Project description:We characterized and compared the proteomic composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts isolated from different donors, employing a multidimensional proteomic approach coupled with label-based quantitative proteomics. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential proliferation and gene expression profiles between the cell types and as compared to TCPS, indicating that the cell-derived ECM influence each cell type in a unique manner.
Project description:In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts isolated from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering.
Project description:We characterized and compared the proteomic composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts isolated from different donors, employing a multidimensional proteomic approach coupled with label-based quantitative proteomics. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential proliferation and gene expression profiles between the cell types and as compared to TCPS, indicating that the cell-derived ECM influence each cell type in a unique manner.
Project description:We reported a specialized protocol to produce human cardiac cell-derived matrices without the introduction of exogenous matrix proteins. Human cardiac fibroblasts cultured under conditions to produced cell-derived matrices were harvested at the beginning, middle and completion of the experiment - corresponding to days 0, 5 and 10. Fetal and adult human cardiac fibroblasts were cultured densely in the presence of ficoll as a macromolecular crowder on the adhesive biopolymer l-polydopamine, and sampled throughout matrix deposition to determine the age-specific responses of primary human cardiac fibroblasts to macromolecular crowders and enhanced matrix deposition.
Project description:The fibroblast-populated 3D collagen matrix has been used to study the effect of mechanical stress on cell fate; this process is relevant to the fields of wound healing and tissue engineering. Gene array data was generated from mechanically stressed vs. stress-released matrices. The parameters of the collagen matrix model were: collagen type = bovine type I; collagen concentration = 1.5 mg/mL; initial matrix volume = 0.2 mL; initial matrix diameter = 11 mm (cultured in 24-well plates); cell type = human foreskin fibroblast, passage <10; initial matrix cell concentration = 1,000,000 cell/mL (200,000 cell/matrix); culture medium = 5% FBS in DMEM with 1 M-BM-5g/mL ascorbate. Matrices (n = 6 per experimental group) were incubated for 24 hr in the attached state; the released groups then underwent matrix detachment from the culture plate (defined as t = 0), while the attached groups were left undisturbed. RNA was isolated from attached and released matrices 6 and 24 hr after t = 0. Gene expression in the attached vs. released condition at 6 or 24 hr then was analyzed by hybridizing the anti-sense RNA derived from attached and released matrices at a given time point onto a single chip. Refer to the attached Figure 1 for the experimental design. The index experiment was defined as the comparison of gene expression in attached vs. released collagen matrices in a single strain of human foreskin fibroblasts at 6 and 24 hr after stress-release (i.e., after t = 0). Each experiment utilized two mechanical conditions (attached and released) at two time points (6 and 24 hr). So with each condition utilizing 6 matrices, each index experiment required a total of 24 matrices. In each index experiment, the chip hybridizations were: (i) 6 hr attached vs. 6 hr released, and (ii) 24 hr attached vs. 24 hr released (i.e., two gene chips per index experiment). Each hybridization was done using a 10K spotted gene chip manufactured in the UNMC Microarray Core Facility. The index experiment was performed on three fibroblast strains, meaning that expressional data was derived from three foreskin donors (nonpooled samples). Dye-swap was not performed; dye assigned to attached vs. released remained constant among all chips. Since the index experiment was performed three times, the total number of gene chips used for this entire dataset was six.