Secondary assays confirmed the pro-survival effects of the ROCK inhibitor HA-1077 on OCT4 positive hESCs.

<p><b>A:</b> 320 candidate small molecules were rescreened in identical conditions to the primary assay. 2 small molecules circled in red, HA-1077 and T6140247, resulted in a significant (Z score ≥3 SD) improvement in viable Calcein positive objects. <b>B:</b> As a secondary assay, the 320 candidate molecules were screened at 1500 cells per well. After two days, cells were stained with the pluripotency marker OCT4 and counterstained with Hoechst 33342, in place of Calcein staining. The compounds identified in (<b>A</b>) are again circled. T6140247 is found clustered with other experimental samples (black) and negative control wells (red), while HA-1077 is found clustered with positive control wells treated with 10 µM HA-1077 (blue).</p

Lower hESC plating density results in greater observed changes in survival using propidium iodide.

<p>H1 OCT4-GFP hESCs were plated at the densities shown and treated with 10 µM HA-1077 (blue) or 0.1% DMSO (red). Colored bars represent average number of PI-positive objects identified ±SD. Values over bars represent fold increase in objects after treatment with HA-1077 relative to DMSO control. Statistical significance of HA-1077 treatment was determined using analysis of variance (ANOVA, ***: p<0.001).</p

Small molecules identified in the OCT4-GFP screening assay were not confirmed in secondary assays.

<p>Three treatments are compared to assess changes in survival of dissociated hESCs: 0.1% DMSO (negative control; <b>A,D</b>), 10 µM HA-1077 (positive control; <b>B,E</b>), and 10 µM AST 5588603 (representative of small molecules identified in OCT4-GFP screening assay; <b>C,F</b>). <b>A-C:</b> Readout from the Acumen microplate cytometer. The wells treated with HA-1077 and AST 5588603 had similar numbers of GFP positive objects (quantified in <b>G</b>). <b>D-F:</b> Alkaline phosphatase staining of cells treated as described. Pluripotent cells are stained with a red color. Wells treated with HA-1077 (<b>E</b>) contain significantly more pluripotent cells than cells treated with DMSO or any other candidate compounds. <b>G:</b> Quantification of GFP signal detected in (<b>A-</b>C). <b>H:</b> Quantification of alkaline phosphatase staining in (<b>D-F</b>) by calculating what percent of the well area stained positive for alkaline phosphatase. Data shown as mean ±SD, ***: p<0.001.</p

A perimeter-based object identification scheme results in the most robust detection of surviving hESCs.

<p>Three methods for quantifying Calcein staining of hESCs were compared: Unfiltered imposed no constraints on the object identification, while Area filtering requires fluorescent objects to have a perimeter ≥50 µm and area ≥125 µm² to be counted and Perimeter filtering requires objects to have a perimeter ≥100 µm. Images compare the two compounds highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054948#pone-0054948-g004" target="_blank">Figure 4</a>.6: HA-1077 (left) and T6140247 (right). For each category, objects identified are highlighted in white and quantified to the right of each image along with the percentage of the Unfiltered object count.</p

Small Molecule Screening with Laser Cytometry Can Be Used to Identify Pro-Survival Molecules in Human Embryonic Stem Cells

<div><p>Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.</p> </div

Altered hESC plating density improves the ability to detect changes in survival using Calcein AM.

<p>H1 OCT4-GFP hESCs were plated at the densities shown and treated with 10 µM HA-1077 (blue) or 0.1% DMSO (red) as a negative control. After 2 days viable cells were quantified. Colored bars represent average number of Calcein-stained objects identified ±SD. Values over bars represent fold increase in objects after treatment with HA-1077 relative to DMSO control. Statistical significance of HA-1077 treatment was determined using analysis of variance (ANOVA, ***: p<0.001).</p

REST KD results in altered signaling including an increase in MEK and WNT activity in embryoid bodies (EBs).

<p><b>A</b>. Western blot showing that REST KD H9 hESCs have increased pMEK1/2 (S217/221) expression compared to control NT H9 hESCs. MEK1/2 and β-ACTIN were used as loading controls. <b>B</b>. QPCR showing that REST KD H9 hESCs have increased expression of CFOS, a down-stream target of pMEK1/2. CFOS expression was examined in cells collected under four conditions: 24 hours after last feeding with media containing knockout serum (hESC); 24 hours after last feeding with media devoid of knockout serum (Starve); 30 minutes after switching from ‘Starve’ condition to knockout serum media; 4 hours after switching from ‘Starve’ condition to knockout serum media. REST KD hESCs had increased CFOS expression under all four conditions. <b>C</b>. Western Blot Analysis of Day 5 EBs for changes in MEK signaling revealed that pMEK 1/2 was increased in REST KD EBs. MEK ½ and GAPDH were used as loading controls. <b>D</b>. In order to evaluate WNT signaling, we evaluated expression of the WNT target genes AXIN2, Beta-CATENIN (B-CAT), FRIZZLED2 (FZD2) AND TROY in NT and REST KD hESCs as well as EBs, in H9 and H1 lines, via qPCR. Significant changes in expression of WNT target genes in REST KD hESCs are shown with a single asterisk (*). For both H1 and H9 REST KD hESCs, FZD2 was statistically significantly decreased (p<0.05). Significant changes in expression of WNT target genes in REST KD Day 5 EBs are shown with two asterisks (**). Across both lines of REST KD Day 5 EBs, we found a statistically significant increase in expression of WNT target genes AXIN2, B-CAT and TROY (p<0.022). Error bars represent SEM of three independent experiments and asterisks denote a p value < .05 using Student’s t-test analysis.</p

