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Human embryonic stem cells (hESCs) hold promise in regenerative medicine but allogeneic immune rejections caused by highly polymorphic human leukocyte antigens (HLAs) remain a barrier to their clinical applications. Here, we used a CRISPR/Cas9-mediated HLA-editing strategy to generate a variety of HLA homozygous-like hESC lines from pre-established hESC lines. We edited four pre-established HLA-heterozygous hESC lines and created a mini library of 14 HLA-edited hESC lines in which single HLA-A and HLA-B alleles and both HLA-DR alleles are disrupted. The HLA-edited hESC derivatives elicited both low T cell- and low NK cell-mediated immune responses. Our library would cover about 40% of the Asian-Pacific population. We estimate that HLA-editing of only 19 pre-established hESC lines would give rise to 46 different hESC lines to cover 90% of the Asian-Pacific population. This study offers an opportunity to generate an off-the-shelf HLA-compatible hESC bank, available for immune-compatible cell transplantation, without embryo destruction. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s12015-020-10113-7.

Many groups are working to improve the results of clinical allogeneic islet transplantation in a primate model. However, few studies have focused on the optimal islet dose for achieving normal glycemia without exogenous insulin after transplantation in primate models or on the relationship between rejection and islet amyloid polypeptide (IAPP) expression. We evaluated the dose (10,000, 20,000, and > 25,000 islet equivalents (IEQ)/kg) needed to achieve normal glycemia without exogenous insulin after transplantation using eleven cynomolgus monkeys, and we analyzed the characteristics exhibited in the islets after transplantation. 10,000 IEQ/kg (N = 2) failed to control blood glucose level, despite injection with the highest dose of exogenous insulin, and 20,000 IEQ/kg group (N = 5) achieved unstable control, with a high insulin requirement. However, 25,000 IEQ/kg (N = 4) achieved normal glycemia without exogenous insulin and maintained it for more than 60 days. Immunohistochemistry results from staining islets found in liver biopsies indicated that as the number of transplanted islets decreased, the amount of IAPP accumulation within the islets increased, which accelerated CD3+ T cell infiltration. In conclusion, the optimal transplantation dose for achieving a normal glycemia without exogenous insulin in our cynomolgus monkey model was > 25,000 IEQ/kg, and the accumulation of IAPP early after transplantation, which depends on the transplanted islet dose, can be considered one factor in rejection.

Many groups are working to improve the results of clinical allogeneic islet transplantation in a primate model. However, few studies have focused on the optimal islet dose for achieving normal glycemia without exogenous insulin after transplantation in primate models or on the relationship between rejection and islet amyloid polypeptide (IAPP) expression. We evaluated the dose (group 1/10,000, group 2/20,000, and group 3/>25,000 islet equivalents (IEQ)/kg) needed to achieve normal glycemia without exogenous insulin after transplantation using eleven cynomolgus monkeys, and we analyzed the characteristics exhibited in the islets after transplantation. Group 1 (n2) failed to control blood glucose level, despite injection with the highest dose of exogenous insulin, and group 2 (n5) achieved unstable control, with a high insulin requirement. However, group 3 (n4) achieved normal glycemia without exogenous insulin and maintained it for more than 60 days. Immunohistochemistry results from staining islets found in liver biopsies indicated that as the number of transplanted islets decreased, the amount of IAPP accumulation within the islets increased, which accelerated immune cell infiltration. In conclusion, the marginal transplantation dose for achieving a normal glycemia without exogenous insulin in our cynomolgus monkey model was >25,000 IEQ/kg, and the accumulation of IAPP early after transplantation, which depends on the transplanted islet dose, can be considered one factor in rejection.

BACKGROUND: Since primates have more biological similarities to humans than do other animals, they are a valuable resource in various field of research, including biomedicine, regenerative medicine, and drug discovery. However, there remain limitations to maintenance and expansion of primary hepatocytes derived from nonhuman primates. To overcome these limitations, we developed a novel culture system for primate cells. METHODS: Primary hepatocytes from Macaca fascicularis (mf-PHs) were isolated from hepatectomized liver. To generate chemically derived hepatic progenitor cells (mf-CdHs), mf-PHs were cultured with reprogramming medium containing A83-01, CHIR99021, and hepatocyte growth factor (HGF). The bi-potent differentiation capacity of mf-CdHs into hepatocytes and biliary epithelial cells was confirmed by treatment with hepatic differentiation medium (HDM) and cholangiocytic differentiation medium (CDM), respectively. RESULTS: mf-PHs cultured with reprogramming medium showed rapid proliferation capacity in vitro and expressed progenitor-specific markers. Moreover, when cultured in HDM, these progenitor cells stably differentiated into hepatocyte-like cells expressing the mature hepatic markers. On the other hand, when cultured in CDM, the differentiated biliary epithelial cells expressed mature cholangiocyte characteristics. CONCLUSION: The results of the present study demonstrate that we successfully induced the formation of hepatic progenitor cells from mf-PHs by culturing them with a combination of small molecules, including growth factors. These results offer a means of expanding nonhuman primate hepatocytes without genetic manipulation for cellular resource, preclinical applications and regenerative medicine for the liver.

