Nathaniel W. Hartman

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.

mTORC1 Targets the Translational Repressor 4E-BP2, but Not S6 Kinase 1/2, to Regulate Neural Stem Cell Self-Renewal In Vivo

The mammalian target of rapamycin complex 1 (mTORC1) integrates signals important for cell growth, and its dysregulation in neural stem cells (NSCs) is implicated in several neurological disorders associated with abnormal neurogenesis and brain size. However, the function of mTORC1 on NSC self-renewal and the downstream regulatory mechanisms are ill defined. Here, we found that genetically decreasing mTORC1 activity in neonatal NSCs prevented their differentiation, resulting in reduced lineage expansion and aborted neuron production. Constitutive activation of the translational repressor 4E-BP1, which blocked cap-dependent translation, had similar effects and prevented hyperactive mTORC1 induction of NSC differentiation and promoted self-renewal. Although 4E-BP2 knockdown promoted NSC differentiation, p70 S6 kinase 1 and 2 (S6K1/S6K2) knockdown did not affect NSC differentiation but reduced NSC soma size and prevented hyperactive mTORC1-induced increase in soma size. These data demonstrate a crucial role of mTORC1 and 4E-BP for switching on and off cap-dependent translation in NSC differentiation.

A circuitry and biochemical basis for tuberous sclerosis symptoms: from epilepsy to neurocognitive deficits.

Tuberous sclerosis complex (TSC) is an autosomal dominant monogenetic disorder that is characterized by the formation of benign tumors in several organs as well as brain malformations and neuronal defects. TSC is caused by inactivating mutations in one of two genes, TSC1 and TSC2, resulting in increased activity of the mammalian Target of Rapamycin (mTOR). Here, we explore the cytoarchitectural and functional CNS aberrations that may account for the neurological presentations of TSC, notably seizures, hydrocephalus, and cognitive and psychological impairments. In particular, recent mouse models of brain lesions are presented with an emphasis on using electroporation to allow the generation of discrete lesions resulting from loss of heterozygosity during perinatal development. Cortical lesions are thought to contribute to epileptogenesis and worsening of cognitive defects. However, it has recently been suggested that being born with a mutant allele without loss of heterozygosity and associated cortical lesions is sufficient to generate cognitive and neuropsychiatric problems. We will thus discuss the function of mTOR hyperactivity on neuronal circuit formation and the potential consequences of being born heterozygous on neuronal function and the biochemistry of synaptic plasticity, the cellular substrate of learning and memory. Ultimately, a major goal of TSC research is to identify the cellular and molecular mechanisms downstream of mTOR underlying the neurological manifestations observed in TSC patients and identify novel therapeutic targets to prevent the formation of brain lesions and restore neuronal function.

CXCL12-Mediated Guidance of Migrating Embryonic Stem Cell-Derived Neural Progenitors Transplanted into the Hippocampus

Stem cell therapies for neurodegenerative disorders require accurate delivery of the transplanted cells to the sites of damage. Numerous studies have established that fluid injections to the hippocampus can induce lesions in the dentate gyrus (DG) that lead to cell death within the upper blade. Using a mouse model of temporal lobe epilepsy, we previously observed that embryonic stem cell-derived neural progenitors (ESNPs) survive and differentiate within the granule cell layer after stereotaxic delivery to the DG, replacing the endogenous cells of the upper blade. To investigate the mechanisms for ESNP migration and repair in the DG, we examined the role of the chemokine CXCL12 in mice subjected to kainic acid-induced seizures. We now show that ESNPs transplanted into the DG show extensive migration through the upper blade, along the septotemporal axis of the hippocampus. Seizures upregulate CXCL12 and infusion of the CXCR4 antagonist AMD3100 by osmotic minipump attenuated ESNP migration. We also demonstrate that seizures promote the differentiation of transplanted ESNPs toward neuronal rather than astrocyte fates. These findings suggest that ESNPs transplanted into the adult rodent hippocampus migrate in response to cytokine-mediated signals.

