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Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated,turning MSCs into variety of cell lineages of mesodermal,ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial,these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently,using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function,as well as with specific signalling pathways that promote neural fate commitment,we have been able to generate neural-like cells from human bone marrow (BM)-derived MSCs (hMSCs). However,the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early-passage hMSCs,comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early- and late-passage hMSCs. The results demonstrated that early-passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs,accompanied by morphological changes into neural-like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early-passage hMSCs could explain their higher plasticity and pliability for neural induction. Copyright textcopyright 2012 John Wiley & Sons,Ltd. View Publication

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol,passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization,centrifugation or drug treatment. It also allows for higher throughput,requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation,colony expansion,cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion,and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages,this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium. View Publication

Hepatocytes play a central and crucial role in cholesterol and lipid homeostasis,and their proper function is of key importance for cardiovascular health. In particular,hepatocytes (especially periportal hepatocytes) endogenously synthesize large amounts of cholesterol and secrete it into circulating blood via apolipoprotein particles. Cholesterol-secreting hepatocytes are also the clinically-relevant cells targeted by statin treatment in vivo. The study of cholesterol homeostasis is largely restricted to the use of animal models and immortalized cell lines that do not recapitulate those key aspects of normal human hepatocyte function that result from genetic variation of individuals within a population. Hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells can provide a cell culture model for the study of cholesterol homeostasis,dyslipidemias,the action of statins and other pharmaceuticals important for cardiovascular health. We have analyzed expression of core components for cholesterol homeostasis in untreated human iPS cells and in response to pravastatin. Here we show the production of differentiated cells resembling periportal hepatocytes from human pluripotent stem cells. These cells express a broad range of apolipoproteins required for secretion and elimination of serum cholesterol,actively secrete cholesterol into the medium,and respond functionally to statin treatment by reduced cholesterol secretion. Our research shows that HLCs derived from human pluripotent cells provide a robust cell culture system for the investigation of the hepatic contribution to human cholesterol homeostasis at both cellular and molecular levels. Importantly,it permits for the first time to also functionally assess the impact of genetic polymorphisms on cholesterol homeostasis. Finally,the system will also be useful for mechanistic studies of heritable dyslipidemias,drug discovery,and investigation of modes of action of cholesterol-modulatory drugs. View Publication

Invasion and metastasis are the major cause of deaths in patients with esophageal cancer. In this study,we isolated cancer stem-like cells from an esophageal squamous cell carcinoma cell line EC109 based on aldehyde dehydrogenase 1A1 (ALDH1A1),and found that ALDH1A1(high) cells possessed the capacities of self-renewal,differentiation and tumor initiation,indications of stem cell properties. To support their stemness,ALDH1A1(high) cells exhibited increased potential of invasion and metastasis as compared with ALDH1A1(low) cells. ALDH1A1(high) esophageal squamous cell carcinoma cells expressed increased levels of mRNA for vimentin,matrix metalloproteinase 2,7 and 9 (MMP2,MMP7 and MMP9),but decreased the level of E-cadherin mRNA,suggesting that epithelial-mesenchymal transition and secretary MMPs may be attributed to the high invasive and metastatic capabilities of ALDH1A1(high) cells. Furthermore,we examined esophageal squamous cell carcinoma specimens from 165 patients and found that ALDH1A1(high) cells were associated with esophageal squamous dysplasia and the grades,differentiation and invasion depth,lymph node metastasis and UICC stage of esophageal squamous cell carcinoma,as well as poor prognosis of patients. Our results provide the strong evidence that ALDH1A1(high) cancer stem-like cells contribute to the invasion,metastasis and poor outcome of human esophageal squamous cell carcinoma. View Publication

Systemic sclerosis (SSc) is a life-threatening chronic connective tissue disease with the characteristics of skin fibrosis,vascular injury,and inflammatory infiltrations. Though inhibition of phosphodiesterase 4 (PDE4) has been turned out to be an effective strategy in suppressing inflammation through promoting the accumulation of intracellular cyclic adenosine monophosphate (cAMP),little is known about the functional modes of inhibiting PDE4 by apremilast on the process of SSc. The present research aimed to investigate the therapeutic effects and underlying mechanism of apremilast on SSc. Herein,we found that apremilast could markedly ameliorate the pathological manifestations of SSc,including skin dermal thickness,deposition of collagens,and increased expression of $\alpha$-SMA. Further study demonstrated that apremilast suppressed the recruitment and activation of macrophages and T cells,along with the secretion of inflammatory cytokines,which accounted for the effects of apremilast on modulating the pro-fibrotic processes. Interestingly,apremilast could dose-dependently inhibit the activation of M1 and T cells in vitro through promoting the phosphorylation of CREB. In summary,our research suggested that inhibiting PDE4 by apremilast might provide a novel therapeutic option for clinical treatment of SSc patients. View Publication

