Pyramidal Neurons Derived from Human Pluripotent Stem Cells Integrate Efficiently into Mouse Brain Circuits In Vivo

Abstract

The study of human cortical development has major implications for brain evolution and diseases but has remained elusive due to paucity of experimental models. Here we found that human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), cultured without added morphogens, recapitulate corticogenesis leading to the sequential generation of functional pyramidal neurons of all six layer identities. After transplantation into mouse neonatal brain, human ESC-derived cortical neurons integrated robustly and established specific axonal projections and dendritic patterns corresponding to native cortical neurons. The differentiation and connectivity of the transplanted human cortical neurons complexified progressively over several months in vivo, culminating in the establishment of functional synapses with the host circuitry. Our data demonstrate that human cortical neurons generated in vitro from ESC/iPSC can develop complex hodological properties characteristic of the cerebral cortex in vivo, thereby offering unprecedented opportunities for the modeling of human cortex diseases and brain repair.

Novel and Robust Transplantation Reveals the Acquisition of Polarized Processes by Cortical Cells Derived from Mouse and Human Pluripotent Stem Cells

“…The induction method of the H9 human ESC line that ubiquitously expresses GFP to a cortical neuron fate using an adherent monoculture system and the detailed characterization of induced neural cells using neural lineage markers were described previously [3]. GFPexpressing ESCs were differentiated into cortical-like cells for 24 days and then dissociated using Accutase (Invitrogen) for subsequent in utero transplantation via the transplantation into epithelial tissue using the calcium depletion (TETCaD) method.…”

“…We next examined whether the TETCaD method is applicable to human cortical progenitors, which can be obtained efficiently from human ESCs or iPSCs [2,3]. At 24 days after the initiation of cortical differentiation from GFPexpressing human ESCs, the cells in the culture dish can be immunostained for the cortical progenitor marker PAX6 and the differentiated neuron marker TUJ1 (Supplementary Fig.…”

“…Indeed, the generation of the different neuronal subtypes found within the six distinct layers of the mammalian cortex from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has been established in adherent monolayer culture systems [1][2][3] and self-organized cultures with polarized cytoarchitecture [4]. With these types of approaches, the entire neurogenic process, from undifferentiated stem cells to terminally differentiated neurons, can be tracked in the defined conditions of a culture dish.…”

“…These culture systems are expected not only to offer an unlimited source of cortical neurons for clinical research into regenerative therapies and pharmacological screening for neurodegenerative diseases but also to provide novel strategies to answer fundamental questions of the brain's development and its disorders [5]. It has been demonstrated that terminally differentiated cortical neurons derived from mouse [6] and human [3] ESCs can integrate into mouse brain circuits. However, it is still largely unknown whether and to what extent undifferentiated neural progenitors or immature neurons generated in vitro integrate into germinal tissue and recapitulate the processes of physiological neurogenesis in vivo.…”

Direct somatic lineage conversion

“…Mostly through the application of small molecules such as dual SMAD inhibitors (blocking both BMP and TGFb pathways) and selective patterning and growth factor signalling cues, differentiation protocols were refined to produce several different neuronal subtypes including excitatory cortical neurons, forebrain inhibitory interneurons and midbrain dopaminergic neurons [63][64][65]. A complication of these differentiation methods is the multi-stage and therefore experimentally involved differentiation procedure and the protracted functional maturation of the differentiating neurons, which often require over eight weeks to exhibit the first signs of synaptic activity [64,66]. Given our observation that just a few transcription factors have the remarkable ability to induce the neuronal lineage from even distantly related cell types, we sought to revisit the effects of proneural transcription factors in human pluripotent stem cells.…”