Function characterization: differentiation potential

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)

1. Isolation and characterization of human embryonic stem cells and future applications in tissue engineering therapies

1.2. Basic characterization of hESCs

ESCs are unique in their ability to self-renew and differentiate into all three embryonic germ layers, in principal forming any fully terminally differentiated cell within the body. In the mouse, ESC pluripotency is defined by the ability to generate chimeric offspring and contribute to the germ line. However, for ethical and practical reasons, in humans and some nonhuman primate species, the ability of ESCs to form chimeras is not a testable property, and alternative protocols on which to base functional pluripotency must be used. In the absence of the natural stem cell niche of the embryo, hESCs are in a dynamic balance between cell fates and are highly susceptible to environmental cues, which can induce spontaneous cell differentiation or, in the correct combination, can be employed to drive a more ‘directed’ cell differentiation. Therefore, pluripotency is measured either in vitro by differentiation of cells as aggregates in suspension culture (called embryoid bodies, EBs) or in vivo by their formation in the mouse as benign tumours called teratomas. In vitro: EBs

Human ESCs can be induced to differentiate in vitro by the process of EB formation (Figure 1.4). The process involves growing hESCs in suspension to form cell aggregates on a nonadhesive substrate to prevent their dissociation. As the EBs mature, hESCs alter their morphological appearance and acquire molecular markers characteristic of differentiated derivatives. Markers specific to each embryonic lineage can include neurofilament 68Kd (ectoderm), β-globin (mesoderm) and a-fetoprotein (endoderm) (Itskovitz-Eldor et al., 2000). However, more markers per germ layer are usually analysed, to illustrate a more global picture of differentiation ability. Initial testing of differentiation capacity is commonly done by spontaneous EB differentiation in medium supplemented with serum. Methods have become more refined, however, using defined number of cells and defined media formulations (Ng et al., 2005). An EB formation assay should always be part of the basic hESC characterization, and should clearly show either upregulation of markers from the three germ layers in the EBs or outgrowth from them.

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Figure 1.4. Simple overview of EB formation from hESCs. EBs from hESCs should contain tissues derived from all three embryonic germ layers. In vivo: teratoma formation

The formation of a teratoma is a formal demonstration of pluripotency of hESCs in vivo. Teratomas are benign tumours that contain different types of developmental tissue derived from all three germ layers. They are formed after injection of undifferentiated hESCs into the hind leg, testis or kidney capsule of immunocompromised mice (i.e. nonobese diabetic severe combined-immunodeficient, NOD/SCID). They are then usually analysed by histological evaluation of the tumour mass for the presence of representatives of all three germ layers (Figure 1.5) (Thomson et al., 1998). On occasion, after injection, hESCs fail to form teratomas; therefore, injection of more than one mouse is often necessary to account for any variability. This may be due to the abnormal environment in which hESCs are placed, residual immune reactivity, the quality of the hESCs or the scientific methodology. Efficiencies can be improved by adding ECMs such as inactivated mouse embryonic fibroblasts (MEFs) or Matrigel with the hESCs and by using more severely immunocompromised mice (Gropp et al., 2012).

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Figure 1.5. Simple overview of teratoma formation in immunocompromised mice. Teratomas from hESCs contain tissues derived from all three embryonic germ layers.

While teratoma formation is an expected part of the basic characterization panel for new hESC lines, a search for less expensive and shorter surrogate assays is ongoing. In particular, more streamlined and time-efficient methods are required for the mass generation of iPSCs (Muller et al., 2011).


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