Optimizing Differentiation Protocols for Producing Dopaminergic Neurons from Human Induced Pluripotent Stem Cells for Tissue Engineering Applications

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder that results when the dopaminergic neurons (DNs) present in the substantia nigra necessary for voluntary motor control are depleted, making patients with this disorder ideal candidates for cell replacement therapy. Human induced pluripotent stem cells (hiPSCs), obtained by reprogramming adult cells, possess the properties of pluripotency and immortality while enabling the possibility of patient-specific therapies. An effective cell therapy for PD requires an efficient, defined method of DN generation, as well as protection from the neuroinflammatory environment upon engraftment. Although similar in pluripotency to human embryonic stem cells (hESCs), hiPSCs differentiate less efficiently into neuronal subtypes. Previous work has shown that treatment with guggulsterone can efficiently differentiate hESCs into DNs. Our work shows that guggulsterone is able to derive DNs from hiPSCs with comparable efficiency, and furthermore, this differentiation can be achieved inside three-dimensional fibrin scaffolds that could enhance cell survival upon engraftment.

Engineering Neural Tissue from Human Pluripotent Stem Cells Using Novel Small Molecule Releasing Microspheres

“…hiPSC Culture and Formation of Engineered Tissues : Experiments using hiPSCs were conducted with the approval of the University of Victoria's Human Ethics Committee under protocol number: 12–187. hiPSCs (iPS(Foreskin)‐1, Lot 1‐DL‐01, WiCell) were cultured in six‐well plates coated with Vitronectin XF in the presence of E8 medium (STEMCELL Technologies) until 80% confluency was reached as previously described . Eleven confluent wells were used for the formation of uniform cell aggregates in AggreWell 800 plate (STEMCELL Technologies) as previously described .…”

“…Analysis of Cell Cultures Using Immunocytochemistry : Cell cultures and engineered tissues were stained for various markers according to the ICC protocol previously published . Table S1 (Supporting Information) shows the antibodies, catalogue number, vendor, and dilution used for ICC analysis on days 15, 28, and 35.…”

Biomaterial Strategies for Delivering Stem Cells as a Treatment for Spinal Cord Injury

“…The effect is not surprising considering that 2D laminin culture is effective for promoting the differentiation of human iPSCs into neural progenitors and neurons [Robinson et al, 2015]. Based on the work associated with collagen hydrogels for the treatment of SCI, it might be possible to functionalize 3D fibrin scaffolds or electrospun nanofibers with peptide sequences taken from laminin to further enhance neuronal differentiation [Fukushima et al, 2008].…”

“…TM media [Robinson et al, 2015]. To induce neural differentiation, these cells were cultured in the presence of STEMdiff TM (NIM; Neural Induction Medium) for 5 days.…”

“…The recombinant GDNF, which was made using good manufacturing practice, was provided by MedGenesis Therapeutix Inc. (Victoria, B.C., Canada). The protocol for preparation of our fibrin scaffolds has been previously reported [Kolehmainen and Willerth, 2012;Montgomery et al, 2015;Robinson et al, 2015]. Undifferentiated human iPSCs were cultured on a Vitronectin XF TM matrix in the presence of TeSR TM -E8…”