Jumi Kim

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.

Enhancement of wound healing by secretory factors of endothelial precursor cells derived from human embryonic stem cells

BACKGROUND AIMS Stem cells have been shown to have a therapeutic effect in several ischemic animal models, including hindlimb ischemia and chronic wound. We examined the wound-healing effect of secretory factors released by human embryonic stem cell (hESC)-derived endothelial precursor cells (EPC) in cutaneous excisional wound models. METHODS hESC-EPC were sorted by CD133/KDR, and endothelial characteristics were confirmed by reverse transcription (RT)-polymerase chain reaction (PCR), Matrigel assay and ac-LDL uptake. Conditioned medium (CM) of hESC-EPC was prepared, and concentrated hESC-EPC CM was applied in a mouse excisional wound model. RESULTS hESC-EPC CM accelerated wound healing and increased the tensile strength of wounds after topical treatment and subcutaneous injection. In addition, hESC-EPC CM treatment caused more rapid re-formation of granulation tissue and re-epithelialization of wounds compared with control vehicle medium and CB-EPC CM-treated wounds. In vitro, hESC-EPC CM significantly improved the proliferation and migration of dermal fibroblasts and epidermal keratinocytes. hESC-EPC CM also increased the extracellular matrix synthesis of fibroblasts. Analysis of hESC-EPC CM with a multiplex cytokine array system indicated that hESC-EPC secreted distinctively different cytokines and chemokines, such as epidermal growth factor (EGF), fibroblast growth factor (bFGF), fractalkine, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-8, platelet-derived growth factor-AA (PDGF-AA) and vascular endothelial growth factor (VEGF), which are well known to be important in normal angiogenesis and wound healing. CONCLUSIONS This study has demonstrated the wound-healing effect of hESC-EPC CM and characterized the spectrum of cytokines released by hESC-EPC that are functionally involved in the wound-healing process. These results suggest that secretory factors released from stem cells could be an important mediator of stem cell therapy in ischemic tissue diseases.

Quantitative proteomic analysis of induced pluripotent stem cells derived from a human Huntington's disease patient.

HD (Huntingtons disease) is a devastating neurodegenerative genetic disorder caused by abnormal expansion of CAG repeats in the HTT (huntingtin) gene. We have recently established two iPSC (induced pluripotent stem cell) lines derived from a HD patient carrying 72 CAG repeats (HD-iPSC). In order to understand the proteomic profiles of HD-iPSCs, we have performed comparative proteomic analysis among normal hESCs (human embryonic stem cells; H9), iPSCs (551-8) and HD-iPSCs at undifferentiated stages, and identified 26 up- and down-regulated proteins. Interestingly, these differentially expressed proteins are known to be involved in different biological processes, such as oxidative stress, programmed cell death and cellular oxygen-associated proteins. Among them, we found that oxidative stress-related proteins, such as SOD1 (superoxide dismutase 1) and Prx (peroxiredoxin) families are particularly affected in HD-iPSCs, implying that HD-iPSCs are highly susceptible to oxidative stress. We also found that BTF3 (basic transcription factor 3) is up-regulated in HD-iPSCs, which leads to the induction of ATM (ataxia telangiectasia mutated), followed by activation of the p53-mediated apoptotic pathway. In addition, we observed that the expression of cytoskeleton-associated proteins was significantly reduced in HD-iPSCs, implying that neuronal differentiation was also affected. Taken together, these results demonstrate that HD-iPSCs can provide a unique cellular disease model system to understand the pathogenesis and neurodegeneration mechanisms in HD, and the identified proteins from the present study may serve as potential targets for developing future HD therapeutics.

Discovery and Characterization of Novel MicroRNAs During Endothelial Differentiation of Human Embryonic Stem Cells

MicroRNAs (miRNAs) are small RNAs that participate in the regulation of genes associated with the differentiation and proliferation. In this study, 5 novel miRNAs were identified from human mesenchymal stem cells and characterized using various analyses. To investigate the potential functions associated with the regulation of cell differentiation, the differences in miRNA expression were examined in undifferentiated and differentiated human embryonic stem (ES) cells using reverse transcription (RT)-PCR analysis. Specifically, 3 miRNAs exhibited decreased expression levels in human umbilical vein endothelial cells (HUVECs) and endothelial cells derived from human ES cells. Putative target genes related to differentiation or maturation of endothelial cells were predicted by seed sequences of 2 novel miRNAs and analyzed for their expression via miRNA-mediated regulation using a luciferase assay. In HUVECs, CDH5 gene expression was directly repressed by hsa-miR-6086. Similarly, hsa-miR-6087 significantly downregulated endoglin expression. Therefore, the roles of these 2 miRNAs may be to directly suppress their target genes, popularly known as endothelial cell markers. Taken together, our results demonstrate that several novel miRNAs perform critical roles in human endothelial cell development.

