Bio-Engineered Pancreas with Human Embryonic Stem Cells and Whole Organ Derived Extracellular Matrix Scaffolds

According to Centers of Disease Control (CDC), 25.8 million Americans were diagnosed with diabetes in 2010, and more than 300 million people were affected worldwide. One potential future treatment for diabetes is transplantation of bioengineered pancreas capable of restoring insulin function. However, bioengineering of the complex pancreas function is a significant engineering feat. It calls for appropriate combinations of cells, with biomaterials that provide structural support and a suitable extracellular environment to maintain cell survival and function in vitro and in vivo. The first objective of this work is to investigate a suitable 3D bioscaffold to support pancreatic cell types. Our result demonstrated that perfusion-decellularization of whole pancreas effectively removes cellular material but retains intricate three-dimensional microarchitecture and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result showed successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to higher insulin gene expression over individual ECM proteins. The second objective of this work is to evaluate the feasibility to repopulate the native organ-derived scaffolds with renewable cell types such as differentiating human pluripotent stem cells (hPSC). We developed an in-house bioreactor to support the regenerative reconstruction of pancreas. Our result demonstrated that hPSCs cultured and differentiated as aggregates are more suitable than the parallel adherent cultures for organ repopulation. Upon continued culture with chemical induction in bioreactor, the seeded PP aggregates grow within the 3D organ scaffolds with homogeneity and mature in situ into monohormonal C-peptide positive cells. The last objective of this work is to evaluate the matrix-specificity of organ-derived ECM. We evaluated this by developing a miniaturized ECM array composed of organ-specific matrices derived from decellularized pancreas, liver and heart. Interestingly, our result showed higher PP cell adhesion and differentiation on liver-ECM over pancreas- and heart-ECM, suggesting that the requirement for ‘like-to-like” basis for tissue engineering approaches may not always be the case. Overall, the findings from this dissertation represent a notable step toward bioengineering of pancreas as an alternative therapeutic solution for diabetes

Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>The investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. High-throughput approaches based on chromatin immunoprecipitation (ChIP) have been widely used for this purpose. However, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and require complex statistical analysis. Advances in this field will greatly benefit from the development and use of simple, fast, sensitive and straightforward ChIP assay and analysis methodologies.</p> <p>Results</p> <p>We initially developed a simplified, basic ChIP protocol that combines simplicity, speed and sensitivity. ChIP analysis by real-time PCR was compared to analysis by densitometry with the ImageJ software. This protocol allowed the rapid identification of known target genes for SOX2, NANOG, OCT3/4, SOX17, KLF4, RUNX2, OLIG2, SMAD2/3, BMI-1, and c-MYC in a human embryonic stem cell line. We then developed a novel Sequential ChIP protocol to investigate <it>in vivo </it>promoter co-occupancy, which is basically characterized by the absence of antibody-antigen disruption during the assay. It combines centrifugation of agarose beads and magnetic separation. Using this Sequential ChIP protocol we found that c-MYC associates with the SOX2/NANOG/OCT3/4 complex and identified a novel RUNX2/BMI-1/SMAD2/3 complex in BG01V cells. These two TF complexes associate with two distinct sets of target genes. The RUNX2/BMI-1/SMAD2/3 complex is associated predominantly with genes not expressed in undifferentiated BG01V cells, consistent with the reported role of those TFs as transcriptional repressors.</p> <p>Conclusion</p> <p>These simplified basic ChIP and novel Sequential ChIP protocols were successfully tested with a variety of antibodies with human embryonic stem cells, generated a number of novel observations for future studies and might be useful for high-throughput ChIP-based assays.</p

Bioengineering thymus organoids to restore thymic function and induce donor-specific immune tolerance to allografts

One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids effectively promoted homing of lymphocyte progenitors and supported thymopoiesis. Nude mice transplanted with thymus organoids promptly rejected skin allografts and were able to mount antigen-specific humoral responses against ovalbumin on immunization. Notably, tolerance to skin allografts was achieved by transplanting thymus organoids constructed with either thymic epithelial cells coexpressing both syngeneic and allogenic major histocompatibility complexes, or mixtures of donor and recipient thymic epithelial cells. Our results demonstrate the technical feasibility of restoring thymic function with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in organ transplantation and regenerative medicine

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al

Immunofluorescence images of ECM biomolecules preserved after decellularization treatment in both groups of EBs.

<p>(A) dSPT EB and (B) dRA EB. Both groups of decellularized EB preserved Collagen I, Collagen IV, Fibronectin, and Laminin after decellularization treatment.</p

Examination of viability, engraftment location and proliferation kinetics of the seeded EB constructs.

<p>(A) Whole mount fluorescence images of seeded EB scaffolds at day 2 depict more ESR1 cells attach and grow on dSPT than dRA EB scaffolds. (B) Alamar blue assay depicts higher proliferation of ESR1 seeded on both decellularized EB scaffolds compared to native ESR1 EB over the course of 6 days. (C) Representative live/dead staining images show the survival of the engrafted ESR1 cells on both dSPT and dRA EB scaffolds after 2 days of culture. (D) Image analysis of live/dead assay to depict the mean percentage of live cells. (E) XY single-plane-two-photon imaging of cell-seeded constructs was done at depths of 33, 64 and 94 µm – this demonstrates that engrafted cells attached on the surface of the scaffolds with minimal infiltration into the ECM scaffold. All values are mean ± SD, n = 3, represents individual seeded EBs in 3 experimental repeats.</p