Distinct differentiation characteristics of individual human embryonic stem cell lines

Abstract

Background: Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic.

Results: The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines.

Conclusion: hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.

Figure 1

Morphology of hESC colonies. Cells grown on MEF (A and C) or HFF (B and D) after enzymatic passaging with collagenase IV. The initially attached colonies are smaller on MEF (A and B, FES 22 cells 36 h after passaging). At three days, the colonies on MEF (C) are larger, better defined and more homogenous than on HFF (D). Scale bar is 100 μm.

Figure 2

Effect of feeders on cell proliferation. Analysis of population doubling time on different types of feeders. Daily cell counts of triplicate wells of FES 22 cells are shown (mean ± SD).

Figure 3

Karyotype analysis. Abnormal karyotypes of two FES lines. FES 22 (48, XY, +12, +17; A) and FES 30 (47, XX, +20; B) cells after prolonged enzymatic passaging on MEF.

Figure 4

Gene expression. RT-PCR analysis of expression of typical genes associated with pluripotency in hESC, together with some primordial germ cell markers in undifferentiated hESCs (FES 22, 29 and 30), embryoid bodies (FEB22, 29 and 30), pooled stage 3 (ST3) differentiated cells and negative control (H₂O).

Figure 5

Analysis of embryoid bodies (EBs) and teratomas. Specific markers of all three germ cell layer derivatives detected in EBs (A-D) and teratomas (E-G) from FES line 29. Phase contrast image of EB (A). Neuroectoderm differentiation is indicated by immunostaining for neurofilament (B, F); mesoderm differentiation by expression of brachyury (C) and desmin (E); endoderm differentiation by positivity for HNF3β (D, G). Original magnification 10× (A), 20× (B, C, E-G), 40× (D).

Figure 6

Analysis of EB formation. Fifty hESC colonies were placed in suspension culture and the number of developing EBs counted after 2, 4 and 7 days. The FES 30 line formed fewer EBs already after 4 days as compared to FES 22 and 29 (p < 0.01 at d 4 and < 0.001 at d 7, mean ± SD, n = 7).

Figure 7

Cell-line dependent differentiation patterns. Spontaneous differentiation is lower in 7-day EBs (A-D) and teratomas (E-F) derived from FES 30 cells (B, D, F) than FE2 22 cells (A, C, E). OCT-4 was detected by immostaining in EBs (A-B, dark brown color) and it remained much higher in FES 30 EBs. In contrast, brachyury expression, as a marker of mesodermal differentiation, was clearly detectable in FES 22 (C) but was not detected in FES 30 EBs (D). Teratomas derived from FES 22 cells showed typical multi-lineage tissue differentiation (E) while a major part of FES 30 teratomas consisted of cystic structures (collapsed cyst wall shown in F). Original magnification 4×.