Different hESC lines exhibit different colony morphologies
Undifferentiated hESC lines BG01V, I3 and I6 that had been maintained on mitomycin C-treated mouse embryonic fibroblasts (MEF) for 4–5 days displayed distinct cell colony morphologies. Cells in the center regions of colonies exhibited prominent nucleoli and a high nucleus to cytoplasm ratio. Cells from BG01V (passage 22) and I6 (passage 50) lines were highly compact with rather vague borders (Figure and and ). Their colonies exhibited a round and sharp edge separating the hESC from surrounding feeder cells. In contrast, colonies of I3 cell line (passage 58) exhibited a mosaic appearance with loosely packed cells (Figure and ). This observation is consistent with previous descriptions [21
Figure 1 (A-C) Morphology of undifferentiated colonies of hESC lines I3, I6, and BG01V. Phase contrast photographs of hESC colonies cultured on mouse embryonic fibroblast feeders for 4 days. The boxes in the center of the colonies indicate areas shown in A1-C1. (more ...)
Differences in growth curves between undifferentiated hESC lines
To examine potential differences in the ability to self-renew between the three hESC lines, the percent increase in cell numbers relative to cell numbers at day 1 were calculated up to 6 days (Figure ). The relative growth was determined, followed by the plating of approxmately 105 cells on a MEF feeder, and trypsinized cells were counted using a Cedex Analysis System. The differences in the cell number between each cell lines should represent the differences in relative growth of hESCs since the fibroblast feeder cells were mitomycin C-treated. The growth curves in Figure show significant differences in percent increase in the number of cells at day 6 in culture between the three cell lines. I6 and BG01V cell colonies reached an average size of 300–400 cells for splitting at 5–6 days after passaging. In contrast, I3 cells grew slower than the other two cell lines and were not ready to passage until culture day 8. It appeared more difficult to maintain the I3 cell line in an undifferentiated state since it had more tendency to differentiate.
MPSS expression analysis of three human ESC lines
After full annotation of over one million sequenced signatures from each ESC line, there were 28, 071 human UniGene clusters detected by MPSS in the three human undifferentiated ESC lines (BGO1V, I3 and I6). I6 expressed the most genes (25,862) and I3 expressed the fewest genes (10, 945); BGO1V cells expressed 19, 858. Although the differences in numbers of total expressed gene numbers among the three ESC lines was significant, the number of core transcriptome expression of the three hESC lines were substantial: 95.7% (10479/10945) and 93.3% (10210/10945) of I3 genes were expressed in I6 and BGO1V, respectively, and 92% (10,057 out of 10, 945) genes of I3 were co-expressed in the other two lines (Figure ). These findings indicate that different hESC lines retain core transcriptome features of embryonic "stemness".
Figure 2 Digital gene expression of the three undifferentiated hESC lines. It shows that around 92% (10057 out of 10945) of the genes expressed in the I3 cells were also expressed in the BG01V and I6 cells. The large overlap in genes expressed among the 3 hESC (more ...)
All undifferentiated hESC lines express pluripotency markers, but their gene expression levels are variable
The undifferentiated state of human ES cells was first characterized by immunocytochemistry. All the three hESC lines expressed glycolipid antigens such as stage-specific embryonic antigen SSEA-4, tumor rejection antigen TRA-1-81, and transcription factors Oct-3/4 and NANOG as described previously [23
]. All cell lines also exhibited alkaline phosphatase (AP) activity (Figure ). To further study the quantitative expression of pluripotency markers, quantitative RT-PCR and MPSS were used to assay the expression of a series of genes in each of the three undifferentiated hESC lines (Figure ) and in their differentiated EB. We cross validated the results of quantitative RT-PCR and MPSS analysis of the expression levels of undifferentiated hESC marker genes (NANOG, POU5F1, and UTF1) in the three hESC lines over time.
Figure 3 Differences in relative expression levels of undifferentiated cell ("stemness") markers among the three undifferentiated hESC lines. (A) Immunofluorescent staining of hESC colonies shows an expression of Oct3/4, NANOG, SSEA4, TRA-1-81 and alkaline phosphatase (more ...)