Purpose: This study aimed to determine the effect of thymosin beta 4 (Tβ4) on human corneal epithelial cell migration and the downstream signaling pathways. Tβ4 has a role in tissue development, ce...

Mesenchymal stromal cells (MSCs) isolated from numerous tissues including human fetal tissue are currently used in cell therapy and regenerative medicine. Among fetal tissues, the umbilical cord (UC) is one of the sources for both MSCs and endothelial cells (ECs). To establish ectopic vascularized bone tissue formation, UC-derived MSCs and ECs were isolated. UC-MSCs expressing human BMP-2 (hBMP-2-MSCs) were generated using an adenoviral system to promote bone formation. These cells were then transplanted with Matrigel into the subcutaneous tissue of an immune deficient NSG mouse, and bone tissue was analyzed after several weeks. The osteogenic differentiation ability of MSCs was elevated by transduction of the hBMP-2 expressing adenoviral system, and vascularization of bone tissue was enhanced by human umbilical vein endothelial cells (HUVEC). In this study, our results provide evidence that MSCs and HUVECs from human umbilical cord are suitable cells to investigate bone tissue engineering. The results also suggest that the co-transplantation of hBMP2-MSCs and HUVECs may be a simple and efficient strategy for improving tissue generation and angiogenesis in bone tissue engineering using stem cells.

Three-dimensional (3D) bioprinting technology is a promising new technology in the field of bioartificial organ generation with regard to overcoming the limitations of organ supply. The cell source for bioprinting is very important. Here, we generated 3D hepatic scaffold with mouse-induced hepatocyte-like cells (miHeps), and investigated whether their function was improved after transplantation in vivo. To generate miHeps, mouse embryonic fibroblasts (MEFs) were transformed with pMX retroviruses individually expressing hepatic transcription factors Hnf4a and Foxa3. After 8-10 days, MEFs formed rapidly growing hepatocyte-like colonies. For 3D bioprinting, miHeps were mixed with a 3% alginate hydrogel and extruded by nozzle pressure. After 7 days, they were transplanted into the omentum of Jo2-treated NOD Scid gamma (NSG) mice as a liver damage model. Real-time polymerase chain reaction and immunofluorescence analyses were conducted to evaluate hepatic function. The 3D bioprinted hepatic scaffold (25 × 25 mm) expressed Albumin, and ASGR1 and HNF4a expression gradually increased for 28 days in vitro. When transplanted in vivo, the cells in the hepatic scaffold grew more and exhibited higher Albumin expression than in vitro scaffold. Therefore, combining 3D bioprinting with direct conversion technology appears to be an effective option for liver therapy.

Background aims Major challenges in de-differentiated liposarcoma (DDLPS) therapy are the high rate of sequential recurrence (>80%) and metastasis (20–30%) following surgical removal. However, well-defined therapeutic strategies for this rare malignancy are lacking and are critically needed. Methods We investigated a new approach to DDLPS therapy with mesenchymal stromal cells expressing herpes simplex virus–thymidine kinase (MSC-TK). In an effort to evaluate this efficacy, in vitro cytotoxicity of MSC-TK against DDLPS cells was analyzed using an apoptosis assay. For pre-clinical study, the MSC-TK–induced reduction in recurrence and metastasis was validated in a recurrent DDLPS model after the macroscopic complete resection and lung metastasis DDLPS model. Results MSC-TK induced apoptosis in DDLPS cells by bystander effects via gap junction intracellular communication (GJIC) of toxic ganciclovir (GCV). Recurrent DDLPS models following no residual tumor/microscopic tumor resection and lung metastasis DDLPS models were established, which suggested clinical relevance. MSC-TK markedly reduced locoregional recurrence rates and prolonged recurrence-free survival, thus increasing overall survival in the recurrent DDLPS model. MSC-TK followed by GCV treatment yielded a statistically significant reduction in early- and advanced-stage lung metastasis. Discussion This therapeutic strategy may serve as an alternative or additional strategy by applying MSC-TK to target residual tumors following surgical resection, thus reducing local relapse and metastasis in these patients.

