and differentiation.

ABSTRACT: Microenvironment extracellular matrices (ECMs) influence cell adhesion, proliferation and differentiation. The ECMs of different microenvironments have distinctive compositions and architectures. This investigation addresses effects ECMs deposited by a variety of cell types and decellularized with a cold-EDTA protocol have on multipotent human mesenchymal stromal/stem cell (hMSC) behavior and differentiation. The cold-EDTA protocol removes intact cells from ECM, with minimal ECM damage and contamination. The decellularized ECMs deposited by cultured hMSCs, osteogenic hMSCs, and two smooth muscle cell (SMC) lines were tested for distinctive effects on the behavior and differentiation of early passage ('naïve') hMSC plated and cultured on the decellularized ECMs. Uninduced hMSC decellularized ECM enhanced naïve hMSC proliferation and cell motility while maintaining stemness. Decellularized ECM deposited by osteogenic hMSCs early in the differentiation process stimulated naïve hMSCs osteogenesis and substrate biomineralization in the absence of added dexamethasone, but this osteogenic induction potential was lower in ECMs decellularized later in the osteogenic hMSC differentiation process. Decellularized ECMs deposited by two smooth muscle cell lines induced naïve hMSCs to become smooth muscle cell-like with distinctive phenotypic characteristics of contractile and synthetic smooth muscle cells. This investigation demonstrates a useful approach for obtaining functional cell-deposited ECM and highlights the importance of ECM specificity in influencing stem cell behavior.

SUBMITTER: Rao Pattabhi S

PROVIDER: S-EPMC4336570 | biostudies-literature | 2014 Nov-Dec

REPOSITORIES: biostudies-literature

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Decellularized ECM effects on human mesenchymal stem cell stemness and differentiation.

Rao Pattabhi Sudhakara S Martinez Jessica S JS Keller Thomas C S TC

Differentiation; research in biological diversity 20141101 4-5

Microenvironment extracellular matrices (ECMs) influence cell adhesion, proliferation and differentiation. The ECMs of different microenvironments have distinctive compositions and architectures. This investigation addresses effects ECMs deposited by a variety of cell types and decellularized with a cold-EDTA protocol have on multipotent human mesenchymal stromal/stem cell (hMSC) behavior and differentiation. The cold-EDTA protocol removes intact cells from ECM, with minimal ECM damage and conta ...[more]

PMID: 25578478

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Project description:Mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) have been reported to be able to differentiate to hepatocyte in vitro with varying degree of hepatocyte maturation. A simple method to decellularize liver scaffold has been established by the Department of Histology, Faculty of Medicine, Universitas Indonesia, in SCTE IMERI lab.15 This study aims to evaluate hepatocyte differentiation from iPSCs compared to MSCs derived in our decellularized liver scaffold. The research stages started with iPSC culture, decellularization, seeding cell culture into the scaffold, and differentiation into hepatocytes for 21 days. Hepatocyte differentiation from iPSCs and MSCs in the scaffolds was characterized using hematoxylin-eosin, Masson Trichrome, and immunohistochemistry staining to determine the fraction of the differentiation area. RNA samples were isolated on days 7 and 21. Expression of albumin, CYP450, and CK-19 genes were analyzed using the qRT-PCR method. Electron microscopy images were obtained by SEM. Immunofluorescence examination was done using HNF4-α and CEBPA markers. The results of this study in hepatocyte-differentiated iPSCs compared with hepatocyte-differentiated MSCs in decellularized liver scaffold showed lower adhesion capacity, single-cell-formation and adhered less abundant, decreased trends of albumin, and lower CYP450 expression. Several factors contribute to this result: lower initial seeding number, which causes only a few iPSCs to attach to certain parts of decellularized liver scaffold, and manual syringe injection for recellularization, which abruptly and unevenly creates pattern of single-cell-formation by hepatocyte-differentiated iPSC in the scaffold. Hepatocyte-differentiated MSCs have the advantage of higher adhesion capacity to collagen fiber decellularized liver scaffold. This leads to positive result: increase trends of albumin and higher CYP450 expression. Hepatocyte maturation is shown by diminishing CK-19, which is more prominent in hepatocyte-differentiated iPSCs in decellularized liver scaffold. Confirmation of mature hepatocyte-differentiated iPSCs in decellularized liver scaffold maturation is positive for HNF4-a and CEBPA. The conclusion of this study is hepatocyte-differentiated iPSCs in decellularized liver scaffold is mature with lower cell-ECM adhesion, spatial cell distribution, albumin, and CYP450 expression than hepatocyte-differentiated MSCs in decellularized liver scaffold.

Dataset Information

Decellularized ECM effects on human mesenchymal stem cell stemness and differentiation.

Publications

Decellularized ECM effects on human mesenchymal stem cell stemness and differentiation.

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