BACKGROUND This study aimed to investigate the role of circulating tumor cells (CTCs) and circulating cancer stem-like cells (cCSCs) before and after one cycle of chemotherapy and assessed the effects of early changes in CTCs and cCSCs on the outcomes of patients with metastatic breast cancer. METHODS Patients with stage IV invasive ductal carcinoma of the breast who received first-line chemotherapy between April 2014 and January 2016 were enrolled. CTCs and cCSCs were measured before the first cycle of chemotherapy (baseline) and on day 21,before the second cycle of chemotherapy commenced; a negative selection strategy and flow cytometry protocol were employed. RESULTS CTC and cCSC counts declined in 68.8 and 45.5% of patients,respectively. Declines in CTCs and cCSCs following the first chemotherapy cycle were associated with superior chemotherapy responses,longer progression-free survival (PFS),and longer overall survival (OS). An early decline in cCSCs remained an independent prognostic indicator for OS and PFS in multivariate analysis. CONCLUSIONS A cCSC decline after one cycle of chemotherapy for metastatic breast cancer is predictive of a superior chemotherapy response and longer PFS and OS,implying that cCSC dynamic monitoring may be helpful in early prediction of treatment response and prognosis. View Publication

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however,although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes,most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here,the authors report a 3D cellular microenvironment plate (3D-CEP),which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG),which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally,global gene expression analyses are used to elucidate small variations among different test environments. Interestingly,the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine. View Publication

The cell line described here was established for a 50-year-old male patient with rapidly progressive non-Hodgkin's lymphoma whose marrow was diffusely infiltrated with large granular lymphocytes (LGL). Immunophenotyping of marrow blasts and peripheral lymphocytes was positive for CD56,CD2 and CD7,and negative for CD3. Cytotoxicity of peripheral blood mononuclear cells at an effector: target (E:T) cell ratio of 50:1 was 79% against K562 cells and 48% against Daudi cells. To establish the line,cells from the peripheral blood were placed into enriched alpha medium containing 12.5% fetal calf serum,12.5% horse serum,10(-4) M beta-mercaptoethanol and 10(-6) M hydrocortisone. Growth of the line (termed NK-92) is dependent on the presence of recombinant IL-2 and a dose as low as 10 U/ml is sufficient to maintain proliferation. Conversely,cells die within 72 h when deprived of IL-2; IL-7 and IL-12 do not maintain long-term growth,although IL-7 induces short-term proliferation measured by 3H-thymidine incorporation. None of the other cytokines tested (IL-1 alpha,IL-6,TNF-alpha,IFN-alpha,IFN-gamma) supported growth of NK-92 cells which have the following characteristics: surface marker positive for CD2,CD7,CD11a,CD28,CD45,CD54,CD56bright; surface marker negative for CD1,CD3,CD4,CD5,CD8,CD10,CD14,CD16,CD19,CD20,CD23,CD34,HLA-DR. DNA analysis showed germline configuration for T-cell receptor beta and gamma genes. CD25 (p55 IL-2 receptor) is expressed on about 50% of all cells when tested at 100 U/ml of IL-2 and its expression correlates inversely with the IL-2 concentration. The p75 IL-2 receptor is expressed on about half of the cells at low density irrespective of the IL-2 concentration. NK-92 cells kill both K562 and Daudi cells very effectively in a 4 h51-chromium release assay (84 and 86% respectively,at an E:T cell ratio of 5:1). The cell line described here thus displays characteristics of activated NK-cells and could be a valuable tool to study their biology. View Publication

Simultaneous ex vivo expansion of different progenitor cell types may be beneficial for cord blood (CB) transplantation,to overcome a potential limitation due to restricted cell numbers. Therefore,1.5 x 10(6) CD34+ cells isolated from fresh or thawed CB samples were inoculated in a large-scale stirred suspension bioreactor and cultured in the presence of Flt3-L,SCF and IL-3. At days 0,7,10,14,21 and 28,the spinner cultures were analyzed for viable cells,colony-forming cells (CFC),including erythroid burst-forming unit (BFU-E),granulocyte-macrophage colony-forming unit (CFU-GM) and granulocyte-erythrocyte-megakaryocyte-monocyte colony-forming unit (CFU-GEMM) as well as long-term culture-initiating cells (LTC-IC). Expansion of thawed CD34+ cells resulted in a substantial amplification of total cells (maximal at day 28: 154 +/- 132-fold),CFC (maximal at day 14: 45 +/- 36-fold),CFU-GM (maximal at day 14: 88 +/- 85-fold),CFU-GEMM (maximal at day 7: 4 +/- 2-fold) and of LTC-IC (maximal at day 10: 8 +/- 3-fold). There was no significant difference between fresh and thawed CD34+ cells. These results demonstrate that simultaneously committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified from CD34+-enriched CB samples in large-scale stirred suspension cultures within 7-14 days without exhausting the proliferative potential and,thus,it may be possible to improve CB transplantation by ex vivo generated cells. View Publication

Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders such as cystic fibrosis. However,limited understanding of human airway patterning has made this goal challenging. Here,we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach,we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases. View Publication

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury,as well as neurodegenerative conditions such as Parkinson's disease,Alzheimer's disease,and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However,the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly,in the present study we demonstrate that tumor necrosis factor α (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore,we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-κB-dependent mechanism. Specifically,we show that NF-κB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-κB inhibitor BAY-117082. Importantly,we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs,indicating that Puma induction is required for NPC death. Consistent with this,we demonstrate that Puma-deficient NPCs exhibit an 13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary,we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions. View Publication