The Y-axis plots the fold change of the undifferentiated I6 and BGO1V cell lines in comparison to the undifferentiated I3 cell line. The expression level of the undifferentiated genes implicates that the I3 hES cells express much lower levels of "stemness" (undifferentiated) genes compared to the I6 and BG01V cell lines (Figure and ).
Differences in BrdU incorporation between undifferentiated I3, I6 and BG01V cells
To examine the potential difference in the ability to proliferate between different the hESC lines, bromodeoxyuridine (BrdU) incorporation assays were performed in colonies of I3, I6 and BG01V cells (Figure ). hESC maintained on the MEF for 4 days under the same conditions were incubated with BrdU for 4 h before being processed for BrdU immunocytochemistry (Figure ). Cells were counterstained with PI which stained nuclei of all cells (Figure ). We indexed proliferation for each cell line by quantifying the proportion of BrdU+ cells versus the total number of cells (PI-stained cells). At 4 days post-passaging, all three cell lines showed active DNA synthesis, but the I3 cell line exhibited significantly smaller colonies and a lower proliferation rate compared to the other two cell lines (Figure ). Unlike the I6 and BG01V cell colonies within which BrdU+ cells were confined, some BrdU+ cells were scattered outside of the I3 colonies, although the majority of BrdU+ cells were located within the colonies (Figure ). The scattered BrdU+ cells may represent individual proliferative I3 cells on the MEF layer before forming a colony, indicating much slower proliferation in the I3 cells compared to the I6 and BG01V cells. Cell counting showed differences in BrdU incorporation between the three cell lines. BG01V cells exhibited highest BrdU incorporation levels (88% ± 12%), while I3 cells had the lowest levels (62% ± 3.1%) (Figure ). The proliferation rate in I6 cells was 78% ± 9.2%. The difference between three cell lines in BrdU incorporation was consistent with the sizes (diameters) of the colonies. At 4 days in culture I3 cells exhibited smaller colonies with a lower proliferation index compared to the BG01V and I6 cells. Colonies of the I6 and BG01V cells exhibited higher numbers of active DNA synthesizing cells compared to the I3 cells.
Figure 4 Differences in the proliferation rate between the undifferentiated I3, I6 and BG01V cells. All three cell lines were maintained on the MEF for 4 days post-passaging under the same culture conditions. A four hour BrdU pulse shows significant differences (more ...)
Embryoid body formation and growth rate vary among the three hESC lines
EB represents a unique tool to investigate in vitro differentiation processes of hESCs. Colonies of the three ESC lines were removed from feeder cells and grown as cell aggregates in a suspension in low attachment dishes without basic fibroblast growth factor. The growth rate of the EBs was calculated by the increase of EB size (total EB area) in every 5 days period from days 1 though 25 (Figure ). The percent increase in size of the cell spheroids was significantly different in the EBs from the three different hESC lines after 10 days in the suspension culture (Figure ). The relative EB growth was greater in the I3 and BG01V cells compared to the I6 cells.
Figure 5 Differences in the embryoid body (EB) growth rates between the hESC lines I3, I6 and BG01V. hESC colonies were removed from feeder monolayers and grown in low attachment dishes. (A) Phase contrast images of the EBs in the three cell lines cultured in (more ...)
All three hESC lines are able to differentiate into cells expressing markers of all three germ layers and neural cells
EB formation is a model of in vitro
embryogenesis in which all three primary embryonic germ cell lineages are generated [24
]. To examine differences in the gene expression of the three germ layer markers in EBs derived from three ESC lines, we assessed the expression of keratin C (ectoderm) and alpha-globin (mesoderm) by qRT-PCR (Figure ), and expression of alpha FP (endoderm) and IGF2 (mesoderm) by MPSS (Figure ). The fold changes (log scale) in the expression levels of keratin C and alpha-Globin for each cell line relative to its own undifferentiated levels showed that the I3 cell line expressed markedly higher levels of these two genes at 7 and 14 days when compared to the I6 and BG01V cell lines (Figure ). MPSS results showed that the I3 cell line expressed higher levels of both Alpha FP and IGF-2 than the I6 and BG01V cell lines (Figure ).