As wound contraction in the cutaneous layer occurs rapidly in mice, mechanical means are typically used to deliberately expose the wound to properly investigate healing by secondary intention. Previously, silicon rings and splinting models were attempted to analyze histological recovery but prevention of surrounding epidermal cell migration and subsequent closure was minimal. Here, we developed an ideal chimney wound model to evaluate epidermal regeneration in murine under hESC-EC transplantation through histological analysis encompassing the three phases of regeneration: migration, proliferation, and remodeling. Human embryonic stem cell derived endothelial cells (hESC-EC) were transplanted due to possessing a well-known therapeutic effect in angiogenesis which also enhances epidermal repair to depict the process of regeneration. Following a standard 1 mm biopsy punch, a chimney manufactured by modifying a 1.7 mL microtube was simply inserted into the excisional wound to complete the modeling process. Under this model, the excisional wound remained fully exposed for 14 days and even after 4 weeks, only a thin transparent layer of epidermal tissue covered the wound site. This approach is able to more accurately depict epidermal repair in relation to histology while also being a user-friendly and cost-effective way to mimic human recovery in rodents and evaluate epithelial repair induced by a form of therapy.

Much of our understanding of human biology and the function of mammalian cells in tissue regeneration have been derived from mechanistically and genetically manipulated rodent models. However, current models examining epidermal wound repair fail to address both the cross-species mechanistic and immunogenic differences simultaneously. Herein, we describe a multifaceted approach intended to better recapitulate human skin recovery in rodent models. First, immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice were intravenously inoculated with human hematopoietic stem cells to become, in essence, humanized, and capable of initiating an adaptive immune response. Next, a chimney-shaped mechanical device was implanted onto the excisional wound site to prevent healing by primary intention (contraction) and expedite cell transplantation. Subsequently, cell therapy was administered by transplanting cord blood–derived endothelial progenitor cells or human pluripotent stem cell–derived endothelial cells into the wound site to examine the regeneration process at a histological level. This study demonstrates human cutaneous repair in a murine model by addressing both the mechanistic and immunogenic differences in the epidermis. We further show human leukocyte recruitment in damaged tissue and improved healing by secondary intention in the transplanted groups, highlighting the need for useful preclinical animal models to better understand leukocyte function in human (tissue repair and) regeneration.

Background Porcine islet xenotransplantation is considered an attractive alternative treatment for type 1 diabetes mellitus. However, it is largely limited because of initial rejection due to Instant Blood-Mediated Inflammatory Reaction (IBMIR), oxidative stress, and inflammatory responses. Recently, soluble tumor necrosis factor-ɑ receptor type I (sTNF-αR) and heme oxygenase (HO)-1 genes (HO-1/sTNF-αR) have been shown to improve the viability and functionality of porcine islets after transplantation. Methods In this study, genetically modified mesenchymal stem cells (MSCs) expressing the HO-1/sTNF-αR genes (HO-1/sTNF-αR-MSC) were developed using an adenoviral system, and porcine islet viability and function were confirmed by in vitro tests such as GSIS, AO/PI, and the ADP/ATP ratio after coculturing with HO-1/sTNF-αR-MSCs. Subsequently, isolated porcine islets were transplanted underneath the kidney capsule of diabetic humanized mice without MSCs, with MSCs or with HO-1/sTNF-αR-MSCs. Results According to the results, the HO-1/sTNF-αR-MSC-treated group exhibited improved survival of porcine islets and could reverse hyperglycemia more than porcine islets not treated with MSCs or islets cotransplanted with MSCs. Moreover, the HO-1/sTNF-αR-MSC group maintained its morphological characteristics and the insulin secretion pattern of transplanted porcine islets similar to endogenous islets in immunocompetent humanized mice. Conclusions Our results suggest that HO-1/sTNF-αR-MSCs are efficient tools for porcine islet xenotransplantation, and this study may provide basic information for pre-clinical animal models and future clinical trials of porcine islet xenotransplantation.

