Soon-Jung Park

Effect of chromosome instability on the maintenance and differentiation of human embryonic stem cells in vitro and in vivo.

The therapeutic potential of human embryonic stem cells (hESCs) has long been appreciated, and the recent FDA approval of hESC derivatives for cell-based therapy encourages the clinical application of hESCs. Here, using CHA3-hESCs with normal and abnormal karyotypes, we report the importance of maintaining normal chromosomes during in vitro culture and the differentiation of hESCs for minimization of posttransplantation complications. We found that undifferentiated CHA3-hESCs with trisomy chromosome 12 undergo abnormal cell division with multiple spindles in comparison to the bipolar cell division of the karyotypically normal CHA3-hESCs. Transplanted karyotypically abnormal CHA3-hESC derivatives formed a tumor-like tissue 6weeks after transplantation in two out of seven mice tested. Our results demonstrate that the preservation of normal chromosomes is indispensable for maintaining the true properties of hESCs in vitro and abolishing adverse effects posttransplantation. Thus, the development of optimized techniques for stabilizing the chromosome state during in vitro hESC culture is a prerequisite for the therapeutic application of hESCs.

The use of aggregates of purified cardiomyocytes derived from human ESCs for functional engraftment after myocardial infarction

Embryonic stem cells (ESCs) have the capacity to undergo directed differentiation into contracting cardiomyocytes. Therefore, functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are potential candidates for cellular cardiomyoplasty to regenerate the myocardium after infarction. However, the directed differentiation of hESCs induces not only contracting cardiomyocytes but also other cell types. Thus, a risk of teratoma formation and oncologic transformation exists following the transplantation of hESC-CMs containing other cell lineages. In addition, the transplantation of hESC-CMs into the infarcted myocardium limits therapeutic efficacy due to low viability and poor engraftment. In this study, we established an efficient preparation method to obtain pure contracting cardiomyocytes from hESCs. We also developed a delivery system to achieve enhanced viability and a functional connection with the host myocardium after transplantation in a myocardial infarction model. A serum-free medium was used to obtain pure contracting cardiomyocytes from other cell lineages after the cardiac differentiation of hESCs. Aggregates of purified hESC-CMs were formed, and then the expression of cardiomyocyte-specific markers and the viability of the aggregated CMs were examined in hypoxic conditions. In addition, we determined whether the viability of the hESC-CMs and their ability to engraft with the host myocardium could be enhanced by transplanting them as aggregates in a myocardial infarction model. The therapeutic efficacy of the cardiomyocytes was examined by immunohistochemical analyses as well as physiological analyses of left-ventricular function. We found that the transplantation of contracting hESC-CM aggregates improved their survival and function in infarcted rat hearts in comparison to the transplantation of dissociated cells. Our method using hESC-CMs can be considered an effective strategy for clinical applications without critical barriers.

A comparison of human cord blood- and embryonic stem cell-derived endothelial progenitor cells in the treatment of chronic wounds

Endothelial progenitor cells (EPCs) promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. Therefore, EPCs have been highlighted as an alternative cell source for wound healing. EPCs can be isolated from various sources, including the bone marrow, cord blood, and adipose tissue. However, several recent studies have reported that isolating EPCs from these sources has limitations, such as the isolation of insufficient cell numbers and the difficulty of expanding these cells in culture. Thus, human embryonic stem cells (hESCs) have generated great interest as an alternative source of EPCs. Previously, we established an efficient preparation method to obtain EPCs from hESCs (hESC-EPCs). These hESC-EPCs secreted growth factors and cytokines, which are known to be important in angiogenesis and wound healing. In this study, we directly compared the capacity of hESC-EPCs and human cord blood-derived EPCs (hCB-EPCs) to benefit wound healing. The number of hESC-EPCs increased during culture and was always higher than the number of hCB-EPCs during the culture period. In addition, the levels of VEGF and Ang-1 secreted by hESC-EPCs were significantly higher than those produced by hCB-EPCs. After transplantation in a mouse dermal excisional wound model, all EPC-transplanted wounds exhibited better regeneration than in the control group. More importantly, we found that the wounds transplanted with hESC-EPCs showed significantly accelerated re-epithelialization. Thus, hESC-EPCs may be a promising cell source for the treatment of chronic wounds.

Development of a Xeno-Free Autologous Culture System for Endothelial Progenitor Cells Derived from Human Umbilical Cord Blood

Despite promising preclinical outcomes in animal models, a number of challenges remain for human clinical use. In particular, expanding a large number of endothelial progenitor cells (EPCs) in vitro in the absence of animal-derived products is the most critical hurdle remaining to be overcome to ensure the safety and efficiency of human therapy. To develop in vitro culture conditions for EPCs derived from human cord blood (hCB-EPCs), we isolated extracts (UCE) and collagen (UC-collagen) from umbilical cord tissue to replace their animal-derived counterparts. UC-collagen and UCE efficiently supported the attachment and proliferation of hCB-EPCs in a manner comparable to that of animal-derived collagen in the conventional culture system. Our developed autologous culture system maintained the typical characteristics of hCB-EPCs, as represented by the expression of EPC-associated surface markers. In addition, the therapeutic potential of hCB-EPCs was confirmed when the transplantation of hCB-EPCs cultured in this autologous culture system promoted limb salvage in a mouse model of hindlimb ischemia and was shown to contribute to attenuating muscle degeneration and fibrosis. We suggest that the umbilical cord represents a source for autologous biomaterials for the in vitro culture of hCB-EPCs. The main characteristics and therapeutic potential of hCB-EPCs were not compromised in developed autologous culture system. The absence of animal-derived products in our newly developed in vitro culture removes concerns associated with secondary contamination. Thus, we hope that this culture system accelerates the realization of therapeutic applications of autologous hCB-EPCs for human vascular diseases.

Repair of Ischemic Injury by Pluripotent Stem Cell Based Cell Therapy without Teratoma through Selective Photosensitivity.

Summary Stem-toxic small molecules have been developed to induce selective cell death of pluripotent stem cells (PSCs) to lower the risk of teratoma formation. However, despite their high efficacies, chemical-based approaches may carry unexpected toxicities on specific differentiated cell types. Herein, we took advantage of KillerRed (KR) as a suicide gene, to selectively induce phototoxicity using visible light via the production of reactive oxygen species. PSCs in an undifferentiated state that exclusively expressed KR (KR-PSCs) were eliminated by a single exposure to visible light. This highly selective cell death in KR-PSCs was exploited to successfully inhibit teratoma formation. In particular, endothelial cells from KR-mPSCs remained fully functional in vitro and sufficient to repair ischemic injury in vivo regardless of light exposure, suggesting that a genetic approach in which KR is expressed in a tightly controlled manner would be a viable strategy to inhibit teratoma formation for future safe PSC-based therapies.

Gene expression profiles in CHA3 and CHA4 human embryonic stem cells and embryoid bodies.

The establishment of the first human embryonic stem cells (hESCs) in 1998 provided a unique tool for studying human development. Although several Western embryoderived hESC lines are well characterized, the biological properties of Asian embryo-derived hESC lines remain unexamined. The aim of this study was to characterize Korean embryo-derived hESC lines and their differentiation potential. In this context, we conducted microarraybased differential gene expression analyses using two Korean embryo-derived hESC lines (CHA3 and CHA4) to identify undifferentiated and spontaneously differentiated (human embryoid body, or hEB) status. These two cell lines showed great similarity in gene expression. By comparing their expression patterns, we determined novel hESC-specific genes and transcriptomes that could serve as reliable hESC markers associated with the “stemness” phenotype. Additionally, we sought to identify hEB markers that could be used to determine the presence of differentiated cells in specific tissues, allowing for the purification of homogeneous cell populations or serving as indicators of hESC differentiation. Novel sets of 68 hESC-specific markers, 12 hESC-specific transcripts and 36 hEB markers were identified and shown by quantitative RTPCR to be similarly expressed in CHA3- and CHA4-hESC lines, as compared to the Western embryo-derived H9-hESC line. Furthermore, our data analysis revealed that the cell cycle, urea cycle, p53 signaling, and metabolism of amino groups are significantly implicated in the regulation of hESC differentiation. These results provide another unique set of hESC/hEB markers and foster a better understanding of the molecular mechanisms underlying hESC biology. These results may thus facilitate studies of human developmental events and provide information regarding Korean embryo-derived hESCs, which could be used to determine differences in developmental events between human races.

