Development of electrophysiological and morphological properties of human embryonic stem cell-derived GABAergic interneurons at different times after transplantation into the mouse hippocampus.

Transplantation of human embryonic stem cell (hESC)-derived neural progenitors is a potential treatment for neurological disorders, but relatively little is known about the time course for human neuron maturation after transplantation and the emergence of morphological and electrophysiological properties. To address this gap, we transplanted hESC-derived human GABAergic interneuron progenitors into the mouse hippocampus, and then characterized their electrophysiological properties and dendritic arborizations after transplantation by means of ex vivo whole-cell patch clamp recording, followed by biocytin staining, confocal imaging and neuron reconstruction software. We asked whether particular electrophysiological and morphological properties showed maturation-dependent changes after transplantation. We also investigated whether the emergence of particular electrophysiological properties were linked to increased complexity of the dendritic arbors. Human neurons were classified into five distinct neuronal types (Type I-V), ranging from immature to mature fast-spiking interneurons. Hierarchical clustering of the dendritic morphology and Sholl analyses suggested four morphologically distinct classes (Class A-D), ranging from simple/immature to highly complex. Incorporating all of our data regardless of neuronal classification, we investigated whether any electrophysiological and morphological features correlated with time post-transplantation. This analysis demonstrated that both dendritic arbors and electrophysiological properties matured after transplantation.

Derivation and isolation of NKX2.1-positive basal forebrain progenitors from human embryonic stem cells.

Gamma aminobutyric acid (GABA)-expressing interneurons are the major inhibitory cells of the cerebral cortex and hippocampus. These interneurons originate in the medial ganglionic eminence (MGE) and lateral ganglionic eminence of the ventral forebrain during embryonic development and show reduced survival and function in a variety of neurological disorders, including temporal lobe epilepsy. We and others have proposed that embryonic stem cell (ESC)-derived ventral forebrain progenitors might provide a source of new GABAergic interneurons for cell-based therapies. While human ESCs (hESCs) are readily differentiated in vitro into dorsal telencephalic neural progenitors, standard protocols for generating ventral subtypes of telencephalic progenitors are less effective. We now report efficient derivation of GABAergic progenitors using an established hESC reporter line that expresses green fluorescent protein (GFP) under the control of an endogenous NKX2.1 promoter. GABAergic progenitors were derived from this hESC line by a modified monolayer neural differentiation protocol. Consistent with sonic hedgehog (SHH)-dependent specification of NKX2.1-positive progenitors in the embryonic MGE, we show a dose-dependent increase in the generation of NKX2.1:GFP-positive progenitors after SHH treatment in vitro. Characterization of NKX2.1:GFP-positive cells confirms their identity as MGE-like neural progenitors, based on gene expression profiles and their ability to differentiate into GABAergic interneurons. We are also able to generate highly enriched populations of NKX2.1:GFP-positive progenitors, including cells with telencephalic identity, by fluorescence-activated cell sorting. These hESC-derived ventral forebrain progenitors are suitable candidates for cell-based therapies that aim at replacing dysfunctional or damaged cortical or hippocampal GABAergic interneurons.

Teratocarcinoma formation in embryonic stem cell-derived neural progenitor hippocampal transplants.

Embryonic stem cells (ESCs) hold great therapeutic potential due to their ability to differentiate into cells of the three primary germ layers, which can be used to repopulate disease-damaged tissues. In fact, two cell therapies using ESC derivatives are currently in phase I clinical trials. A main concern in using ESCs and their derivatives for cell transplantation is the ability of undifferentiated ESCs to generate tumors in the host. Positive selection steps are often included in protocols designed to generate particular cell types from ESCs; however, the transition from ESC to progenitor cell or terminally differentiated cell is not synchronous, and residual undifferentiated cells often remain. In our transplants of ESC-derived neural progenitors (ESNPs) into the adult mouse hippocampus, we have observed the formation of teratocarcinomas. We set out to reduce teratocarcinoma formation by enrichment of ESNPs using fluorescence-activated cell sorting (FACS) and have found that, although enrichment prior to transplant reduces the overall rate of teratocarcinoma formation, the tumorigenicity of cell batches can vary widely, even after FACS enrichment to as much as 95% ESNPs. Our data suggest that this variability may be due to the percentage of residual ESCs remaining in the transplant cell population and to the presence of pluripotent epiblast-like cells, not previously identified in transplant batches. Our data emphasize the need for stringent characterization of transplant cell populations that will be used for cell replacement therapies in order to reduce the risk of tumor formation.

Differentiation and functional incorporation of embryonic stem cell-derived GABAergic interneurons in the dentate gyrus of mice with temporal lobe epilepsy.

