Saik-Kia Goh

Characterization and Functionality of Cardiac Progenitor Cells in Congenital Heart Patients

Background— Human cardiac progenitor cells (hCPCs) may promote myocardial regeneration in adult ischemic myocardium. The regenerative capacity of hCPCs in young patients with nonischemic congenital heart defects for potential use in congenital heart defect repair warrants exploration. Methods and Results— Human right atrial specimens were obtained during routine congenital cardiac surgery across 3 groups: neonates (age, <30 days), infants (age, 1 month to 2 years), and children (age, >2 to ≤13 years). C-kit+ hCPCs were 3-fold higher in neonates than in children >2 years of age. hCPC proliferation was greatest during the neonatal period as evidenced by c-kit+ Ki67+ expression but decreased with age. hCPC differentiation capacity was also greatest in neonatal right atrium as evidenced by c-kit+, NKX2–5+, NOTCH1+, and NUMB+ expression. Despite the age-dependent decline in resident hCPCs, we isolated and expanded right atrium–derived CPCs from all patients (n=103) across all ages and diagnoses using the cardiosphere method. Intact cardiospheres contained a mix of heart-derived cell subpopulations that included cardiac progenitor cells expressing c-kit+, Islet-1, and supporting cells. The number of c-kit+–expressing cells was highest in human cardiosphere-derived cells (hCDCs) grown from neonatal and infant right atrium. Furthermore, hCDCs could differentiate into diverse cardiovascular lineages by in vitro differentiation assays. Transplanted hCDCs promoted greater myocardial regeneration and functional improvement in infarcted myocardium than transplanted cardiac fibroblasts. Conclusions— Resident hCPCs are most abundant in the neonatal period and rapidly decrease over time. hCDCs can be reproducibly isolated and expanded from young human myocardial samples regardless of age or diagnosis. hCPCs are functional and have potential in congenital cardiac repair.

Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network 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 shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches.

The adult human heart as a source for stem cells: repair strategies with embryonic-like progenitor cells.

Adequate cell-based repair of adult myocardium remains an elusive goal because most cells that are used cannot generate mature myocardium sufficient to promote large functional improvements. Embryonic stem cells can generate both mature cardiocytes and vasculature, but their use is hampered by associated teratoma formation and the need for an allogeneic source. The detection of sca-1+, c-kit+, or isl-1+ cardiac precursors and the creation of cardiospheres from adult heart tissues suggest that a persistent population of immature progenitor cells is present in the mature myocardium. These cell populations probably represent stages along a continuum of cardiac stem cell development and differentiation. We report isolation from ventricle of uncommitted cardiac progenitor cells, which appear to resemble the more immature, common pool of embryonic lateral plate mesoderm progenitors that yield both myocardial and endocardial cells during normal cardiac development. Under controlled in vitro conditions and in vivo, these cells can differentiate into endothelial, smooth muscle, and cardiomyocyte lineages and can be isolated and expanded to clinically relevant numbers from adult rat myocardial tissue. In this article, we discuss the potential for autologous repair or even cardiac regeneration with cells that follow a developmental pathway similar to embryonic cardiac precursors but without the inherent limitations associated with undifferentiated embryonic stem cells.

A Strong Regenerative Ability of Cardiac Stem Cells Derived From Neonatal Hearts

Background— Human cardiac stem cells (CSCs) promote myocardial regeneration in adult ischemic myocardium. The regenerative capacity of CSCs in very young patients with nonischemic congenital heart defects has not been explored. We hypothesized that isolated neonatal-derived CSCs may have a higher regenerative ability than adult-derived CSCs and might address the structural deficiencies of congenital heart disease. Methods and Results— Human specimens were obtained during routine cardiac surgical procedures from right atrial appendage tissue discarded from 2 age groups: neonates and adults patients. We developed a reproducible isolation method that generated cardiosphere-derived cells (CDCs), regardless of starting tissue weight or age. Neonatal-derived CDCs demonstrated increased number of cardiac progenitor cells expressing c-kit+, flk-1, and Islet-1 by flow cytometry and immunofluorescence. When transplanted into infarcted myocardium, neonatal-derived CDCs had a significantly higher ability to preserve myocardial function, prevent adverse remodeling, and enhance blood vessel preservation and/or formation when compared with adult-derived CDCs. Last, neonatal-derived CDCs were more cardiomyogenic than adult-derived CDCs when cocultured with neonatal cardiomyocytes and displayed enhanced angiogenic function compared with adult-derived CDCs. Conclusions— Neonatal-derived CDCs have a strong regenerative ability when compared with adult-derived CDCs that may depend on angiogenic cytokines and an increase prevalence of stem cells. This has important implications in the potential use of CDCs in future clinical trials.

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.

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.

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

BackgroundThe 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.ResultsWe 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 in vivo 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.ConclusionThese 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.

A novel combination of bioresorbable polymeric film and expanded polytetrafluoroethylene provides a protective barrier and reduces adhesions

OBJECTIVEnA bioresorbable polymeric film reduces the extent and severity of postoperative adhesions in infants undergoing repeat sternotomy. Resorption of the bioresorbable polymeric film, however, leaves no barrier between the sternum and the epicardium. A sheet of expanded polytetrafluoroethylene is used by many surgeons to create a physical barrier between the sternum and the cardiac structures. We hypothesized that placing bioresorbable polymeric film beneath an expanded polytetrafluoroethylene pericardial membrane would both decrease pericardial adhesions and provide a physical barrier.nnnMETHODSnA novel combination of bioresorbable polymeric film underneath an expanded polytetrafluoroethylene membrane was tested in an established rabbit model of pericardial adhesion formation. After sternotomy, a portion of pericardium was resected and the epicardium was abraded. Rabbits (n = 36) were randomly assigned to 4 treatment groups: control group, no bioresorbable polymeric film or expanded polytetrafluoroethylene; bioresorbable polymeric film group; expanded polytetrafluoroethylene group; and bioresorbable polymeric film + expanded polytetrafluoroethylene group. At 4 weeks post-sternotomy, pericardial adhesions were scored grossly for area and density of adhesions using an established 4-point (0-3) grading system.nnnRESULTSnThe bioresorbable polymeric film group had a significant reduction in mean adhesion score compared with the control group (control = 2.86 ± 0.37 vs bioresorbable polymeric film = 0.57 ± 0.53, P < .0001) and expanded polytetrafluoroethylene group (expanded polytetrafluoroethylene = 2.75 ± 0.46 vs bioresorbable polymeric film = 0.57 ± 0.53, P < .0001). The bioresorbable polymeric film + expanded polytetrafluoroethylene group had a low adhesion profile similar to the bioresorbable polymeric film group (bioresorbable polymeric film + expanded polytetrafluoroethylene = 1.0 ± 0, vs bioresorbable polymeric film = 0.57 ± 0.53), but a considerably lower mean adhesion score than the expanded polytetrafluoroethylene group (expanded polytetrafluoroethylene = 2.75 ± 0.46, vs bioresorbable polymeric film + expanded polytetrafluoroethylene = 1.0 ± 0, P < .0001).nnnCONCLUSIONSnPlacement of bioresorbable polymeric film resulted in minimal pericardial adhesions compared with controls. The placement of bioresorbable polymeric film underneath expanded polytetrafluoroethylene at the time of sternal closure provides a novel combination to reduce the extent and severity of pericardial adhesions while providing a physical barrier between the sternum and the cardiac structures.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the inxa0vitro reproduction of specific inxa0vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to inxa0vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering.