Substrate Dependent Galvanotaxis of Directly Reprogrammed Human Neural Precursor Cells

Background: Neural precursor cells (NPCs) hold great promise for neural repair. Endogenous NPCs, found in the subventricular zone of the adult brain, proliferate and migrate toward lesion sites; however, it is not sufficient for neural repair. NPCs are electrosensitive cells that undergo directed migration in an electric field (EF). Here, we examined the EF-induced migration of a clinically relevant human NPC population. Materials & Methods: We examined the effects of different substrates and microenvironments on human NPC galvanotaxis. Results: Human NPCs increased their migration speed in the presence of an EF, and the direction of migration (anodal vs. cathodal) varied between substrates. The secretome and extracellular pH were not significant factors in EF-induced migration; however, our results are consistent with substrate stiffness playing a role in the direction of cell migration. Conclusion: These findings provide insight into the importance of the microenvironment on modulating human NPC migration and highlight substrate-dependent considerations for neurorepair.

Human fetal neural precursor cells can up-regulate MHC class I and class II expression and elicit CD4 and CD8 T cell proliferation

Human neural precursor cells continue to proliferate and exhibit low cell death after transplantation to the injured rat spinal cord

Human cytomegalovirus infection inhibits the differentiation of human hippocampus neural precursor cells into astrocytes

Roof plate secretion of bone morphogenetic proteins (BMPs) directs the cellular fate of sensory neurons during spinal cord development, including the formation of the ascending sensory columns, though their biology is not well understood. Type-II BMP receptor (BMPRII), the cognate receptor, is expressed by neural precursor cells during embryogenesis; however, an in vitro method of enriching BMPRII+ human neural precursor cells (hNPCs) from the fetal spinal cord is absent. Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRII and leukemia inhibitory factor (LIF). Regions of highest BMPRII+ immunofluorescence localized to sensory columns. Parenchymal and meningeal-associated BMPRII+ vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers, CD34/CD39. LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons, mirroring the expression of LIF receptor/CD118. A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages, while synergistically increasing the proportion of neurospheres with a stratified, cytoarchitecture. These neurospheres were characterized by BMPRII+/MAP2ab+/-/βIII-tubulin+/nestin-/vimentin-/GFAP-/NeuN- surface hNPCs surrounding a heterogeneous core of βIII-tubulin+/nestin+/vimentin+/GFAP+/MAP2ab-/NeuN- multipotent precursors. Dissociated cultures from tripotential neurospheres contained neuronal (βIII-tubulin+), astrocytic (GFAP+), and oligodendrocytic (O4+) lineage cells. Fluorescence-activated cell sorting-sorted BMPRII+ hNPCs were MAP2ab+/-/βIII-tubulin+/GFAP-/O4- in culture. This is the first isolation of BMPRII+ hNPCs identified and characterized in human fetal spinal cords. Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres, characterized by surface BMPRII+ hNPCs. Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for > 10 passages. Investigations of the role BMPRII plays in spinal cord development have primarily relied upon mouse and rat models, with interpolations to human development being derived through inference. Because of significant species differences between murine biology and human, including anatomical dissimilarities in central nervous system (CNS) structure, the findings made in murine models cannot be presumed to apply to human spinal cord development. For these reasons, our human in vitro model offers a novel tool to better understand neurodevelopmental pathways, including BMP signaling, as well as spinal cord injury research and testing drug therapies.

Multiple Sclerosis (MS) is a chronic, autoimmune disease of the central nervous system for which there is no cure. Defects in neurological function are a result of demyelination and axonal loss caused by infiltrating immune cells. Transplantation of neural precursor cells (NPCs) is a promising therapeutic strategy to treat neurological diseases. However, studies evaluating NPC transplantation often employ a syngeneic donor or immune suppressed subjects. Therefore, it is important to consider how transplanted cells elicit an immune response. We report that human neural precursor cells (hNPCs) induce antigen specific Tregs which influence endogenous repair pathways to promote remyelination in murine models of MS. We observed remyelination and decreased neuroinflammation in mice receiving an intra-spinal transplant of syngeneic NPCs, which replaced damaged cells. Alternatively, mice receiving transplants of xenogeneic human NPCs displayed remyelination, a decrease in neuroinflammation, and an increase in CD4+CD25+FoxP3+ Tregs. Recovery was not a result of direct cell replacement as hNPCs underwent xenograft rejection. Importantly, ablation of Tregs abrogated histopathological improvement. Furthermore, hNPCs promoted generation of Tregs in vitro. These findings support an emerging role for Tregs in promoting tissue regeneration, distinct from immune modulation.

Growth factors are currently evaluated as therapeutics in stroke and neurodegeneration. Besides direct neurotrophic effects, they promote proliferation, survival, and differentiation of both transplanted and endogenous neural precursor cells (NPCs). In the current study, we investigated whether NPCs expressing Vascular Endothelial Growth Factor VEGF-A165 are a useful vehicle for growth factor delivery after transplantation into the caudate putamen of the rat brain. We found an increased survival of adenovirally transfected NPCs after 11 days, but not after 24 hours or 4 days. Additional brain immunohistochemistry revealed increased expression of the endothelial cell marker PECAM-1 (CD31) after 24 hours, 4 day, and 11 days after transplantation. In conclusion, we show that the graft itself is a useful vehicle for growth factor delivery, promoting the survival of NPCs. Moreover, transplantation of VEGF-expressing NPCs supports angiogenesis in the brain, which may contribute to potential brain repair.

