Sung-Hwan Moon

Improvement of Postnatal Neovascularization by Human Embryonic Stem Cell–Derived Endothelial-Like Cell Transplantation in a Mouse Model of Hindlimb Ischemia

Background— We established an efficient preparation method to obtain endothelial-like cells (ECs) from human embryonic stem cells (hESCs) and tested whether these hESC-ECs would show therapeutic potential for treatment of hindlimb ischemia. Methods and Results— ECs differentiated from hESCs were obtained by mechanical isolation and cell sorting for von Willebrand factor. The isolated hESC-ECs maintained endothelial cell–specific characteristics such as endothelial marker expression and capillary formation. One day after surgical induction of hindlimb ischemia in athymic mice, hESC-ECs were injected intramuscularly into ischemic limbs. Four weeks after treatment, hESC-EC treatment significantly increased limb salvage (36%) compared with treatment with medium (0%). In addition, laser Doppler imaging showed that the ratio of blood perfusion (ischemic to normal limb) was increased significantly (P<0.01) by hESC-EC treatment (0.511±0.167) compared with medium injection (0.073±0.061). Capillary and arteriole densities were 658±190/mm2 and 30±11/mm2 in the hESC-EC group, respectively, whereas those in the medium group were 392±118/mm2 and 16±8/mm2, respectively (P<0.01). Reverse-transcription polymerase chain reaction with human-specific primers revealed mRNA expression of human endothelial markers and human angiogenic factors in ischemic mouse tissues. The transplanted hESC-ECs were localized as capillaries near muscle tissues in ischemic regions or incorporated in the vessels between muscle tissues, as confirmed by human nuclear antigen staining with platelet/endothelial cell adhesion molecule or von Willebrand factor. Conclusions— This study demonstrates that hESC-EC transplantation improves blood perfusion and limb salvage by facilitating postnatal neovascularization in a mouse model of hindlimb ischemia. Thus, hESC-ECs might be useful as an alternative cell source for angiogenic therapy.

Identification of Developmental Pluripotency Associated 5 Expression in Human Pluripotent Stem Cells

Pluripotent embryonic germ cells (EGCs) can be derived from the culture of primordial germ cells (PGCs). However, there are no reports of gonocytes, following the stage of PGC development, becoming stem cell lines. To analyze the gene expression differences between PGCs and gonocytes, we performed cDNA subtractive hybridization with mouse gonads containing either of the two cell populations. We confirmed that developmental pluripotency associated 5 (Dppa5), originally found in mouse embryonic stem cells (ESCs) and mouse embryonic carcinoma cells (ECCs), was strongly expressed in mouse PGCs and the expression was rapidly downregulated during germ cell development. A human sequence homologous to Dppa5 was identified by bioinformatics approaches. Interestingly, human Dppa5 was expressed only in human PGCs, human EGCs, and human ESCs and was not detected in human ECCs. Its expression was downregulated during induced differentiation of human ESCs. These findings confirmed that Dppa5 is specifically and differentially expressed in human cells that have pluripotency. The results strongly suggest that Dppa5 may have an important role in stemness in human ESCs and EGCs and also can be used as a marker of pluripotent stem cells. Human pluripotent stem cells may have their own ways to be pluripotent, as opposed to the much uniform mouse stem cells.

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.

Mussel-Inspired Cell-Adhesion Peptide Modification for Enhanced Endothelialization of Decellularized Blood Vessels

Enhanced endothelialization of tissue-engineered blood vessels is essential for vascular regeneration and function of engineered vessels. In this study, mussel-inspired surface chemistry of polydopamine (pDA) coatings are applied to functionalize decellularized vein matrix (DVM) with extracellular matrix-derived cell adhesion peptides (RGD and YIGSR). DVMs engineered with pDA-peptides enhance focal adhesion, metabolic activity, and endothelial differentiation of human endothelial progenitor cells (EPCs) derived from cord blood and embryonic stem cells compared with EPCs on non-coated or pDA-coated DVMs. These results indicate that pDA-peptide functionalization may contribute to enhanced, rapid endothelialization of DVM surfaces by promoting adhesion, proliferation, and differentiation of circulating EPCs. Ultimately, this approach may be useful for improving in vivo patency and function of decellularized matrix-based blood vessels.

