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Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general,but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies,however,is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is,however,limited and thereby further optimization in terms of time,efficiency,and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts,at least in part,in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1,Inhba and Grem1. Hence,we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. View Publication

OBJECTIVE: The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation,expansion,and differentiation. MATERIALS AND METHODS: We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives),together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses,Affymetrix profiling,real-time PCR,and immunoprecipitation. RESULTS: Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g.,POU5F1,NANOG,SOX2,FOXD3). siRNA analyses identified two retinoblastoma binding proteins (RBBP4,RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells,and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1,NANOG,SOX2,and/or FOXD3 plus certain cell cycle genes (e.g.,CCNA2,CCNB1),while increasing expression of genes involved in organogenesis (particularly neurogenesis). CONCLUSIONS: These results reveal new candidate positive regulators of human PS cells,providing evidence of their ability to regulate expression of pluripotency,cell cycle,and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency. View Publication

Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population,but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44(high)/CD24(low/-) subpopulation,increased ability of cells to form mammospheres,and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells,which express high basal levels of TG2,shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together,these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells. View Publication

Genome stability of human embryonic stem cells (hESC) is an important issue because even minor genetic alterations can negatively impact cell functionality and safety. The incorrect repair of DNA double-stranded breaks (DSBs) is the ultimate cause of the formation of chromosomal aberrations. Using G2 radiosensitivity assay,we analyzed chromosomal aberrations in pluripotent stem cells and somatic cells. The chromatid exchange aberration rates in hESCs increased manifold 2 hours after irradiation as compared with their differentiated derivatives,but the frequency of radiation-induced chromatid breaks was similar. The rate of radiation-induced chromatid exchanges in hESCs and differentiated cells exhibited a quadratic dose response,revealing two-hit mechanism of exchange formation suggesting that a non-homologous end joining (NHEJ) repair may contribute to their formation. Inhibition of DNA-PK,a key NHEJ component,by NU7026 resulted in a significant decrease in radiation-induced chromatid exchanges in hESCs but not in somatic cells. In contrast,NU7026 treatment increased the frequency of radiation-induced breaks to a similar extent in pluripotent and somatic cells. Thus,DNA-PK dependent NHEJ efficiently participates in the elimination of radiation-induced chromatid breaks during the late G2 in both cell types and DNA-PK activity leads to a high level of misrejoining specifically in pluripotent cells. View Publication

By providing access to affected neurons,human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease,defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system,causing relentless progression toward severe mental retardation. Partially digested proteoglycans,which affect fibroblast growth factor signaling,accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages,patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures,undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions,whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together,these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues,which possibly affect Golgi organization,cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development,once HS proteoglycan accumulation becomes prominent in the affected child brain. View Publication

AMP-activated protein kinase (AMPK) is an energy-sensing kinase that has recently been shown to regulate the differentiation of preadipocytes and osteoblasts. However,the role of AMPK in stem cell differentiation is largely unknown. Using in vitro culture models,the present study demonstrates that AMPK is a critical regulatory factor for osteogenic differentiation. We observed that expression and phosphorylation of AMPK were increased during osteogenesis in human adipose tissue-derived mesenchymal stem cells (hAMSC). To elucidate the role of AMPK in osteogenic differentiation,we investigated the effect of AMPK inhibition or knockdown on mineralization of hAMSC. Compound C,an AMPK inhibitor,reduced mineralized matrix deposition and suppressed the expression of osteoblast-specific genes,including alkaline phosphatase (ALP),runt-related transcription factor 2 (RUNX2),and osteocalcin (OCN). Knockdown of AMPK by shRNA-lentivirus infection also reduced osteogenesis. In addition,inhibition or knockdown of AMPK during osteogenesis inhibited ERK phosphorylation,which is required for osteogenesis. Interestingly,inhibition of AMPK induced adipogenic differentiation of hAMSC,even in osteogenic induction medium (OIM). These results provide a potential mechanism involving AMPK activation in osteogenic differentiation of hAMSC and suggest that commitment of hAMSC to osteogenic or adipogenic lineage is governed by activation or inhibition of AMPK,respectively. View Publication

A practical synthesis of the Rho-Kinase inhibitor Y-27632 and two new fluoro derivatives was achieved in seven steps and with a good overall yield of 45% starting from commercially available (R)-1-phenylethylamine. Compared to Y-27632 the new fluoro derivatives showed reduced or no effect on hPSC vitality and expansion after dissociation in human pluripotent stem cells. View Publication

