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PURPOSE: To investigate the distribution of CD44(+)/CD24(-) cells in breast cancers in relation to tumor size before and after the administration of neoadjuvant chemotherapy. METHODS: CD44(+)/CD24(-) tumor cells obtained from breast cancer specimens were characterized in vivo and in vitro using tumor formation assays and mammosphere generation assays,respectively. The distribution of CD44+/CD24- tumor cells in 78 breast cancer specimens following administration of neoadjuvant chemotherapy was also evaluated using immunofluorescence assays,and this distribution was compared with the extent of tumor invasion predicted by Response Evaluation Criteria in Solid Tumours (RECIST). RESULTS: In 27/78 cases,complete remission (CR) was identified using RECIST. However,18 of these CR cases were associated with a scattered distribution of tumor stem cells in the outline of the original tumor prior to neoadjuvant chemotherapy. After neoadjuvant chemotherapy,24 cases involved cancer cells that were confined to the tumor outline,and 21 cases had tumor cells or tumor stem cells overlapping the tumor outline. In addition,there were 6 patients who were insensitive to chemotherapy,and in these cases,both cancer cells and stem cells were detected outside the contours of the tumor volume imaged prior to chemotherapy. CONCLUSION: CD44+/CD24- tumor cells may be an additional parameter to evaluate when determining the extent of breast cancer invasion. View Publication

Low oxygen (O(2)) levels characterize the microenvironment of both stem cells and rapidly growing tumors. Moreover,hypoxia is associated with the maintenance of stem cell-like phenotypes and increased invasion,angiogenesis and metastasis in cancer patients. Metastatic cancers,such as breast cancer and melanoma,aberrantly express the embryonic morphogen Nodal,and the presence of this protein is correlated with metastatic disease. In this paper,we demonstrate that hypoxia induces Nodal expression in melanoma and breast cancer cells concomitant with increased cellular invasion and angiogenic phenotypes. Of note,Nodal expression remains up-regulated up to 48 h following reoxygenation. The oxygen-mediated regulation of Nodal expression occurs via a combinatorial mechanism. Within the first 24 h of exposure to low O(2),there is an increase in protein stability. This increase in stability is accompanied by an induction of transcription,mediated by the HIF-1α-dependent activation of Notch-responsive elements in the node-specific enhancer of the Nodal gene locus. Finally,Nodal expression is maintained upon reoxygenation by a canonical SMAD-dependent feed-forward mechanism. This work provides insight into the O(2)-mediated regulation of Nodal,a key stem cell-associated factor,and reveals that Nodal may be a target for the treatment and prevention of hypoxia-induced tumor progression. View Publication

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus,hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks,however,conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology,retained a normal karyotype,and expressed characteristic hESC markers (OCT4,SSEA3,SSEA4 and TRA-1-60). Moreover,the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus,the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells. View Publication

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits,FXS patients exhibit hyperactivity,attention deficits,social difficulties,anxiety,and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing,the epigenetic modifications observed at the FMR1 locus,and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations,we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases,iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance,reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific,FXS iPSC models,we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall,these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons,these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology. View Publication

Genetic comparison between human embryonic stem cells and induced pluripotent stem cells has been hampered by genetic variation. To solve this problem,we have developed an isogenic system that allows direct comparison of induced pluripotent stem cells (hiPSCs) to their genetically matched human embryonic stem cells (hESCs). We show that hiPSCs have a highly similar transcriptome to hESCs. Global transcriptional profiling identified 102-154 genes (textgreater2 fold) that showed a difference between isogenic hiPSCs and hESCs. A stringent analysis identified NNAT as a key imprinted gene that was dysregulated in hiPSCs. Furthermore,a disproportionate number of X-chromosome localized genes were over-expressed in female hiPSCs. Our results indicate that despite a remarkably close transcriptome to hESCs,isogenic hiPSCs have alterations in imprinting and regulation of X-chromosome genes. View Publication

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation,purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages. View Publication

