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It has been recently reported that the regulatory circuitry formed by OCT4,miR-302,and NR2F2 controls both pluripotency and neural differentiation of human embryonic stem cells (hESCs). We show here that JMJD1C,a histone 3 lysine 9 (H3K9) demethylase expressed in hESCs,directly interacts with this circuitry. hESCs with stable knockdown of JMJD1C remain pluripotent while having reduced miR-302 expression,decreased BMP signaling,and enhanced TGF$\$ JMJD1C binds to the miR-302 promoter and reduces H3K9 methylation. Withdrawal of basic fibroblast growth factor (bFGF) from the culture induces neural differentiation of the knockdown,but not the control,cells within 3 days,accompanied by elevated NR2F2 expression. This can be attenuated with miR-302 mimics or an H3K9 methytransferase inhibitor. Together,our findings suggest that JMJD1C represses neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression and that JMJD1C knockdown is sufficient to trigger neural differentiation upon withdrawal of exogenous bFGF. View Publication

Many solid cancers display cellular hierarchies with self-renewing,tumorigenic stemlike cells,or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies,they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs,we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays,but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines,including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion,and targeting IL-17A signaling impaired CIC growth. Notably,IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer. View Publication

Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse,and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically,following interaction with Pin1,Notch1 and Notch4,key regulators of cell fate,escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7$$. Functionally,we show that Fbxw7$$ acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity,but the establishment of a Notch/Pin1 active circuitry opposes this effect,thus promoting breast CSCs self-renewal,tumor growth and metastasis in vivo. In human breast cancers,despite Fbxw7$$ expression,high levels of Pin1 sustain Notch signaling,which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes,through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers. View Publication

A defined xeno-free system for patient-specific iPSC derivation and differentiation is required for translation to clinical applications. However,standard somatic cell reprogramming protocols rely on using MEFs and xenogeneic medium,imposing a significant obstacle to clinical translation. Here,we describe a well-defined culture system based on xeno-free media and LN521 substrate which supported i) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages into hiPSCs with a 15-30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisable polycistronic vector and optimized culture conditions,we achieved up to 0.15%-0.3% reprogramming efficiencies. Subsequently,transgene-free hiPSCs were obtained by Cre-mediated excision of the reprogramming factors. The derived iPSCs maintained long-term self-renewal,normal karyotype and pluripotency,as demonstrated by the expression of stem cell markers and ability to form derivatives of three germ layers both in vitro and in vivo. Importantly,we demonstrated that Parkinson's patient transgene-free iPSCs derived using the same system could be directed towards differentiation into dopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robust platform for the generation of patient-specific iPSCs and derivatives for clinical and translational applications. textcopyright 2013 Elsevier Ltd. View Publication

Ring chromosomes are structural aberrations commonly associated with birth defects,mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome,and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes,no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division,ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations,enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of /`chromosome therapy/' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition,our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control,which is of critical relevance to human development and disease. View Publication

Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear,however,whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells,among which is E2F2,a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1,in addition to proliferation of hESC,indicated by a higher proportion of cells in G1,fewer cells in G2/M phase,and a reduced capacity to generate hESC colonies in vitro. Nonetheless,E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly,E2F2 knockdown in hESC significantly inhibited tumor growth in vivo,which was considerably smaller than tumors generated from control hESC,although displaying typical teratoma traits,a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness,providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells. View Publication

The purpose of this study was to investigate the chondrogenic features of human induced pluripotent stem cells (hiPSCs) and examine the differences in the chondrogenesis between hiPSCs and human bone marrow-derived MSCs (hBMMSCs). Embryoid bodies (EBs) were formed from undifferentiated hiPSCs. After EBs were dissociated into single cells,chondrogenic culture was performed in pellets and alginate hydrogel. Chondro-induced hiPSCs were implanted in osteochondral defects created on the patellar groove of immunosuppressed rats and evaluated after 12 weeks. The ESC markers NANOG,SSEA4 and OCT3/4 disappeared while the mesodermal marker BMP-4 appeared in chondro-induced hiPSCs. After 21 days of culture,greater glycosaminoglycan contents and better chondrocytic features including lacuna and abundant matrix formation were observed from chondro-induced hiPSCs compared to chondro-induced hBMMSCs. The expression of chondrogenic markers including SOX-9,type II collagen,and aggrecan in chondro-induced hiPSCs was comparable to or greater than chondro-induced hBMMSCs. A remarkably low level of hypertrophic and osteogenic markers including type X collagen,type I collagen and Runx-2 was noted in chondro-induced hiPSCs compared to chondro-induced hBMMSCs. hiPSCs had significantly greater methylation of several CpG sites in COL10A1 promoter than hBMMSCs in either undifferentiated or chondro-induced state,suggesting an epigenetic cause of the difference in hypertrophy. The defects implanted with chondro-induced hiPSCs showed a significantly better quality of cartilage repair than the control defects,and the majority of cells in the regenerated cartilage consisted of implanted hiPSCs. ?? 2014 Elsevier Ltd. View Publication

Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive,result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs,we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate,formulated as a hypertonic solution,gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium,retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together,this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology,high levels (textgreater80%) of pluripotency markers OCT4,SSEA-4,TRA-1-60 andTRA-1-81,a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers,both in vivo and in vitro. View Publication

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy,drug screening,physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods,techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results,we adopted a polycistronic cassette encoding Thomson's cocktail OCT4,NANOG,SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones,based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes,ONSL and OCT4,SOX2,KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly,in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM,we indeed observed a remarkable synergy,yielding a reprogramming efficiency of textgreater2%. We present here a drastic improvement of the reprogramming efficiency,which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore,non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency. View Publication

OBJECTIVE: In vitro production of a definitive endoderm (DE) is an important issue in stem cell-related differentiation studies and it can assist with the production of more efficient endoderm derivatives for therapeutic applications. Despite tremendous progress in DE differentiation of human embryonic stem cells (hESCs),researchers have yet to discover universal,efficient and cost-effective protocols. MATERIALS AND METHODS: In this experimental study,we have treated hESCs with 200 nM of Stauprimide (Spd) for one day followed by activin A (50 ng/ml; A50) for the next three days (Spd-A50). In the positive control group,hESCs were treated with Wnt3a (25 ng/ml) and activin A (100 ng/ml) for the first day followed by activin A for the next three days (100 ng/ml; W/A100-A100). RESULTS: Gene expression analysis showed up regulation of DE-specific marker genes (SOX17,FOXA2 and CXCR4) comparable to that observed in the positive control group. Expression of the other lineage specific markers did not significantly change (ptextless0.05). We also obtained the same gene expression results using another hESC line. The use of higher concentrations of Spd (400 and 800 nM) in the Spd-A50 protocol caused an increase in the expression SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration of activin A (25 ng/ml) was not able to up regulate the DE-specific marker genes. Then,A50 was replaced by inducers of definitive endoderm; IDE1/2 (IDE1 and IDE2),two previously reported small molecule (SM) inducers of DE,in our protocol (Spd-IDE1/2). This replacement resulted in the up regulation of visceral endoderm (VE) marker (SOX7) but not DE-specific markers. Therefore,while the Spd-A50 protocol led to DE production,we have shown that IDE1/2 could not fully replace activin A in DE induction of hESCs. CONCLUSION: These findings can assist with the design of more efficient chemically-defined protocols for DE induction of hESCs and lead to a better understanding of the different signaling networks that are involved in DE differentiation of hESCs. View Publication

The ability of human embryonic stem cells and induced pluripotent stem cells to differentiate into all adult cell types greatly facilitates the study of human development,disease pathogenesis,and the generation of screening systems to identify novel therapeutic agents. Autologous cell therapies based on patient-derived induced pluripotent stem cells also hold great promise for the treatment and correction of many inherited and acquired diseases. The full potential of human pluripotent stem cells can be unleashed by genetically modifying a chosen locus with minimal impact on the remaining genome,which can be achieved by targeting genes by homologous recombination. This chapter will describe a protocol for gene modification of pluripotent stem cells by homologous recombination and several methods for the screening and identification of successfully modified clones. View Publication

Efficient approaches for the precise genetic engineering of stem cells can enhance both basic and applied stem cell research. Adeno-associated virus (AAV) vectors have demonstrated high-efficiency gene delivery and gene targeting to numerous cell types,and AAV vectors developed specifically for gene delivery to stem cells have further increased gene targeting frequency compared to plasmid construct techniques. This chapter details the production and purification techniques necessary to generate adeno-associated viral vectors for use in high-efficiency gene targeting of adult or pluripotent stem cell applications. Culture conditions used to achieve high gene targeting frequencies in rat neural stem cells and human pluripotent stem cells are also described. View Publication