Differentiation of mES cells into muscle cell lineages

When mES cells are induced to form aggregates in vitro, in the absence of those self-renewal signals normally provided by mitotically inactivated feeder layers or LIF, cell differentiation is initiated (see Chapter 5). If aggregates are allowed to form spontaneously in suspension by seeding a culture onto a non-adhesive substratum (typically, a bacteriological-grade Petri dish), the number of cells incorporated into each aggregate is variable. But this number can be controlled through the use of a 'hanging-drop' technique, in which a defined number of cells are suspended in droplets and thereby induced to coalesce. As aggregates differentiate and develop into EBs, primary germ-like layers are produced spontaneously. Initially, an outer layer of endoderm-like cells forms around the EB, followed over a period of a few days by the development of an ectodermal 'rim' and subsequent specification of mesodermal cells. For differentiation into muscle cell lineages, EBs are most efficiently generated by the hanging-drop technique (Figure 1) but mass culture techniques also can be used. Differentiation into muscle cell lineages may be promoted by addition of drugs and growth factors. Several morphogens have proven useful in this respect, including the retinoids, all-trans retinoic acid (RA) and 9-cis RA, and bipolar compounds such as dimethyl sulphoxide (DMSO). Furthermore, k opioid receptor agonists have been shown specifically to promote cardiomyogenesis (11), whilst induction of vascular smooth muscle (VSM) may require use of dibutyryl-cyclic AMP (db-cAMP) and transforming growth factor b1 (TGF b1).

3.1 Differentiation of mES cells into cardiac muscle cells (Figure 2A, B)

Cardiomyocytes differentiated from mES cells display properties similar to those observed both in vivo and in primary culture. Depending on their stage of differentiation or maturation, they: (i) express cardiac gene products in a develop-mentally controlled manner; (ii) display characteristic sarcomeric structures; and (iii) undergo spontaneous contractions triggered by cardiac-specific ion currents and membrane-bound ion channels (8). Electrophysiological analyses have also revealed in differentiated cultures those phenotypes typical of primary myocardium and, with continued differentiation, atrial-, ventricular-, Purkinje-, and pacemaker-like cell types (12).

Experiments with serum-free medium supplemented with non-protein additives, hormones, and growth factors show significant promise for the generation of cardiomyocytes under defined conditions (see Chapter 5). In mES cell cultures differentiated in serum-replacement medium containing insulin and transferrin, for example, the expression of cardiac-restricted genes (e.g. a— and b — MHC) is increased, and the addition of either platelet-derived growth factor BB (PDGF-BB) or sphingosine 1 phosphate enhances cardiogenesis (15).

Mouse ES cells on feeder layers +

2 5 iffer h

EBs in bacteriological plates " for Suspension Culture

Hanging Drops on lid of a bacteriological plate

-RT-PCR or qRT-PCR analyses -In situHybridizations -Immunofluorescence -Electrophysiology -Confocalimage analysis Differentiation-dependent expression of genes, proteins, action potentials and ion channels

Cardiac muscle

Skeletal muscle

Smooth muscle

Cardiac muscle

Skeletal muscle

Smooth muscle

Figure 1 'Hanging-drop' method for the generation EBs for muscle-cell differentiation. The 'hanging-drop' method generates aggregates of defined cell number and size. Main stages include: the aggregation of mES cells, induced by their culture in hanging drops for 2d; continued culture of EBs in suspension for 5d; and plating of EBs on an adhesive substratum on day 7 postaggregation, followed by incubation for >30d further. (For example of EB, scale bar = 50mm.) Depending on the precise culture conditions, cardiac-, striated-, and smooth-muscle cells differentiate from the mES cells (scale bars = 10mm). These differentiated derivatives, either within EB outgrowths or following their isolation, can be analysed for muscle-restricted gene expression, or for the presence and function of muscle-related proteins.

3.2 Differentiation of mES cells into skeletal myocytes (Figure 2C, D)

Despite the availability of skeletal-muscle cell lines (e.g. C2, L6, L8), and the ease of isolation of the myogenic stem-cell pool (i.e. satellite cells) from muscle, the mES-cell differentiation system has proven especially valuable for the study of skeletal muscle owing to the recapitulation of developmental processes. For

Actinnin Staing

Figure 2 Immunofluorescence staining of muscle-cell derivatives from plated EBs. (A, B) Cardiac differentiation in the R1 line of mES cells. (A) Staining of cardiomyocytes for a-actinin, 10d after EB plating at day 7, i.e. 7 +10d postaggregation (scale bar = 20mm). (B) Staining of cardiomyocytes for ANP (a cardiac cell-restricted protein), 6d after EB plating at day 7 (scale bar = 20mm). (C) Staining of myotubes, obtained using the BLC6 line of mES cells, for Titin (a Z-band epitope) 31d after EB plating at day 5 (scale bar = 10mm). (D) Staining of skeletal myoblast/myotube, obtained using R1 cells, for a-actinin 6d after EB plating at day 7 (scale bar = 20 mm). (E, F) Smooth-muscle differentiation in the D3 line of mES cells, induced by treatment of plated EBs with RA plus db-cAMP between 7 and 11d after plating at day 7 (for methods see Protocol 5, or (18)). (E) Staining for smooth muscle a-actin, 7d after plating (scale bar = 1 mm). (F) Staining for smooth-muscle MHC, 14d after plating (scale bar = 1 mm).

Figure 2 Immunofluorescence staining of muscle-cell derivatives from plated EBs. (A, B) Cardiac differentiation in the R1 line of mES cells. (A) Staining of cardiomyocytes for a-actinin, 10d after EB plating at day 7, i.e. 7 +10d postaggregation (scale bar = 20mm). (B) Staining of cardiomyocytes for ANP (a cardiac cell-restricted protein), 6d after EB plating at day 7 (scale bar = 20mm). (C) Staining of myotubes, obtained using the BLC6 line of mES cells, for Titin (a Z-band epitope) 31d after EB plating at day 5 (scale bar = 10mm). (D) Staining of skeletal myoblast/myotube, obtained using R1 cells, for a-actinin 6d after EB plating at day 7 (scale bar = 20 mm). (E, F) Smooth-muscle differentiation in the D3 line of mES cells, induced by treatment of plated EBs with RA plus db-cAMP between 7 and 11d after plating at day 7 (for methods see Protocol 5, or (18)). (E) Staining for smooth muscle a-actin, 7d after plating (scale bar = 1 mm). (F) Staining for smooth-muscle MHC, 14d after plating (scale bar = 1 mm).

example, when EBs are plated onto an adhesive substratum and cultured for 1-2 weeks, myoblasts are produced that subsequently fuse and form recognizable myocytes, which are characteristic of developing skeletal muscle. Moreover, mES cell-derived skeletal-muscle cells display many properties similar to those observed in established skeletal-muscle cell lines, including the expression of skeletal-muscle gene transcripts (e.g. Myf-5, Myogenin, and MyoD) in a manner that mimics the temporal activation seen in normal embryogenesis (16).

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