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
1.4. Future applications in tissue-engineering therapies
Protocols need to be devised that can recapitulate the embryology of a specific cell type in vitro. It is unlikely to be possible to produce just one phenotype, so methods have to be in place to recognize the cell of interest and enrich the population for that particular cell type. This all needs to be done with enough cells to allow the required function to be elicited from their transplantation into the patient.
Figure 1.8. Simplified research and development pathway for future therapies from ES/iPSCs to differentiated, potentially therapeutically applicable cells, highlighting factors that can affect cancer risk (in red), as described in this chapter.
In the developing embryo, differentiation of pluripotent cells is spatially, temporally and chemically regulated. When cellular transplantation from a multipotent or terminally differentiated source is used for clinical purposes, the cells already have the required function and can be identified easily through tissue location and marker expression. When hPSC derivatives are used, they first have to be differentiated in vitro. For example, in order to replace foetal dopaminergic neuron treatment of Parkinson’s disease with hPSC derivatives, this process has been mimicked with a defined protocol that drives differentiation in the correct direction. Use of small-molecule inhibitors (SMIs) to block or stimulate specific signalling pathways has allowed the development of well-defined culture conditions that do not require co-culture with secondary cell types (Ali et al., 2013).
When intrastriatal transplantation of foetal neurons is carried out, the ventral mesencephalon of the foetus is dissected and dissociated before it is implanted into the courdate and putamen of the patient. When this is done, knowledge of dissection location combined with approximately 15% of cells expressing tyrosine hydroxylase is enough to be confident of cell type (Widner et al., 1992). The range of different derivatives that can be produced from hPSCs raises the possibility that contaminating phenotypes, even with comparable marker expression, may be present in any differentiated culture. For this reason, the dopaminergic differentiation protocol for hPSCs can be used in concert with serum-free culture methods that remove any extrinsic signalling from the system, reducing contaminating cell types and enriching the final cell population for the required phenotype. These cells are able to respond to the axon guidance cues that allow innervation and connection to host neurons (Cord et al., 2010) and have been shown to alleviate symptoms of Parkinson’s disease in Rat models (Ali et al., 2013). All of this work has brought us to a stage where there is a real possibility of clinical trials using hPS derived neurons to treat Parkinson’s disease.
Some cell types require more than clever combinations of signalling molecules and SMIs for successful differentiation from hPSCs. Production of glucose-sensitive, insulin-producing β cells for use in the treatment of diabetes has been a much sought-after goal ever since the isolation of the first hESCs. Human PSCs can be induced to form definitive endoderm by Activin A/Nodal signalling, or more efficiently by using the SMIs IDE1 and IDE2.
Following this, there are a number of different methods for differentiation and enrichment of the culture for pancreatic cells, assessed on the basis of the pancreatic marker PDX1 (Aguayo-Mazzucato and Bonner-Weir, 2010). Insulin-expressing cells have been produced in this manner, paving the way for a cell-replacement therapy for diabetes, but none of these methods appears to be capable of producing cells that secrete insulin in a glucose-sensitive manner. This is a characteristic that they share with foetal β cells, perhaps identifying them as a nascent counterpart to the fully functioning cell required for clinical use. The failure to achieve this last step of β-cell maturation has prevented a cell-replacement therapy for diabetes from reaching clinical trials and is driving research into methods for better recapitulating pancreatic development in vitro by spatially organizing the cells into 3D structures, with the hope that polarization of the cells will allow them to mature (Greggio et al., 2013).
Despite the establishment of specific and selective methods for producing a desired cell type, such protocols will rarely be able to exclude all other cell types that might develop from hPSCs. Because of this, most protocols, especially those for clinical applications, will include steps for purification (i.e. antibody-based magnetic-activated cell sorting). Importantly, undifferentiated hPSCs that can form teratomas and cells that might in any way negatively affect the treatment must be removed from a final cell-therapy product.