Highly Efficient Neural Conversion of Human Pluripotent Stem Cells in Adherent and Animal-Free Conditions

Abstract

Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal‐free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal‐free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic. Stem Cells Translational Medicine 2017;6:1217–1226

Transplantation of feeder‐free human induced pluripotent stem cell–derived cortical neuron progenitors in adult male Wistar rats with focal brain ischemia

“…We obtained a comparable cortical neuron induction with previous feeder‐maintained hiPSC neural induction (Gomi et al, ; Motono et al, ). The previous study showed that xeno‐free neural induction could be performed with insulin, transferrin, and sodium selenite and taurine cocktail (Lukovic et al, ), but there was no cortical neuron formation. The majority of postmitotic neurons were GABAergic, which are physiological distinct from cortical neurons (glutamatergic) (Lukovic et al, ).…”

“…The previous study showed that xeno‐free neural induction could be performed with insulin, transferrin, and sodium selenite and taurine cocktail (Lukovic et al, ), but there was no cortical neuron formation. The majority of postmitotic neurons were GABAergic, which are physiological distinct from cortical neurons (glutamatergic) (Lukovic et al, ). The neuron is a highly specified cell that developed within the specific region of neuroaxis; therefore, each region yielded distinct neurons.…”

Culturing Adult Stem Cells for Cell-Based Therapeutics: Neuroimmune Applications

“…The most current iPSC protocols for neural differentiation require GFs or embryoid body formation, decreasing yields and limiting medical applications. Our lab recently developed a simple animal-free medium formula based on the inclusion of insulin and human extracellular matrix components leading to direct conversion of >98% of iPSCs into expandable and functional neural progenitors with neural rosette characteristics [111]. Further differentiation into dopaminergic and spinal motoneurons as well as oligodendrocytes and astrocytes supports the proposal that these neural progenitors retain responsiveness to environmental cues supporting applicability of the protocol for the treatment of neurodegenerative diseases.…”

“…Human NSPCs have been isolated from the fetal brain and spinal cord of aborted fetuses [110] and postmortem tissue, but actually NSPCs can also be derived from human iPSCs [111].…”

“…Further differentiation into dopaminergic and spinal motoneurons as well as oligodendrocytes and astrocytes supports the proposal that these neural progenitors retain responsiveness to environmental cues supporting applicability of the protocol for the treatment of neurodegenerative diseases. The fact that this protocol avoids embryoid body formation makes it suitable for the clinic [111].…”