Custom-engineered hydrogels for delivery of human iPSC-derived neurons into the injured cervical spinal cord.

Cervical damage is the most prevalent type of spinal cord injury clinically, although few preclinical research studies focus on this anatomical region of injury. Here we present a combinatorial therapy composed of a custom-engineered, injectable hydrogel and human induced pluripotent stem cell (iPSC)-derived deep cortical neurons. The biomimetic hydrogel has a modular design that includes a protein-engineered component to allow customization of the cell-adhesive peptide sequence and a synthetic polymer component to allow customization of the gel mechanical properties. In vitro studies with encapsulated iPSC-neurons were used to select a bespoke hydrogel formulation that maintains cell viability and promotes neurite extension. Following injection into the injured cervical spinal cord in a rat contusion model, the hydrogel biodegraded over six weeks without causing any adverse reaction. Compared to cell delivery using saline, the hydrogel significantly improved the reproducibility of cell transplantation and integration into the host tissue. Across three metrics of animal behavior, this combinatorial therapy significantly improved sensorimotor function by six weeks post transplantation. Taken together, these findings demonstrate that design of a combinatorial therapy that includes a gel customized for a specific fate-restricted cell type can induce regeneration in the injured cervical spinal cord.

Maternal immune activation dysregulation of the fetal brain transcriptome and relevance to the pathophysiology of autism spectrum disorder.

Maternal immune activation (MIA) via infection during pregnancy is known to increase risk for autism spectrum disorder (ASD). However, it is unclear how MIA disrupts fetal brain gene expression in ways that may explain this increased risk. Here we examine how MIA dysregulates rat fetal brain gene expression (at a time point analogous to the end of the first trimester of human gestation) in ways relevant to ASD-associated pathophysiology. MIA downregulates expression of ASD-associated genes, with the largest enrichments in genes known to harbor rare highly penetrant mutations. MIA also downregulates expression of many genes also known to be persistently downregulated in the ASD cortex later in life and which are canonically known for roles in affecting prenatally late developmental processes at the synapse. Transcriptional and translational programs that are downstream targets of highly ASD-penetrant FMR1 and CHD8 genes are also heavily affected by MIA. MIA strongly upregulates expression of a large number of genes involved in translation initiation, cell cycle, DNA damage and proteolysis processes that affect multiple key neural developmental functions. Upregulation of translation initiation is common to and preserved in gene network structure with the ASD cortical transcriptome throughout life and has downstream impact on cell cycle processes. The cap-dependent translation initiation gene, EIF4E, is one of the most MIA-dysregulated of all ASD-associated genes and targeted network analyses demonstrate prominent MIA-induced transcriptional dysregulation of mTOR and EIF4E-dependent signaling. This dysregulation of translation initiation via alteration of the Tsc2-mTor-Eif4e axis was further validated across MIA rodent models. MIA may confer increased risk for ASD by dysregulating key aspects of fetal brain gene expression that are highly relevant to pathophysiology affecting ASD.

Lithium regulates adult hippocampal progenitor development through canonical Wnt pathway activation.

Neural stem cells give rise to new hippocampal neurons throughout adulthood, and defects in neurogenesis may predispose an individual to mood disorders, such as major depression. Our understanding of the signals controlling this process is limited, so we explored potential pathways regulating adult hippocampal progenitor (AHP) proliferation and neuronal differentiation. We demonstrate that the mood stabilizer lithium directly expands pools of AHPs in vitro, and induces them to become neurons at therapeutically relevant concentrations. We show that these effects are independent of inositol monophosphatase, but dependent on Wnt pathway components. Both downregulation of glycogen synthase kinase-3beta, a lithium-sensitive component of the canonical Wnt signaling pathway, and elevated beta-catenin, a downstream component of the same pathway produce effects similar to lithium. In contrast, RNAi-mediated inhibition of beta-catenin abolishes the proliferative effects of lithium, suggesting that Wnt signal transduction may underlie lithium's therapeutic effect. Together, these data strengthen the connection between psychopharmacologic treatment and the process of adult neurogenesis, while also suggesting the pursuit of modulators of Wnt signaling as a new class of more effective mood stabilizers/antidepressants.

Adult neurogenesis: a compensatory mechanism for neuronal damage.

It is now evident that the adult vertebrate brain including the human brain is efficiently and continuously generating new neurons. In the first part we describe the current view of how neurons are generated in the adult brain and the possible compensatory reactions to pathological situations in which neuronal damage might stimulate neural stem cell activity. In the second part, we discuss the current knowledge on the signals and cells involved in the process of neurogenesis. This knowledge is important because any neuronal replacement strategy depends on our ability to induce or modulate each step on the way to a new neuron: stem cell proliferation, cell fate determination, progenitor migration, and differentiation into specific neuronal phenotypes. Identification of the molecular signals that control these events are essential for the application of neural stem cell biology to develop repair strategies for neurodegenerative disorders.