Angiomyolipomas (AMLs) are relatively rare hamartomatous or benign tumors that occasionally occur as part of tuberous sclerosis complex (TSC). Mutations in either of the two genes, TSC 1 and TSC 2, have been attributed to the development of TSC. Between 1994 and January 2009, 83 patients were diagnosed with AML at the Samsung Medical Center. In that group of patients, 5 (6%) had AML with TSC (AML-TSC). Mutational analysis of the TSC 2 gene was performed using 7 samples from the 5 AML-TSC patients and 14 samples from 14 patients with sporadic AML without TSC (AML-non-TSC). From this analysis, mutations in TSC genes were identified in 5 samples from the AML-TSC patients (mutation detection rate = 71%) and 3 samples from AML-non-TSC patients (mutation detection rate = 21%). In the case of AML-TSC, 6 mutations were found including 3 recurrent mutations and 3 novel mutations, while in the case of AML-non-TSC, 4 mutations were identified once, including 1 novel mutation. Also MLPA analysis of the TSC2 gene showed that TSC2 exon deletion is more frequently observed in AML-TSC patients than in AML-non-TSC patients. This is the first mutation and multiplex ligation-dependent probe amplification (MLPA) analyses of TSC 2 in Korean AMLs that focus on TSC. This study provides data that are representative of the distribution of mutations and exon deletions at TSC genes in clinically diagnosed AML-TSC cases of the Korean population.

// Ju-Ae Hwang 1,5,* , Heung-Mo Yang 1,* , Doo-Pyo Hong 1 , Sung-Yeon Joo 1,2 , Yoon-La Choi 4 , Joo-Hung Park 5 , Alexander J. Lazar 6 , Raphael E. Pollock 7 , Dina Lev 6 and Sung Joo Kim 1,3,8 1 Transplantation Research Center, Samsung Biomedical Research Institute, Seoul, Republic of Korea 2 Samsung Advanced Institute for Health Sciences & Technology, Graduate School, Department of Health Sciences & Technology, Sungkyunkwan University 3 Department of Surgery, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 4 Department of Pathology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 5 Department of Biology, Changwon National University, Changwon, Kyungnam, Republic of Korea 6 Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 7 Division of Surgical Oncology, James Comprehensive Cancer Center, Ohio State University, Columbus, OH, USA 8 Sarcoma Research Center, Samsung Medical Center, Seoul, Republic of Korea * These authors are contributed equally to this work Correspondence: Sung Joo Kim, email: // Dina-Lev , email: // Keywords : Liposarcoma, 2-DE, Gankyrin, Predictive factor, Tumorigenesis Received : May 25, 2014 Accepted : August 20, 2014 Published : August 21, 2014 Abstract Liposarcoma is one of the most common histologic types of soft tissue sarcoma and is frequently an aggressive cancer with poor outcome. Hence, alternative approaches other than surgical excision are necessary to improve treatment of well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). For this reason, we performed a two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry/mass spectrometry (MALDI-TOF/MS) analysis to identify new factors for WDLPS and DDLPS. Among the selected candidate proteins, gankyrin, known to be an oncoprotein, showed a significantly high level of expression pattern and inversely low expression of p53/p21 in WDLPS and DDLPS tissues, suggesting possible utility as a new predictive factor. Moreover, inhibition of gankyrin not only led to reduction of in vitro cell growth ability including cell proliferation, colony-formation, and migration, but also in vivo DDLPS cell tumorigenesis, perhaps via downregulation of the p53 tumor suppressor gene and its p21 target and also reduction of AKT/mTOR signal activation. This study identifies gankyrin, for the first time, as new potential predictive and oncogenic factor of WDLPS and DDLPS, suggesting the potential for service as a future LPS therapeutic approach.

The epithelium-specific ETS transcription factor-1 (ESE-1) is physiologically important in the pathogenesis of various diseases. Recently, OCT4, a transcription factor involved in stem cell pluripotency, has been implicated in tumorigenesis. In this study, we invested the molecular mechanism by which ESE-1 regulates transcription of OCT4 in NCCIT human embryonic carcinoma cells. Real-time PCR analysis revealed that OCT4 levels were high in undifferentiated NCCIT cells but significantly decreased upon retinoic acid-mediated differentiation, concomitant with up-regulation of ESE-1 expression. OCT4 mRNA level rose following shRNA-mediated knockdown of ESE-1, but declined when ESE-1 was overexpressed, suggesting that the expression levels of OCT4 and ESE-1 may be coordinated in an opposite manner. Promoter-reporter assays revealed that induced OCT4 promoter activity in NCCIT cells was significantly down-regulated by ESE-1 overexpression in a dose-dependent manner. The inhibitory effect of ESE-1 on OCT4 promoter activity was relieved by co-expression of an ESE-1 mutant lacking the transactivation domain, but not by mutants lacking other domains. Serial deletion and site-directed mutagenesis of the OCT4 promoter revealed that a potential ETS binding site (EBS) is present in the conserved region 2 (CR2). ESE-1 interacted with the EBS element in CR2 and enrichment of CR2 significantly increased upon RA-mediated differentiation of NCCIT cells, suggesting that this binding is likely to be involved in ESE-1-mediated repression of OCT4 promoter activity upon differentiation. Taken together, the results of this study reveal the molecular details of the mechanism by which the oncogenic factor ESE-1 regulates expression of the stem cell transcription factor OCT4 in pluripotent NCCIT cells.

