(55d) Biomaterial Scaffolds for Scalable Differentiation and Transplantation of Hpsc-Derived Cells for Cell Replacement Therapy in the Central Nervous System

Three major challenges facing effective cell replacement therapy in the central nervous system (CNS)- scalable differentiation, graft survival, and graft integration- may be addressed by optimally engineered biomaterials. Neurodegenerative disorders and traumatic injuries to the CNS, including Parkinson’s disease (PD), Huntington’s disease (HD) and Spinal Cord Injury (SCI), affect more than 5M people in the US alone, cause a significant socio-economic burden, and currently lack effective long-term treatments or cures. Cell replacement therapy (CRT), using cells derived from human pluripotent stem cells (hPSCs), is a promising strategy to treat neurodegeneration and CNS trauma. Typically, a large number of cells are required for effective CRT in the clinic; however, current standards of generating cells on conventional 2D platforms lack scalability and reproducibility. Additionally, effective CRT is often hindered by poor post-transplantation survival of the relevant cell type and poor graft dispersion and integration with the host tissue. These challenges need to be addressed in order to facilitate clinical translation of CRT.

Towards this end, we rapidly and reproducibly generated large numbers of hPSC-derived cells in a fully-defined, scalable, thermoreversible poly-N-isopropyl acrylamide-co-poly-ethylene glycol (PNIPAAm-PEG)-based 3D biomaterial. Specifically, we generated ~2-5 fold higher yields (relative to cells differentiated on conventional 2D surfaces) of midbrain dopaminergic (mDA) neurons, medium spiny neurons (MSNs) and oligodendrocyte progenitor cells (OPCs), which are the relevant cell types for CRT in PD, HD and SCI, respectively. Interestingly, cells derived in 3D demonstrated potential for rapid functional maturation, which may facilitate downstream applications such as drug screening and disease modeling. Research into the mechanistic effects of the 3D biomaterial on stem cell biology, using bulk and single-cell RNAseq, is ongoing.

Next, to address the problem of low post-transplantation cell survival, we developed a biodegradable, bio-functionalized hyaluronic acid (HA)-based transplantation scaffold, with tunable stiffness and capable of rapid gelation. For these subsequent studies, we focused on improving CRT for PD. In vitro, HA hydrogels functionalized with RGD and heparin enhanced the neurite extension of mDA neurons. Following striatal transplantation of hydrogel-encapsulated hPSC-derived mDA neurons in rats, we obtained a 5-fold increase in cell survival, relative to unencapsulated cells transplanted as a suspension.

Subsequently, to address the challenge of poor graft dispersion and integration, we first used an in vitro screen to identify biomolecules that can disperse hPSC-derived mDA neurons. We then validated that these factors dispersed hPSC-derived mDA neurons encapsulated within HA-based hydrogels; furthermore, we discovered that stiffer hydrogels increased dispersion. Next, we developed a controlled release platform by physically incorporating dispersion factors into HA hydrogels. Controlled release of factors from HA-based hydrogels mediated effective dispersion of co-encapsulated hPSC-derived mDA neurons for at least two weeks in vitro. Finally, mDA neurons co-transplanted with dispersion factors within a protective HA hydrogel, optimized for controlled factor release and cell dispersion, rapidly alleviated disease symptoms in PD model rats 4 weeks after implantation. Importantly, treatment benefits correlated with increased mDA neuron survival, dispersion, and integration, with no graft overgrowth.

Overall, we developed a method for rapid generation of large numbers of hPSC-derived cells in a scalable biomaterial platform, for potential applications in cell replacement therapy, drug screening, and disease modeling. Additionally, we show that an optimally engineered cell-instructive transplantation platform holds promise for enhancing CRT in PD, and potentially in a range of other degenerative diseases or trauma.