Vascular niche for adult hippocampal neurogenesis.

The thin lamina between the hippocampal hilus and granule cell layer, or subgranule zone (SGZ), is an area of active proliferation within the adult hippocampus known to generate new neurons throughout adult life. Although the neuronal fate of many dividing cells is well documented, little information is available about the phenotypes of cells in S-phase or how the dividing cells might interact with neighboring cells in the process of neurogenesis. Here, we make the unexpected observation that dividing cells are found in dense clusters associated with the vasculature and roughly 37% of all dividing cells are immunoreactive for endothelial markers. Most of the newborn endothelial cells disappear over several weeks, suggesting that neurogenesis is intimately associated with a process of active vascular recruitment and subsequent remodeling. The present data provide the first evidence that adult neurogenesis occurs within an angiogenic niche. This environment may provide a novel interface where mesenchyme-derived cells and circulating factors influence plasticity in the adult central nervous system.

Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord.

The existence of multipotent progenitor populations in the adult forebrain has been widely studied. To extend this knowledge to the adult spinal cord we have examined the proliferation, distribution, and phenotypic fate of dividing cells in the adult rat spinal cord. Bromodeoxyuridine (BrdU) was used to label dividing cells in 13- to 14-week-old, intact Fischer rats. Single daily injections of BrdU were administered over a 12 d period. Animals were killed either 1 d or 4 weeks after the last injection of BrdU. We observed frequent cell division throughout the adult rodent spinal cord, particularly in white matter tracts (5-7% of all nuclei). The majority of BrdU-labeled cells colocalized with markers of immature glial cells. At 4 weeks, 10% of dividing cells expressed mature astrocyte and oligodendroglial markers. These data predict that 0.75% of all astrocytes and 0.82% of all oligodendrocytes are derived from a dividing population over a 4 week period. To determine the migratory nature of dividing cells, a single BrdU injection was given to animals that were killed 1 hr after the injection. In these tissues, the distribution and incidence of BrdU labeling matched those of the 4 week post injection (pi) groups, suggesting that proliferating cells divide in situ rather than migrate from the ependymal zone. These data suggest a higher level of cellular plasticity for the intact spinal cord than has previously been observed and that glial progenitors exist in the outer circumference of the spinal cord that can give rise to both astrocytes and oligodendrocytes.

The search for neural progenitor cells: prospects for the therapy of neurodegenerative disease.

The etiology of many neurodegenerative diseases has been identified in recent years. Treatment of central nervous system (CNS) disease could focus on one or more steps that lead to cell loss. In the past decade, cell therapy and/or ex vivo gene therapy have emerged as possible strategies for the treatment of neurodegenerative diseases. The ability to grow CNS-derived neural progenitor cells using growth factors has been extremely useful to study diverse phenomena including lineage choice, commitment and differentiation. By virtue of their biological properties and their presence in the adult CNS, neural progenitors represent good candidates for multiple cell-based therapies for neural diseases. Further identification of the molecules that direct the differentiation of adult neural progenitors may allow their activation in vivo to induce self-repair. This review addresses the nature, distribution and regulation of neural stem cells and the potential for applying these cells to both structural CNS repair and gene therapy.

Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS.

During development of the mammalian brain, both neurons and glia are generated from multipotent neural stem cells. Although neurogenesis ceases in most areas at birth, stem cells continue to generate neurons within the subventricular zone and hippocampal dentate gyrus throughout adult life. In this work, we provide the first demonstration that precursors native to regions of the adult brain that generate only glia can also generate neurons after exposure to FGF-2 in vitro. When progenitors isolated from hippocampal tissue were directly compared with cells isolated from the neocortex, both populations were able to initiate a program of proliferative neurogenesis. Genetic marking and lineage analysis showed that a majority of the cells able to generate neurons were multipotent precursors; however, progeny from these precursors acquired the competence to differentiate into neurons only after exposure to FGF-2. The recruitment of similar FGF-2-responsive cells from the adult optic nerve, a structure well isolated from the neurogenic zones within the brain, confirmed that neuron-competent precursors naturally exist in widely divergent tissues of the adult brain.

Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain.