Objective— Allogeneic transplantation of human embryonic stem cell (hESC) derivatives has the potential to elicit the patient’s immune response and lead to graft rejection. Although hESCs and their derivatives have been shown to have advantageous immune properties in vitro, such observations could not be determined experimentally in vivo because of ethical and technical constraints. However, the generation of humanized mice (hu-mice) harboring a human immune system has provided a tool to perform in vivo immunologic studies of human cells and tissues. Using this model, we sought to examine the therapeutic potential of hESC-derived endothelial cells, human embryonic fibroblasts, and cord blood–derived endothelial progenitor cells in a human immune system environment. Approach and Results— All cell types transplanted in hu-mice showed significantly reduced cell survival during the first 14 days post-transplantation compared with that observed in immunodeficient mice. During this period, no observable therapeutic effects were detected in the hindlimb ischemic mouse models. After this point, the cells demonstrated improved survival and contributed to a long-term improvement in blood perfusion. All cell types showed reduced therapeutic efficacy in hu-mice compared with NOD scid IL2 receptor gamma chain knockout mice. Interestingly, the eventual improvement in blood flow caused by the hESC-derived endothelial cells in hu-mice was not much lower than that observed in NOD scid IL2 receptor gamma chain knockout mice. Conclusions— These findings suggest that hESC derivatives may be considered a good source for cell therapy and that hu-mice could be used as a preclinical in vivo animal model for the evaluation of therapeutic efficacy to predict the outcomes of human clinical trials.

Mesenchymal stem cells (MSCs) hold great promise for the field of tissue regeneration. Because only a limited number of MSCs can be obtained from each donor site, it is important to establish standard methods for MSC expansion using growth and trophic factors. Thymosin β4 (Tβ4) is a novel trophic factor that has antimicrobial effects and the potential to promote tissue repair. Tβ4 is a ubiquitous, naturally-occurring peptide in the wound bed. Therefore, the relationship between Tβ4 and MSCs, especially adjacent adipose tissue-derived stem cells (ASCs), merits consideration. Exogenous Tβ4 treatment enhanced the proliferation of human ASCs, resulting in prominent nuclear localization of PCNA immunoreactivity. In addition, exogenous Tβ4 also increased IL-8 secretion and blocking of IL-8 with neutralizing antibodies decreased Tβ4-induced ASC proliferation, suggesting that IL-8 is a critical mediator of Tβ4-enhanced proliferation. Moreover, Tβ4 activated phosphorylation of ERK1/2 and increased the nuclear translocation of NF-κB. These observation provide that Tβ4 promotes the expansion of human ASCs via an IL-8-dependent mechanism that involves the ERK and NF-κB pathways. Therefore, Tβ4 could be used as a tool for MSC expansion in cell therapeutics.

Mesenchymal stromal cells (MSC) from several tissues have immunomodulatory properties that involve various immunosuppressive molecules. An example is human leukocyte antigen (HLA)-G, a non-classical major histocompatibility complex (MHC) class I molecule that induces tolerance via interactions with inhibitory receptors present on major immune effector cells. Recently, the molecular mechanisms that regulate MSC-mediated immunosuppression have come under investigation. Our goal was to determine whether HLA-G plays a crucial role in immunosuppression and whether human adipose tissure (hAT) MSC can be used as a tool for biologic immunosuppression with HLA-G in transplantation.MSC were characterized by fluorescence-activated cell sorting (FACS) analysis, reverse transcriptase (RT)-polymerase chain reaction (PCR) and staining for differentiation. The immunogenicity and immunomodulatory effects of MSC were monitored by peripheral blood mononuclear cell (PBMC) proliferation assay with or without phytohemagglutinin (PHA) stimulation. Stable expression of HLA-G1 in MSC was done using a lentiviral system. Results. MSC from different tissues had similar morphology, immunophenotypic characters and differentiation potential. We also found that the immunosuppressive effect of MSC was monitored along with their endogenous HLA-G mRNA and protein levels. Stable expression of HLA-G1 appeared to enhance the immunosuppressive effect of hAT MSC, and the function of HLA-G1 was significantly decreased by HLA-G antagonistic antibody in PBMC proliferation assays.Although the HLA-G molecule is not the sole factor for MSC-mediated immunosuppression, our data provide evidence that HLA-G plays an important role in immunosuppression and that hAT MSC can be used as a tool for biologic immunosuppression during transplantation procedures.