Comparative Analysis of Human Mesenchymal Stem Cells Derived From Bone Marrow, Placenta, and Adipose Tissue as Sources of Cell Therapy.

Various source‐derived mesenchymal stem cells (MSCs) with multipotent capabilities were considered for cell therapeutics of incurable diseases. The applicability of MSCs depends on the cellular source and on their different in vivo functions, despite having similar phenotypic and cytological characteristics. We characterized MSCs from different sources, including human bone marrow (BM), placenta (PL), and adipose tissue (AT), in terms of the phenotype, surface antigen expression, differentiation ability, proteome reference map, and blood flow recovery in a hindlimb ischemic disease model. The MSCs exhibit different differentiation potentials depending on the cellular source despite having similar phenotypic and surface antigen expression. We identified approximately 90 differentially regulated proteins. Most up‐ or down‐regulated proteins show cytoskeletal or oxidative stress, peroxiredoxin, and apoptosis roles according to their functional involvement. In addition, the PL‐MSCs retained a higher therapeutic efficacy than the BM‐ and AT‐MSCs in the hindlimb ischemic disease model. In summary, we examined differentially expressed key regulatory factors for MSCs that were obtained from several cellular sources and demonstrated their differentially expressed proteome profiles. Our results indicate that primitive PL‐MSCs have biological advantages relative to those from other sources, making PL‐MSCs a useful model for clinical applications of cell therapy. J. Cell. Biochem. 117: 1112–1125, 2016.

Proteomic validation of multifunctional molecules in mesenchymal stem cells derived from human bone marrow, umbilical cord blood and peripheral blood.

Mesenchymal stem cells (MSCs) are one of the most attractive therapeutic resources in clinical application owing to their multipotent capability, which means that cells can differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle and marrow stroma. Depending on the cellular source, MSCs exhibit different application potentials according to their different in vivo functions, despite similar phenotypic and cytological characteristics. To understand the different molecular conditions that govern the different application or differentiation potential of each MSC according to cellular source, we generated a proteome reference map of MSCs obtained from bone marrow (BM), umbilical cord blood (CB) and peripheral blood (PB). We identified approximately 30 differentially regulated (or expressed) proteins. Most up-regulated proteins show a cytoskeletal and antioxidant or detoxification role according to their functional involvement. Additionally, these proteins are involved in the increase of cell viability, engraftment and migration in pathological conditions in vivo. In summary, we examined differentially expressed key regulatory factors of MSCs obtained from several cellular sources, demonstrated their differentially expressed proteome profiles and discussed their functional role in specific pathological conditions. With respect to the field of cell therapy, it may be particularly crucial to determine the most suitable cell sources according to target disease.

Proteomic analysis of pregnancy-related proteins from pig uterus endometrium during pregnancy

Many important molecular events associated with implantation and development occur within the female reproductive tract, especially within the uterus endometrium, during pregnancy periods. The endometrium includes the mucosal lining of the uterus, which provides a suitable site for implantation and development of a fertilized egg and fetus. To date, the molecular cascades in the uterus endometrium during pregnancy periods in pigs have not been elucidated fully. In this study, we compared the functional regulated proteins in the endometrium during pregnancy periods with those in non-pregnant conditions and investigated changes in expression patterns during pregnancy (days 40, 70, and 93) using two-dimensional gel electrophoresis (2-DE) and western blotting. The functional regulated proteins were identified and discovered from differentially expressed proteins in the uterus endometrium during pregnancy. We discovered 820 protein spots in a proteomic analysis of uterus endometrium tissues with 2-DE gels. We identified 63 of the 98 proteins regulated differentially among non-pregnant and pregnant tissues (matched and unmatched spots). Interestingly, 10 of these 63 proteins are development-, cytoskeleton- and chaperon-related proteins such as transferrin, protein DJ-1, transgelin, galectin-1, septin 2, stathmin 1, cofilin 1, fascin 1, heat shock protein (HSP) 90β and HSP 27. The specific expression patterns of these proteins in the endometrium during pregnancy were confirmed by western blotting. Our results suggest that the expressions of these genes involved in endometrium function and endometrium development from early to late gestation are associated with the regulation of endometrium development for maintaining pregnancy.

