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BACKGROUND Rhodiola crenulata is a Tibetan mountainous plant,commonly used in Eastern alternative medicine. Many phytochemicals possess estrogenic activity,a critical regulator of proliferation in mammary epithelial cells. We have previously characterized anti-cancer properties of R. crenulata in aggressive triple negative breast cancer cells,lacking the expression of estrogen receptor. Currently,it is unknown whether R. crenulata exerts estrogenic effects and as such consumption may be a concern for women with estrogen receptor positive breast cancer that use Rhodiola sp. to relieve mild to moderate depression. PURPOSE In this study,we wished to determine whether a hydroalcoholic fraction of the R. crenulata root extract exhibits estrogenic activity in estrogen receptor positive (ER+) breast cancer cells in vitro and whether it affects normal mammary epithelial ER target gene expression in vivo. METHODS ER transcriptional activity was analyzed in MCF7 cells expressing an ERE reporter construct and confirmed via qPCR of endogenous ER target genes. We also monitored cellular proliferation over time. Additionally,to assess stem-like properties in MCF7 cells,we performed a tumorsphere formation assay under anchorage independent conditions. We examined whether R. crenulata treatment reduced $$-catenin levels via Western blotting and measured $$-catenin transcriptional activity by a reporter assay. To examine the effects of R. crenulata on normal mammary epithelial cells,we performed immunohistochemical staining of ER and PR in the mammary glands of mice fed R. crenulata for 12 weeks. RESULTS We show an initial activation of ER transcriptional activity by dual reporter assay,qPCR and proliferation of MCF7 ER+ cells in response to 24 h of R. crenulata treatment. However,upon longer treatment basal and R. crenulata induced transcriptional activity was suppressed. There was a decrease in cell doubling times and a decrease in tumorsphere formation. In association with these changes,ER$$ transcript levels were decreased and active $$-catenin levels were reduced in the cells treated for 2 weeks. Finally,we show no change in estrogen targets in normal mammary cells in vivo. CONCLUSION These data suggest that the R. crenulata extract contains components with estrogenic activity. However,R. crenulata treatment could still be protective in ER+ breast cancer cells,as longer treatment reduced the transcriptional activity of $$-catenin and ER responses leading to reduced proliferation and tumorsphere formation. Furthermore,administration of 20 mg/kg/day R. crenulata to mice did not have an observable effect on mammary epithelial ER$$ target gene expression in vivo. View Publication

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However,insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs),which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore,we investigated the effects of sodium fluoride (NaF) on the proliferation,differentiation and viability of H9 hESCs. For the first time,we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology,mitochondrial membrane potential (MMP),caspase activities,cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway,coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a pJNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs. View Publication

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells,only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study,we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1,a critical transcription factor for pancreatic development,leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore,in the presence of biochemical factors,200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin,glucagon,or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ,suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells. View Publication

Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4(+)FOXP3(+) T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10,IFN-γ and IL-17A in FOXP3(+) cells. Further,CD161 and HELIOS separated the FOXP3(+) cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3(+) cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3(+) cells. Together,these data show that S. aureus potently induces FOXP3(+) cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3(+) cells to inflammation and repression of immune-suppression upon encounter with S. aureus. View Publication

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4high cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation,efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved,while retaining their pluripotency. When added during the reprogramming process,CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus,CD30-LV may serve as novel tool for the selective gene transfer into pluripotent stem cells with broad applications in basic and therapeutic research. View Publication

Human embryonic stem cell (hESC) line was derived from abnormal blastocyst donated by Marfan syndrome patient after preimpantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that the hESC line carried the heterozygous deletion mutation,c.3536delA,of FBN1 gene. Characteristic tests proved that the hESC line presented typicalmarkers of pluripotency and had the capability to formthe three germlayers both in vitro and in vivo. View Publication

Under defined differentiation conditions,human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate,the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening,a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification,and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2,increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus,we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE. View Publication

