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Peripheral blood was collected from a 39-year-old unaffected female carrier of an X-linked recessive mutation of Iduronate 2-sulfatase gene (NM000202.7(IDS):c.85CtextgreaterT) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC showed normal karyotype. The line offers a good platform to study MPS II pathophysiology,for drug testing,early biomarker discovery and gene therapy studies. View Publication

Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins,there are elevated concerns regarding their possible use as bioterrorism agents. Moreover,their widespread use for cosmetic purposes,and as medical treatments,has increased the potential risk of accidental overdosing and environmental exposure. Hence,there is an urgent need to develop novel modalities to counter BoNT intoxication. Mammalian motoneurons are the main target of BoNTs; however,due to the difficulty and poor efficiency of the procedures required to isolate the cells,they are not suitable for high-throughput drug screening assays. Here,we explored the suitability of embryonic stem (ES) cell-derived motoneurons as a renewable,reproducible,and physiologically relevant system for BoNT studies. We found that the sensitivity of ES-derived motoneurons to BoNT/A intoxication is comparable to that of primary mouse spinal motoneurons. Additionally,we demonstrated that several BoNT/A inhibitors protected SNAP-25,the BoNT/A substrate,in the ES-derived motoneuron system. Furthermore,this system is compatible with immunofluorescence-based high-throughput studies. These data suggest that ES-derived motoneurons provide a highly sensitive system that is amenable to large-scale screenings to rapidly identify and evaluate the biological efficacies of novel therapeutics. View Publication

We recently demonstrated that lack of type I IFN signaling (IFNAR knockout) in lymphocyte-deficient mice (IFrag(-/-)) results in bone marrow (BM) failure after Pneumocystis lung infection,whereas lymphocyte-deficient mice with intact IFNAR (RAG(-/-)) had normal hematopoiesis. In the current work,we performed studies to define further the mechanisms involved in the induction of BM failure in this system. BM chimera experiments revealed that IFNAR expression was required on BM-derived but not stroma-derived cells to prevent BM failure. Signals elicited after day 7 postinfection appeared critical in determining BM cell fate. We observed caspase-8- and caspase-9-mediated apoptotic cell death,beginning with neutrophils. Death of myeloid precursors was associated with secondary oxidative stress,and decreasing colony-forming activity in BM cell cultures. Treatment with N-acetylcysteine could slow the progression of,but not prevent,BM failure. Type I IFN signaling has previously been shown to expand the neutrophil life span and regulate the expression of some antiapoptotic factors. Quantitative RT-PCR demonstrated reduced mRNA abundance for the antiapoptotic factors BCL-2,IAP2,MCL-1,and others in BM cells from IFrag(-/-) compared with that in BM cells from RAG(-/-) mice at day 7. mRNA and protein for the proapoptotic cytokine TNF-α was increased,whereas mRNA for the growth factors G-CSF and GM-CSF was reduced. In vivo anti-TNF-α treatment improved precursor cell survival and activity in culture. Thus,we propose that lack of type I IFN signaling results in decreased resistance to inflammation-induced proapoptotic stressors and impaired replenishment by precursors after systemic responses to Pneumocystis lung infection. Our finding may have implications in understanding mechanisms underlying regenerative BM depression/failure during complex immune deficiencies such as AIDS. View Publication

The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected,patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression,avoiding the risk of insertional mutagenesis by therapeutic vectors,and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However,gene targeting in human pluripotent cells has remained challenging and inefficient. Recently,engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs,raising the prospect of using this technology to correct disease causing mutations. Here,we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions,we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient,transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications. View Publication

Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs),yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast,highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations,one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)). The CD48(-)EPCR(+)ALDH(dim) cells are virtually all in G(0) and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48(-)EPCR(+)ALDH(int) cells are virtually all in G(1),have little intravenous engraftment potential,and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However,varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G(1) with an intravenous engraftment defect. View Publication

Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate along specific lineage pathways. Many of the transcriptional networks that drive reprogramming of a self-renewing ESC to a differentiating cell have been identified. However,fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. USP22 is induced as ESCs differentiate and is necessary for differentiation into all three germ layers. We further report that USP22 is a transcriptional repressor of the locus encoding the core pluripotency factor sex-determining region Y-box 2 (SOX2) in ESCs,and this repression is required for efficient differentiation. USP22 occupies the Sox2 promoter and hydrolyzes monoubiquitin from ubiquitylated histone H2B and blocks transcription of the Sox2 locus. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs,allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs. View Publication

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined,low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements,express neural precursor markers,generate neurons and glia in vitro,and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment. View Publication