C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However,Δ32 donors are scarce,heterologous bone marrow transplantation is not exempt of risks,and genetic engineering of autologous hHSCs is not trivial. Here,we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34(+) cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS. View Publication

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts,perceptions,and emotions,usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however,most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells,derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient,presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS),when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia,contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening. View Publication

Lipid droplets,also called lipid bodies (LB) in inflammatory cells,are important cytoplasmic organelles. However,little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here,we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system,the maturing MCs,derived from 18 different donors,invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore,the MCs transcribe the genes for perilipins (PLIN)1-4,but not PLIN5,and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation,the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion,and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary,we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions,with particular emphasis on AA metabolism,eicosanoid biosynthesis,and subsequent release of proinflammatory lipid mediators from these cells. View Publication

Avian species are important model animals for developmental biology and disease research. However,unlike in mice,where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function,clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs),the first nonmammalian iPSCs,which were clonally isolated and propagated,important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes,indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes,oligodendrocytes,and neurons. Ultimately,qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers,extraembryonic tissues,and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals. View Publication

Many human diseases share a developmental origin that manifests during childhood or maturity. Aneuploid syndromes are caused by supernumerary or reduced number of chromosomes and represent an extreme example of developmental disease,as they have devastating consequences before and after birth. Investigating how alterations in gene dosage drive these conditions is relevant because it might help treat some clinical aspects. It may also provide explanations as to how quantitative differences in gene expression determine phenotypic diversity and disease susceptibility among natural populations. Here,we aimed to produce induced pluripotent stem cell (iPSC) lines that can be used to improve our understanding of aneuploid syndromes. We have generated iPSCs from monosomy X [Turner syndrome (TS)],trisomy 8 (Warkany syndrome 2),trisomy 13 (Patau syndrome) and partial trisomy 11;22 (Emanuel syndrome),using either skin fibroblasts from affected individuals or amniocytes from antenatal diagnostic tests. These cell lines stably maintain the karyotype of the donors and behave like embryonic stem cells in all tested assays. TS iPSCs were used for further studies including global gene expression analysis and tissue-specific directed differentiation. Multiple clones displayed lower levels of the pseudoautosomal genes ASMTL and PPP2R3B than the controls. Moreover,they could be transformed into neural-like,hepatocyte-like and heart-like cells,but displayed insufficient up-regulation of the pseudoautosomal placental gene CSF2RA during embryoid body formation. These data support that abnormal organogenesis and early lethality in TS are not caused by a tissue-specific differentiation blockade,but rather involves other abnormalities including impaired placentation. View Publication

The ability to stimulate mammalian cells with light has significantly changed our understanding of electrically excitable tissues in health and disease,paving the way toward various novel therapeutic applications. Here,we demonstrate the potential of optogenetic control in cardiac cells using a hybrid experimental/computational technique. Experimentally,we introduced channelrhodopsin-2 into undifferentiated human embryonic stem cells via a lentiviral vector,and sorted and expanded the genetically engineered cells. Via directed differentiation,we created channelrhodopsin-expressing cardiomyocytes,which we subjected to optical stimulation. To quantify the impact of photostimulation,we assessed electrical,biochemical,and mechanical signals using patch-clamping,multielectrode array recordings,and video microscopy. Computationally,we introduced channelrhodopsin-2 into a classic autorhythmic cardiac cell model via an additional photocurrent governed by a light-sensitive gating variable. Upon optical stimulation,the channel opens and allows sodium ions to enter the cell,inducing a fast upstroke of the transmembrane potential. We calibrated the channelrhodopsin-expressing cell model using single action potential readings for different photostimulation amplitudes,pulse widths,and frequencies. To illustrate the potential of the proposed approach,we virtually injected channelrhodopsin-expressing cells into different locations of a human heart,and explored its activation sequences upon optical stimulation. Our experimentally calibrated computational toolbox allows us to virtually probe landscapes of process parameters,and identify optimal photostimulation sequences toward pacing hearts with light. ?? 2011 Biophysical Society. View Publication