Injectable 3D microcultures enable intracerebral transplantation of mature neurons directly reprogrammed from patient fibroblasts

Abstract

Direct reprogramming of somatic cells into induced neurons (iNs) has become an attractive strategy for the generation of patient-specific neurons for disease modeling and regenerative neuroscience. To this end, adult human dermal fibroblasts (hDFs) present one of the most relevant cell sources. However, iNs generated from adult hDFs using two-dimensional (2D) cultures poorly survive transplantation into the adult brain in part due to the need for enzymatic or mechanical cellular dissociation before transplantation. Three-dimensional (3D) culturing methodologies have the potential to overcome these issues but have largely been unexplored for the purposes of direct neuronal reprogramming. Here we report a strategy for direct in vitro reprogramming of adult hDFs inside suspension 3D microculture arrays into induced DA neurospheroids (iDANoids). We show that iDANoids express neuronal and DA markers and are capable of firing mature action potentials and releasing dopamine. Importantly, they can be gently harvested and transplanted into the brain of a Parkinsons disease rat model to reproducibly generate functionally integrated neuron-rich grafts. The 3D culturing approach presented here thus eliminates a major bottleneck in direct neuronal reprogramming field and, due to its simplicity and versatility, could readily be adapted as a culturing platform used for a broad range of transplantation studies as well as disease modeling.