Project description:Stem cells have received substantial interest both for their potential as in vitro tools to study development and as potential therapeutic agents in a range of degenerative diseases of the nervous system. We have generated clonal neural stem cell lines from human foetal spinal cord conditionally immortalised with 4-hydroxy tamoxifen inducible cMyc (cMycERTAM), and performed a detailed assessment of their identity in terms of their expression of homeodomain transcription factors and their capacity to generate particular neuronal subtypes of the spinal cord. These lines retain a ventral spinal cord progenitor phenotype and give rise to electrically active neuronal subtypes characteristic of specific ventral progenitor subdomains. Upon grafting into lesioned rat spinal cord these cells differentiate into ChAT+ve motorneurons and show robust survival after 4 months. Total RNA was obtained from three proliferative undifferentiated neural stem cell lines at 75% confluence in culture. Two replicates of each clonal line were generated for microarray analysis.
Project description:Stem cells have received substantial interest both for their potential as in vitro tools to study development and as potential therapeutic agents in a range of degenerative diseases of the nervous system. We have generated clonal neural stem cell lines from human foetal spinal cord conditionally immortalised with 4-hydroxy tamoxifen inducible cMyc (cMycERTAM), and performed a detailed assessment of their identity in terms of their expression of homeodomain transcription factors and their capacity to generate particular neuronal subtypes of the spinal cord. These lines retain a ventral spinal cord progenitor phenotype and give rise to electrically active neuronal subtypes characteristic of specific ventral progenitor subdomains. Upon grafting into lesioned rat spinal cord these cells differentiate into ChAT+ve motorneurons and show robust survival after 4 months.
Project description:Comparison of genomic data from neural progenitor cells derived from mouse embryonic stem cells under different experimetnal conditions in vitro and invivo. We conducted genome-wide RNA sequencing of immunoprecipitated specific ribosome-associated mRNA using RiboTag methods from: (i) mouse embryonic stem cell (ESC), (ii) derived neural progenitor cells, (iii) differentiated neural progenitor cells (in vitro), (iv) grafted neural progenitor cells (recovered from different in vivo tissue enivornments - healthy spinal cord, spinal cord injury lesions) and (v) host astrocytes using GFAp-Cre RiboTag mice.
Project description:Human astrocytes have reported to reprogram into neurons, but they have yet to be induced to three-dimensional (3D) neural tissue for neural organogenesis. Here, we demonstrate a remarkable strategy for 3D organoid generation by direct reprogramming human astrocytes. By combining overexpression OCT4, suppression p53 and small molecules CHIR99021, SB431542, RepSox and Y27632, termed as Op53-CSBRY, we successfully reprogramed human astrocytes into neural ectodermal cells and further induced human 3D-brain organoid. Those organoids can be finally induced into spinal cord organoids by activating FGF, SHH and BMP signaling. The grafts of Human astrocyte derived spinal cord organoids (hADSC-Organs) can survived, differentiated into spinal cord neurons, migrated long distance, formed synaptic connectivity with host neurons, bridged complete injury spinal cord tissue in mice. This method indicates that human astrocytes could be directly triggered neural organogenesis, and may hold a great promising to support local astrocytes in situ organogenesis after brain damages, such as stroke and spinal cord injury.
Project description:The DNA methylation profiles of Glioma Stem Cell (GSC) lines were investigated in order to find the stem cell signature associated to glioblastoma (GBM). This goal was achieved through the comparison of GSC methylation data with FFPE-GBM biopsies and human foetal Neural Stem Cell (NSC) lines profiles. GSC lines: 3 (GBM2, G144, G166). FFPE-GBM biopsy pool: FFPE-GBM pool: 1 pool from 5 GBM biopsies. Human foetal NSC lines: 2 (CB660 from forebrain; CB660SP form spinal cord). Methylated DNA from each sample was enriched with the immunoprecipitation method using 5-methylcytosine antibody (Eurogentec). Immunoprecipitated DNA (IP-DNA) and total DNA were labeled and hybridized on Agilent Human CpG Island ChIP-on-Chip Microarray 244K. IP-DNA were labeled with Cy5 while the matching total DNA were labeled with Cy3.
Project description:Neonatal spinal cord tissues exhibit remarkable regenerative capabilities compared to adult tissues following injury. Although some cellular signaling pathways involved in the process have been identified, the specific role of extracellular matrix (ECM) responsible for neonatal spinal cord regeneration has remained elusive. Here we revealed that early developmental spinal cord contained a higher abundance of ECM proteins associated with neural development and axon growth but fewer inhibitory proteoglycans compared to adult spinal cord. Decellularized spinal cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserve the major difference of native spinal cord tissues in both stages. Compared to DASCM, DNSCM promoted proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs), as well as facilitated the long-distance axonal outgrowth and axon regeneration of spinal cord organoids. Pleiotrophin (PTN) and Tenascin (TNC) in DNSCM were identified as contributors to the remarkable neural regeneration ability. Furthermore, DNSCM demonstrated superior performance when used as a delivery vehicle for NPCs and organoids in rats with spinal cord injury (SCI). It suggests that ECM cues derived from different development stage might contribute to the distinct regeneration ability of spinal cord.