Figure 6 Differences in expression of three germ layer markers in the embryoid bodies (EBs) derived from three hES cell lines BG01V, I3 and I6. (A) Quantitative RT-PCR analysis of expression of keratin C (ectoderm marker) and alpha-Globin (mesoderm marker) at (more ...)
Since a primitive neural stem cell stage can be acquired though a default mechanism [25
], we assessed the expression levels of the neural markers, Nestin and Musashi 1 (neural progenitor), MAP2 (mature neurons) and S100B (astrocytes) by qRT-PCR and MPSS (Figure ). qRT-PCR analysis showed differences in expression levels of these genes between the three hES cell lines. The I3 and I6 cells expressed higher levels of neural progenitor-specific genes (Nestin and Musashi) (Figure ), as well as neuronal gene (MAP2) and glial gene (S100B) (Figure ) than BG01V cells. Both MAP2 and S100B genes were upgraded from day 7 to Day 21 in culture in the I3 and I6 cells.
Figure 7 Quantitative RT-PCR analysis shows differences in expression levels of neural cell lineage-specific genes between three hES cell lines. The Y-axis plots the fold change for each cell line when compared to its own undifferentiated cells (Day 0). (A) shows (more ...)
Differences in expression of neural phenotypes and genes in directed neural differentiation between three hESC lines
Differences in pluripotency between the three cell lines were also examined during the directed neural differentiation. To test the possible differences we used a reliable step-wise differentiation protocol (Figure ) which generated highly pure neural progenitors from the I3 and I6 cells [18
]. The hESC colonies were removed from MEF feeders and cultured in suspension in low attachment dishes with hESC medium without bFGF for 5 days (Figure ). Differentiating EBs were transferred into the neural differentiation medium for 10 days. At days 15–17 of differentiation, EBs were plated on poly-D-lysine/laminin-coated 35 mm dishes. Neural rosettes were visualized after plating of the EBs on the substrate (Figure ). Neuroectodermal cells in neural rosettes were stained for Sox1 and Nestin (not shown), and further differentiated into neural progenitors and their progeny (Figure ). Under the same neural differentiation-promoting condition, we quantified the percentage of hESC-differentiated neural progenitors by immunostaining for Nestin, together with nuclear DAPI counterstaining (Figure ). Nestin+ cells were manually counted and were expressed as a percentage of the total DAPI labeled cells (Figure ). A significant difference was found in the percentage increase in number of Nestin+ cells differentiated from the three cell lines after 3 days post plating (Figure ). The percentage increase in Nestin+ cells generated from the I3 cells was greater than that of the I6 cells. The BG01V generated undetectable or an insignificant number of neural progenitors which were lightly scattered among differentiated cells. In addition, we tracked the appearance of neural rosettes during neural differentiation and found that the I3-derived rosettes were generated 5–9 days earlier than the I6-derived neural rosettes. The BG01V-derived neural rosettes were barely detected.
Figure 8 The comparison of the neural progenitor derivation from the I3, I6 and BG01V hESC lines over time under the same neural differentiation-promoting condition. (A) The protocol used to direct hESCs into neural cell lineages. (B-E) Phase contrast images show (more ...)
Figure 9 Difference in directed neural differentiation between hESC lines I3, I6 and BG01V. Two days after transferring EBs to a poly-D-lysine/laminin substrate, parallel immunocytochemistry and quantitative RT-PCR were performed in hESC-derived cell populations. (more ...)
To assess differences in the gene expression during neural differentiation between the three cell lines, total RNA was harvested from the EBs at 2 days post plating on poly-D-lysine/laminin substrates. The qRT-PCR analysis of the expression levels for the neural progenitor markers SOX1 and MSI1, the mature neuron marker MAP2, and the astrocyte marker GFAP showed that the all these genes were strongly expressed in the I3 cells (Figure ). The Y-axis plots the fold changes of gene expression for each cell line when compared to its own undifferentiated levels at Day 0. The high levels of expression for the neural progenitor genes SOX1 and MSI1 in the I3 and I6 cells were consistent with their high expression of nestin immunoreactivity (Figure ).