Efficient cryopreservation of human pluripotent stem cells (hPSCs) in chemically defined,xeno-free conditions is highly desirable for medical research and clinical applications such as cell-based therapies. Here we present a simple and effective slow freezing-rapid thawing protocol for the cryopreservation of feeder-free,single hPSCs. This cryopreservation protocol involves the supplementation of 10 % dimethyl sulfoxide (DMSO) and 10 $$M Rho-associated kinase inhibitor Y-27632 into two types of xeno-free,defined media supplements (Knockout Serum Replacement and TeSR2). High post-thaw cell recovery (˜90 %) and cell expansion (˜70 %) can be achieved using this protocol. The cryopreserved single cells retain the morphological characteristics of hPSCs and differentiation capabilities of pluripotent stem cells. View Publication

We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types,such as induced pluripotent stem cells (iPSCs) and fibroblasts,reducing the protocol time by one full day. To preserve cell physiology during gene transfer,we designed a microfluidic strategy,which facilitates significant gene delivery in a short transfection time (textless1 min) for several human cell types. This fast,optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. View Publication

BACKGROUND Humanized mouse models are still under development,and various protocols exist to improve human cell engraftment and function. METHODS Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34(+) hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM),spleen,and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS Most of the NSG mice tolerated the regimen well. The percentage of hCD45(+) and CD19(+) cells rose significantly in a time-dependent manner. The median percentage of hCD45(+)cells in the BM was 55.5% at week 8,and 67.2% at week 12. The median percentage of hCD45(+) cells in the spleen at weeks 8 and 12 was 42% and 51%,respectively. The median percentage of hCD19(+) cells in BM at weeks 8 and 12 was 21.5% and 39%,respectively (P=0.04). Similarly,the median percentage of hCD19(+) cells in the spleen at weeks 8 and 12 was 10% and 24%,respectively (P=0.04). The percentage of hCD19(+) B cells in PB was 23% at week 12. At week 8,hCD3(+) T cells were barely detectable,while hCD7(+) was detected in the BM and spleen. The percentage of hCD3(+) T cells was 2‒3% at week 12 in the BM,spleen,and PB of humanized NSG mice. CONCLUSION We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45(+) cells than earlier studies without any significant toxicity. And human CD45(+) cell engraftment at week 8 was comparable to that of week 12. View Publication

Antibodies to donor specific HLA antigens (DSA) detected by single antigen bead (SAB) analysis prior to kidney transplant have been associated with inferior graft outcomes. However,studies of pre-transplant DSA specifically in the setting of a negative flow cytometry crossmatch (FCXM) without desensitization therapy are limited. 660 kidney and kidney/pancreas recipients with a negative pre-transplant FCXM from 09/2007 to 08/2012 without desensitization therapy were analyzed with a median follow-up of 4.2 years. All patients underwent cell-based FCXM and SAB analysis on current and historic sera prior to transplantation. 162 patients (24.5%) had DSA detected prior to transplant. One-year acute rejection rates were similar in DSA(+) vs. DSA(-) patients (15.4% vs. 11.4% respectively,p=0.18) and were higher in those with DSA MFI≥3000 in multivariable analysis (p=0.046). eGFR at 3 and 4 years was lower in the DSA(+) vs. DSA(-) group (p=0.050 at 3 years) without an impact on 5-year death-censored graft survival (89.0% vs. 90.6% respectively,p=0.53). Timing (current or historic) of DSA detection did not alter these findings. In conclusion,pre-transplant DSA in the setting of a negative FCXM confers minimal immunologic risk in the intermediate-term,does not necessitate desensitization therapy,and should not represent a barrier to renal transplant. This article is protected by copyright. All rights reserved. View Publication

Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently,murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4,Sox2,Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors,we have derived iPS cells from fetal,neonatal and adult human primary cells,including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency,and establish a method whereby patient-specific cells might be established in culture. View Publication