Journal of Translational Neurosciences

Description

Human Neural Stem Cell (NSC) production from differentiation of Human Pluripotent Stem Cell (hPSC) is complicated by the time-consuming procedure, complex medium composition, and purification step, despite significant advancements in commercially available media and kits and differentiation approaches. The scarcity of human brain tissues has hampered brain research for a long time. One of the most important facts is that, in contrast to the majority of organs, where tissue samples can be obtained through biopsy, the human brain is not suitable for this procedure. As a result, the majority of studies on neurodevelopmental disorders must use small animals. Sadly, it is frequently discovered that experiments conducted on small animals do not accurately reflect human circumstances. Due to their ability to produce neurons and glia subtypes through advanced differentiation processes, Human Pluripotent Stem Cells (hPSCs), which include Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs), have recently emerged as an excellent renewable source of brain tissue.

Cell-Based Replacement Therapies and the Screening of New Drugs for the Nervous System

Human Neural Stem Cells (NSCs) that have been differentiated from hPSCs, for instance, may be an important tool for modeling the development of the brain and neurological disorders. Specifically, iPSCs technology can be utilized to generate patient-specific NSCs. Therefore, cell-based replacement therapies and the screening of new drugs for the nervous system require the effective production, expansion, and differentiation of NSCs from hPSCs. To this point, extensive efforts have been put into developing a protocol for the efficient generation of human NSCs through differentiation of hPSC. A minimum of four to seven days of the Embryoid Body (EB) formation stage is typically reported as the first step in a stepwise NSC generation procedure. The EB culture can even take longer to produce NSCs in some procedures. For instance, the Liu group reported that they were able to generate NSCs from hPSCs by starting with ten days of EB culture in a complicated medium that included the conditioned medium of a stromal cell line, Rock inhibitor, growth factors, PA6, and adherent culture for four to seven days before cell sorting. The neural rosettes were produced by hESCs seeded in a monolayer culture environment after approximately one month. Despite their high costs, neural differentiation media and kits have recently become commercially available. Direct lineage specification of hPSCs into a specific neural cell type, such as a dopaminergic neuron, motor neuron, astrocyte, or oligodendrocyte, has been developed as an alternative. Finding small molecules that can use intracellular signaling pathways to boost the efficiency of NSC differentiation is another strategy. The NSC differentiation medium can be supplemented with a number of small molecules to induce NSC generation. Dorsomorphin, CHIR99021, SB431542, insulin, transferrin, sodium selenite, fibronectin, noggin, selenous acid, EGF, and bFGF are among these molecules. Multiple small molecules can be used to directly generate neurons from hPSC, avoiding the need for completed NSC production processes. Neural lineage specification is promoted by inhibitors that can suppress bone morphogenic protein and TGF/activin/nodal signaling. Through Notch signaling, the transmembrane protein Dlk1 that is related to Notch can help neural progenitors derived from hESCs differentiate. As a result, the production of neural progenitor cells can be significantly enhanced by including Dlk1. Using two inhibitors of SMAD signaling simultaneously can facilitate neural conversion of hPSCs. Multiple small molecules can direct cell lineage specification from hESCs without the EB formation stage, based on the knowledge that human primitive neural precursors can be generated from hESCs if Glycogen Synthase Kinase 3 (GSK3), Transforming Growth Factor (TGF-), and Notch pathways are inhibited. However, the majority of these differentiation procedures were time-consuming and laborious because they either required EB formation as the first step of lineage restriction or used multiple small molecules and growth factors to mimic early human embryogenesis.

Strategy for Producing Self-Renewable NSCs

In addition, numerous studies have utilized mechanical isolation of neural rosettes, which is ineffective and results in a heterogeneous population of neural cells containing unknown derivatives and undifferentiated hPSC remnants. By suspension culture of hESCs and iPSCs in a differentiation medium containing heparin and a high concentration of EGF and bFGF, the Ebert group, for instance, developed a method for generating and expanding pre-rosette neural progenitors. An automated tissue chopping apparatus made in the United Kingdom by Mickle Laboratory Engineering Co. Ltd was used to move the cell aggregates. The automated tissue chopping method, on the other hand, does not pass pro-rosettes that could be Neural Stem Cells (NSCs) or Neural Progenitor Cells (NPCs) selectively because it requires specialized equipment. To meet the needs of research into brain development and neurological diseases, a simplified NSC generation protocol must be developed. In this study, we discuss our recent findings that ectoderm lineage specification from hPSCs may be facilitated by a brief suspension culture period. Using a simple and inexpensive culture medium, NSCs can be produced from hPSCs without the need for signaling molecules or an EB formation step. A straightforward strategy for producing self-renewable NSCs that can facilitate a wide range of scientific research applications for human brain studies, this new method is also time-effective and enables rapid production of NSCs. Using a simplified medium formulation and procedure, we demonstrated a straightforward method for producing NSCs from hPSCs effectively. Ectoderm lineage specification from hPSC differentiation is facilitated by a dynamic change in cell-substrate matrix interactions during a brief suspension culture period, according to our findings. This method makes it possible to rapidly produce NSCs with a consistent identity and the potential to produce a variety of cells.