Cell therapies for neurological disorders require an extensive knowledge of disease-associated neuropathology and procedures for generating neurons for transplantation. In many patients with severe acquired temporal lobe epilepsy (TLE), the dentate gyrus exhibits sclerosis and GABAergic interneuron degeneration. Mounting evidence suggests that therapeutic benefits can be obtained by transplanting fetal GABAergic progenitors into the dentate gyrus in rodents with TLE, but the scarcity of human fetal cells limits applicability in patient populations. In contrast, virtually limitless quantities of neural progenitors can be obtained from embryonic stem (ES) cells. ES cell-based therapies for neurological repair in TLE require evidence that the transplanted neurons integrate functionally and replace cell types that degenerate. To address these issues, we transplanted mouse ES cell-derived neural progenitors (ESNPs) with ventral forebrain identities into the hilus of the dentate gyrus of mice with TLE and evaluated graft differentiation, mossy fiber sprouting, cellular morphology, and electrophysiological properties of the transplanted neurons. In addition, we compared electrophysiological properties of the transplanted neurons with endogenous hilar interneurons in mice without TLE. The majority of transplanted ESNPs differentiated into GABAergic interneuron subtypes expressing calcium-binding proteins parvalbumin, calbindin, or calretinin. Global suppression of mossy fiber sprouting was not observed; however, ESNP-derived neurons formed dense axonal arborizations in the inner molecular layer and throughout the hilus. Whole-cell hippocampal slice electrophysiological recordings and morphological analyses of the transplanted neurons identified five basic types; most with strong after-hyperpolarizations and smooth or sparsely spiny dendritic morphologies resembling endogenous hippocampal interneurons. Moreover, intracellular recordings of spontaneous EPSCs indicated that the new cells functionally integrate into epileptic hippocampal circuitry.

Region-specific differentiation of embryonic stem cell-derived neural progenitor transplants into the adult mouse hippocampus following seizures.

Embryonic stem (ES) cells can generate neural progenitors and neurons in vitro and incorporate into the adult central nervous system (CNS) following transplantation, suggesting their therapeutic potential for treating neurological disorders. However, our understanding of the conditions that direct ES-derived neural progenitor (ESNP) migration and differentiation within different regions of the adult CNS is incomplete. Rodents treated with the chemoconvulsant kainic acid (KA) experience seizures and display hippocampal sclerosis, as well as enhanced hippocampal neurogenesis, similar to pathological findings in patients with temporal lobe epilepsy (TLE). To examine the potential for ESNPs to incorporate into the adult hippocampus and differentiate into hippocampal neurons or glia following seizure-induced damage, we compared the fates of ESNPs after they were transplanted into the CA3 region or fimbria 1 week following KA-induced seizures. After 4-8 weeks, ESNPs grafted into the CA3 region had migrated to the dentate gyrus (DG), where a small subset adopted neural stem cell fates and continued to proliferate, based on bromodeoxyuridine uptake. Others differentiated into neuroblasts or dentate granule neurons. In contrast, most ESNPs transplanted into the fimbria migrated extensively along existing fiber tracts and differentiated into oligodendrocytes or astrocytes. Hippocampal grafts in mice not subjected to seizures displayed a marked tendency to form tumors, and this effect was more pronounced in the DG than in the fimbria. Taken together, these data suggest that seizures induce molecular changes in the CA3 region and DG that promote region-specific neural differentiation and suppress tumor formation.

Migration of F9 parietal endoderm cells is regulated by the ERK pathway.

Cell migration is regulated by the action of many signaling pathways that are activated in specific regions of migrating cells. Extracellular regulated kinase 1/2 (ERK) signaling can modulate the migration of cells by controlling the turnover of focal adhesions and the dynamics of actin polymerization. Focal adhesion turnover is necessary for cell migration, and the formation of strong actin stress fibers and mature focal adhesions puts the brakes on cell migration. We used F9 wild-type and vinculin null (vin-/-) parietal endoderm (PE) outgrowth to study the role of the ERK signaling pathway in cell migration. Upon plating of F9 embryoid bodies (EBs) onto laminin-coated dishes, PE cells migrate away from the EBs, providing an in vitro model for studying directed migration of this embryonic cell type. Our results suggest that the ERK pathway regulates PE cell migration by affecting the formation of focal adhesions and lamellipodia through the action of myosin light chain kinase (MLCK).

CD9-alpha6beta1 interactions in migratory parietal endoderm cells.

Tetraspanins modulate the function of a variety of membrane proteins, including integrin receptors. We show here that the tetraspanin CD9 preferentially coimmunoprecipitates with the alpha6beta1 integrin heterodimer in F9-derived parietal endoderm cells in comparison to F9 stem cells. We also show that CD9 function-blocking antibody inhibits parietal endoderm migration in an embryoid body outgrowth assay. In addition, both CD9 and alpha6beta1 colocalize with vinculin to apparent focal adhesion sites in parietal endoderm cells. The data presented here suggests a role for CD9 in localizing the integrin to the focal adhesion. In addition, the data suggest a role for CD9 in alpha6beta1 mediated migration of parietal endoderm.