Human embryonic stem cell technology: large scale cell amplification and differentiation

Abstract

Embryonic stem cells (ESC) hold the promise of overcoming many diseases as potential sources of, for example, dopaminergic neural cells for Parkinson's Disease to pancreatic islets to relieve diabetic patients of their daily insulin injections. While an embryo has the innate capacity to develop fully functional differentiated tissues; biologists are finding that it is much more complex to derive singular, pure populations of primary cells from the highly versatile ESC from this embryonic parent. Thus, a substantial investment in developing the technologies to expand and differentiate these cells is required in the next decade to move this promise into reality. In this review we document the current standard assays for characterising human ESC (hESC), the status of 'defined' feeder-free culture conditions for undifferentiated hESC growth, examine the quality controls that will be required to be established for monitoring their growth, review current methods for expansion and differentiation, and speculate on the possible routes of scaling up the differentiation of hESC to therapeutic quantities.

Figure 1

A hypothetical model of the action of different ligands in maintaining hESC pluripotency. BMP4 which is present in serum and produced by hESC binds to BMPR I and II receptors to activate Smad 1/5/8 leading to differentiation. Noggin produced by feeders can sequester BMP preventing its action. Alternatively Activin A, which belongs to the BMP family can compete for the BMPR I and II receptors to activate Smad 2/3/4 leading to self-renewal and block the actions of Smad 1/5/8 via Smad 6/7. FGF-2 is postulated to bind to FGFR1 receptors and activate the Akt/PI3K pathway which is required for self-renewal.

Figure 2

Attempts at growing hESC in suspension (a) attached to feeders on Cytodex 3 microcarriers (b) immobilized in alginate (c) in 0.5% Dextran 8000 (d) in 0.5% PEG 200 (e) in 0.5% PEI 2000 (f) in 0.5% Pluronic respectively, were unsuccessful. (g) and (h) shows control cultures of hESC grown on feeders and feeder free conditions. All pictures were taken at a magnification of 40× except for (a) which was taken at 200×.

Figure 3

A schematic of possible future manufacturing systems in the expansion and differentiation of various lineages from hESC. The process would begin with generating large quantities of hESC which have an indefinite capability to expand. These resulting cells could be initiated to differentiate in suspension cultures as human embryoid bodies (hEBs) for a period of time. The hEBs would be separated to remove residual hESC and the target progenitor population selected for expansion. Then depending on whether the cell type required grows in suspension (e.g. islets) these would be grown in bioreactors (a), or if they require a combination of suspension culture and anchorage to a surface (e.g. cardiomyocytes), they would be grown accordingly (b). If the neural lineage is required, neurospheres could be cultured, undergo a separation step of enrichment for neural progenitors and eventually specific dopaminergic neurons grown on plastic culture surfaces (c). At the end of the expansion processes, ideally only the final phenotype is purified.