Development Of Human Spinal Cord Research Articles

Decoding the spatiotemporal regulation of transcription factors during human spinal cord development

The spinal cord is a crucial component of the central nervous system that facilitates sensory processing and motor performance. Despite its importance, the spatiotemporal codes underlying human spinal cord development have remained elusive. In this study, we have introduced an image-based single-cell transcription factor (TF) expression decoding spatial transcriptome method (TF-seqFISH) to investigate the spatial expression and regulation of TFs during human spinal cord development. By combining spatial transcriptomic data from TF-seqFISH and single-cell RNA-sequencing data, we uncovered the spatial distribution of neural progenitor cells characterized by combinatorial TFs along the dorsoventral axis, as well as the molecular and spatial features governing neuronal generation, migration, and differentiation along the mediolateral axis. Notably, we observed a sandwich-like organization of excitatory and inhibitory interneurons transiently appearing in the dorsal horns of the developing human spinal cord. In addition, we integrated data from 10× Visium to identify early and late waves of neurogenesis in the dorsal horn, revealing the formation of laminas in the dorsal horns. Our study also illuminated the spatial differences and molecular cues underlying motor neuron (MN) diversification, and the enrichment of Amyotrophic Lateral Sclerosis (ALS) risk genes in MNs and microglia. Interestingly, we detected disease-associated microglia (DAM)-like microglia groups in the developing human spinal cord, which are predicted to be vulnerable to ALS and engaged in the TYROBP causal network and response to unfolded proteins. These findings provide spatiotemporal transcriptomic resources on the developing human spinal cord and potential strategies for spinal cord injury repair and ALS treatment.

Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin

The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5–12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.

BMPRII+ neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development.

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.

Expression of L1 Cell Adhesion Molecule (L1CAM) in extracellular vesicles in the human spinal cord during development.

L1 cell adhesion molecule (L1CAM) is a glycoprotein characterized by three components: an extracellular region, a transmembrane segment, and a cytoplasmic tail. L1CAM is expressed in multiple human cells, including neurons. The neural cell adhesion molecule L1 has been implicated in a variety of neurologic processes, including neuritogenesis and cerebellar cell migration. The presence of L1CAM on the surface of nerve cells allows the adhesion of neurons among them. Furthermore, when it is bound to itself or to other proteins, L1-CAM induces signals inside the cell. The aim of this work was to study L1CAM expression in the human spinal cord during development, at different gestational ages, through immunohistochemistry. Immunohistochemical analysis for L1CAM was performed in five human spinal cord samples, including three embryos and two fetuses of different gestational ages, ranging from 8 to 12 weeks. L1CAM expression was detected in all 5 spinal cords examined in this study. The adhesion molecule was found in the vast majority of cells. The highest levels of immunoreactivity for L1CAM were detected at the periphery of the developing organs, in the spinal cord zones occupied by sensory and motor fibers. In the alar and basal columns, immunoreactivity for L1CAM was characterized by a reticular pattern, being mainly expressed in axons. Strong reactivity of L1CAM was also found in extracellular vesicles. This extracellular localization might indicate the ability of L1CAM to mediate the transduction of extracellular signals that support axon outgrowth. The high reactivity of L1cam in the axons of developing neurons in the fetal spinal cord confirms previous studies on the ability of L1CAM to promote axon sprouting and branching in the developing nervous system. In this work, a new actor is reported to have a role in the complex field of human spinal cord development: L1CAM, whose expression is highly found in the developing neuronal and glial precursors.

Single-cell analysis reveals dynamic changes of neural cells in developing human spinal cord.

During central nervous system development, neurogenesis and gliogenesis occur in an orderly manner to create precise neural circuitry. However, no systematic dataset of neural lineage development that covers both neurogenesis and gliogenesis for the human spinal cord is available. We here perform single-cell RNA sequencing of human spinal cord cells during embryonic and fetal stages that cover neuron generation as well as astrocytes and oligodendrocyte differentiation. We also map the timeline of sensory neurogenesis and gliogenesis in the spinal cord. We further identify a group of EGFR-expressing transitional glial cells with radial morphology at the onset of gliogenesis, which progressively acquires differentiated glial cell characteristics. These EGFR-expressing transitional glial cells exhibited a unique position-specific feature during spinal cord development. Cell crosstalk analysis using CellPhoneDB indicated that EGFR glial cells can persistently interact with other neural cells during development through Delta-Notch and EGFR signaling. Together, our results reveal stage-specific profiles and dynamics of neural cells during human spinal cord development.