REST KD hESCs have increased survival.

<p><b>A</b>. REST KD and NT hESCs (H1 and H9) were evaluated for BrdU incorporation by FACS analysis for TRA-1-81 and BrdU double positive cells. <b>B</b>. Percentage of TRA-1-81and BrdU positive cells were not statistically significantly different in REST KD compared to control NT cells. Error bars represent standard error of three independent experiments (performed at p52, p54 and p60 for H9 cells, and at p47, p49, and p50 for H1 cells). <b>C</b>. REST KD and NT hESCs (H1 and H9) were evaluated for the percentage of Annexin V and DAPI positive cells by FACS analysis. <b>D</b>. FACS analysis for Annexin V and DAPI staining demonstrated that REST KD hESCs have statistically significant improvements in survival as demonstrated by reduced levels of apoptotic cells (p<0.032 in H9 and p<0.002 in H1) compared to control NT hESCs. Error bars represent standard error of three independent experiments (performed at p55, p57 and p59 for H9 cells, and at p55, p56, and p57 for H1 cells) and asterisks denote a p value < .05 using Student’s t-test analysis.</p

REST KD cells have increased mesendoderm lineage differentiation bias <i>in vitro</i>.

<p><b>A-B</b>. Evaluation of <i>in vitro</i> differentiation potential in Day 10 embryoid bodies (EBs). QPCR analysis revealed an increase in expression of endoderm and/or mesoderm markers in both H9 (<b>A</b>.) and H1 (<b>B</b>.) REST KD day 10 EBs. Shown are representative graphs of lineage marker analysis for each of the three germ layers. Error bars represent standard error of the mean (SEM) from three technical replicates. ND = Not detected. <b>C-E</b>. Evaluation of protein changes using FACS analysis across two independent REST KD lines (H9, H1) revealed an increase in expression of the mesendoderm marker BRACHYURY compared to control NT lines but no change in the ectoderm marker PAX6 or endoderm marker SOX17. Error bars represent SEM of three independent experiments and asterisks denote a p value < .05 using Student’s t-test analysis.</p

Molecular Recognition Enables Nanosubstrate-Mediated Delivery of Gene-Encapsulated Nanoparticles with High Efficiency

Substrate-mediated gene delivery is a promising method due to its unique ability to preconcentrate exogenous genes onto designated substrates. However, many challenges remain to enable continuous and multiround delivery of the gene using the same substrates without depositing payloads and immobilizing cells in each round of delivery. Herein we introduce a gene delivery system, nanosubstrate-mediated delivery (NSMD) platform, based on two functional components with nanoscale features, including (1) DNA⊂SNPs, supramolecular nanoparticle (SNP) vectors for gene encapsulation, and (2) Ad-SiNWS, adamantane (Ad)-grafted silicon nanowire substrates. The multivalent molecular recognition between the Ad motifs on Ad-SiNWS and the β-cyclodextrin (CD) motifs on DNA⊂SNPs leads to dynamic assembly and local enrichment of DNA⊂SNPs from the surrounding medium onto Ad-SiNWS. Subsequently, once cells settled on the substrate, DNA⊂SNPs enriched on Ad-SiNWS were introduced through the cell membranes by intimate contact with individual nanowires on Ad-SiNWS, resulting in a highly efficient delivery of exogenous genes. Most importantly, sequential delivery of multiple batches of exogenous genes on the same batch cells settled on Ad-SiNWS was realized by sequential additions of the corresponding DNA⊂SNPs with equivalent efficiency. Moreover, using the NSMD platform <i>in vivo</i>, cells recruited on subcutaneously transplanted Ad-SiNWS were also efficiently transfected with exogenous genes loaded into SNPs, validating the <i>in vivo</i> feasibility of this system. We believe that this nanosubstrate-mediated delivery platform will provide a superior system for <i>in vitro</i> and <i>in vivo</i> gene delivery and can be further used for the encapsulation and delivery of other biomolecules