3D Biomaterials for Accelerated Generation and Transplantation of Human Pluripotent Stem Cell Derived Midbrain Dopaminergic Neurons to Treat Parkinson’s Disease

Authors: 
Adil, M. M., University of California, Berkeley
Rodrigues, G. M. C., Instituto Superior Técnico, University of Lisbon
Kulkarni, R. U., University of California Berkeley
Miller, E. W., University of California Berkeley


3D
biomaterials for accelerated generation and transplantation of human
pluripotent stem cell derived midbrain dopaminergic neurons to treat
Parkinson�s disease

Maroof
M. Adil1,
Gon�alo MC Rodrigues2, Rishikesh U. Kulkarni, Tandis Vazin,
Badriprasad Ananthanarayanan, Evan W. Miller, Sanjay Kumar and David V.
Schaffer1-4

1Department of
Chemical and Biomolecular Engineering; 2Department of
Bioengineering; 3Department of Molecular and Cell Biology; 4Helen
Wills Neuroscience Institute; University of California, Berkeley, CA

Cell
replacement therapies have considerable biomedical potential; however, low
survival of cells post-transplantation is a major hurdle hampering their
efficacy. For example, cell replacement therapies have strong promise for the
treatment of neurodegenerative diseases such as Parkinson�s, which is a
currently incurable, progressively debilitating disorder affecting ~10M people
in the world. However, following striatal transplantation of midbrain
dopaminergic (mDA) neurons for the treatment of Parkinson�s disease (PD), less
than 5% of the neurons typically survive pre-clinically. Such poor neuronal
survival requires the resource-intensive generation and implantation of larger
numbers of mDA neurons to compensate, and the widespread death of the majority
of the graft could reduce the efficacy of the cells remaining. We took a
two-pronged approach to resolve this issue by developing methods to first
increase the yield of human pluripotent stem cell (hPSC) derived mDA neurons
generated in vitro, and second, to increase the post-transplantation survival
of neurons in vivo.

Human
pluripotent stem cells (hPSCs) were cultured and differentiated into mDA
neurons in 3D pNIPAAm-PEG gels. The authenticity of resulting neurons
thoroughly characterized by immunocytochemistry, qPCR and voltage-sensitive dye
based electrophysiology, and benchmarked against mDA neuronal development on 2D
surfaces. Subsequently, a biodegradable, hyaluronic acid (HA) based hydrogel functionalized
with ECM derived factors was developed for maturation and transplantation of
mDA neurons generated on pNIPAAm-PEG. Post-transplantation survival of
hydrogel-encapsulated neurons was then investigated 4.5 months after striatal
implantation into rats.�

mDA
neuron production typically involves undefined components and difficult to
scale-up 2D culture formats. Here, we used a fully-defined, 3D thermoresponsive
biomaterial platform to derive mDA neurons from PSCs at substantially greater
yield, quality, and maturity compared to 2D Matrigel coated surfaces. In
particular, after 25 days of differentiation, ~40% of cells differentiated in
3D expressed tyrosine hydroxylase (TH), the rate limiting enzyme for dopamine
production and a hallmark of mDA neurons, compared to 20% on 2D. Crucial for
functional mDA neurons, ~ 5-fold higher proportion of neurons differentiated in
3D fired mDA-like action potentials. Interestingly, FOXA2 and EN1, markers
known to improve survival of mDA neurons, were expressed at significantly
higher levels in cells generated in the 3D platform compared to control 2D
surfaces.

Low
post-transplantation cell survival is often attributed to adverse biochemical,
mechanical, and immunological stresses cells experience during and
post-transplantation. To address these challenges, we developed a
functionalized hyaluronic acid (HA) based hydrogel first to mature pluripotent
stem cell (PSC) derived neural progenitors towards functional neurons in vitro
in a biomimetic 3D environment, and second to effectively transplant
hydrogel-encapsulated neurons into the central nervous system. We demonstrated
that incorporating adhesive peptides to promote cell adhesion increased
extended neurite density, and adding neurotrophic factors increased
dopaminergic differentiation in our 3D HA gels. Furthermore, we observed higher
survival post-harvest and injection in vitro for mDA neurons matured within
hydrogels compared to those on 2D surfaces. By using an optimized 3D HA
biomaterial matrix for neuronal maturation and transplantation, we
significantly increased the post-transplantation survival of hPSC derived mDA
neurons in the rat striatum by 5 fold compared to the current standard method
of bolus injections of unencapsulated neurons.

The
combination of a defined, scalable, and resource-efficient cell culture
platform and an optimally engineered transplantation biomaterial significantly
increased the yield of mDA neurons generated in vitro and improved their post-transplantation
viability in vivo, thereby addressing crucial challenges in treatment of
neurodegenerative disease.