Human embryonic stem cells (hESCs) are emerging as an attractive alternative source for cell replacement therapy since the cells can be expanded in culture indefinitely and differentiated into any cell types in the body. In order to optimize cell-to-cell interaction, cell proliferation and differentiation into specific lineages as well as tissue organization, it is important to provide a microenvironment for the hESCs which mimics the stem cell niche. One approach is to provide a three-dimensional (3D) environment such as encapsulation. We present an approach to culture and differentiate hESCs into midbrain dopamine (mdDA) neurons in a 3D microenvironment using alginate microcapsules for the first time. A detailed gene and protein expression analysis during neuronal differentiation showed an increased gene and protein expression of various specific DA neuronal markers, particularly tyrosine hydroxylase (TH) by >100 folds after 2weeks and at least 50% higher expression after 4weeks respectively, compared to cells differentiated under conventional two-dimensional (2D) platform. The encapsulated TH+ cells co-expressed mdDA neuronal markers, forkhead box protein A-2 (FOXA2) and pituitary homeobox-3 (PITX3) after 4weeks and secreted approximately 60pg/ml/106 cells higher DA level when induced. We propose that the 3D platform facilitated an early onset of DA neuronal generation compared to that with conventional 2D system which also secretes more DA under potassium-induction. It is a very useful model to study the proliferation and directed differentiation of hESCs to various lineages, particularly to mdDA neurons. This 3D system also allows the separation of feeder cells from hESCs during the process of differentiation and also has potential for immune-isolation during transplantation studies.
Bibliographical noteFunding Information:
This work was funded by the National Health and Medical Research Council Program Grant (Perminder Sachdev) and Faculty of Medicine, University of New South Wales, Stem Cell Initiative .