![]() To increase the efficiency of hESC derivation using blastomere-derived blastocysts, a modified approach using culture medium supplemented with laminin was employed, which resulted in increasing the efficiency of this process to levels similar to hESC derivation from whole blastocysts (15). Development of an ICM from isolated blastomeres was found to be inefficient because the blastomere-derived aggregates mostly gave rise to trophectoderm-like vesicles. To address this issue, many attempts have been made to generate hESCs from earlier stages of embryonic development without destruction of the embryo, and the derivation of hESCs from a single blastomere at the 8-cell stage has been reported through co-culturing of isolated blastomeres with hESCs (13, 14). With regard to ethical and political concerns, hESCs derived from the ICM at the blastocyst stage have been criticized because destruction of the embryo results from this process. It is unclear which approach is more efficient, but it is certain that derivation of xeno-free hESC lines will be suitable for future clinical application of hESCs. Human ESC lines can be derived from outgrowths of the ICM after plating intact blastocysts on feeder cells, without isolating the ICM (12). ![]() The derivation of hESC lines from abnormal human pre-implantation genetic diagnosis (PGD) embryos using laser dissection for the isolation of ICMs has been reported (11). Mechanical dissection (9) and mechanical isolation of the ICM using flexible metal needles with sharpened tips (10) has been introduced as a method for hESC derivation. To avoid the contamination of animal-derived components during the derivation procedure, several methods have been used instead of immunosurgery. However, these reagents may include animal pathogens and molecules, and the exposure of animal-derived products to the ICM and its derivatives (8), hESCs, during immunosurgery may bring about immune responses in patients after transplantation. ![]() In the initial stages of hESC research, immunosurgery procedure employing anti-human serum and guinea pig complement was used for isolation of the ICM (4- 7). The most important process involved in hESC derivation might be the isolation of the ICM from the TE. The successful derivation of a mouse embryonic stem cell line from ICM was first reported in 1981, and subsequently, advances in mESC culture techniques were applied to generate ESC lines in non-human primates and humans. In mammalian embryonic development, the blastocyst consists of an inner cell layer called the inner cell mass (ICM) and an outer cell layer called the trophoectoderm (TE). Then, we will introduce several cryopreservation methods for hESCs, which is important for the production of a cell bank.ĭerivation conditions for human embryonic stem cells In this review, we describe the derivation and culture conditions of hESCs based on recent advances. ![]() Efforts on the part of several stem cell registries to share information regarding various hESC lines, and requests for the development of large banks of hESC lines have been put forth. Since the first report of hESC establishment, approximately 1,000 hESC lines have been established worldwide (International stem cell registry, University of Massachusetts medical school ). Guided-differentiation protocols of hESCs into a given functional cell type must be established so that the resulting cells are homogeneous and do not form teratomas or cause cancer (3), and immune responses/rejection caused by the transplantation of hESCs or their differentiated derivatives should be prevented. In gerneral, all processes related to the derivation, maintenance and differentiation of hESCs should be accomplished under xeno-free culture condition using good manufacturing practice (GMP) systems (1, 2). However, there are prerequisites for clinical application of hESCs. Their unique properties are expected to improve investigations in the field of human development and provide new materials for cell/tissue transplantation therapies and drug screening. Human embryonic stem cells (hESCs) are derived from inner cell mass of blastocyst-stage embryos, and exhibit unlimited proliferation ability and pluripotency to differentiate into various cell types originated from the threegerm layers. ![]()
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