Successful Occlusion of Spontaneous Portosystemic Shunts Leading to Encephalopathy in a Non-cirrhotic Patient by Using the Amplatzer Vascular Plug

A 55-year-old woman presented with recurrent, recently aggravated encephalopathic episodes. However, the patient had no evidence of liver cirrhosis, and her serum ammonia level was high. An abdominal computed tomography (CT) scan revealed two portal-systemic venous shunts and, accordingly, she was diagnosed as having non-cirrhotic portal-systemic encephalopathy due to portal-systemic shunts. The shunts were successfully occluded using Amplatzer vascular plugs, and this led to a normalization of her blood ammonia levels immediately after occlusion. Over a 3-month follow-up, the patient experienced no complications or symptoms, and no shunt recanalization was detected by CT. The described case demonstrates that the Amplatzer vascular plug offers a useful option for occluding portal-systemic shunts responsible for encephalopathy.

Pertussis Toxin Enhances Colony Organization of Enzymatic-Dissociated Single Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) self-renew indefinitely as highly organized pluripotent colonies. Unlike mouse pluripotent stem cell colonies, human colonies form a uniform, flat, epithelium-like monolayer. Interestingly, it has been reported that colony morphology is closely correlated with the maintenance of pluripotency. However, the molecular mechanisms that underlie human pluripotent colony formation and organization are poorly understood. In this study, we used real-time imaging tools to examine the in vitro colony formation of enzymatically dissociated single hESCs under feeder-free conditions. We demonstrate that colony formation consists of 4 stages: attachment, migration, aggregation, and colony formation, which are facilitated in an intracellular, calcium-dependent manner. Moreover, we found that blocking G(i)-coupled G protein-coupled receptor (GPCR) signaling results in enhanced cell-cell interactions and plays an integral role in promoting the survival of hESCs in culture. From the imaging results, we identified the conditions required for colony formation, and we identified the importance of blocking G(i)-coupled GPCR by pertussis toxin in modulating hESC colony formation and organization. These results will likely be useful for engineering hESCs to further study the mechanisms involved in their function.

Photodynamic Approach for Teratoma-Free Pluripotent Stem Cell Therapy Using CDy1 and Visible Light

Pluripotent stem cells (PSC) are promising resources for regeneration therapy, but teratoma formation is one of the critical problems for safe clinical application. After differentiation, the precise detection and subsequent elimination of undifferentiated PSC is essential for teratoma-free stem cell therapy, but a practical procedure is yet to be developed. CDy1, a PSC specific fluorescent probe, was investigated for the generation of reactive oxygen species (ROS) and demonstrated to induce selective death of PSC upon visible light irradiation. Importantly, the CDy1 and/or light irradiation did not negatively affect differentiated endothelial cells. The photodynamic treatment of PSC with CDy1 and visible light irradiation confirmed the inhibition of teratoma formation in mice, and suggests a promising new approach to safe PSC-based cell therapy.

Modification of a Purification and Expansion Method for Human Embryonic Stem Cell-Derived Cardiomyocytes

Objective: This study aimed to develop a simple and efficient purification method for human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) using a low-glucose culture system. In addition, we investigated whether intercellular adhesion between single hESC-CMs plays a critical role in enhancing proliferation of purified hESC-CMs. Method: hESCs were cultured in suspension to form human embryoid bodies (hEBs) from which ∼15% contracting clusters were derived after 15-20 days in culture. To purify CMs from contracting hEBs, we first manually isolated contracting clumps that were re-cultured on gelatin-coated plates with media containing a low concentration of glucose. The purified hESC-CMs were cultured at different densities to examine whether cell-cell contact enhances proliferation of hESC-CMs. Results: Purified CMs demonstrated spontaneous contraction and strongly expressed the CM-specific markers cardiac troponin T and slow myosin heavy chain. We investigated the purification efficiency by examining the expression levels of cardiac-related genes and the expression of MitoTracker Red dye. In addition, purified hESC-CMs in low-glucose culture demonstrated a 1.5-fold increase in their proliferative capacity compared to those cultured as single hESC-CMs. Conclusion: A low level of glucose is efficient in purifying hESC-CMs and intercellular adhesion between individual hESC-CMs plays a critical role in enhancing hESC-CM proliferation.