In vitro differentiation of hES cells

4.1 Differentiation via the formation of embryoid bodies

Human ES cells, like their mouse and non-human primate counterparts (see Chapters 5, 6, 8, 9, and 12), can be induced to enter a program of in vitro differentiation through the primary germ layers via the formation of embryoid bodies (EBs) (45). The basic principles for EB production using mES cells may be applied successfully also to hES cells. For example aggregation may be promoted by seeding hES cells into bacteriological-grade Petri dishes, to which these cells are non-adherent; or the hanging-drop technique may be used, to confer relative control over the number of hES cells per forming aggregate and hence the eventual size and uniformity of EBs produced. By analogy with the mouse system (see Chapter 5) it was anticipated that the use of serum-containing medium would promote mesoderm differentiation and cyst formation, whilst the use of KnockOutā„¢ SR or other serum replacement inhibits those processes, stimulating instead neuronal differentiation (6; and personal observations); and our protocols for inducing mesodermal and haematopoietic differentiation of hES cells consequently utilize fetal calf serum (FCS or FBS), amongst other factors. As yet, techniques for EB differentiation are less well developed for hES cells than for mES cells, in terms of the range of differentiated derivatives obtained.

Figure 2 Time-course of development of EBs from hES cells In suspension culture. Photomicrographs of EBs derived using the I9 line of hES cells, demonstrating their gradual growth and morphological development starting with (A) complete aggregation of cells to form simple EBs by day 2, followed by (B) the appearance of distinct layers within EBs on day 3, (C) clear cavitation of EBs on days 7-10, and finally (D) development of fully cystic EBs after 2 weeks of differentiation. Scale bars = 100mm

Figure 2 Time-course of development of EBs from hES cells In suspension culture. Photomicrographs of EBs derived using the I9 line of hES cells, demonstrating their gradual growth and morphological development starting with (A) complete aggregation of cells to form simple EBs by day 2, followed by (B) the appearance of distinct layers within EBs on day 3, (C) clear cavitation of EBs on days 7-10, and finally (D) development of fully cystic EBs after 2 weeks of differentiation. Scale bars = 100mm

The differentiation of hES cells in EBs has been shown to reproduce aspects of early embryogenesis, and occurs in sequential stages (45, 46). Figure 2 shows a time-course of EB development from seeding a culture of hES cells onto a non-adhesive substratum, where the day of seeding is 'day 0': by day 2 the hES cells have coalesced, resulting in the formation of simple EBs; this is followed by cavitation of some EBs as early as day 3 (and in all EBs between days 7 and 10); and development and growth of cystic EBs between days 7 and 14. Subsequently a more complex, three-dimensional cellular 'architecture' is elaborated within the EBs, where cell-cell and cell-ECM interactions are considered to influence the observed differentiation of the three embryonic germ layers and their derivatives (Figure 3). Thus, the hES cell/EB system offers opportunities for the in vitro investigation of cellular interactions normally occurring during early embryonic development, and of mechanisms of lineage determination. This potential is illustrated by a large-scale, complementary DNA (cDNA) analysis that was performed on hES cell-derived EBs at different stages of differentiation using DNA microarrays, which revealed sets of temporally expressed genes that could be related to the sequential stages of human embryonic development (47). Using the same technique, it was further demonstrated that many genes known

Figure3 (seePlatell) Ultrastructure of EBs derived from hES cells. (A-C) Haematoxilln- and eosin-stained sections of EBs cultured in suspension for 1 month from aggregation, demonstrating cells of a variety of morphological types. (A) A group of EBs at lower magnification; (B) and (C), individual EBs at higher magnification. Different cell types with characteristic organization can be observed within the EBs, including: neuronal rosettes (solid arrows), vascular structures (i.e. blood vessels; dashed arrows), an epithelial tube (arrowhead), and connective tissue (asterisks). Scale bars = 100mm.

Figure3 (seePlatell) Ultrastructure of EBs derived from hES cells. (A-C) Haematoxilln- and eosin-stained sections of EBs cultured in suspension for 1 month from aggregation, demonstrating cells of a variety of morphological types. (A) A group of EBs at lower magnification; (B) and (C), individual EBs at higher magnification. Different cell types with characteristic organization can be observed within the EBs, including: neuronal rosettes (solid arrows), vascular structures (i.e. blood vessels; dashed arrows), an epithelial tube (arrowhead), and connective tissue (asterisks). Scale bars = 100mm.

to be involved in human vascular development are activated also during the differentiation of EBs (48). Consequently it is anticipated that EBs derived from hES cells may be utilized for determining the cellular basis of certain embryonic defects, and for interpreting knockout phenotypes; and that they may be especially appropriate for the analysis of mutations where comparative studies in the mouse are complicated by early embryonic lethality.

4.2 Vascular morphogenesis within EBs

When hES cell-derived EBs reach a certain state of maturation, they display distinct vascular structures that are formed by differentiated endothelial cells (ECs) and vascular smooth-muscle cells (v-SMC) (15, 16). A convenient way to visualize these three-dimensional structures is by confocal microscopy, which enables inspection of the EB in toto (Figure 4). When such vascular structures in EBs are stained by immunofluorescence (Protocol 4) for the EC markers, CD31 and CD34, and the v-SMC markers, SMA (alpha smooth-muscle actin) and SM-MHC (smooth-muscle myosin heavy chain), it is observed that most of the CD31+ or SM-MHC+ cells are contained within these three-dimensional structures. The CD34+ population comprises cells that are elongated into an organized structure (Figure 4A) and cells that are heaped and rounded,

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