Enhancement of differentiation efficiency of hESCs into vascular lineage cells in hypoxia via a paracrine mechanism

Hypoxia is one way of inducing differentiation due to the activation of the key regulatory factor, Hypoxia-inducible factor 1 alpha (HIF-1α). However, the action of HIF-1α on the differentiation of hESCs was unclear until now. To investigate the effect of hypoxia on the differentiation of hESCs, we compared the differentiation efficacy into vascular lineage cells under normoxic and hypoxic conditions. We observed HIF-1α expression and the related expression of pro-angiogenic factors VEGF, bFGF, Ang-1 and PDGF in hEBs cultured under hypoxic conditions. Along with this, differentiation efficacy into vascular lineage cells was improved under hypoxic conditions. When HIF-1α was blocked by echinomycin, both angiogenic factors and the differentiation efficacy were down-regulated, suggesting that the enhancement of differentiation efficacy was caused by intrinsic up-regulation of HIF-1α and these pro-angiogenic factors under hypoxic condition. This response might be primarily regulated by the HIF-1α signal pathway, and hypoxia might be the key to improving the differentiation of hESCs into vascular lineage cells. Therefore, this study demonstrated that microenvironmental changes (i.e., hypoxia) can improve differentiation efficacy of hESCs into a vascular lineage without exogenous factors via cell-intrinsic up-regulation of angiogenic factors. These facts will contribute to the regulation of stem cell fate.

Comparative proteomic analysis of endothelial cells progenitor cells derived from cord blood- and peripheral blood for cell therapy

Vasculopathy due to ischemia in damaged tissues is a major cause of morbidity and mortality. To treat these conditions, endothelial progenitor cells (EPCs) from various sources, such as umbilical cord or peripheral blood, have been the focus of the regenerative medicine field due to their proliferative and vasculogenic activities. However, the fundamental, molecular-level differences between EPCs obtained from different cellular sources have rarely been studied. In this study, we established endothelial progenitor cells derived from cord blood- and peripheral blood (CB- and PB-EPCs) and investigated their fundamental differences at the cellular and molecular levels through a combination of stem cell biology techniques and proteomic analysis. Our results suggest that specifically up-regulated factors such as STMIN 1, CFL 1, PARK 7, NME 1, GLO 1, HSP 27 and PRDX 2 in CB-EPCs as key elements that could be functionally active in ischemic regions. We also discussed functional behaviors important for inducing and maintaining long-lasting blood vessels under ischemic conditions. As a result, CB-EPCs retained a higher anti-oxidant and migration ability than PB-EPCs in vitro. Furthermore, CB-EPCs retained a higher therapeutic efficacy than PB-EPCs in a hindlimb ischemic disease model. The up-regulated expression pattern of STMIN 1, CFL 1, PARK 7, NME 1, GLO 1, HSP 27 and PRDX 2 was confirmed under several conditions in vitro and in vivo, indicating that the up-regulation of these molecules in CB-EPCs may be critical to the mechanism of healing in ischemic conditions and that CB-EPCs may be more appropriate for inducing neo-vessels. Thus, these results may aid in predetermining which cell sources will be of value for cell-based therapies of pathological conditions and identify several candidate molecules that may be involved in the therapeutic mechanism for ischemia.

Stem cell recruitment factors secreted from cord blood-derived stem cells that are not secreted from mature endothelial cells enhance wound healing

Wounds are one of the most frequently occurring medical complication. Stem cells were recently highlighted as a novel therapeutic approach to treating wounds, although some negative aspects of allogenic stem cell transplantation were observed, such as cellular source limitations and unknown side effects in vivo. To address and eliminate these side effects, we examined the wound healing effect of secretory factors released from human cord blood-derived stem cells (hCB-SCs) and human umbilical vascular endothelial cells (HUVECs) on cutaneous excisional wound models. The hCB-SCs retained endothelial progenitor cell characteristics and expressed MSC markers such as CD73, CD105, and CD44. Analysis of hCB-SC-conditioned medium (CM) indicated that hCB-SCs secrete distinctly unique cytokines and chemokines such as TGF-β, PDGF, bFGF, EGF, KGF, and VEGF, which are well known to be important in normal angiogenesis and wound healing. Furthermore, hCB-SCs also secreted stem cell-recruiting factors such as G-CSF and GM-CSF, whereas HUVECs did not. When CB-SC-CM was applied to wound sites, hCB-SC-CM accelerated the wound healing rate compared with HUVEC-CM- and control medium-treated groups. In addition, hCB-SC-CM treatment caused a more rapid re-formation of granulation tissue and re-epithelialization of wounds, which indicates that the therapeutic effect of hCB-SC-CM is due to secreted stem cell-recruiting factors from stem cells, not just from endothelial lineage cells. Taken together, these results suggest that secretory factors released from stem cells, not just from endothelial cells, could be an important mediator of stem cell therapy in ischemic tissue diseases.