This study investigates the variation and spatial distribution of nitrogen dioxide() concentrations measured with passive diffusive samplers at 40 curbside points in Daejeon. Average concentration was (n=1,127) and the significant difference in concentrations by regional groups (Dong-gu, Jung-gu, Seo-gu, Daedeok-gu, Yuseong-gu) was not observed. The frequency distribution of concentration was found to be a normal distribution with the high frequency in the concentration range of 30 to 40 ppb (20 to 25%). Average concentration measured during the rainy periods was lower than that measured during the non-rainy periods and the decrease of concentration by rainfall was about 16% (7 ppb). The variation of concentrations measured by passive diffusive samplers during the sampling period was similar to that continuously measured at the air quality monitoring station.

As wound contraction in the cutaneous layer occurs rapidly in mice, mechanical means are typically used to deliberately expose the wound to properly investigate healing by secondary intention. Previously, silicon rings and splinting models were attempted to analyze histological recovery but prevention of surrounding epidermal cell migration and subsequent closure was minimal. Here, we developed an ideal chimney wound model to evaluate epidermal regeneration in murine under hESC-EC transplantation through histological analysis encompassing the three phases of regeneration: migration, proliferation, and remodeling. Human embryonic stem cell derived endothelial cells (hESC-EC) were transplanted due to possessing a well-known therapeutic effect in angiogenesis which also enhances epidermal repair to depict the process of regeneration. Following a standard 1 mm biopsy punch, a chimney manufactured by modifying a 1.7 mL microtube was simply inserted into the excisional wound to complete the modeling process. Under this model, the excisional wound remained fully exposed for 14 days and even after 4 weeks, only a thin transparent layer of epidermal tissue covered the wound site. This approach is able to more accurately depict epidermal repair in relation to histology while also being a user-friendly and cost-effective way to mimic human recovery in rodents and evaluate epithelial repair induced by a form of therapy.

Nanog is a newly identified divergent homeodomain protein that directs the infinite propagation and sustains the pluripotency of embryonic stem cells. It has been reported that murine Nanog has two potent transactivation domains in N-terminal and C-terminal regions. Human Nanog (hNanog) polypeptide shares about 58% and 87% identity to the open reading frame and homeodomain of murine Nanog, respectively. However, the functional domains and molecular mechanisms of hNanog are poorly understood. In this study, for the first time, we presented that only C-terminus of hNanog contains a potent transactivation domain. Based on the amino acid sequences of homeobox domain, we roughly divided hNanog open reading frame into the three regions such as N-terminal, homeodomain and C-terminal regions and constructed either the fusion proteins between hNanog individual and Gal4 DNA binding domain or the context of native hNanog protein. Reporter assays by using reporter plamid containing Gal4 or Nanog binding site revealed that the only C-terminal region exhibited the significant fold induction of transactivation. However, interestingly, there was no significant activation through N-terminal region unlike murine Nanog, suggesting that C-terminal region may have more critical roles in the transcriptional activation of target genes. Taken together, the finding of a putative transactivation domain in hNanog may contribute to the further understanding of molecular mechanism on the regulation of downstream genes involved in self-renewal and pluripotency of human stem cells.

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.

Octamer-binding transcription factor-4 (Oct4), a member of the POU domain transcription factors, is crucial for both early embryonic development and the maintenance of stem cell pluripotency. The human Oct4 (hOct4) 5' upstream sequence contains four conserved regions (CR1, 2, 3, 4) that are homologous in the murine. In this study, we constructed a series of deletion mutants of the hOct4 5' upstream region and identified cis-regulatory elements that may be important determinants for the transcriptional activity of the hOct4 promoter. Our studies showed that CR2, 3, and 4 each acted as positive cis-regulatory elements in hOct4 promoter activity. We also newly identified a putative negative cis-acting element located between CR1 and CR2. In addition, the sequence -380/-1 at CR1 that contains a GC box was sufficient to provide the minimal promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays revealed the GC box located in the -380/-1 region may play a critical role in controlling the transcriptional activity of hOct4 by the direct binding of Sp1 or Sp3 transcription factors to the GC box. An overexpression study showed that Sp1 and Sp3 positively and negatively regulate hOct4 promoter activity. Thus, the hOct4 promoter upstream region contains multiple regulatory elements, one of which, the GC box, may be an important cis-regulatory element that regulates the transcription of the hOct4 promoter by the binding of Sp family transcription factors.

