273883 Whole Organ 3D Microenvironment As a Regulatory Cue for Pancreatic Differentiation of Embryonic Stem Cells

Wednesday, October 31, 2012: 12:48 PM
Somerset East (Westin )
Saik Kia Goh1, Suzanne Bertera2, Phillip Olsen1, Lei Yang3 and Ipsita Banerjee4, (1)Bioengineering, University of Pittsburgh, Pittsburgh, PA, (2)Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, PA, (3)Developmental Biology, University of Pittsburgh, Pittsburgh, PA, (4)Chemical and Petroleum Engg, University of Pittsburgh, Pittsburgh, PA

Introduction: Type I Diabetes affects over 1 million people in the United States. While islet transplantation has proved to be a promising therapeutic strategy, it is still hindered by lack of donor tissue. Embryonic stem cells (ESCs) have emerged as an alternative cell source owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types, including islet-cells.  However, ESC derived islet-cells are currently limited in their yield and functionality. Current differentiation strategies primarily involve various growth factor/ inducer/ repressor concoctions with less emphasis on the substrate. Governed by the understanding that the extracellular matrix (ECM) proteins and architecture significantly influences specific cellular behaviors, we hypothesize that organ specific ECM microenvironment plays an important role in organogenesis. In this project we are testing our hypothesis by inducing pancreatic differentiation of hESCs in 3 different whole organ derived ECM scaffold: native organ – pancreas; alternate endoderm derived organ – liver; mesoderm derived organ – heart.

Materials and Method: Cadaveric pancreata, livers and hearts were isolated from adult mice (n=8) and mounted on a perfusion apparatus. Ionic detergent, 0.1% SDS was perfused until tissues were translucent and white in color (2 hours). Cell nuclei post-decellularization was quantified via DAPI staining and PicoGreen DNA assay. Immunofluorescence and histology were done to characterize the retention of the extracellular matrix (ECM) protein before and after decellularization. Ultrastructure and the macroscopic three-dimensional architecture of organ-derived ECM scaffolds were characterized using electron microscopy. To examine cell-matrix interaction, whole organ decellularized pancreas, liver or heart were seeded with hESC-derived endodermal cells (3x106 cells) and cultured in pancreatic differentiation medium with growth factors induction. Recell constructs (n=3) were then mounted on a perfusion apparatus to allow dynamic culture for 4 days and 12 days. Whole organ recell constructs were analyzed using immunohistochemistry, TUNEL, and qRT-PCR.

Results and Discussion: Perfusion decellularization with 0.1% SDS resulted in complete decellularization of pancreas, liver and heart. DAPI staining and DNA quantification confirmed the removal of cells and residual DNA. Microstructure and major ECM components such as collagen I, collagen IV, fibronectin and laminin were preserved. In all the repopulated whole organ constructs, hESC derived DE cells were found engrafted - evidenced by positive hNuclei staining from IHC. Low apoptotic cells were detected (<10% TUNEL positive) in all three repopulated constructs. qRT-PCR of hESC differentiated on pancreatic matrix showed 52.7±10.8 fold increase in PDX1, pancreatic gene expression, and 92.7±12.7 fold increase in insulin gene expression compared to Matrigel (Fig. 1). Cells found engrafted in the pancreatic matrix were positive for PDX1 and C-peptide from immunostaining - confirming the differentiation of engrafted hESC into insulin-expressing cells. Compared to pancreatic matrix, qPCR of recellularized liver and heart construct gave lower expression of pancreatic gene expression. Immunostaining results were consistent with qPCR result – with low PDX-1 and C-peptide expression found in liver and heart recell constructs.

Conclusions: Perfusion-decellularization of cadaveric pancreas, liver and heart efficiently removes cells while retaining ECM protein and microstructure. Tissue-specific three-dimensional spatial organization and ECM composition drives cell fate differently with pancreatic matrix being the preferred ECM substrate for pancreatic differentiation. Further proteomic studies are underway to establish comprehensive and mechanistic differences between the ECM composition of endoderm organs (pancreas and liver) and mesoderm organs (heart) to better understand the cell-matrix interactions that contributed to the specific differentiation of hESC.

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