Derivation of Neural Precursor Cells from Human Embryonic Stem Cells for DNA Methylomic Analysis

Abstract

Embryonic stem cells are self-renewing pluripotent cells with competency to differentiate into all three-germ lineages. Many studies have demonstrated the importance of genetic and epigenetic molecular mechanisms in the maintenance of self-renewal and pluripotency. Stem cells are under unique molecular and cellular regulations different from somatic cells. Proper regulation should be ensured to maintain their unique self-renewal and undifferentiated characteristics. Understanding key mechanisms in stem cell biology will be important for the successful application of stem cells for regenerative therapeutic medicine. More importantly practical use of stem cells will require our knowledge on how to properly direct and differentiate stem cells into the necessary type of cells. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have tested commercial neural differentiation system and successfully derived neural precursor cells exhibiting key molecular features of neural stem cells, which will be useful for experimental application.

iPSCs-Based Neural 3D Systems: A Multidimensional Approach for Disease Modeling and Drug Discovery

“…Human ESC lines were first isolated in 1998 [1] and differentiation protocols towards multiple tissues were soon designed, aiming to eventually develop allogeneic cell-based therapies to several degenerative diseases. As for neural disease modeling, ESCs were successfully differentiated to neural precursors [2] and many neuronal subtypes, e.g., dopaminergic neurons [3] and motor neurons [4], as well as astrocytes [5], oligodendrocytes [6] and microglia [7]. However, ESCs advantages were offset by the need of genetic manipulation to introduce disease-relevant mutations and their limited supply [8].…”

Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells

“…To establish a model system to examine the molecular effects of EtOH on neural differentiation and maintenance of neural stem cells, we used a commercial culture system developed for neural differentiation of human embryonic stem cells (STEMCELL Technologies Inc., Vancouver, Canada) as we have previously reported [ 23 , 24 ]. Human embryonic stem cells (hESCs) exponentially growing on mouse embryonic fibroblast (MEF) feeder were first adapted to the feeder-free culture system using mTeSR1 medium (STEMCELL Technologies Inc., Vancouver, Canada) and subjected to embryoid body formation in neural differentiation medium as described in Materials and Methods.…”

“…Human embryonic stem cells (H1 and H9 lines) obtained through license agreement with WiCell Research Institute (Madison, WI) were cultured on mouse embryonic fibroblast (MEF) feeder layer and were transferred to mTeSR1 serum free human embryonic stem cell (hESC) culture system (STEMCELL Technologies Inc., Vancouver, Canada). Neural differentiation of hESCs was performed by using STEMdiff Neural System (STEMCELL Technologies Inc., Vancouver, Canada) according to the manufacturer’s instruction as described in our previous publications [ 23 , 24 ]. After 7 day differentiation, morphological assessment and scoring of neural rosettes were done to ensure 50% or more of the area of each aggregate was filled with neural rosettes (as shown in Fig 1 ).…”

“…We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have successfully derived neural precursor cells exhibiting key molecular features of neural stem cells, which will be useful for experimental application [ 23 ]. Our established model has further been used to assess the molecular effect of alcohol treatment on neural stem cell population.…”