Integrated chemical genomics reveals modifiers of survival in human embryonic stem cells

Abstract

Understanding how survival is regulated in human embryonic stem cells (hESCs) could improve expansion of stem cells for production of cells for regenerative therapy. There is great variability in comparing the differentiation potential of multiple hESC lines. One reason for this is poor survival upon dissociation, which limits selection of homogeneous populations of cells. Understanding the complexity of survival signals has been hindered by the lack of a reproducible system to identify modulators of survival in pluripotent cells. We therefore developed a high-content screening approach with small molecules to examine hESC survival. We have identified novel small molecules that improve survival by inhibiting either Rho-kinase or protein kinase C. Importantly, small molecule targets were verified using short hairpin RNA. Rescreening with stable hESCs that were genetically altered to have increased survival enabled us to identify groups of pathway targets that are important for modifying survival. Understanding how survival is regulated in hESCs could overcome severe technical difficulties in the field, namely expansion of stem cells to improve production of cells and tissues for regenerative therapy.

Figure 1

Design of high-content screen for compounds that increase OCT4-positive hESCs. (A): hESCs were dissociated and plated at low density onto mouse embryonic fibroblasts, and small molecule inhibitors were added in duplicate 384-well plates. After 4 days in culture, hESCs were stained with OCT4, a marker for hESC pluripotency (green) and nuclei (Hoechst, blue). Red lines are drawn to a larger image from two of the wells, showing more hESC OCT4-positive (upper) and a low or average number of hESC OCT4-positive cells in control wells (lower). (B): A representative heat map from a Biomol Enzyme plate showing targets that have increased numbers of OCT4-positive hESCs. Red and black cells represent increased hESC numbers relative to controls and green cells represent decreased or unchanged numbers of hESCs. (C): Cluster analysis was used to identify positive hits that improve hESC growth based on W2 (number of OCT4-positive cells or percentage of W2 compared with control wells; Materials and Methods). Abbreviation: hESC, human embryonic stem cell.

Figure 2

Data mining reveals small molecule targets that increase OCT4-positive hESCs. (A): Scatter plot from the combined libraries (Biomol and Prestwick) showing targets that increase the percentage of W2 (OCT4)-positive hESCs. (B, C): Control hESCs (B) or hESCs treated with the Rho-kinase inhibitor, HA (C). In the presence of 10 μM HA, there was a dramatic increase in OCT4-positive hESCs as shown by immunofluorescence, with OCT4 in green and nuclei (Hoechst) in blue. (D): Cluster view of a representative Biomol Enzyme plate showing targets that improve hESC growth on the basis of percentage of positive W2 (OCT4). (E): Comparison of HSF1 hESC growth using fluorescence-activated cell sorting analysis, using the pluripotency marker, SSEA4, in the presence of 10 μM HA or H7; two hits from the small molecule screen. Treatment with 10 μM HA or H7 resulted in statistically significant numbers of SSEA4-positive hESCs at p ≤ .05. All studies were performed on mouse embryonic fibroblasts (MEFs). We have also validated that the H7 and HA small molecules can also promote survival on Matrigel (data not shown), suggesting that these targets can also regulate hESC survival independently of MEFs. Abbreviations: HA, HA-1077; hESC, human embryonic stem cell; SSEA4, stage-specific embryonic antigen 4.

