Guided differentiation

With the newly identified, potential therapeutic uses of hES-cell derivatives in medicine, much attention currently is being focused on methods for achieving guided differentiation to generate highly purified populations of the desired cell types. The success of these particular differentiation systems ultimately will rely on a high level of understanding of the wide range of biochemical and physical cues that are involved during lineage specification in human embryogenesis. For the interim, methods are available to guide differentiation towards preferred pathways.

• 24-well dishes (Cat. No. 142475, Nunc), precoated with gelatin (Protocol 1)

• Inverted phase-contrast microscope

• Multiarray electrode systems (MEAs; Multi Channel Systems MCS GmbH)

Undifferentiated hES cells

(a) Culture on type IV collagen in alpha MEM with 10% FCS for 6 d

(b) Guided lineage differentiation Re-culture on type IV collagen in VDM with either hVEGF165 or hPDGF-BB

Harvesting cells and filtration

(c) 3D gel assay for tube formation Aggregation and culture in either collagen or Matrigel™

Vasculogenesis

Inhibition

Vasculogenesis

Inhibition

Figure 5 Experimental strategy for guided, vascular differentiation of hES cells. By this two-step differentiation strategy (Protocol 7), a culture of undifferentiated hES cells is first induced to undergo mesodermal differentiation in monolayer culture, in step (a). In step (b), the mesodermal progenitor cells are guided towards maturation into v-SMCs and ECs by further incubation in VDM supplemented with hVEGF165 (i) or hPDGF (ii), respectively. In step (c), mesodermal progenitor cells are assayed for vasculogenesis and angiogenesis by aggregation and culture in collagen or Matrigel™ (Protocol8): aggregates in Matrigel™ show vigorous sprouting of vascular structures (iii), which process is inhibited in the presence of anti-VE-cad monoclonal antibody (iv), which was previously suggested to be a potent inhibitor of angiogenesis (67, 68).

5.1 Haematopoietic differentiation

The induction of haematopoiesis in hES cells is a guided differentiation system currently under investigation (Protocol 6): here, single-cell suspensions of differentiated hES cells are cultured in either liquid or semisolid medium supplemented with specific cytokines, resulting in further differentiation into haematopoietic colony forming units (CFU) (17, 18).

5.2 Non-EB-mediated hES-cell differentiation: vascular development

For murine and primate ES cells, differentiation into certain cell types can now be achieved in monolayer culture-without recourse to EB formation-by

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