Project description:We generated iPSCs from human intervertebral disc cells which were obtained during spine fusion surgery of patients with spinal cord injury. The disc cell-derived iPSCs (diPSCs) showed similar characteristics to human embryonic stem cells (hESCs) and were efficiently differentiated into neural progenitor cells (NPCs) with the capability of differentiation into mature neurons in vitro. To examine whether the transplantation of NPCs derived from the diPSCs showed therapeutic effects, the NPCs were transplanted into mice at 9 days post-spinal cord injury. We detected a significant amelioration of hind limb dysfunction during the follow up recovery periods. Histological analysis at 5 weeks post-transplantation, we could identify undifferentiated human NPCs (Nestin+) as well as early (TUJ1+) and mature neurons (MAP2+) derived from the NPCs. Furthermore, the NPC transplantation demonstrated a preventive effect on the spinal cord degeneration resulting from the secondary injury. This study revealed that the intervertebral disc, a M-bM-^@M-^\to-be-wasteM-bM-^@M-^] tissue, removed from the surgical procedure, could provide a unique opportunity to study iPSCs derived from hardly accessible somatic cells in normal situation and also be a useful therapeutic resource to generate autologous neural cells to treat patients suffering from spinal cord injury. Total RNA was isolated using the NucleoSpin RNA II Kit (Macherey-Nagel, Duren, Germany, www.mn-net.com) according to the manufacturerM-bM-^@M-^Ys suggestions and was utilized for a genome-wide gene expression profiling experiment using the Illumina array (Illumina, San Diego, CA, USA, www.illumina.com) at Macrogen (Macrogen, Seoul, Korea, www.macrogen.com).
Project description:Neural stem cells (NSCs) can be isolated from different regions of the central nervous system. There has been controversy whether regional differences amongst stem and progenitor cells are cell intrinsic and whether these differences are maintained during expansion in culture. The identification of inherent regional differences has important implications for the use of these cells in neural repair. Here, we compared neural stem cells derived from the spinal cord and embryonic cortex. We found that while cultured cortical and spinal cord derived neural stem cells respond similarly to mitogens and are equally neuronogenic, they retain and maintain through multiple passages gene expression patterns indicative of the region from which they were isolated. Further microarray analysis identified 229 genes that were differentially expressed between cortical and spinal cord derived neurospheres. Experiment Overall Design: Cortex and and spinal cords were isolated from embryonic day 14 cd1 mice and cultured ad neurospheres for 2 passages in EGF and bFGF. 3 independent sets of cultures serve as biological replicates with a dye flip control for each hybridization, for a total of 6 arrays.
Project description:Salamanders have the remarkable ability to functionally regenerate after spinal cord transection. In response to injury, GFAP+ glial cells in the axolotl spinal cord proliferate and migrate to replace the missing neural tube and create a permissive environment for axon regeneration. In this paper we show that miR-200a acts to repress expression of Brachyury in sox2 positive progenitor cells in the axoltol spinal cord after spinal cord injury but after tail amputation when multiple tissue types must be regenerated then mir-200a is downregualted allowing progenitor cells in the spinal cord to naturally become bipotent progenitors which can give rise to muscle and neural cell types. When miR-200a is inhibited after spinal cord injury then these cells also express BRachyury cna can form muscle.
Project description:Neonatal spinal cord tissues exhibit remarkable regenerative capabilities compared to adult tissues following injury. Although some cellular signaling pathways involved in the process have been identified, the specific role of extracellular matrix (ECM) responsible for neonatal spinal cord regeneration has remained elusive. Here we revealed that early developmental spinal cord contained a higher abundance of ECM proteins associated with neural development and axon growth but fewer inhibitory proteoglycans compared to adult spinal cord. Decellularized spinal cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserve the major difference of native spinal cord tissues in both stages. Compared to DASCM, DNSCM promoted proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs), as well as facilitated the long-distance axonal outgrowth and axon regeneration of spinal cord organoids. Pleiotrophin (PTN) and Tenascin (TNC) in DNSCM were identified as contributors to the remarkable neural regeneration ability. Furthermore, DNSCM demonstrated superior performance when used as a delivery vehicle for NPCs and organoids in rats with spinal cord injury (SCI). It suggests that ECM cues derived from different development stage might contribute to the distinct regeneration ability of spinal cord.