(453a) Scalable Production of Dopaminergic Neuron Progenitors for Treating Parkinson's Disease

Introduction: Cell replacement therapies (CRTs) for Parkinson’s disease (PD) based on human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising.  Prior studies with human fetal tissues containing dopaminergic neuron progenitors (DA) have shown this promise, but they have problems with scale and ethics, which hPSCs can solve.  hPSCs have the capacities for indefinite in vitro expansion and, due to recent advances, can be efficiently differentiated into DA progenitors or neurons.  However, a GMP compliant and cost effective culture system is required to produce these cells at various scales before they can be broadly utilized in the clinic.  It is becoming clear that current 2D-based cell culture system cannot generate the massive numbers of cells needed for such therapeutic application. As an illustrative calculation, in several clinical trials for PD, patient benefit was associated with the survival of ~200,000 transplanted dopamine neurons, and the survival of hPSC-derived DA neurons in animal models is often ~5% or less. At a typical density of ~5,000 DA neurons/cm² in 2D culture system, and with over a million Americans with PD, culturing sufficient numbers of DA neurons would require ~0.5 km² of cell culture surface (equivalent to ~100 football fields), not to mention the area required to expand the parent hPSCs.  In this presentation, we will introduce a simple, defined, scalable and GMP compliant 3D culture system for producing DA progenitors.  We will also present data on using these cells for treating PD with rodent model.

Materials and Methods: hPSCs were first expanded to a target number before differentiation into DA progenitors, both in the 3D system.   PSCs were dissociated into single cells, and cultured in the 3D system with a fully defined medium for 5 days before the next passage.  Cells were continuously expanded until the target number was reached.  Following expansion, a DMEM/F12 medium supplied with a small molecule blend was used to convert the PSCs into DA progenitors, which were then implanted into the striatum of rat to evaluate their safety, maturation, and integration.  The whole process was thus done in a fully defined system with only recombinant growth factors and no matrix proteins

Results and Discussion: With the 3D culture system, hPSCs could be efficiently expanded with a high replication rate (20-fold over 5-day per passage), yield (~2.0x10⁷ cells/ml), and purity (~95% Oct4+) through single cell inoculation.  We developed a small molecule cocktail to convert these hPSCs into DA progenitors with efficiency close to a published protein-based protocol that has been used in 2D.  This approach successfully yielded DA progenitors in the new 3D culture system, resulting in a much higher yield (~8x10⁷ cells/ml) than the conventional 2D culture system.  The ability of these cells to survive, mature into TH+ DA neuron and function in vivo will also be presented. 

Conclusions:  In summary, we developed an efficient GMP compliant system along with a small molecule based protocol for producing DA progenitors.  This system has the capacity to produce sufficient DA progenitors at scales necessary for clinical translation and application.