Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (>200 fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, non-viral methods for reprogramming human somatic cells.

Using a high-throughput chemical screen, we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action, we discovered an essential role of E-cadherin signaling for ESC survival. Specifically, we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling, which then leads to a fatal perturbation of integrin signaling. Furthermore, we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally, we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology, growth factor requirement, and sensitivity to enzymatic cell dissociation.human embryonic stem cell survival | cell-cell adhesion | cell-ECM adhesion C onventional murine and human embryonic stem cells (hESCs) derived from blastocysts can be propagated indefinitely and have the ability to generate all cell types (1-3). They express pluripotency transcription factors, including the ones that can reprogram somatic cells back to pluripotent states: Oct4, Sox2, Nanog, and Klf4. However, murine and human ESCs respond very differently to several key signaling pathways in selfrenewal or differentiation. For example, murine ESCs (mESCs) self-renew under leukemia inhibitory factor (LIF) and bone morphogenic protein (BMP) (4, 5), whereas human ESCs (hESCs) appear dependent on FGF, and TGFβ/Activin/Nodal pathway activity for self-renewal (6-11). These studies clearly suggest that there exist two distinct self-renewal mechanisms. In addition, hESCs grow in vitro as large flattened 2D colonies, whereas mESCs display characteristic small-domed 3D appearances of compact colonies. Moreover, unlike mESCs, hESCs are very vulnerable to single-cell dissociation. Massive cell death occurs after complete single-cell dissociation, which has been a significant hurdle for rapid expansion and genetic manipulation of hESCs. To address this critical challenge and understand the molecular mechanisms that govern hESC survival, we used a high-throughput chemical screening approach and identified two small molecules with distinct mechanisms of action that significantly increase hESC survival after single-cell dissociation. In depth characterizations of compounds' mechanism of action revealed that hESC survival and self-renewal is regulated by the interplay between two cell adhesion systems: cell-cell adhesion and cell-ECM adhesion. Our studies also uncovered a common mechanism that underlies and integrates two seemingly distinct self-renewal states represented by conventional murine and human ESCs.

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Carbohydrates are ubiquitous biological polymers that are important in a broad range of biological processes. However, owing to their branched structures and the presence of stereogenic centres at each glycosidic linkage between monomers, carbohydrates are harder to characterize than are peptides and oligonucleotides. Methods such as nuclear magnetic resonance spectroscopy can be used to characterize glycosidic linkages, but this technique requires milligram amounts of material and cannot detect small amounts of coexisting isomers. Mass spectrometry, on the other hand, can provide information on carbohydrate composition and connectivity for even small amounts of sample, but it cannot be used to distinguish between stereoisomers. Here, we demonstrate that ion mobility-mass spectrometry--a method that separates molecules according to their mass, charge, size, and shape--can unambiguously identify carbohydrate linkage-isomers and stereoisomers. We analysed six synthetic carbohydrate isomers that differ in composition, connectivity, or configuration. Our data show that coexisting carbohydrate isomers can be identified, and relative concentrations of the minor isomer as low as 0.1 per cent can be detected. In addition, the analysis is rapid, and requires no derivatization and only small amounts of sample. These results indicate that ion mobility-mass spectrometry is an effective tool for the analysis of complex carbohydrates. This method could have an impact on the field of carbohydrate synthesis similar to that of the advent of high-performance liquid chromatography on the field of peptide assembly in the late 1970s.