Ki-Sung Hong

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.

Perivascular Progenitor Cells Derived From Human Embryonic Stem Cells Exhibit Functional Characteristics of Pericytes and Improve the Retinal Vasculature in a Rodent Model of Diabetic Retinopathy

Diabetic retinopathy (DR) is the leading cause of blindness in working‐age people. Pericyte loss is one of the pathologic cellular events in DR, which weakens the retinal microvessels. Damage to the microvascular networks is irreversible and permanent; thus further progression of DR is inevitable. In this study, we hypothesize that multipotent perivascular progenitor cells derived from human embryonic stem cells (hESC‐PVPCs) improve the damaged retinal vasculature in the streptozotocin‐induced diabetic rodent models. We describe a highly efficient and feasible protocol to derive such cells with a natural selection method without cell‐sorting processes. As a cellular model of pericytes, hESC‐PVPCs exhibited marker expressions such as CD140B, CD146, NG2, and functional characteristics of pericytes. Following a single intravitreal injection into diabetic Brown Norway rats, we demonstrate that the cells localized alongside typical perivascular regions of the retinal vasculature and stabilized the blood‐retinal barrier breakdown. Findings in this study highlight a therapeutic potential of hESC‐PVPCs in DR by mimicking the role of pericytes in vascular stabilization.

An improved method for the derivation of high quality iPSCs in the absence of c-Myc

Induced pluripotent stem cells (iPSCs) hold tremendous potential for the development of new regenerative medicine therapies and the study of molecular mechanisms of pluripotency and development. However, reactivation of c-Myc, which results in tumor formation in chimeric mice, is a major roadblock in the translation of iPSCs into therapies. Although ectopic expression of c-Myc is not absolutely required for somatic reprogramming, in the absence of c-Myc, the overall efficiency of reprogramming is drastically reduced and the reprogramming time is increased. Subtle, abnormal epigenetic modifications in iPSCs derived in the absence of c-Myc have also been documented. Therefore, we developed a reprogramming method without c-Myc to generate high-quality iPSCs, a prerequisite to harnessing the full potential of iPSCs. In this study, we determined that serum replacement (SR)-based culture conditions dramatically increased the transcription factor-mediated reprogramming of mouse embryonic fibroblast cells (MEFs). The process was shortened to approximately 8 days when Oct4/Sox2/Klf4 (3F)-transduced MEFs were first cultured for 3 days under low serum conditions (LS protocol). The 3F-derived iPSCs that were generated by this method resembled mouse ES cells (mESCs) in morphology, gene expression, and in vitro differentiation. Finally, we observed that 3F-derived iPSC colonies were able to reach definite pluripotency in terms of molecular signatures when the catalytic function of c-Myc was tolerated. The 3F induction of pluripotency described here should facilitate the use of iPSCs and may also facilitate the mechanistic dissection of somatic reprogramming.

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.

GEMINI: Initial behavioral results after full severance of the cervical spinal cord in mice.

Background: The GEMINI spinal cord fusion protocol has been developed to achieve a successful cephalosomatic anastomosis. Here, we report the preliminary data on the use of a fusogen [polyethylene glycol (PEG)] after full cervical cord transection in mice to facilitate the fusion of both ends of a sharply transected spinal cord. Methods: Cervical laminectomy and a complete, visually confirmed cervical cord (C 5) transection was performed on female albino mice (n = 16). In Group 1 (n = 8), a fusogen, (PEG) was used to bridge the gap between the cut ends of the spinal cord. Group 2 received the same spinal cord transection but was treated with saline. Outcome was assessed daily using a standard scale (modified 22-point Basso-Beattie-Bresnahan scale) and filmed on camera. Results: The PEG group (group 1) showed partial restoration of motor function after 4 weeks of observation; group 2 (placebo) did not recover any useful motor activity. Conclusion: In this preliminary experiment, PEG, but not saline, promoted partial motor recovery in mice submitted to full cervical transection.

Improved efficacy and in vivo cellular properties of human embryonic stem cell derivative in a preclinical model of bladder pain syndrome

Interstitial cystitis/bladder pain syndrome (IC/BPS) is an intractable disease characterized by severe pelvic pain and urinary frequency. Mesenchymal stem cell (MSC) therapy is a promising approach to treat incurable IC/BPS. Here, we show greater therapeutic efficacy of human embryonic stem cell (hESC)-derived multipotent stem cells (M-MSCs) than adult bone-marrow (BM)-derived counterparts for treating IC/BPS and also monitor long-term safety and in vivo properties of transplanted M-MSCs in living animals. Controlled hESC differentiation and isolation procedures resulted in pure M-MSCs displaying typical MSC behavior. In a hydrochloric-acid instillation-induced IC/BPS animal model, a single local injection of M-MSCs ameliorated bladder symptoms of IC/BPS with superior efficacy compared to BM-derived MSCs in ameliorating bladder voiding function and histological injuries including urothelium denudation, mast-cell infiltration, tissue fibrosis, apoptosis, and visceral hypersensitivity. Little adverse outcomes such as abnormal growth, tumorigenesis, or immune-mediated transplant rejection were observed over 12-months post-injection. Intravital confocal fluorescence imaging tracked the persistence of the transplanted cells over 6-months in living animals. The infused M-MSCs differentiated into multiple cell types and gradually integrated into vascular-like structures. The present study provides the first evidence for improved therapeutic efficacy, long-term safety, and in vivo distribution and cellular properties of hESC derivatives in preclinical models of IC/BPS.