Adult rat-derived hippocampal progenitor cells express many of the molecules implicated in midbrain dopaminergic determination, including FGF receptors 1, 2 and 3, the sonic hedgehog receptor components Smo and Ptc, and the region-specific transcription factors Ptx3 and Nurr1. Here we use undifferentiated progenitors to probe the events leading to the dopaminergic phenotype and find that the influences of Nurr1 can be temporally and mechanistically uncoupled from the patterning influences of sonic hedgehog and FGF-8 or the more generic process of neuronal differentiation itself. In gain-of-function experiments, Nurr1 is able to activate transcription of the tyrosine hydroxylase gene by binding a response element within a region of the tyrosine hydroxylase promoter necessary for midbrain-specific expression. This activation is mediated through a retinoid X receptor independent mechanism and occurs in all precursors, regardless of differentiation status. Overexpression of Nurr1 does not affect proliferation or stimulate neuronal differentiation and has no influence on the expression of other dopaminergic markers. This uncoupling of tyrosine hydroxylase expression from other dopaminergic markers suggests that the midbrain dopaminergic identity is dictated by a combination of pan-dopaminergic (e.g., Shh/FGF-8) and region-specific (Nurr1) mechanisms.

Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures.

The adult rat hippocampus contains fibroblast growth factor 2-responsive stem cells that are self-renewing and have the ability to generate both neurons and glia in vitro, but little is known about the molecular events that regulate stem cell differentiation. Hippocampus-derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation. Exposure to RA caused an immediate up-regulation of NeuroD, increased p21 expression, and concurrent exit from cell cycle. These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons. An accompanying effect of RA was to sustain or up-regulate trkA, trkB, trkC, and p75NGFR expression. Without RA treatment, cells were minimally responsive to neurotrophins (NTs), whereas the sequential application of RA followed by brain-derived neurotrophic factor or NT-3 led to a significant increase in neurons displaying mature y-a-minobutyric acid, acetylcholinesterase, tyrosine hydroxylase, or calbindin phenotypes. Although NTs promoted maturation, they had little effect on the total number of neurons generated, suggesting that RA and neurotrophins acted at distinct stages in neurogenesis. RA first promoted the acquisition of a neuronal fate, and NTs subsequently enhanced maturation by way of RA-dependent expression of the Trk receptors. In combination, these sequential effects were sufficient to stimulate stem cell-derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes.

Multipotent progenitor cells in the adult dentate gyrus.

Neurogenesis persists in the adult dentate gyrus of rodents throughout the life of the organism. The factors regulating proliferation, survival, migration, and differentiation of neuronal progenitors are now being elucidated. Cells from the adult hippocampus can be propagated, cloned in vitro, and induced to differentiate into neurons and glial cells. Cells cultured from the adult rodent hippocampus can be genetically marked and transplanted back to the adult brain, where they survive and differentiate into mature neurons and glial cells. Although multipotent stem cells exist in the adult rodent dentate gyrus, their biological significance remains elusive.

Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina.

Adult rat hippocampus-derived neural progenitor cells (AHPC) show considerable adaptability following grafting to several brain regions. To evaluate the plasticity of AHPCs within the optic retina, retrovirally engineered AHPCs were grafted into the vitreous cavity of the adult and newborn rat eye. Within the adult eye, AHPCs formed a uniform nondisruptive lamina in intimate contact with the inner limiting membrane. Within 4 weeks of grafting to the developing eye, the AHPCs were well integrated into the retina and adopted the morphologies and positions of Müller, amacrine, bipolar, horizontal, photoreceptor, and astroglial cells. Although the cells expressed neuronal or glial markers, none acquired end-stage markers unique to retinal neurons. This suggests that the adult-derived stem cells can adapt to a wide variety of heterologous environments and express some but not all features of retinal cells when exposed to the cues present late in retinal development.

The adult rat hippocampus contains primordial neural stem cells.

Adult-derived hippocampal progenitors generate neurons, astrocytes, and oligodendrocytes in vitro and following grafting into the adult brain. Although these progenitors have a considerable capacity for in vitro self renewal, it is not known if each lineage is generated by separate committed precursors or by multipotent stem cells. By genetic marking, we have followed individual cells through the process of proliferative expansion, commitment, and differentiation. All three lineages are generated by single marked cells and the relative proportions of each lineage can be strongly influenced by environmental cues. Differentiation is accompanied by a characteristic progression of lineage-specific markers and can be potentiated by retinoic acid, elevated cyclic AMP, or neurotrophic factors. The ability to genetically mark and clone normal diploid hippocampal progenitors provides the first definitive evidence that multipotent neural stem cells exist outside of the adult striatal subventricular zone and supports the hypothesis that FGF-2-responsive neural stem cells may be broadly distributed in the adult brain.

Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.

