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Advancing pluripotent stem cell technologies for modelling haematopoietic stem cell development and blood therapies requires identifying key regulators of haematopoietic commitment from human pluripotent stem cells (hPSCs). Here,by screening the effect of 27 candidate factors,we reveal two groups of transcriptional regulators capable of inducing distinct haematopoietic programs from hPSCs: pan-myeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1). In both cases,these transcription factors directly convert hPSCs to endothelium,which subsequently transform into blood cells with pan-myeloid or erythro-megakaryocytic potential. These data demonstrate that two distinct genetic programs regulate the haematopoietic development from hPSCs and that both of these programs specify hPSCs directly to haemogenic endothelial cells. In addition,this study provides a novel method for the efficient induction of blood and endothelial cells from hPSCs via the overexpression of modified mRNA for the selected transcription factors. View Publication

Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications,there is a need to develop scalable cell culture platforms to propagate hESCs. In this study,we encapsulated hESCs in calcium alginate microfibers as single cells,for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (textgreater10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM),which did not affect hESCs viability,was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical,as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line,HUES7,for up to five passages while maintaining pluripotency. Immunohistochemistry,polymerase chain reaction,and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells,possessed normal karyotype,and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively,our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions. View Publication

Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes,both in vitro and in vivo. Beyond uses in cell replacement therapy,patient-specific cardiomyocytes may find applications in drug testing,drug discovery,and disease modeling. Recently,approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs),adult heart-derived cardiac progenitor cells (CPCs),and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts,highlighting potential applications and future challenges. View Publication

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. View Publication

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here,we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore,in vitro,iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice,human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together,our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons. View Publication

Crystalline Mg-Zinc (Zn)-Strontium (Sr) ternary alloys consist of elements naturally present in the human body and provide attractive mechanical and biodegradable properties for a variety of biomedical applications. The first objective of this study was to investigate the degradation and cytocompatibility of four Mg-4Zn-xSr alloys (x=0.15,0.5,1.0,1.5wt%; designated as ZSr41A,B,C,and D respectively) in the direct culture with human umbilical vein endothelial cells (HUVEC) in vitro. The second objective was to investigate,for the first time,the early-stage inflammatory response in cultured HUVECs as indicated by the induction of vascular cellular adhesion molecule-1 (VCAM-1). The results showed that the 24-h in vitro degradation of the ZSr41 alloys containing a β-phase with a Zn/Sr at% ratio ∼1.5 was significantly faster than the ZSr41 alloys with Zn/Sr at% ∼1. Additionally,the adhesion density of HUVECs in the direct culture but not in direct contact with the ZSr41 alloys for up to 24h was not adversely affected by the degradation of the alloys. Importantly,neither culture media supplemented with up to 27.6mM Mg(2+) ions nor media intentionally adjusted up to alkaline pH 9 induced any detectable adverse effects on HUVEC responses. In contrast,the significantly higher,yet non-cytotoxic,Zn(2+) ion concentration from the degradation of ZSr41D alloy was likely the cause for the initially higher VCAM-1 expression on cultured HUVECs. Lastly,analysis of the HUVEC-ZSr41 interface showed near-complete absence of cell adhesion directly on the sample surface,most likely caused by either a high local alkalinity,change in surface topography,and/or surface composition. The direct culture method used in this study was proposed as a valuable tool for studying the design aspects of Zn-containing Mg-based biomaterials in vitro,in order to engineer solutions to address current shortcomings of Mg alloys for vascular device applications. STATEMENT OF SIGNIFICANCE Magnesium (Mg) alloys specifically designed for biodegradable implant applications have been the focus of biomedical research since the early 2000s. Physicochemical properties of Mg alloys make these metallic biomaterials excellent candidates for temporary biodegradable implants in orthopedic and cardiovascular applications. As Mg alloys continue to be investigated for biomedical applications,it is necessary to understand whether Mg-based materials or the alloying elements have the intrinsic ability to direct an immune response to improve implant integration while avoiding cell-biomaterial interactions leading to chronic inflammation and/or foreign body reactions. The present study utilized the direct culture method to investigate for the first time the in vitro transient inflammatory activation of endothelial cells induced by the degradation products of Zn-containing Mg alloys. View Publication

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular,this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells),hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here,we described a new platform which enables,rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular,we devised a protocol that,combining the expression of the CRISPR components with neurogenic factors,generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy,fast and that does not require the generation of stable isogenic clones,practice that is time consuming and for some genes not feasible. View Publication

Human engineered heart tissues have potential to revolutionize cardiac development research,drug-testing,and treatment of heart disease; however,implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment,we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward,ontomimetic approach,imitating the process of development,requires only a single cell-handling step,provides reproducible results for a range of tested geometries and size scales,and overcomes inherent limitations in cell maintenance and maturation,while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation,mimicking heart development,and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue. View Publication

Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields,cellular heterogeneity,and limited scalability. In the present study,we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes,secreted hepatic proteins such as Albumin,Alpha Fetoprotein,and Fibrinogen,metabolized ammonia,and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes,such as LDL storage and uptake,ICG uptake and release,and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure,providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. 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

Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of dextran (DEX) onto polyethylene glycol (PEG)-covered cells though a glass capillary needle connected to a pneumatic pump. This technique is applied to purify colonies of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblast (MEF) feeder cultures and inefficiently induced iPSC colonies by selectively dissociating the iPSCs with proteases. 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