S Phase Entry of Neural Progenitor Cells Correlates with Increased Blood Flow in the Young Subventricular Zone

The postnatal subventricular zone (SVZ) contains proliferating neural progenitor cells in close proximity to blood vessels. Insults and drug treatments acutely stimulate cell proliferation in the SVZ, which was assessed by labeling cells entering S phase. Although G1-to-S progression is metabolically demanding on a minute-to-hour time scale, it remains unknown whether increased SVZ cell proliferation is accompanied by a local hemodynamic response. This neurovascular coupling provides energy substrates to active neuronal assemblies. Transcardial dye perfusion revealed the presence of capillaries throughout the SVZ that constrict upon applications of the thromboxane A2 receptor agonist U-46119 in acute brain slice preparations. We then monitored in vivo blood flow using laser Doppler flowmetry via a microprobe located either in the SVZ or a mature network. U-46119 injections into the lateral ventricle decreased blood flow in the SVZ and the striatum, which are near the ventricle. A 1-hour ventricular injection of epidermal and basic fibroblast growth factor (EGF and bFGF) significantly increased the percentage of Sox2 transcription factor-positive cells in S phase 1.5 hours post-injection. This increase was accompanied by a sustained rise in blood flow in the SVZ but not in the striatum. Direct growth factor injections into the cortex did not alter local blood flow, ruling out direct effects on capillaries. These findings suggest that an acute increase in the number of G1-to-S cycling SVZ cells is accompanied by neurometabolic-vascular coupling, which may provide energy and nutrient for cell cycle progression.

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.

Switching on mTORC1 induces neurogenesis but not proliferation in neural stem cells of young mice.

Recent evidence reported that activation of the mechanistic target of rapamycin complex 1 (mTORC1) induces terminal differentiation of neural stem cells (NSCs) in the neonatal subventricular zone (SVZ), but did not affect their proliferation. Here, we investigated whether such an effect of hyperactive mTORC1 would be recapitulated in young adults following removal of the negative mTORC1 regulator TSC1as seen in the neurological disorder tuberous sclerosis complex, TSC. Conditional mTORC1 activation in NSCs of 3-4 weeks old mice resulted in the generation of proliferative (Ki67+) cells and newborn neuroblasts. However, hyperactive mTORC1 did not induce NSCs to proliferate, consistent with the findings that mTORC1 induces symmetric division and differentiation of slow-cycling NSCs into proliferative daughter cells. Taken together these data suggest that hyperactivity of mTORC1 could lead to the progressive loss of NSCs over time.

Embryonic stem cell-derived neural progenitors transplanted to the hippocampus migrate on host vasculature

This study describes the migration of transplanted ESNPs either injected directly into the hippocampus of a mouse, seeded onto hippocampal slices, or under in vitro culture conditions. We show that transplanted mouse ESNPs associate with, and appear to migrate on the surface of the vasculature, and that human ESNPs also associate with blood vessels when seeded on hippocampal slices, and migrate towards BECs in vitro using a Boyden chamber assay. This initial adhesion to vessels is mediated, at least in part, via the integrin α6β1, as observed for SVZ neural progenitor cells. Our data are consistent with CXCL12, expressed by the astroglial-vasculature niche, playing an important role in the migration of transplanted neural progenitors within and outside of the hippocampus.

Migration of Transplanted Neural Stem Cells in Experimental Models of Neurodegenerative Diseases

Significant progress has been made toward development of stem cell–based therapies to treat neurodegenerative diseases. Transplanted cell populations can incorporate and, in some cases, restore function in rodent models. Therapeutic efficacy is dependent upon the ability to both direct the migration of endogenous or transplanted progenitors to locations of cellular degeneration and promote the maturation of desired cell types. A more complete understanding of the molecular and cellular guidance cues for neuronal and glial migration in the embryonic and adult brain is necessary for developing effective cell replacement therapies that direct newly introduced stem cells to the specific brain structures devastated by degeneration. When compared with currently utilized treatments, the ability to physically replace previously degenerated neurons within neural circuits is more likely to provide functional recovery. Therefore, stem cell therapies targeted for the treatment of adult brain injuries must invoke the mechanisms that govern normal migration of neural progenitors during embryogenesis and adult neurogenesis. In addition, these new cells must overcome the barriers to migration that develop after traumatic injury to the adult brain and spinal cord. This chapter reviews current knowledge regarding neuronal and glial migration in development and adulthood, as well as the factors that promote or limit cell migration in degenerative disorders, including demyelinating diseases, stroke, and epilepsy.

Directing Differentiation of Embryonic Stem Cells into Distinct Neuronal Subtypes

There is great interest in testing the efficacy of treating neurodegenerative diseases using embryonic stem cell derivatives. A first step towards this goal is demonstrating that embryonic stem cells can produce, in culture, the specific cell types lost in the various diseases. We describe here currently used approaches for generating neural stem cells, as well as specific neuronal subtypes, from mouse and human embryonic stem cells. Based upon their demonstrated role in neurodegenerative disease and the reports documenting their derivation from embryonic stem cells, we focus on dopaminergic neurons, GABAergic interneurons, spinal cord motor neurons, serotonergic neurons, and basal forebrain cholinergic neurons. Protocols are all based upon what has been learned about the conditions that promote emergence of these lineages in the embryo.