The dentate gyrus of the hippocampus is one of the few areas of the adult brain that undergoes neurogenesis. In the present study, cells capable of proliferation and neurogenesis were isolated and cultured from the adult rat hippocampus. In defined medium containing basic fibroblast growth factor (FGF-2), cells can survive, proliferate, and express neuronal and glial markers. Cells have been maintained in culture for 1 year through multiple passages. These cultured adult cells were labeled in vitro with bromodeoxyuridine and adenovirus expressing beta-galactosidase and were transplanted to the adult rat hippocampus. Surviving cells were evident through 3 months postimplantation with no evidence of tumor formation. Within 2 months postgrafting, labeled cells were found in the dentate gyrus, where they differentiated into neurons only in the intact region of the granule cell layer. Our results indicate that FGF-2 responsive progenitors can be isolated from the adult hippocampus and that these cells retain the capacity to generate mature neurons when grafted into the adult rat brain.

FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain.

Neurogenesis is restricted to discrete germinal zones within the developing and the adult central nervous systems. With few exceptions, cells that migrate away from these zones and into the parenchyma no longer participate in the generation of new neurons. In this work, we have found that basic fibroblast growth factor is able to stimulate the proliferation of neuronal and glial progenitors isolated from the septum and striatum of adult rats. These progenitors are indistinguishable from those isolated from the adult hippocampus and subventricular zone, two regions that generate neurons well into adult life. Although a variety of cell types are initially isolated from each brain region, the progenitor-like cells from all four regions are capable of considerable proliferation and, with limited serial passage, can be cultured as enriched populations of immature cells that are capable of differentiating into mature glia and neurons following density arrest and growth factor withdrawal. The fact that cells isolated from the septum and striatum proliferate and have the ability to differentiate into neurons once they are removed from their local environment indicates that neurogenesis may be restricted to discrete areas of the developing and the adult brain by regional differences in regulatory signals rather than from an absence of progenitors capable of responding to neurogenic cues.

Efficient expression of a protein coding gene under the control of an RNA polymerase I promoter.

In mammalian cells, RNA polymerase I transcripts are uncapped and retain a polyphosphate 5' terminus. It is probably for this reason that they are poorly translated as messenger RNA. We show in this report that insertion of an Internal Ribosome Entry Site (IRES) into the 5' leader of an RNA polymerase I transcript overcomes the block to translation, presumably by substituting for the 5' trimethyl G cap. Addition of an SV40 polyA addition signal also enhances protein production from the RNA polymerase I transcript. RNA Polymerase I driven expression vectors containing both elements produce protein at levels comparable to that produced from RNA polymerase II driven expression vectors which utilize a retroviral LTR. RNA Polymerase I driven expression vectors may have a variety of uses both for basic research and for practical expression of recombinant proteins.

High-level human adenosine deaminase expression in dog skin fibroblasts is not sustained following transplantation.

Primary skin fibroblasts are an attractive target tissue for retroviral-mediated gene therapy; however, transient expression of therapeutic genes has been a recurrent problem in several rodent models. The gradual decrease in gene expression could be tissue or species specific. To investigate the phenomenon further, human adenosine deaminase (ADA) expression was monitored in genetically modified skin fibroblasts transplanted in beagle dogs. In culture, transduced canine fibroblasts expressed high levels of human ADA activity (33.6 mumoles adenosine metabolized per hour per milligram of soluble protein) in comparison to canine ADA in untreated control cells (1.3 mumol/hr.mg protein) and for 2 weeks following transplantation, the graft contained up to four-fold more enzyme activity from human ADA than the endogenous canine enzyme. However, by 10 weeks, human ADA expression was undetectable. At the time when human ADA expression was greatly reduced, DNA analysis showed the presence of vector sequences. These results directly parallel those observed in rodent models and suggest retroviral vector inactivation is a tissue-specific rather than species-specific mechanism.

Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.

Genetically engineered fibroblasts have been successfully used to produce therapeutic proteins in animals, but sustained production of the proteins has not been achieved. This limits the potential of fibroblast-mediated gene therapy in humans. We have studied the phenomenon of decreased production in rats by using retroviral vectors carrying genes encoding human adenosine deaminase and neomycin phosphotransferase. While transplanted skin fibroblasts containing vector sequences persisted at constant levels for at least 8.5 mo, vector expression decreased by greater than 1500-fold after 1 mo. Cellular or antibody-mediated immune responses were not detected in transplanted animals, and expression could not be restored in fibroblasts recultivated from the grafts. This phenomenon is reminiscent of sequence-specific gene inactivation observed in other cell types. Because genetic manipulation and expression of foreign proteins did not affect survival of the transplanted cells, effective long-term therapy may be possible with the use of alternative gene regulatory elements.