To illustrate the efficacy of the rapid hESC enumeration procedure and to exemplify the importance of optimal seeding density for the maintenance of undifferentiated hESC growth, we seeded hESCs in clumps at varying cell densities of 0.05, 0.1, 0.5, 1, and 2 × 105 hESCs/well (based on the PI hESC quantification method) and subsequently cultured these for 10 days. Figure 2A demonstrates the density of MEL1 colonies at days 2, 6, and 10 that were plated at varying cell densities based on cell counts using the PI assay. MEL1 cells grew as a monolayer in every condition except the 2 × 105 cell seeding density, wherein overcrowding of the cells was evident at day 10. Early morphological analysis (day 2 after seeding) of the lower-density cultures (0.05–0.1 × 105 cells/well) showed cells of an elongated or triangular morphology (Figure 2, B and C, black arrows) whereas cells seeded at higher densities (≥0.5 × 105) displayed a typical round morphology with a high nucleus-to-cytoplasm ratio that is characteristic of undifferentiated hESCs (Figure 2D). Next, the hESCs were analyzed for the expression of the pluripotency marker CD9 by direct immunofluorescence and expression of both CD9 and GCTM2 by flow cytometry. In agreement with the apparent reduction in the fraction of undifferentiated hESCs by morphological criteria (Figure 2, BB–D), the two lowest cell seeding densities (0.05 and 0.1 × 105 cells/well) showed reduced expression of CD9 compared with the higher seeding densities (0.5, 1, and 2 × 105 cells/well, Figure 2A). In contrast, however, the CV for the spread of pluripotency markers was decreased for lower-seeded populations (0.05 × 105 cells/well, GCTM2 CV = 54.5±1.9, CD9 CV = 52.1±1.5) compared with higher-density cultures (0.1 × 105 cells/well, GCTM2 CV = 67.9±7, CD9 CV = 69.7±8.7) indicating a more uniform cell type in the lower cell densities. We hypothesize that seeding cells at low densities, while leading to increased differentiation (Figure 2, A–C), also selects for a subset of cells capable of survival that results in a purer population of undifferentiated cells with a narrower distribution of pluripotency markers (Figure 3A). In contrast, cells seeded at higher densities allow for the proliferation and survival of a larger subset of undifferentiated cells.
Although our data indicate that the PI assay can be used to accurately seed hESCs at different densities and highlight the importance of determining appropriate seeding densities for hESC culture, it should be noted that undifferentiated cell growth is dependent not only on cell numbers/well but also clump size (9) and that this varies between different culture systems and harvesting techniques. The counting method described here performs equally well on hESCs with various clump sizes (unpublished data) and thus will allow hESC culture optimization provided the clump size within a specific culture protocol is kept consistent.
To illustrate the power of the hESC enumeration assay described in this paper in high-throughput multifactorial analyses, we next seeded hESC at identical densities, based on our PI assay (verified with manual cell counting) on Geltrex- or ECM-coated wells and determined cell numbers after 10 days using the PI assay. Figure 3B shows that there was very little interwell variation in hESC cell numbers and that the PI assay was able to successfully differentiate between wells of different cell densities with high accuracy (P<0.05, Student's two-tailed t-test). Finally, to further demonstrate the power of the assay in an end point analysis, growth curves for MEL2 grown on Geltrex in StemPro were determined using three independent cell number determinants: manual hemocytometer counts, an Incucyte cell imager (Essen Instruments) and the PI assay. Figure 3C shows a comparative growth curve of the PI assay and the manual cell counts. The Incucyte growth curve data was not directly comparable to the other two methods as it is based on confluence rather than overall cell number. However, it was possible to directly compare population doubling times (PDT) determined by all three methods (Figure 3C). Although the PDT calculated using the automated cell imager was closer to the PDT determined using the hemocytometer cell counting method, there are a number of concerns using this type of automated analysis in hESC cultures. First, the errors are significantly greater than either the manual or PI cell counting methods (±4.21 h compared with ±1.04 and ±1.57 h, respectively). Second, the PDT is determined using percent confluency and not overall cell numbers, which, in heterogeneous hESC cultures that differentiate into cells with dramatically different surface area, can introduce further errors or overestimated PDTs. These and other automated systems (11) are limited by their ability to perform confluency measurements only on a two-dimensional (surface when it is well established that hESCs often grow in three-dimensional colonies. Thirdly, our method is cheap and does not require advanced instrumentation when compared with automated imaging systems.
Our data indicate that the assay performs well when compared with manual counting and has the added advantage of removing operator-mediated counting errors and judgment calls when scoring cell numbers with a hemocytometer. The assay requires access to a standard fluorescence plate reader and can in principle be adapted to the use of other DNA-binding fluorophores such as picogreen or Cy-5. We calculate that when using this assay it is possible to accurately enumerate the cell number of up to 96 hESC clump suspensions in 45 min (15 min pipetting + 30 min read) while the same outcome using a manual counting method would require over 8 h (dissociation 3 min, hemocytometer counting 2 min = 5 min; 5 min × 96 suspensions = 480 min = >8 h. In each case, the time taken for harvesting the cells from the plates was not considered. Adoption of this cost-effective and rapid technique will allow for high-throughput screening of hESC numbers for relative end point analysis of cell proliferation, growth curves, and consistent seeding of cells.