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Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature,with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries,including planar 2-D films,macroporous 3-D sponges,and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However,synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion,viability,proliferation,self-renewal,and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover,the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment,where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus,synthetic substrates with specific 3-D microgeometries can support hESC colony formation,self-renewal,and directed differentiation to multiple lineages while obviating the stringent needs for complex,exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions. View Publication

The advance in stem cell research relies largely on the efficiency and biocompatibility of technologies used to manipulate stem cells. In our previous study,we had designed an amphipathic peptide RV24 that can deliver proteins into cancer cell lines efficiently without significant side effects. Encouraged by this observation,we moved forward to test whether RV24 could be used to deliver proteins into human embryonic stem cells and human induced pluripotent stem cells. RV24 successfully mediated protein delivery into these pluripotent stem cells,as well as their derivatives including neural stem cells and dendritic cells. Based on NMR studies and particle surface charge measurements,we proposed that hydrophobic domain of RV24 interacts with ??-sheet structures of the proteins,followed by formation of peptide cage" to facilitate delivery across cellular membrane. These findings suggest the feasibility of using amphipathic peptide to deliver functional proteins intracellularly for stem cell research. ?? 2012 Elsevier Inc." View Publication

The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular,in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers,a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts,allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells,but that specific genes,including positive and negative selection markers,regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies. View Publication

Pluripotency is a unique state in which cells can self-renew indefinitely but also retain the ability to differentiate into other cell types upon receipt of extracellular cues. Although it is clear that stem cells have a distinct transcriptional program,little is known about how alterations in post-transcriptional mechanisms,such as mRNA turnover,contribute to the achievement and maintenance of pluripotency. Here we have assessed the rates of decay for the majority of mRNAs expressed in induced pluripotent stem (iPS) cells and the fully differentiated human foreskin fibroblasts (HFFs) they were derived from. Comparison of decay rates in the two cell types led to the discovery of three independent regulatory mechanisms that allow coordinated turnover of specific groups of mRNAs. One mechanism results in increased stability of many histone mRNAs in iPS cells. A second pathway stabilizes a large set of zinc finger protein mRNAs,potentially through reduced levels of miRNAs that target them. Finally,a group of transcripts bearing 3' UTR C-rich sequence elements,many of which encode transcription factors,are significantly less stable in iPS cells. Intriguingly,two poly(C)-binding proteins that recognize this type of element are reciprocally expressed in iPS and HFF cells. Overall,our results highlight the importance of post-transcriptional control in pluripotent cells and identify miRNAs and RNA-binding proteins whose activity may coordinately control expression of a wide range of genes in iPS cells. View Publication

Differentiation of embryonic stem cells (ESCs) into insulin-producing cells for cell replacement therapy of diabetes mellitus comprises the stepwise recapitulation of in vivo developmental stages of pancreatic organogenesis in an in vitro differentiation protocol. The chemical compounds IDE-1 and (-)-indolactam-V can be used to direct mouse and human ESCs through these stages to form definitive endoderm via an intermediate mesendodermal stage and finally into pancreatic endoderm. Cells of the pancreatic endoderm express the PDX1 transcription factor and contribute to all pancreatic cell types upon further in vitro or in vivo differentiation. Even though this differentiation approach is highly effective and reproducible,it generates heterogeneous populations containing PDX1-expressing pancreatic progenitors amongst other cell types. Thus,a technique to separate PDX1-expressing cells from this mixture is very desirable. Recently it has been reported that PDX1-positive pancreatic progenitors,derived from human embryonic stem cells,express the surface marker CD24. Therefore were subjected mouse and human ESCs to a small molecule differentiation approach and the expression of the surface marker CD24 was monitored in undifferentiated cells,cells committed to the definitive endoderm and cells reminiscent of the pancreatic endoderm. We observed that both mouse and human ESCs expressed CD24 in the pluripotent state,during the whole process of endoderm formation and upon further differentiation towards pancreatic endoderm. Thus CD24 is not a suitable cell surface marker for identification of PDX1-positive progenitor cells. View Publication

