Human Neural Tissues from Neural Stem Cells Using Conductive Biogel and Printed Polymer Microelectrode Arrays for 3D Electrical Stimulation

Abstract

Electricity is important in the physiology and development of human tissues such as embryonic and fetal development, and tissue regeneration for wound healing. Accordingly, electrical stimulation (ES) is increasingly being applied to influence cell behavior and function for a biomimetic approach to in vitro cell culture and tissue engineering. Here, the application of conductive polymer (CP) poly(3,4‐ethylenedioxythiophene)‐polystyrenesulfonate (PEDOT:PSS) pillars is described, direct‐write printed in an array format, for 3D ES of maturing neural tissues that are derived from human neural stem cells (NSCs). NSCs are initially encapsulated within a conductive polysaccharide‐based biogel interfaced with the CP pillar microelectrode arrays (MEAs), followed by differentiation in situ to neurons and supporting neuroglia during stimulation. Electrochemical properties of the pillar electrodes and the biogel support their electrical performance. Remarkably, stimulated constructs are characterized by widespread tracts of high‐density mature neurons and enhanced maturation of functional neural networks. Formation of tissues using the 3D MEAs substantiates the platform for advanced clinically relevant neural tissue induction, with the system likely amendable to diverse cell types to create other neural and non‐neural tissues. The platform may be useful for both research and translation, including modeling tissue development, function and dysfunction, electroceuticals, drug screening, and regenerative medicine.

Conducting Polymer Mediated Electrical Stimulation Induces Multilineage Differentiation with Robust Neuronal Fate Determination of Human Induced Pluripotent Stem Cells

“…Notably, early to late expression of Tuj1 by differentiating cells is consistent with Tuj1 being present in mitotically active neuronal precursors, as well as newly formed immature postmitotic neurons and more differentiated neurons [16]. Finally, initial studies of conventional electrical stimulation of the iPSCs within neural induction medium affirms published reports of augmented cell differentiation using electrical stimulation contemporaneously with cell-specific induction media [5][6][7][8].…”

“…Here, we describe the use of electrical stimulation for differentiating human iPSCs and expound the versatility of electroactive PPy:DBS. Initial CV and impedance studies critically demonstrated the conductivity and stability of PPy:DBS films necessary for iPSC stimulation, and affirm the method used for their preparation [5,13]. In addition, immunophenotyping of the iPSCs confirmed pluripotent cell state, with undifferentiated cells ubiquitously expressing pluripotency markers OCT4, SOX2, SSEA4, TRA-1-60, and TRA-1-81.…”

“…Notwithstanding earlier recognition of the significance of bioelectricity and therapeutic stimulation, researchers have only recently begun to explore the potential of electric stimulus in vitro. Potential applications include augmented tissue engineering and tissue maturation, and in vitro modelling of in vivo electroceuticals [5]. Beyond laboratory-based research and development, resulting products-be they tissues or devices-may be clinically-amenable for tissue replacement and/or regeneration.…”

“…We and others have investigated electrical stimulation for modulating cell behaviour initially under conventional two-dimensional (2D)/planar culture and more recently for three-dimensional (3D) tissue engineering [5][6][7][8]. The 2D studies demonstrated stimulation biased native human neural stem cells (NSCs) and murine embryonic stem cells (ESCs) towards neuronal lineages [6,7].…”

“…The ESC differentiation was more rapid compared to traditional growth-factor based methods. Novel 3D electrical stimulation of human NSCs within a conductive bio-gel using printed polymer poly(3,4-ethylenedioxythiophene)-polystyrenesulfonate (PEDOT:PSS) pillar microelectrodes enabled the development and maturation of 3D human neural tissues with enhanced neuronal cell function and increased response to drug-induced disinhibition [5].…”