Alternative Xeno-Free Biomaterials Derived from Human Umbilical Cord for the Self-Renewal Ex-vivo Expansion of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are attractive candidates for novel cell-therapy applications. However, the in vitro expansion of MSCs typically depends on the presence of fetal bovine serum (FBS) and coating materials derived from animal sources, which may cause contamination in clinical applications. In this study, we investigated whether human umbilical cord extract (UCE) could serve as a serum replacement and whether collagen purified from umbilical cord (UC-collagen) could act as an extracellular matrix (ECM) for the in vitro culture of MSCs derived from human UC (UC-MSCs). A total of 5.61 ± 0.54 mg UCE and 18.41 ± 2.42 mg collagen were extracted, and 1.3 ± 0.2 × 10⁵ cells were isolated from 1 g of UC, as determined by the expression of typical MSC surface markers. Importantly, the proliferation and stemness of the UC-MSCs cultured with the UCE media were similar to those cultured under FBS conditions on UC-collagen-treated plates for eight passages. Based on these results, we suggest that UCs, which are discarded as medical waste, represent a viable alternative source of xeno-free biomaterials to replace animal-derived serum and ECM materials for the cultivation of various cell types, including UC-MSCs, adipose tissue-derived MSCs, bone marrow-derived MSCs, and fibroblasts. This innovative xeno-free MSCs culture system can overcome many of the problems associated with immunogenicity, and it will further contribute to the enhancement of treatment efficiency.

A novel Fbxo25 acts as an E3 ligase for destructing cardiac specific transcription factors

Alterations in ubiquitin-proteasome system (UPS) have been implicated in the etiology of human cardiovascular diseases. Skp1/Cul1/F-box (SCF) ubiquitin E3 ligase complex plays a pivotal role in ubiquitination of cardiac proteins. However, a specific ubiquitin E3 ligase responsible for the destruction of cardiac transcription factors such as Nkx2-5, Isl1, Mef2C, and Tbx5 remains elusive to date. Here, we show that a novel F-box containing Fbxo25 is cardiac-specific and acts as an ubiquitin E3 ligase for cardiac transcription factors. Fbxo25 expression was nuclei-specific in vitro and cardiomyocytes. Expression level of Fbxo25 was higher in a fetal heart than an adult. Moreover, Fbxo25 expression was increased along with those of cardiac-specific genes during cardiomyocyte development from ESCs. Fbxo25 expression facilitated protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C. Especially, Fbxo25 ubiquitinated Nkx2-5, Isl1, and Hand1. Altogether, Fbxo25 acts as an ubiquitin E3 ligase to target cardiac transcription factors including Nkx2-5, Isl1, and Hand1, indicating that cardiac protein homeostasis through Fbxo25 has a pivotal impact on cardiac development.

Assessment of Differentiation Aspects by the Morphological Classification of Embryoid Bodies Derived from Human Embryonic Stem Cells

In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; ∼10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.

Encapsulation of Bone Morphogenic Protein-2 with Cbfa1-Overexpressing Osteogenic Cells Derived from Human Embryonic Stem Cells in Hydrogel Accelerates Bone Tissue Regeneration

Bone tissue defects caused by trauma and disease are significant problems in orthopedic surgery. Human embryonic stem cells (hESCs) hold great promise for the treatment of bone tissue disease in regenerative medicine. In this study, we have established an effective method for the differentiation of osteogenic cells derived from hESCs using a lentiviral vector containing the transcription factor Cbfa1. Differentiation was initiated in embryoid body formation of Cbfa1-expressing hESCs, resulting in a highly purified population of osteogenic cells based on flow cytometric analysis. These cells also showed characteristics of osteogenic cells in vitro, as determined by reverse-transcription (RT)-polymerase chain reaction and immunocytochemistry using osteoblast-specific markers. We also evaluated the regenerative potential of Cbfa1-expressing cells derived from hESCs (hESC-CECs) compared with hESCs and the osteogenic effects of bone morphogenic protein-2 (BMP2) encapsulated in thermoreversible hydrogel in vivo. hESC-CECs were embedded in hydrogel constructs enriched with BMP2 to promote bone regeneration. We observed prominent mineralization and the formation of nodule-like structures using von Kossa and alizarin red S staining. In addition, the expression patterns of osteoblast-specific genes were verified by RT-polymerase chain reaction, and immunohistochemical analysis revealed that collagen type 1 and Cbfa1 were highly expressed in hESC-CECs compared with other cell types. Taken together, our results suggest that encapsulation of hESC-CECs with BMP2 in hydrogel constructs appears to be a promising method to enhance the in vitro osteoblastic differentiation and in vivo osteogenic activity of hESC-CECs.