Neural differentiation of human embryonic (ES) and induced pluripotent (iPS) stem cell lines has been used for research in early human development,drug discovery,and cell replacement therapies. It is critical to establish generic differentiation protocols to compare the neural specification potential of each individually derived pluripotent stem cell line and identify the efficacious lines for research and therapeutic use. Here,we describe a reproducible and quantitative protocol to assess the neural progenitor (NP) generation of human pluripotent stem cell lines. This method includes a robust and well-defined neural inducing platform for Pax6(+) neural rosette (neuroectodermal cells) generation,propagation,and subsequent differentiation into nestin(+) NPs. A side-by-side comparison under common culture conditions among three human ES cell lines,TE03,TE06,and BG01V,and one iPS cell line,HD02,showed highly variable efficiency in their differentiation into NPs. View Publication

The differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) towards functional neurons particularly hold great potential for the cell-based replacement therapy in neurodegenerative diseases. Here,we describe a stepwise differentiation protocol that mimics the early stage of neural development in human to promote the generation of neuroprogenitors at a high yield. Both the hESCs and hiPSCs are initially cultured in an optimized feeder-free condition,which offer an efficient formation of aggregates. To specify the neuroectodermal specification,these aggregates are differentiated in a defined neural induction medium to develop into neural rosettes-like structures. The rosettes are expanded into free-floating sphere and can be further propagated or developed into variety of neuronal subtypes. View Publication

Embryonic stem cells (ESCs) are unique cells,which have the ability to differentiate into all cell types that comprise the adult organism. Furthermore,ESCs can infinitely self-renew under optimized conditions. These features place human ESCs (hESCs) in a position where these cells can be exploited for tissue engineering and regenerative medicine approaches in treating human degenerative disorders. However,cell therapy approaches will require large amounts of clinically useable cells,not typically achievable using standard static cell culture methods. Here,we describe a method wherein clinically relevant numbers of hESCs can be generated in a cost and time effective manner. View Publication

More than 600 human embryonic stem cell (hESC) lines have been reported today at the human European Embryonic Stem Cell Registry ( http://www.hescreg.eu/ ). Despite these high numbers,there are currently no general protocols for derivation,culture,and characterization of hESC. Moreover,data on the culture of the embryo used for the derivation (medium,day of ICM isolation) are usually not available but can have an impact on the derivation rate. We present here the protocols for derivation,culture and characterization as we applied them for the 22 hESC lines (named VUB-hESC) in our laboratory. View Publication

Human embryonic stem cells (hESCs) are self-renewing,pluripotent cells derived from the inner cell mass of blastocysts,early-stage embryos,or blastomeres. hESCs can be propagated indefinitely in an undifferentiated state in vitro and have the ability to differentiate into all cell types of the body. Therefore,these cells can potentially provide an unlimited source of cells and hold promise for transplantation therapy,regenerative medicine,drug screening and discovery,and basic scientific research. Surplus human embryos donated for hESC derivation are extremely valuable,and inefficient derivation of hESCs would be a terrible waste of human embryos. Here,we describe a method for isolating hESC lines from human blastocysts with high efficiency. We also describe the methods for excising differentiated areas from partially differentiated hESC colonies and re-isolating undifferentiated hESCs from extremely differentiated hESC colonies. View Publication

Although since 1998 more than 1,200 different hESC lines have been established worldwide,there is still a recognized interest in the establishment of new lines of hESC,particularly from HLA types and ethnic groups underrepresented among the currently available lines. The methodology of hESC derivation has evolved significantly since the initial derivations using human LIF (hLIF) for maintenance of pluripotency. However,there are still a number of alternative strategies for the different steps involved in establishing a new line of hESC. We have analyzed the different strategies/parameters used between 1998 and 2010 for the derivation of the 375 hESC lines able to form teratomas in immunocompromised mice deposited in two international stem cell registries. Here we describe some trends in the methodology for establishing hESC lines,discussing the developments in the field. Nevertheless,we describe a much greater heterogeneity of strategies for hESCs derivation than what is used for murine ESC lines,indicating that optimum conditions have not been identified yet,and thus,hESC establishment is still an evolving field of research. View Publication

Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end,we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption,but in parallel functional integration of all probes mandatory for process monitoring,that is,for pO₂ and pH,experiments were performed in 100 mL culture volume in a mini reactor platform" consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×10�?� hiPSCs/100 mL were generated in a single process run in 7 days. Expression of pluripotency markers and ability of cells to differentiate into derivates of all three germ layers in vitro was maintained� View Publication