Derivation of High Purity Neuronal Progenitors from Human Embryonic Stem Cells

The availability of human neuronal progenitors (hNPs) in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs) in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use

Automated analysis of remyelination therapy for spinal cord injury

Demyelination- the loss of myelin sheath that insulates axons, is a prominent feature in many neurological disorders resulting in spinal cord injury (SCI). The lost myelin sheath can be replaced by remyelination, used in SCI treatment. In this paper, we pro-pose an algorithm for efficient automated analysis of remyelination therapy. We use a robust, shape-independent algorithm based on iso-contour analysis of the image at progressively increasing inten-sity levels for detecting cell boundaries. The detected boundaries of spatially clustered cells are then separated using Delaunay triangu-lation based contour separation method. The therapeutically impor-tant oligodendrocyte-remyelinated axons (OR-axons) are identified and a density map is generated for efficacy analysis of the ther-apy. Our efficient automated remyelination analysis significantly reduces error due to human subjectivity. We corroborate the ac-curacy of our results by extensive cross-verification by the domain experts. 1

Human Motor Neuron Progenitor Transplantation Leads to Endogenous Neuronal Sparing in 3 Models of Motor Neuron Loss

Motor neuron loss is characteristic of many neurodegenerative disorders and results in rapid loss of muscle control, paralysis, and eventual death in severe cases. In order to investigate the neurotrophic effects of a motor neuron lineage graft, we transplanted human embryonic stem cell-derived motor neuron progenitors (hMNPs) and examined their histopathological effect in three animal models of motor neuron loss. Specifically, we transplanted hMNPs into rodent models of SMA (Δ7SMN), ALS (SOD1 G93A), and spinal cord injury (SCI). The transplanted cells survived and differentiated in all models. In addition, we have also found that hMNPs secrete physiologically active growth factors in vivo, including NGF and NT-3, which significantly enhanced the number of spared endogenous neurons in all three animal models. The ability to maintain dying motor neurons by delivering motor neuron-specific neurotrophic support represents a powerful treatment strategy for diseases characterized by motor neuron loss

Human motor neuron progenitor transplantation leads to endogenous neuronal sparing in 3 models of motor neuron loss.

Motor neuron loss is characteristic of many neurodegenerative disorders and results in rapid loss of muscle control, paralysis, and eventual death in severe cases. In order to investigate the neurotrophic effects of a motor neuron lineage graft, we transplanted human embryonic stem cell-derived motor neuron progenitors (hMNPs) and examined their histopathological effect in three animal models of motor neuron loss. Specifically, we transplanted hMNPs into rodent models of SMA (Δ7SMN), ALS (SOD1 G93A), and spinal cord injury (SCI). The transplanted cells survived and differentiated in all models. In addition, we have also found that hMNPs secrete physiologically active growth factors in vivo, including NGF and NT-3, which significantly enhanced the number of spared endogenous neurons in all three animal models. The ability to maintain dying motor neurons by delivering motor neuron-specific neurotrophic support represents a powerful treatment strategy for diseases characterized by motor neuron loss

Human Motor Neuron Progenitor Transplantation Leads to Endogenous Neuronal Sparing in 3 Models of Motor Neuron Loss

Motor neuron loss is characteristic of many neurodegenerative disorders and results in rapid loss of muscle control, paralysis, and eventual death in severe cases. In order to investigate the neurotrophic effects of a motor neuron lineage graft, we transplanted human embryonic stem cell-derived motor neuron progenitors (hMNPs) and examined their histopathological effect in three animal models of motor neuron loss. Specifically, we transplanted hMNPs into rodent models of SMA (Δ7SMN), ALS (SOD1 G93A), and spinal cord injury (SCI). The transplanted cells survived and differentiated in all models. In addition, we have also found that hMNPs secrete physiologically active growth factors in vivo, including NGF and NT-3, which significantly enhanced the number of spared endogenous neurons in all three animal models. The ability to maintain dying motor neurons by delivering motor neuron-specific neurotrophic support represents a powerful treatment strategy for diseases characterized by motor neuron loss

Culture manipulation altered the percentage of neuronal subtype derivates.

<p>In RA free conditions with the addition of factors (A), DARPP32 expression was detected in a subset of cells (B), demonstrating the ability to become GABA-responsive neurons. In RA free conditions with the addition of factors (C), 5-HT expression was detected in a subset of cells (D), demonstrating the ability to become serotonergic neurons. In RA free conditions with the addition of BDNF, ChAT expression was detected in a subset of cells (E), demonstrating the ability to become cholinergic neurons. F) In RA free conditions with the addition of BDNF, 5-HT and ChAT colocalize. G) Alteration of growth factors in RA-free conditions changes ChAT expression. In RA free conditions with the addition of FGF2 (H), TH expression was detected in a subset of cells (I), demonstrating the ability to become dopaminergic neurons. J) In RA-containing conditions, few or no TH positive cells were present.</p

Differentiation of hNPs 3 months after transplantation to spinal cord injury sites.

<p>A) Human nuclei-positive cells (green) expressed TUJ1 (red), consistent with a neuronal phenotype. B) Human nuclei-positive cells (green) expressed doublecortin (red), consistent with a young neuronal phenotype. C) Human nuclei-positive cells (green) expressed p75 (red), consistent with young motor, sensory and sympathetic neurons. D) Human nuclei-positive cells (green) expressed GAD 65–67 (red), consistent with an interneuronal phenotype. E) Human nuclei-positive cells (brown) expressed ChAT (gray), consistent with a cholinergic phenotype (nuclear counterstain in purple). F) Human specific NCAM (green) and synaptophysin (red) immunolabeling suggests integration of transplanted cells with the host environment.</p