Intact wound repair activity of human mesenchymal stem cells after YM155 mediated selective ablation of undifferentiated human embryonic stem cells

BACKGROUND Risk of teratoma formation during human pluripotent stem cell (hPSC)-based cell therapy is one of the technical hurdles that must be resolved before their wider clinical application. To this end, selective ablation of undifferentiated hPSCs has been achieved using small molecules whose application should be safe for differentiated cells derived from the hPSCs. OBJECTIVE However, the functional safety of such small molecules in the cells differentiated from hPSCs has not yet been extensively validated. METHOD We used the survivin inhibitor YM155, which induced highly selective cell death of hPSCs for ablating undifferentiated hESCs after differentiation to human mesenchymal stem cells (hMSCs) and examined whether hMSCs remained fully functional after being exposed by YM155. RESULTS We demonstrated that human mesenchymal stem cells (hMSCs) derived from human embryonic stem cells (hESCs) remained fully functional in vitro and in vivo, while hESCs were selectively ablated. CONCLUSION These results suggest that a single treatment with YM155 after differentiation of hMSCs would be a valid approach for teratoma-free cell therapy.

Fbxo25 controls Tbx5 and Nkx2–5 transcriptional activity to regulate cardiomyocyte development

The ubiquitin-proteasome system (UPS) plays an important role in protein quality control, cellular signalings, and cell differentiation through the regulated turnover of key transcription factors in cardiac tissue. However, the molecular mechanism underlying Fbxo25-mediated ubiquitination of cardiac transcription factors remains elusive. We report that an Fbxo25-mediated SCF ubiquitination pathway regulates the protein levels and activities of Tbx5 and Nkx2-5 based on our studies using MG132, proteasome inhibitor, and the temperature sensitive ubiquitin system in ts20 cells. Our data indicate that Fbxo25 directly interacts with Tbx5 and Nkx2-5 in vitro and in vivo. In support of our findings, a dominant-negative mutant of Fbxo25, Fbxo251-236, prevents Tbx5 degradation and increases Tbx5 transcriptional activity in a Tbx5 responsive luciferase assay. Therefore, Fbxo25 facilitates Tbx5 degradation in an SCF-dependent manner. In addition, the silencing of endogenous Fbxo25 increases Tbx5 and Nkx2-5 mRNA levels and suppresses mESC-derived cardiomyocyte differentiation. Likewise, the exogenous expression of FBXO25 downregulates NKX2-5 level in human ESC-derived cardiomyocytes. In myocardial infarction model, Fbxo25 mRNA decreases, whereas the mRNA and protein levels of Tbx5 and Nkx2-5 increase. The protein levels of Tbx5 and Nkx2-5 are regulated negatively by Fbxo25-mediated SCF ubiquitination pathway. Thus, our findings reveal a novel mechanism for regulation of SCFFbox25-dependent Nkx2-5 and Tbx5 ubiquitination in cardiac development and provide a new insight into the regulatory mechanism of Nkx2-5 and Tbx5 transcriptional activity.

Ground-State Conditions Promote Robust Prdm14 Reactivation and Maintain an Active Dlk1-Dio3 Region during Reprogramming

Induced pluripotent stem cells (iPSCs) are capable of unlimited self-renewal and can give rise to all three germ layers, thereby providing a new platform with which to study mammalian development and epigenetic reprogramming. However, iPSC generation may result in subtle epigenetic variations, such as the aberrant methylation of the Dlk1-Dio3 locus, among the clones, and this heterogeneity constitutes a major drawback to harnessing the full potential of iPSCs. Vitamin C has recently emerged as a safeguard to ensure the normal imprinting of the Dlk1-Dio3 locus during reprogramming. Here, we show that vitamin C exerts its effect in a manner that is independent of the reprogramming kinetics. Moreover, we demonstrate that reprogramming cells under 2i conditions leads to the early upregulation of Prdm14, which in turn results in a highly homogeneous population of authentic pluripotent colonies and prevents the abnormal silencing of the Dlk1-Dio3 locus.

Improved Transfection Efficiency and Metabolic Activity in Human Embryonic Stem Cell Using Non-Enzymatic Method

Human embryonic stem cells (hESCs) are pluripotent cells widely used in conventional and regenerative medicine due to their ability to self-renew, proliferate and differentiate. Recently, genetic modification of stem cells using genome editing is the most advanced technique for treating hereditary diseases. Nevertheless, the low transfection efficiency of hESCs using enzymatic methods is still limited in in vitro preclinical research. To overcome these limitations, we have developed transfection methods using non-enzymatic treatments on hESCs. In this study, hESCs were transfected following enzymatic (TrypLE and trypsin) and non-enzymatic treatment ethylenediaminetetraacetic acid (EDTA) to increase transfection efficiency. Flow cytometric analysis using an enhanced green fluorescent protein vector showed a significantly increased transfection efficiency of EDTA method compared to standard enzyme method. In addition, the EDTA approach maintained stable cell viability and recovery rate of hESCs after transfection. Also, metabolic activity by using Extracellular Flux Analyzer revealed that EDTA method maintained as similar levels of cell functionality as normal group comparing with enzymatic groups. These results suggest that transfection using EDTA is a more efficient and safe substitute for transfection than the use of standard enzymatic methods.