FOXM1 is an important cell cycle regulator and regulates cell proliferation. In addition,FOXM1 has been reported to contribute to oncogenesis in various cancers. However,it is not clearly understood how FOXM1 contributes to acute myeloid leukemia (AML) cell proliferation. In this study,we investigated the cellular and molecular function of FOXM1 in AML cells. The FOXM1 messenger RNA (mRNA) expressed in AML cell lines was predominantly the FOXM1B isoform,and its levels were significantly higher than in normal high aldehyde dehydrogenase activity (ALDH(hi)) cells. Reduction of FOXM1 expression in AML cells inhibited cell proliferation compared with control cells,through induction of G(2)/M cell cycle arrest,a decrease in the protein expression of Aurora kinase B,Survivin,Cyclin B1,S-phase kinase-associated protein 2 and Cdc25B and an increase in the protein expression of p21(Cip1) and p27(Kip1). FOXM1 messenger RNA (mRNA) was overexpressed in all 127 AML clinical specimens tested (n = 21,56,32 and 18 for M1,M2,M4 and M5 subtypes,respectively). Compared with normal ALDH(hi) cells,FOXM1 gene expression was 1.65- to 2.26-fold higher in AML cells. Moreover,the FOXM1 protein was more strongly expressed in AML-derived ALDH(hi) cells compared with normal ALDH(hi) cells. In addition,depletion of FOXM1 reduced colony formation of AML-derived ALDH(hi) cells due to inhibition of Cdc25B and Cyclin B1 expression. In summary,we found that FOXM1B mRNA is predominantly expressed in AML cells and that aberrant expression of FOXM1 induces AML cell proliferation through modulation of cell cycle progression. Thus,inhibition of FOXM1 expression represents an attractive target for AML therapy. View Publication

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases,yet current therapeutic treatments are inadequate due to a complex disease pathogenesis. The plant polyphenol apigenin has been shown to have anti-inflammatory and neuroprotective properties in a number of cell and animal models; however a comprehensive assessment has not been performed in a human model of AD. Here we have used a human induced pluripotent stem cell (iPSC) model of familial and sporadic AD,in addition to healthy controls,to assess the neuroprotective activity of apigenin. The iPSC-derived AD neurons demonstrated a hyper-excitable calcium signalling phenotype,elevated levels of nitrite,increased cytotoxicity and apoptosis,reduced neurite length and increased susceptibility to inflammatory stress challenge from activated murine microglia,in comparison to control neurons. We identified that apigenin has potent anti-inflammatory properties with the ability to protect neurites and cell viability by promoting a global down-regulation of cytokine and nitric oxide (NO) release in inflammatory cells. In addition,we show that apigenin is able to protect iPSC-derived AD neurons via multiple means by reducing the frequency of spontaneous Ca(2+) signals and significantly reducing caspase-3/7 mediated apoptosis. These data demonstrate the broad neuroprotective action of apigenin against AD pathogenesis in a human disease model. View Publication

Differentiation of pluripotent cells into endoderm-related cell types initially requires in vitro gastrulation into the definitive endoderm (DE). Most differentiation protocols are initiated from colonies of pluripotent cells complicating their adaption due to insufficiently defined starting conditions. The protocol described here was initiated from a defined cell number of dispersed single cells and tested on three different human embryonic stem cell lines and one human induced pluripotent stem cell line. Combined activation of ActivinA/Nodal signaling and GSK3 inhibition for the first 24 h,followed by ActivinA/Nodal signaling efficiently induced the DE state. Activation of ActivinA/Nodal signaling alone was not effective. Efficient GSK3 inhibition allowed the reduction of the ActivinA concentration during the entire protocol. A feeder-independent cultivation of pluripotent cells was preferred to achieve the high efficiency and robustness since feeder cells hindered the differentiation process. Additionally,inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway was not required,nonetheless yielding high cell numbers efficiently committed toward the DE. Finally,the endoderm generated could be differentiated further into PDX1-positive pan-pancreatic cells and NGN3-positive endocrine progenitors. Thus,this efficient and robust DE differentiation protocol is a step forward toward better reproducibility due to the well-defined conditions based on dispersed single cells from feeder-free-cultivated human pluripotent cells. View Publication

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro,increase our understanding of human embryonic development,and provide clinically relevant cell types for transplantation,drug testing,and toxicology studies. Since their discovery,numerous advances have been made in order to eliminate issues such as vector integration into the host genome,low reprogramming efficiency,incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally,we illustrate methods by which to validate pluripotency of the resulting stem cell population. View Publication

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. View Publication