Figure 3

hESCs in HA or shRNA to Rho-kinase have similar numbers of hESCs after trypsin dissociation. (A): hESCs were trypsinized, grown in regular hESC medium on mouse embryonic fibroblasts (MEFs), and stained by immunofluorescence (IF) using an antibody against OCT4 (green) and nuclei (Hoechst, blue). (B): hESCs in the presence of HA (10 μM) were trypsinized and stained by IF using an antibody against OCT4 (green) and Nuclei (Hoechst, blue). (C): Quantitative reverse transcription polymerase chain reaction analysis of HSF1 hESC only, NT HSF1 hESCs compared with hESCs plus shRNA to ROCK (RI or R1) or ROCK 2 (RII or R2). (D): Western blot showing protein levels of NT control HSF1 hESCs compared with levels of RI and RII (at 160 kDa) in the shRNA-Rho-kinase hESC line. GAPDH was used as a loading control. (E): hESCs were grown on MEFs in the following conditions and then subjected to fluorescence-activated cell sorting analysis of SSEA4+hESCs: in hESC medium only, NT control HSF1 in hESC medium only, hESC medium plus 10 μM HA, NT control HSF1 in HA (10 μM), or shRNA to RI and RII (SH-Rho-kinase) in hESC medium. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, HA-1077; hESC, human embryonic stem cell; NT, nontarget; ROCK, Rho kinase; shRNA, short-hairpin RNA; SSEA4, stage-specific embryonic antigen 4.

Figure 4

Short hairpin RNA (shRNA)-Rho-kinase human embryonic stem cells (hESCs) are pluripotent and karyotypically stable. (A, B): Karyotype analysis of HSF1P62 (A) or HSF1P79 human embryonic stem cells (hESCs) with shRNA to Rho-kinase (B). (C): Example embryoid body formation using shRNA-Rho-kinase hESCs. (D): Hematoxylin and eosin (H&E) section of a teratoma formed from HSF1 hESCs with shRNA to Rho-kinase (×5). Differentiation of the SH-Rho-kinase hESCs into representative lineages (endo-, ecto-, and mesoderm) is shown at 10X in neural rosette (E), bone/cartilage (F), muscle (G), and gut-like structures (H).

Figure 5

hESCs grown in HA-1077 (HA) or short hairpin RNA (shRNA) to Rho-kinase can be used to screen for compounds that decrease hESC survival. (A): A representative Biomol Enzyme plate showing increased OCT4-positive hESCs in the screening assay in the presence of HA or shRNA to Rho-kinase. Red lines are drawn to larger images from three of the wells, showing both decreased hESC and mouse embryonic fibroblasts (upper), increased or average hESCs (middle), and decreased hESC only (lower). (B): A representative heat map from a Biomol Enzyme plate showing that most of the wells have increased numbers of surviving hESCs. Red to black represents higher hESC numbers, and green represents decreased or lower numbers of hESCs. (C): Cluster analysis was used to identify positive hits that either improve or decrease hESC growth on the basis of percentage of positive W2 (OCT4). Abbreviation: hESC, human embryonic stem cell.

Figure 6

Data mining reveals small molecule targets that decrease OCT4-positive human embryonic stem cells (hESCs). (A): Scatter plot from the combined libraries (Biomol and Prestwick) showing targets from the HA-treated hESCs. (B): Scatter plot from the combined libraries (Biomol and Prestwick) showing targets from the shRNA-hESCs. (A, B): Arrows represent potential clusters of targets that decrease OCT4-positive hESCs. (C, D): Treatment of HSF1 hESCs with 10 μM simvastatin results in decreased SSEA4-positive hESCs. Comparison of wild-type (C) and shRNA (D) treated with HA and/or simvastatin revealed increased Annexin-positive (cell death) hESCs upon simvastatin treatment, whereas treatment with HA or shRNA-hESC lines had reduced Annexin-positive cells. Abbreviations: HA, HA-1077; shRNA, short hairpin RNA; SSEA4, stage-specific embryonic antigen 4.

Figure 7

hESC survival is mediated by alteration in components of mitogen-activated protein kinase (MAPK) and p53 activity. Examination of MAPK transcription factor activity in the shRNA- or HA-treated cells as demonstrated using Eppendorf's MAPK Transcription Factor chromatin immunoprecipitation kit. Activity of p53 but not other MAPK components was decreased in the HA-treated or shRNA-Rho-kinase hESCs. *, The decrease in p53 activity was statistically significant at p ≤ .0085. Abbreviations: HA, HA-1077; hESC, human embryonic stem cell; shRNA, short hairpin RNA.