FREDERICK, Md.—A group of researchers led by the Cell Biology and Stem Cell Sciences division of Life Technologies have discovered a new way to grow neural stem cells with the hope that they may be used to accelerate drug discovery and development for brain injuries and disease. Publishing their findings in STEM CELLS Translational Medicine, the team believes their new method will go far in resolving some of the time-consuming, labor-intensive steps currently involved in inducing neural stem cells (NSCs).
Joined by colleagues at the Buck Institute for Age Research in Novato, Calif., the Methodist Hospital Research Institute in Houston and the U.S. National Institutes of Health in Bethesda, Md., Life Technologies has developed a simple, one-step protocol with a good manufacturing practice (GMP)-manufactured medium that is rapid and efficient in the derivation of neural stem cells from human pluripotent stem cells (hPSCs) that retain the cues to differentiate into different disease relevant neurons.
Although hPSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), show great promise in regenerative medicine due to their ability to be “coaxed” into becoming different specific types of cells, the current methods for inducing neural stem cells involve the time-consuming, labor-intensive steps of an embryoid body generation or co-culture with stromal cell lines that result in low-efficiency derivation of NSCs—a process that can take up to three weeks. To enable the transition of hPSCs for cell therapy applications, more efficient and reproducible differentiations methods for desired lineages—such as neurons—are needed.
In the new study, however, the researchers report a highly efficient, serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs in only one week, obviating the need for time-consuming, laborious embryoid body generation or rosette picking.
“With current methods, we’re talking about a 20-day procedure where you start with 1 million pluripotent stem cells, and you may get less than 1 million neural stem cells,” says Dr. Mohan C. Vemuri, director of research and development for Cell Biology and Stem Cell Sciences at Life Technologies. “With the new method, you start with 1 million healthy pluripotent cells and get 40 million neural stem cells in seven days. "You can get 20 million neural stem cells, even from iPSC lines derived from Parkinson’s disease patient samples.".”
The research team’s method starts with any pluripotent stem cell, using a commercially available cGMP-manufactured culture medium that eliminates variability, ensuring lot-to-lot consistency and scalable production of neural stem cells. The method enables the derivation of NSCs in a primitive state that retains the regional positional cues in brain development, making it easier for researchers to generate neural subtypes from all regions of the brain. The entire neural induction process involves a seven-day culture process initiated by plating specific cell densities of PSCs with culture medium changes every other day. On Day 7 of the culture period, NSCs can be harvested by simple enzymatic dissociation. The type of morphology transition to neural fate from PSCs allows culture of cells at much higher densities compared with cells grown as embryoid body followed by rosette formation and picking, the study concludes.
NSCs derived by this method still displayed multipotentiality, with successful differentiation to generic neurons, GABA neurons, dopaminergic neurons, motor neurons, astrocytes, and oligodendrocytes. This differentiation capacity was retained even after expansion of NSCs, demonstrating the competence of the derived NSCs and the robustness of the neural induction method. Based on pairwise comparison of global gene expression, a close correlation between the expression patterns of NSCs grown as rosette-derived versus rosette-free-derived was observed. In addition, cells grown and expanded in neural induction media for longer passages continued to show a close correlation, suggesting minimal genotypic changes upon extended passaging.
Gene-expression data comparing these NSCs with other NSCs, such as rosette-derived and human fetal brain-derived NSCs, was consistent with reported transcript profiles. Based on these results, the researchers concluded there is no significant drift in global gene expression with extended passaging in this medium system with scalable production of primitive NSCs that have the ability to both self-renew and differentiate into neural subtypes.
The study demonstrates, as a proof of principle, that a few subtypes of neurons can be generated, such as GABA neurons from the forebrain, dopaminergic neurons from the midbrain and motor neurons from the hindbrain. The new method offers scope to generate exact neural cell types needed for central nervous system diseases, such as Parkinson’s disease, amyotrophic lateral sclerosis and Huntington’s disease.
Additional studies are warranted to generate region-specific multiple neural types in human brain to fully explore function, genotype changes and disease modeling of these neural cells, says Vemuri.
“We have to be creative in terms of creating stability for this product,” he says. “Then it has to run through a one-year shelf life in our process before the product is rolled out. Right now, we have a 14-month shelf life completed. The goal here is to get a really scalable and robust process for neural stem cell induction to use in drug screening and discovery.”
The study, “Efficient and Rapid Derivation of Primitive Neural Stem Cells and Generation of Brain Subtype Neurons from Human Pluripotent Stem Cells,” was published online in STEM CELLS Translational Medicine on Oct. 10.
