ABSTRACT
The differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) towards functional neurons particularly hold great potential for the cell-based replacement therapy in neurodegenerative diseases. Here, we describe a stepwise differentiation protocol that mimics the early stage of neural development in human to promote the generation of neuroprogenitors at a high yield. Both the hESCs and hiPSCs are initially cultured in an optimized feeder-free condition, which offer an efficient formation of aggregates. To specify the neuroectodermal specification, these aggregates are differentiated in a defined neural induction medium to develop into neural rosettes-like structures. The rosettes are expanded into free-floating sphere and can be further propagated or developed into variety of neuronal subtypes.
Subject(s)
Cell Culture Techniques/methods , Neurons/cytology , Pluripotent Stem Cells/cytology , Stem Cells/cytology , Cell Differentiation/physiology , HumansABSTRACT
OBJECTIVE: A challenge in using human embryonic stem cells (hESCs) as the source of surrogate ß cells is the establishment of methods that could effectively direct their differentiation into functional ß cells. The aim of this study was to assess the effect of NANOG gene suppression in differentiating hESCs as a mean of increasing the efficiency with which endoderm-derived pancreatic cells could be generated. METHODS: A homogenous cell population with stable suppression of NANOG was generated in hESC ENVY line using plasmid-based siRNA approach. Pancreatic differentiation was undertaken according to the ontology-based in vitro selection protocol and followed by transplantation into immunodeficiency mice to mature in vivo. RESULTS: We observed up-regulation of definitive endoderm genes, which expand the role of NANOG in blocking definitive endoderm differentiation. The ontology-based differentiation protocol resulted in increased expression of markers essential for pancreatic epithelium development. Transplantation of these cells further revealed a homogenous pancreatic exocrine-like morphology that stained positively for amylase. CONCLUSIONS: The suppression of NANOG displayed an effective differentiation toward endoderm and pancreatic progenitors. Investigation of the factors required for endocrine formation combined with a prolonged in vivo culturing could be further used to increase the ratio of endocrine-exocrine cells fate.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Endoderm/cytology , Homeodomain Proteins/metabolism , Pancreas/cytology , Amylases/metabolism , Animals , Blotting, Western , Cells, Cultured , Embryonic Stem Cells/metabolism , Endoderm/metabolism , Homeodomain Proteins/genetics , Humans , Immunohistochemistry , Mice , Mice, Inbred NOD , Mice, SCID , Microscopy, Phase-Contrast , Nanog Homeobox Protein , Pancreas/metabolism , Pancreas, Exocrine/cytology , Pancreas, Exocrine/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Transplantation/methods , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
Alzheimer's disease (AD) is a leading cause of age-related dementia that is characterized by an extensive loss of neurons and synaptic transmission. The pathological hallmarks of AD are neurofibrillary tangles and deposition of ß-amyloid (Aß) plaques. Previous research has investigated how Aß fragments disrupt synaptic mechanisms in the vulnerable regions of the brain. There is a tremendous potential for stem cell technology to extend upon this research, not only in terms of developing therapeutic applications, but also in modeling AD. Indeed, the advent of induced pluripotent stem cell technology has opened up exciting new avenues for generating patient and disease-specific cell lines from somatic cells that may be used to model AD. Amyloid precursor protein (APP) is a key protein in neuronal development and this article reviews the role of APP in AD. Stem cell technology offers the opportunity to make use of APP in the directed differentiation of induced pluripotent stem cells into functional neurons, a process that may help generate a model of AD and thereby facilitate an understanding of the mechanisms underlying this disease.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/metabolism , Neurogenesis , Pluripotent Stem Cells/physiology , Amyloid beta-Protein Precursor/physiology , Animals , Disease Models, Animal , Humans , Plaque, Amyloid/metabolism , Pluripotent Stem Cells/metabolism , Protein Processing, Post-TranslationalABSTRACT
Successful gene targeting in human embryonic stem (hES) cells requires the use of primary fibroblast feeder layers, which assist in the maintenance of the pluripotent state of hES cells. Such feeder layers must also survive any further selection strategy for hES cells. Here we report the production of a novel transgenic human fetal fibroblast (tHFF) as a feeder layer that is resistant to puromycin and can be used for gene targeting and selection of positive clones in hES cells. tHFFs survive under a wide range of puromycin concentrations (0.5-2 microg/ml) and also supports the undifferentiated growth of hES cells. We have demonstrated here that tHFFs are suitable for selecting Envy-hES cells that were transfected with a green fluorescent protein-small interfering RNA (GFP-siRNA) plasmid construct to induce GFP gene down-regulation. The later studies were designed to isolate and propagate stably knockdown cells. tHFFs thus can be used for targeting other genes that would serve as a model to select and understand the differentiation process in hES cells.