BackgroundTau is encoded by MAPT and abnormal aggregates of tau are a hallmark of many neurodegenerative diseases. MAPT is highly expressed in neurons but much more lowly expressed in other cell types, including neural precursor cells (NPCs), suggesting that its expression may be controlled by neuron‐specific transcription factors and cis‐regulatory elements. Tau reduction, even of endogenous tau, is a current therapeutic strategy in both model systems and humans; therefore, understanding MAPT regulation may point to new strategies to achieve tau reduction. Additionally, genome sequencing studies continue to identify disease‐associated variants in non‐coding regions of the genome. These variants are likely located in regulatory regions and affect the expression of target genes whose function contributes to neurodegeneration. Therefore, an additional benefit of this study is that high confidence regulatory regions for MAPT may provide a way to identify important non‐coding variants associated with disease.MethodsThree‐dimensional chromatin conformation capture (Capture‐C), single‐cell multiomics (RNAseq + ATACseq), and chromatin marks including H3K27ac, H3K4me1, and DNase hypersensitivity were used to identify candidate enhancers of MAPT in human NPCs, NPCs differentiated to neurons, and pure inhibitory or excitatory neuron cultures (all lines were H1/H1). Approaches included nomination of elements with signals specific to neurons or neuronal subtypes. The nominated regions were then assessed using luciferase assays to test sufficiency for regulatory activity as well as CRISPR‐dCas9‐KRAB to assess the effect of nominated elements on MAPT expression.ResultsWe identified both proximal and distal regulatory elements for MAPT with function confirmed by up to four orthogonal approaches (chromatin capture, single cell multiomics, a reporter assay, and element inhibition using dCas9‐KRAB). Several of these elements were specific to neurons. One particularly important region was centromeric to MAPT beyond the H2 inversion breakpoint.ConclusionsOverall, these results indicate that MAPT regulation is likely controlled by neuron‐specific cis‐regulatory elements, including both proximal and distal elements. Next steps include study of the effect of trans‐regulatory elements (i.e., transcription factors) that may control recruitment of these cis‐regulatory elements for expression of MAPT and assessment of the effects of the disease‐associated genetic variation on cis‐regulatory element function and expression of MAPT.

Skin-derived precursor cells undergo substrate-dependent galvanotaxis that can be modified by neighbouring cells

In the stem cell niche, neural stem cells (NSCs) are in close contact with the specialized blood-brain barrier (BBB) endothelial cells (ECs) that modulate their proliferation and differentiation behavior. NSCs are also an attractive source for cell transplantation and neural tissue repair after central nervous system injury. After systemic grafting, they are confronted with the BBB before they can enter the brain parenchyma. We investigated the interactions of human fetal neural precursor cells (hfNPCs) with human brain ECs in an in vitro model using primary cultures. We demonstrated that hfNPCs efficiently differentiate to neurons, astrocytes, and oligodendrocytes and move to the subendothelial space of human BBB endothelium, but not to pulmonary artery ECs. Effective differentiation was found to be dependent on the chemokine CCL2/MCP-1, but not on CXCL8/IL-8. Our findings suggest that neural precursor cells specifically interact with the BBB endothelium and differentiate in the subendothelial niche into astrocytes, neurons, and oligodendrocytes, under the influence of the chemokine CCL2/MCP-1.

No any effective treatments can prevent from the motor neuron degeneration in amyotrophic lateral sclerosis (ALS) at present. In order to modulating the endogenous neural precursor cells (NPCs) to repairing the degenerative motor neurons in ALS, we studied the alteration of endogenous vimentin-containing NPCs in olfactory bulb (OB) at the different stages of SOD1 wlid-type and G93A transgenic mice. The results showed that the vimentin-containing cells (VCCs) were mainly distributed in the glomerular layer (Gl), the accessory OB (AOB), the OB core, the granular cell layer (GRO) and the mitral cell layer (MI)+the internal plexiform layer (IPL) of the OB of adult mice. Almost all VCCs in Gl, OB core and GRO were the GFAP positive cells. Almost all VCCs in AOB were the Oligo-2 positive cells. Fewer VCCs in MI+IPL were the NeuN positive cells. VCCs significantly increased in the OB core and Gl of adult OB at the pre-onset, onset and progression stages of ALS-like G93A transgenic disease, particularly in OB core. All increased VCCs were the GFAP positive cells. Our data suggested that there extensively existed the endogenous vimentin-containing NPCs in the OB of adult mice, which was a potential resource of neural stem cells, they could differentiate into astrocyte, oligodendrocyte and neuron cells, were a potential astrocyte neuroregenerative response in adult OB in the ALS-like disease, were a potential pathway to repair the degenerated motor neurons.

This unit describes the preparation and transplantation of human neural precursor cells (hNPCs) and mouse neural precursor cells (mNPCs) into the thoracic region of the mouse spinal cord. The techniques in this unit also describe how to prepare the mouse for surgery by performing a laminectomy to expose the spinal cord for transplantation. NPCs genetically labeled with eGFP transplanted into the spinal cord of a mouse following viral-mediated demyelination can efficiently be detected via eGFP expression. Transplantation of these cells into the spinal cord is an efficacious way to determine their effects in neurological disorders such as multiple sclerosis, Alzheimer's disease, and spinal cord injury.

IL8 and CXCL13 are potent chemokines for the recruitment of human neural precursor cells across brain endothelial cells

Combined use of BDNF, ascorbic acid, low oxygen, and prolonged differentiation time generates tyrosine hydroxylase-expressing neurons after long-term in vitro expansion of human fetal midbrain precursor cells

Theory and numerical simulation of field-induced director dynamics in confined nematics investigated by nuclear magnetic resonance