The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers

Abstract

Background: Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions.

Methodology/principal findings: HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions.

Conclusions/significance: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.

Figure 1. Sorting strategy for hESCs.

Bright field images of A) H9 hESCs grown on feeders at passage 42 prior to sorting and B) H9 hESCs grown feeder-free at passage 33 prior to sorting. Cells were stained with SSEA-1, SSEA-3, and TRA-1-81 fluorochrome-conjugated antibodies. C) Single cells were first identified by light scatter gating, D) SSEA-1⁻ cells were gated out and SSEA-3⁺TRA-1-81⁺ cells were gated and then sorted. E) Gating hierarchy and F) post-sort purity is shown. Scale bars in (A) and (B) are 100 µm.

Figure 2. Morphology of hESCs after sorting without and with application of Y-27632.

Bright field images of H9 hESCs at various days after sorting without and with Y-27632. A) HESCs grown on feeders (cells were passage 42 prior to sorting) B) HESCs grown feeder-free (cells were passage 33 prior to sorting). (*) indicates the day at which cells were passaged after the sort. Scale bars are 100 µm.

Figure 3. Recovery of hESCs grown feeder-free after sorting and passaging without and with Y-27632.

Percent recovery of H9 hESCs at day 1 after A) sorting or B) passaging with Accutase. Percent recovery equals the number of cells recovered at day 1 after passage or sorting divided by the number of cells plated. A total of two (Trial 3) to three wells (Trials 1, 2 and 4) were counted for each trial group with error bars representing standard deviation. C) Fold increase in the recovery of cells at day 1 upon addition of Y-27632 after sorting. The number of cells recovered without Y-27632 was set at 1. D) Percent recovery at day 1 post-sort after pre-treatment of cells with Y-27632 and Y-27632 post-sort titration. A total of three wells were counted for each trial group with error bars representing standard deviation.

Figure 4. Characterization of hESCs upon extended passaging after sorting with application of Y-27632.

A) Bright field image of H9 hESCs cultured on feeders at passage 26 post-sort and day 4 after passaging. B–D) Immunofluorescence labeling of hESCs cultured in feeder-growth conditions at passage 26 post-sort. E) Bright field image of hESCs cultured in feeder-free growth conditions at passage 10 post-sort and day 3 after passaging. F–H) Immunofluorescence labeling of hESCs in feeder-free growth conditions at passage 10 post-sort. Post-sort marker expression of H9 hESCs as measured by flow cytometry of I) Passage 14 post-sort on feeders with Y-27632 and passage 13 post-sort without Y-27632 and J) Passage 13 post-sort feeder-free hESCs with and without Y-27632. The percent positive expression from flow cytometry experiments after extended passage post-sort with and without Y-27632 is shown (data from flow analysis experiments is shown in Fig. S4). Scale bars in (A–H) are 100 µm.

Figure 5. In vitro differentiative capacity of hESCs upon extended passaging after sorting with Y-27632.

Bright field images of A) day 5 embryoid bodies from hESCs on feeders at passage 13 post-sort and E) day 6 embryoid bodies from hESCs feeder-free at passage 12 post-sort. Immunofluorescence of differentiated hESCs shows labeling for mesoderm (B, F) (GATA4 and cTnI and Desmin), ectoderm (C, G) (Nestin and Sox1), and endoderm (D, H) (FoxA2, Sox17). For feeder conditions (B–D), hESCs are at passage 14 post-sort (B); passage 13 post-sort (C); and passage 16 post-sort (D). For feeder-free conditions (F–H), hESCs are at passage 12 post-sort (F); passage 12 post-sort (G); and passage 13 post-sort (H). Scale bars are 100 µm.

Figure 6. In vivo differentiative capacity of hESCs upon extended passaging after sorting with Y-27632.

Hematoxylin and Eosin staining of cell-injected spinal cords. Teratomas are from hESCs cultured in feeder-dependent growth conditions passage 21 post-sort (A–F) and feeder-independent growth conditions at passage 12 post-sort (G–K). Individual germ layers were identified by the presence of ectoderm (neural rosettes) (E, H; arrows), endoderm (simple columnar epithelia or goblet cells) (B, J; arrows) and mesoderm ((loose connective tissue of early muscle (F, I ; arrows), the honey-comb like vacuoles of adipocytes (C, K; asterisks) and newly formed blood vessels (D) derivatives). Scales bars: (A,G): 300 µm; (H): 50 µm; (J, K): 30 µm; (B, C, D, E, F and I): 20 µm).

Figure 7. Karyotype analysis of hESCs upon extended passaging after sort with the application of Y-27632.

Post-sort karyotype of H9 hESCs grown on A) feeders at passage 55 (passage 18 post-sort) with Y-27632 and B) feeder-free at passage 50 (passage 21 feeder-free and passage 16 post-sort) with Y-27632.