RNA leukemia viruses induce T-cell lymphoblastic lymphomas or myeloid leukemias. Infection of cells with Moloney murine leukemia virus (M-MuLV) up-regulates the expression of a number of cellular genes, including those involved in T-lymphocyte activation. Previously, we demonstrated that this up-regulation occurs via the trans-activation activity of the M-MuLV long terminal repeat (LTR) sequences which produce an LTR-encoded transcript. Sequence analysis of the LTR revealed a potential transcription unit for RNA polymerase III (Pol III) within the U3 region that is actively occupied by Pol II factors. Here, we provide the direct evidence of involvement of Pol III in the trans-activation process and demonstrate the precise localization of the intragenic control elements for accurate and active Pol III transcription. Deletions of a copy of the directed repeats and further immediate upstream sequences significantly abrogated the generation of LTR-encoded transcript and abolished the trans-activational activity, whereas the deletion of a copy of directed repeats alone proportionally reduced the transcript size, but still retained moderately high trans-activational activity. In electrophoretic mobility shift assay, the fraction containing a multiple transcription factor TFIIIC complex strongly bound to the LTR-U3 probe containing the essential control elements. The specificity of the DNA-TFIIIC interaction was confirmed by conducting competition assays with DNA fragments containing a genuine Pol III-transcribed gene, VA1, and by vaccinia virus infection which stimulates the expression of Pol III factors. However, a deletion mutant lacking an essential control element bound to the TFIIIC complex poorly, consequently resulting in weak Pol III transcription as assessed by an IRES-GFP reporter system. This correlation strongly supports the possibility that the generation of LTR-encoded transcript is directed by Pol III. Therefore, this finding suggests the involvement of Pol III transcription in the retrovirus-induced activation of cellular genes, potentially contributing to leukemogenesis.

Background and ObjectiveThe aim of this study was to characterize primary cells from extrauterine carcinosarcoma (CS) and to establish a primary CS xenograft mouse model.MethodsPrimary cells were isolated from a patient with CS and cultured in vitro. Primary CS cells were verified for their ability to consecutively generate tumorigenesis in NOD/SCID mice. The properties of xenograft tumor and explants cells were investigated by immunohistochemistry, cytogenetic, and FACS analysis. Anticancer drug susceptibility of primary CS was analyzed using CCK-8.ResultsPrimary CS cells greater than 27 passages in vitro showed an ability of a series of xenograft tumorigenesis in vivo having the same marker expression and cytogenetic character as that of original tumor. In addition, explants of xenograft tumors retained their original characteristics in the in vitro culture system. Finally, the analysis of the susceptibility to anticancer drug revealed that primary CS cells were susceptible to both doxorubicin and nilotinib, which are tyrosine kinase inhibitors.ConclusionsThe primary CS cells and the primary CS xenograft tumorigenesis introduce a new therapeutic model for targeting cancer and also explore a deeper understanding of generation of the tumor itself.

OBJECTIVE To overcome the therapeutic limitations of malignant fibrous histiocytoma (MFH), we evaluated human adipose tissue-derived mesenchymal stromal cells (MSCs) that secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on metastatic MFH. BACKGROUND MFH is a highly malignant and metastatic type of sarcoma but surgical removal is the only effective method for treating MFH. MSCs are easily transduced to express a high level of transgene and can migrate toward cancer. For this reason, MSCs are a promising candidate for metastatic MFH therapies. METHODS In vitro sustainability of MSC-TRAIL against MFH-ino was analyzed by apoptosis assay. For preclinical study, anti-MFH effects of MSC-TRAILs were validated in murine models for local tumorigenesis and metastasis. Furthermore, a time-interval metastasis model of MFH was applied to confirm antimetastatic ability of MSC-TRAIL for preestablished metastatic MFH. RESULTS We found that MFH-ino is highly susceptible to recombinant TRAIL and MSC-TRAIL, which selectively induce apoptosis via caspase-8 activation in vitro. Moreover, not only MFH-ino but xenograft explants were also significantly inhibited by MSC-TRAIL in local tumorigenesis. In particular, the metastatic ability of MFH-ino was considerably reduced by MSC-TRAIL in metastasis murine model, particularly for preestablished metastatic MFH. CONCLUSIONS These results suggest that MSC-TRAIL is sufficiently effective in inhibiting MFH-ino metastasis and the application using MSC-TRAIL could be extended to other sarcomas and recurrent metastatic cancers for cell-mediated cancer therapy.

