Biomanufacturing of patterned human neural tube tissues using microfluidic pluripotent stem cell cultures

Introduction: The human nervous system is arguably the most complex but highly organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube (NT), the embryonic precursor to the human nervous system. Patterning of the NT results in distinct classes of neuronal progenitors specified in the NT along both the rostral-caudal (R-C) and dorsal-ventral (D-V) axes. Studying human neurodevelopment is important for prevention and treatment of NT defects and neurodevelopmental disorders. Historically, studies of NT patterning rely on animal models. However, animal models do not reveal some human-specific aspects of neurodevelopment. Recently, human pluripotent stem cell (hPSC)-based neurodevelopmental models, including neural organoids and bioengineered neural tube development models, are emerging as promising tools to study human neurodevelopment. However, existing models fail to recapitulate neural patterning along two orthogonal axes in a 3D tubular geometry, a hallmark of NT development. To overcome these limitations, we developed a novel microfluidic platform that can precisely control the tissue geometry and chemical environments of hPSCs. We further developed the first fully patterned human NT model by applying this microfluidic platform to guide the differentiation and morphogenesis of hPSCs to recapitulated neural patterning of the entire NT along both the R-C and D-V axes, including both brain and spinal cord (SC) regions.

Materials and

Methods: To achieve microfluidic NT-like structures (or μNTLS), rectangular monolayer hPSC colonies were generated in the central channel of the microfluidic device using microcontact printing (Fig. 1). Under a 3D neural induction environment, monolayer hPSC colonies self-organize to form 3D elongated tubular structures, mimicking the tubular morphology of human NT. To achieve R-C patterned μNTLS, caudal-to-rostral gradients of CHIR, FGF8 and retinoic acid (RA) are established along the length of μNTLS (Fig. 2a). To achieve R-C and D-V patterned μNTLS, an antiparallel BMP and RA/SHH gradients along the width of μNTLS are established following the R-C patterning (Fig. 3a).

Results, Conclusions, and Discussions: μNTLS recapitulates important early human NT development landmarks, including a single continuous central lumen enclosed by neuronal progenitor cells, patterned HOX gene expression along R-C axis, in vivo-like D-V patterning in both brain and SC regions, and emergence of the isthmic organizer (IsO) and neuromesodermal progenitors (NMPs) (Fig. 2&3). Transcriptomic analysis using scRNA-seq further showed that various NT regions along the R-C and D-V axes emerged in the µNTLs, including forebrain (FB), midbrain (MB), IsO, hindbrain (HB), SC, NMP, neural crest (NC), roof plate (RP), floor plate (FP), and neuron, indicating that the µNTLS is a complete human NT model (Fig. 4). Depending on their R-C identities, NC cells in μNTLS develop into various NC lineages, including mesenchymal cells, neurons, Schwann cells, and melanoblasts. In addition to its technological breakthroughs, this novel complete NT model enables new knowledge that is difficult to study using other systems. Specially, we revealed the pre-patterning of axial identities of NC before their delamination and the functional roles of NMPs in trunk NC development. CHARGE syndrome is a genetic disease that causes various craniofacial abnormalities, and we have little understanding of the disruptions in the development processes that underpin those defects. By using CHARGE disease patient-derived human induced pluripotent stem cells (hiPSCs), we find that CHARGE iPSC-μNTLS showed defective NC migration and abnormal brain development, partially explaining the defective craniofacial development in CHARGE patients (Fig. 5).
In summary, the µNTLS offer 3D lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, and they are promising for studying human neurodevelopment and disease.

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