Abstract
Initial steps in establishing an optimal strategy for functional bioengineered tissues is generation of three-dimensional constructs containing cells with the appropriate organization and phenotype. To effectively utilize rhesus monkey decellularized kidney scaffolds, these studies evaluated two key parameters: (1) residual scaffold components after decellularization including proteomics analysis, and (2) the use of undifferentiated human embryonic stem cells (hESCs) for recellularization in order to explore cellular differentiation in a tissue-specific manner. Sections of kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis. Proteomics analysis revealed the presence of growth factors and antimicrobial proteins as well as stress proteins and complement components. Immunohistochemistry of recellularized kidney scaffolds showed the generation of Cytokeratin+ epithelial tubule phenotypes throughout the scaffold that demonstrated a statistically significant increase in expression of kidney-associated genes compared to baseline hESC gene expression. Recellularization of lung scaffolds showed that cells lined the alveolar spaces and demonstrated statistically significant upregulation of key lung-associated genes. However, overall expression of kidney and lung-associated markers was not statistically different when the kidney and lung recellularized scaffolds were compared. These results suggest that decellularized scaffolds have an intrinsic spatial ability to influence hESC differentiation by physically shaping cells into tissue-appropriate structures and phenotypes, and that additional approaches may be needed to ensure consistent recellularization throughout the matrix.
Highlights
There is an ongoing interest in using native extracellular matrix (ECM) scaffolds derived from decellularized tissues as a potential method for repair of organs damaged by disease
Decellularized Kidney and Lung Scaffold Characterization Our prior studies have shown intact cells are removed by the decellularization process as determined by the absence of Hematoxylin and Eosin (H&E) and DAPI staining of cell nuclei in decellularized kidney scaffolds [7]
Optimization of decellularization demonstrated that 1% SDS for kidney and 0.1% SDS for lung yielded scaffolds with preserved ECM structure while removing cells including Major Histocompatibility Complex (MHC) class I (HLAE) and II (HLA-DR) antigens as assessed by IHC (Figure 2)
Summary
There is an ongoing interest in using native extracellular matrix (ECM) scaffolds derived from decellularized tissues as a potential method for repair of organs damaged by disease. The clinical outcome of recent studies, such as those involving transplantation of tissue engineered decellularized airways, present issues such as the biomechanical collapse of the graft [1,2,3]. These findings indicate a more exhaustive in vitro understanding of the components of decellularized tissues is required before translational applications can be fully realized and achieved. ECM proteins provide physical biomechanical structure and interactive ligandmediated feedback to guide and alter the fate of cells These features must be carefully considered in order to better understand and effectively utilize the native ECM architecture provided by decellularized scaffolds for tissue repair
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