The mitochondrial contribution to the maintenance of human embryonic stem cell (hESC) pluripotency and culture homeostasis remains poorly understood. Here,we sought to determine whether hESC adaptation to different feeder-free culture conditions is linked to a mitochondrial adaptation. The expression of ESC pluripotency factors and parameters of mitochondrial contribution including mitochondrial membrane potential,mtDNA content,and the expression of master mitochondrial genes implicated in replication,transcription,and biogenesis were determined in 8 hESC lines maintained in 2 distinct human feeders-conditioned media (CM): human foreskin fibroblast-CM (HFF-CM) and mesenchymal stem cell-CM (MSC-CM). We show a robust parallel trend between the expression of ESC pluripotency factors and the mitochondrial contribution depending on the culture conditions employed to maintain the hESCs,with those in MSC-CM consistently displaying increased levels of pluripotency markers associated to an enhanced mitochondrial contribution. The differences in the mitochondrial status between hESCs maintained in MSC-CM versus HFF-CM respond to coordinated changes in mitochondrial gene expression and biogenesis. Importantly,the culture conditions determine the mitochondrial distribution within the stage-specific embryonic antigen 3 positive (SSEA3(+)) and negative (SSEA3(-)) isolated cell subsets. hESC colonies in MSC-CM display an intrinsic" high mitochondrial status which may suffice to support undifferentiated growth� View Publication

The mixed lineage leukemia (MLL) proto-oncogenic protein is a histone-lysine N-methyltransferase that is produced by proteolytic cleavage and self-association of the respective functionally distinct subunits (MLL(N) and MLL(C)) to form a holocomplex involved in epigenetic transcriptional regulation. On the basis of studies in Drosophila it has been suggested that the separated subunits might also have distinct functions. In this study,we used a genetically engineered mouse line that lacked MLL(C) to show that the MLL(N)-MLL(C) holocomplex is responsible for MLL functions in various developmental processes. The stability of MLL(N) is dependent on its intramolecular interaction with MLL(C),which is mediated through the first and fourth plant homeodomain (PHD) fingers (PHD1 and PHD4) and the phenylalanine/tyrosine-rich (FYRN) domain of MLL(N). Free MLL(N) is destroyed by a mechanism that targets the FYRN domain,whereas free MLL(C) is exported to the cytoplasm and degraded by the proteasome. PHD1 is encoded by an alternatively spliced exon that is occasionally deleted in T-cell leukemia,and its absence produces an MLL mutant protein that is deficient for holocomplex formation. Therefore,this should be a loss-of-function mutant allele,suggesting that the known tumor suppression role of MLL may also apply to the T-cell lineage. Our data demonstrate that the dissociated MLL subunits are subjected to distinct degradation pathways and thus not likely to have separate functions unless the degradation mechanisms are inhibited. View Publication

Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin. B-Ep,together with fluorescently tagged streptavidin,was found to specifically detect Ep-receptor-bearing cells by flow cytometry. This was demonstrated in several ways. First,approximately 90% of nucleated spleen cells from phenylhydrazine-treated mice were clearly fluorescent after staining with b-Ep and streptavidin-phycoerythrin,whereas only background fluorescence was detected using spleen cells from untreated mice. In addition,Ep-receptors were detected on 5% to 10% of normal mouse bone marrow cells,and these cells could be identified as erythroid in nature by separating the cells into subpopulations based on light-scatter properties. Third,Ep-receptor expression was found to correlate positively with expression of transferrin receptors,confirming the erythroid nature of these cells. B-Ep was also used to isolate Ep-receptors from monkey COS cells transfected with the murine Ep-receptor cDNA. In these experiments a cell-surface-bound protein of approximately 65 Kd and an intracellular protein of approximately 60 Kd were isolated from these cells. The procedures described in this report for detecting Ep-receptor expressing cells and for isolating the Ep-receptor should be valuable for purifying erythroid cells from heterogeneous cell populations,for elucidating the structure of the Ep-receptor,and for studying the biological activities of Ep at the cellular and molecular level. View Publication

Oligomerization is an important regulatory mechanism for many proteins,including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity,suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges,resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations,which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl,reduced cell proliferation,and increased caspase-3/7 activity and DNA segmentation. Importantly,the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. View Publication