Abstract
Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs.
Highlights
In our previous work, human amnion epithelial cells served as feeder cells and successfully maintained the undifferentiated growth of mESCs and Human embryonic stem cells (hESCs)
Derivation and characterization of hESC lines on human amnion epithelial cells (hAECs). hESC lines are usually derived via immunosurgery to isolate the inner cell mass (ICM) from the human blastocyst [14,15]
Hundreds of hESC lines have been established since the first successful attempt was reported by Thomson et al [1,23]
Summary
Human amnion epithelial cells (hAECs) served as feeder cells and successfully maintained the undifferentiated growth of mESCs and hESCs. It was found to be possible to reprogram mESCs maintained on hAECs to adopt naïve-like pluripotent traits [12] These findings suggest that suitable conditions are important to maintain the pluripotency of hESCs. In order to use hESCs for therapeutic applications, such as regenerative medicine, it is important to develop quality humanized culture environments that support derivation, expansion, and differentiation. HAECs have many advantages over mitotically inactivated MEFs. In order to use hESCs for therapeutic applications, such as regenerative medicine, it is important to develop quality humanized culture environments that support derivation, expansion, and differentiation. HAECs grow slowly and do not express telomerase [13] For this reason, neither mitomycin C nor gamma irradiation treatments are necessary. Characterization of these hESC lines, including visualization of their fine structures was performed
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