To overcome the limitations of allogeneic hematopoietic stem cell transplantation (HSCT), we conducted a study to identify a strategy for enhancing hematopoietic stem cell (HSC) engraftment during HSCT. Co-transplantation experiments with mesenchymal stem cells (MSCs) derived from adult human tissues including bone marrow (BM), adipose tissue (AT), and umbilical cord blood (CB) were conducted. We showed that AT-MSCs and CB-MSCs enhanced the engraftment of HSCs as effectively as BM-MSCs in NOD/SCID mice, suggesting that AT-MSCs and CB-MSCs can be used as alternative stem cell sources for enhancing the engraftment and homing of HSCs. CB-MSCs derived from different donors showed different degrees of efficacy in enhancing the engraftment of HSCs. The most effective CB-MSCs showed higher proliferation rates and secreted more MCP-1, RANTES, EGF, and VEGF. Our results suggest that AT-MSCs and CB-MSCs could be alternative stem cell sources for co-transplantation in HSCT. Furthermore, in terms of MSCs’ heterogeneity, characteristics of each population of MSCs are considerable factors for selecting MSCs suitable for co-transplantation with HSC.

Oct4 encodes a transcription factor that is involved in the maintenance of self-renewal in stem cells. Recently, the molecular mechanisms that regulate Oct4 expression have come under investigation. In this study, we demonstrate that the orphan nuclear receptor steroidogenic factor-1 (SF-1) behaves as a transcriptional activator of human Oct4 (hOct4) through direct interaction with a SF-1 binding element in the hOct4 proximal promoter. We found that Oct4 and SF-1 were co-expressed in undifferentiated human embryonal carcinoma NCCIT cells and downregulated during retinoic acid-mediated differentiation. We examined the functional role played by SF-1 in regulation of hOct4 transcription using a luciferase reporter assay and Western blot analysis. Overexpression of SF-1 increased up to about threefold hOct4 promoter activity and endogenous hOct4 protein expression. Sequence analysis of the hOct4 promoter revealed that the transcriptional activity was closely linked to Conserved Regions 1 (CR1) and 2 (CR2), which contain three putative SF-1-binding sites (1st, 2nd, and 3rd SF-1). Binding assays and mutagenesis of binding sites indicated that the 1st and 2nd SF-1 elements (in CR1 and CR2, respectively) might be important cis-regulatory elements in hOct4 promoter activity. However, differences in response to SF-1 overexpression between wild-type and mutant hOct4 promoters revealed that the 1st SF-1 element is the key binding site for SF-1-mediated transcriptional activation. Thus, our data indicate that SF-1 plays a crucial role in the regulation of hOct4 transcription through direct binding to the 1st SF-1 in CR1 of the hOct4 proximal promoter.

Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.

Erratum to doi: 10.1038/emm.2005.5 AB -The authors would like to amend a reference (Lee et al., 2003) that was cited in "Cell culture" section of "Materials and Methods". Instead of "(Lee et al., 2003)", we would like to change the reference to "(Kim et al., 2003)". In "References", it also needs to include the following reference. Kim YY, Seol HW, Ahn HJ. Temporal expression of differentiation markers in embryoid bodies from various human embryonic stem cell line. International Society for Stem Cell Research 1st Annual Meeting, Washington, DC. U.S.A. June 8-11, 2003, Abstract No. 35. The authors apologize for any inconvenience.

Background and Objectives:By measuring the coronary flow reserve (CFR) and echocardiographic left ventricular function, the purpose of this study was to evaluate the effect of pre-infarction angina (PA) on myocardial protection in patients with acute myocardial infarction (AMI). Subjects and Methods:Sixty-two patients (mean 54±10 years, 51 males) with first anterior AMI were studied. CFR, defined as the ratio of hyperemic (hAPV) to baseline APV (bAPV), was measured at least 24 hours after the onset of AMI at the left anterior descending artery (mean 7±4 days) with a Doppler wire. Echocardiography was performed at admission (baseline) and during follow-up periods (mean 9±7 month). All patients were divided into two groups according to the presence of PA within 72 hours prior to AMI:group A (with PA, n=27) and group B (without PA, n=35). Results:Between the two groups, CFR were higher in group A (2.1±0.5 vs.1.6±0.5, p

We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236–252 and 249–267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.