Human Cerebrospinal Fluid Promotes Neuronal Circuit Maturation of Human Induced Pluripotent Stem Cell-Derived 3D Neural Aggregates

Abstract

Summary Human induced pluripotent stem cell (hiPSC)-derived in vitro neural and organoid models resemble fetal, rather than adult brain properties, indicating that currently applied cultivation media and supplements are insufficient to achieve neural maturation beyond the fetal stage. In vivo , cerebrospinal fluid molecules are regulating the transition of the immature fetal human brain into a mature adult brain. By culturing hiPSC-3D neural aggregates in human cerebrospinal fluid (hCSF) obtained from healthy adult individuals, we demonstrate that hCSF rapidly triggers neurogenesis, gliogenesis, synapse formation, neurite outgrowth, suppresses proliferation of residing neural stem cells, and results in the formation of synchronously active neuronal circuits in vitro within 3 days. Thus, a physiologically relevant and adult brain-like milieu triggers maturation of hiPSC-3D neural aggregates into highly functional neuronal circuits in vitro . The approach presented here opens a new avenue to identify novel physiological factors for the improvement of hiPSC neural in vitro models.

Engineering in vitro human neural tissue analogs by 3D bioprinting and electrostimulation

“… 23,206,207 With this in mind, Izsak et al found that substituting adult human cerebral spinal fluid (hCSF) for common differentiation media in a 3D model of human iPSC derived neural aggregates resulted in rapid gliogenesis, neurogenesis, synapse formation, and neurite outgrowth, with the formation of mature and synchronously active neural networks, as seen in native brain tissue. 208 Limited accessibility aside, with the aim of constructing a controlled and replicable in vitro tissue model, the undefined components of an individual's CSF are not desirable. However, for the formation of patient-specific and translatable cell models for in vitro analysis and/or therapeutic applications, it could be beneficial.…”

TGF-β1 Suppresses Proliferation and Induces Differentiation in Human iPSC Neural in vitro Models

“…The TGF-β1 impact on human neural cells has not yet been reported, therefore we evaluated whether TGF-β1 treatment affects electrophysiological function of human neurons. Since human iPSC-derived neurons are more functional in an 3D neural environment (see Izsak et al, 2019Izsak et al, , 2020, we prepared hiPSC-3D-NA cultures in the absence or presence of 20 ng/ml TGF-β1. We performed cell-attached and whole cell recordings in cells localized at the edges of 3D-NAs, where MAP2AB + neurons are present (Figure 5A).…”

“…During fetal brain development, CSF factors might be crucial to promote differentiation of NSCs into neurons, while in the adult brain, CSF factors rather promote proliferation of ventricular NSCs (Silva-Vargas et al, 2016). Interestingly, human iPSC-NSCs differentiate into neurons and astrocytes when exposed to adult CSF samples (Izsak et al, 2020).…”

“…Recently, we demonstrated that human cerebrospinal fluid (CSF) obtained from healthy adult individuals caused several maturation processes, including the rapid transition of NSCs into neurons and astrocytes in a human iPSC 3D neural aggregate in vitro model (Izsak et al, 2020). This demonstrates that a human iPSC 3D neural aggregate in vitro model can adopt rather mature properties when exposed to an appropriate and physiologically relevant environment.…”

Using organoids to study human brain development and evolution

“…This work produced the hypothesis that neuronal circuits play a role in the maturation of the intrinsic properties of thalamic neurons, because it was found that fusion of thalamic organoids with cortical organoids increased the frequency of electrophysiologically mature neurons in the thalamic tissue (Xiang et al, 2019). Furthermore, other studies found that neurons in organoids show spontaneous network formation producing periodic and regular oscillatory events dependent on glutamatergic and GABAergic signaling, reaffirming that neurons in organoids develop axons, synapses, and mature functional properties (Birey et al, 2017;Izsak et al, 2020;Trujillo et al, 2019;Xiang et al, 2017Xiang et al, , 2019. A recent study has pushed the boundaries of using organoids to model neural circuit formation even further by making cortico-motor assembloids that comprised of three organoids fused together (cortical-spinalmuscle).…”