Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells

Abstract

Three-dimensional (3D) in vitro cell and tissue culture models, particularly for the central nervous system, allow for the exploration of mechanisms of organ development, cellular interactions, and disease progression within defined environments. Here, we describe the development and characterization of human 3D tissue models that promote the differentiation and long-term survival of functional neural networks. This work builds upon previous work where primary rodent neurons were successfully grown in a similar 3D system. The model was adapted to human induced pluripotent stem cells, allowing for a more direct exploration of the human condition. These tissue cultures show diverse cell populations, including neurons and astroglial cells, interacting in 3D and exhibit spontaneous neural activity confirmed through electrophysiological recordings and calcium imaging over at least nine months. This approach allows for the direct integration of pluripotent stem cells into the 3D construct, bypassing early neural differentiation steps (embryoid bodies and neural rosettes). The streamlined process, in combination with the longevity of the cultures, provides a system that can be manipulated to support a variety of experimental applications, including the study of network development, maturation, plasticity, and/or degeneration. This tissue model was tested with stem cells derived from healthy individuals as well as Alzheimer’s and Parkinson’s disease patients. We observed similar growth and gene expression, which indicates the feasibility of generating patient-derived brain tissue models. These could be used to uncover early stage biomarkers of the disease state, in turn supporting earlier diagnosis and improving understanding of disease progression. With additional model development, this approach would have potential use for investigating drug targets in neurodegenerative diseases.

A Long‐Living Bioengineered Neural Tissue Platform to Study Neurodegeneration

“…Next, as we had not observed β‐amyloid (Aβ) plaques within the 10‐month time frame of our previous study, [ 19 ] we sought to evaluate the possibility that long‐term culture conditions would widen the observational window during which we might detect Alzheimer's markers such as an increased ratio of Aβ42 to Aβ40 [ 20,21 ] within media samples as detected by enzyme‐linked immunosorbent assays (ELISAs). Additionally, we examined the ratio of phosphorylated Tau protein (p‐tau) to total Tau (t‐tau) within the media—another clinically‐relevant biomarker associated with Alzheimer's disease.…”

“…ALZ samples (Total n = 29) were not stained or imaged as their availability was limited relative to CON samples (Total n = 92); however, our previous report confirmed the presence of similar structures in ALZ samples at 10 months. [ 19 ] Instead, our limited ALZ samples were subjected to molecular and functional assays and compared to healthy controls (CON).…”

“…For example, ELISAs were performed using cell media rather than cell lysates to multiplex each sample. Whereas we had previously reported that hiPSC source (donor) influenced gene expression, [ 19 ] we did not pursue a similar characterization here; an increased sample size would have permitted a thorough genetic characterization over time within each hiPSC source group. Implementations of any long‐term culture system, including the present model, would benefit from the foresight of a specific, hypothesis‐driven experimental design which would dictate the number of generated samples a priori.…”

“…Building upon our existing tissue model system, we demonstrated that human induced pluripotent stem cells (hiPSCs) differentiated to neurons and glia could be incorporated into the scaffolds and remain functionally active for up to 9 months. [ 19 ] As it was unclear whether the tissues would remain viable and active indefinitely, we continued to generate batches of samples ( Figure 1 A) at irregular intervals over time (Figure S1, Supporting Information) using hiPSCs from donors who were healthy controls (CON) or diagnosed with a sporadic form of Alzheimer's disease (ALZ). The model system would be determined stable and suitable for neurodegeneration research if, as we had predicted, networks would remain structurally and functionally stable over years.…”

Innovations in 3D Tissue Models of Human Brain Physiology and Diseases

“…Limited capability for high-throughput screening applications Porous scaffolds [330][331][332][333][334][335][336][337][338][339][340][341][342]346,351,352,358] Long-term culture capability of larger scale tissue models Challenging to scale-up for high-throughput screening applications . This master pattern is then used as a mold for casting liquid PDMS, curing the polymer, and peeling the resultant replica with a negative relief from the master.…”