Principles of stem cell biology and cancer: future applications and therapeutics. Edited by T. Regad, T. J. Sayers and R. C. Rees. John Wiley & Sons (2015)
Isolation and characterization of human embryonic stem cells and future applications in tissue engineering therapies
1.1. Derivation of human embryonic stem cells from the ICM
Success in the derivation of hESCs depends in part on the quality of the human embryos used (usually blastocysts from days 5 to 8), although cell lines have been generated from morphologically poor embryos. Numerous hESC lines have been derived (Figure 1.1) from normal, aneuploid and mutant embryos from patients undergoing treatment for assisted conception (IVF, ICSI) or preimplantation genetic diagnosis (PGD) who consent to donate them for stem cell research. Some of these cell lines have been extensively characterized and compared, enabling international standards to be established (Adewumi et al., 2007).
Figure 1.1. (A) Outgrowth of hESCs over 10 days of culture from ICM. In this instance, a clearly defined colony was observed by 10 days, which was mechanically passaged. (B) hESC line Shef1 plated on ECM.
220.127.116.11. Evolution to a more efficient and better-defined derivation method: drivers and technologies
Over the last 15 years, continuous improvements have been made in the process of deriving and maintaining hESC lines. The emphasis initially was on improving efficiency and consistency in the stem cell laboratory. But as hESC lines have become readily available for research in many countries, the focus has changed to devising methods for deriving clinical-grade cell lines that comply with health care regulatory authorities (e.g. Federal Drug Administration, FDA; European Medicines Agency, EMA), which can be used as starting materials for potential cell-therapy trials. Xeno-free methods (free of nonhuman animal components) are preferable as they minimize the risk of cross-species contamination with adventitious agents. An important early improvement was the replacement of FCS with a serum extract (knockout serum replacement, KOSR) to reduce hESC differentiation. This modification also minimized batch variation (inherent in FCS) between culture media, and allowed consistency in the proliferation of the cells after passaging (transfer of cells to a new culture vessel). Subsequently, more defined culture media (xeno-free) have been devised, which, in combination with a variety of extracellular matrix (ECM) compositions, facilitate the proliferation and passage of pluripotent hESCs in the absence of feeder cells (mouse or human), which otherwise remain an ill-defined and inconsistent component of the cell culture. Manipulation of the embryo has also changed over time. Initially, the ICM was isolated according to mouse protocols using enzymatic (protease) removal of the zona pellucida (ECM surrounding blastocyst) and immunosurgical lysis of TE with antitrophoblast antibody to prevent TE culture outgrowth from inhibiting early ESC proliferation. However, xeno-free methods using laser-assisted removal of the zona and plating of the intact blastocyst or the ICM on to a defined matrix (e.g. laminin 521) with a defined culture medium is the method of choice, leading to successful feeder/xeno-free cell line production in ~20 – 40% of attempts with good-quality human embryos (Hasegawa et al., 2010). With further improvements to the cell adhesion matrix and cell medium, the efficiency of hESC line derivation is likely to increase further, although the quality of the embryo used to develop ICM cells remains a crucial factor.
Another important consideration is the genetic character and stability of the hESC line. Generally, most hESC outgrowths and initial cell lines derived from unselected embryos (i.e. not PGD selected) are determined to be karyotypically normal within the precision of the chromosomal analysis. However, hESCs acquire genetic mutations in culture, which may endow them with a selective cell culture advantage, so that mutated cells predominate (Baker et al., 2007). Since derivation and ESC passage represent key stress events for ESC cultures, minimization of selective pressure on cells at these stages may help to maintain their normal karyotype. For example, the proliferation of cells by mechanical division of hESC colonies into smaller aggregates may be preferable to enzymatic disaggregation to single cells, which will initiate apoptotic stress pathways unless inhibited from doing so by a chemical inhibitor (i.e. ROCK inhibitor).