Human ex vivo spinal cord slice culture as a useful model of neural development, lesion, and allogeneic neural cell therapy

BackgroundThere are multiple promising treatment strategies for central nervous system trauma and disease. However, to develop clinically potent and safe treatments, models of human-specific conditions are needed to complement in vitro and in vivo animal model-based studies.MethodsWe established human brain stem and spinal cord (cross- and longitudinal sections) organotypic cultures (hOCs) from first trimester tissues after informed consent by donor and ethical approval by the Regional Human Ethics Committee, Stockholm (lately referred to as Swedish Ethical Review Authority), and The National Board of Health and Welfare, Sweden. We evaluated the stability of hOCs with a semi-quantitative hOC score, immunohistochemistry, flow cytometry, Ca2+ signaling, and electrophysiological analysis. We also applied experimental allogeneic human neural cell therapy after injury in the ex vivo spinal cord slices.ResultsThe spinal cord hOCs presented relatively stable features during 7–21 days in vitro (DIV) (except a slightly increased cell proliferation and activated glial response). After contusion injury performed at 7 DIV, a significant reduction of the hOC score, increase of the activated caspase-3+ cell population, and activated microglial populations at 14 days postinjury compared to sham controls were observed. Such elevation in the activated caspase-3+ population and activated microglial population was not observed after allogeneic human neural cell therapy.ConclusionsWe conclude that human spinal cord slice cultures have potential for future structural and functional studies of human spinal cord development, injury, and treatment strategies.

Three-dimensional induction of dorsal, intermediate and ventral spinal cord tissues from human pluripotent stem cells.

ABSTRACTThe spinal cord contains more than 20 distinct subclasses of neurons that form well-organized neural circuits capable of sensing the environment and generating motor behavior. Although recent studies have described the efficient in vitro generation of spinal motor neurons, the induction of the spinal cord as a whole tissue has not been achieved. In the present study, we demonstrate three-dimensional (3D) induction of dorsal spinal cord-like tissues from human pluripotent stem cells. Our 3D spinal cord induction (3-DiSC) condition recapitulates patterning of the developing dorsal spinal cord and enables the generation of four types of dorsal interneuron marker-positive cell populations. By activating Shh signaling, intermediate and ventral spinal cord-like tissues are successfully induced. After dissociation of these tissues, somatosensory neurons and spinal motor neurons are detected and express neurotransmitters in an in vivo manner. Our approach provides a useful experimental tool for the analysis of human spinal cord development and will contribute to research on the formation and organization of the spinal cord, and its application to regenerative medicine.

Peculiarities of spinal cord segments structural organization in human fetuses with anencephaly of 17–18 weeks of intrauterine development

The principles of the human spinal cord development constitute the basis for understanding of appearance of congenital abnormalities of the CNS development, which make 7 % of the total number of malformations in the embryonic period of human’s development.Purpose – to find out the cytoarchitectonic structure of the spinal cord segments of human fetuses with anencephaly, their morphometric parameters and the degree of the proliferation of neural stem cells.Materials and methods – anatomical and histological, immunohistochemical and morphometric research was carried out related to the spinal cord of 4 human fetuses with anencephaly of the gestational period of 17–18 weeks.Results. The length of the spinal cord of 17–18 weeks anencephalic fetuses corresponds to the length of human fetuses without malformations of 11–12 weeks, the rest macrometric parameters – 8–9 weeks. The largest area of grey matter was that of segments at the level of lumbosacral and cervical enlargements, the least one at the level of thoracic segments. The specific gravity of neuroepithelium in the grey matter of cervical, thoracic, lumbar and sacral segments was 0.4, 0.1, 0.6, 1.6 % respectively. The expression of the proliferation protein in the spinal cord neuroepithelium segments of anencephalic fetuses is approaching 0 %. However, in the ventral horns near the location of neural complexes single mitoses of glial cells occur. In quantitative terms, in the ventral horns 1.5 % of glial cells reacted, while in the dorsal ones – 0.6 %. The expression of vimentin in the mantle and marginal layers is poor. On the border between the ventral and dorsal horns an insignificant number of fibers have a radial direction. Moderate expression of synaptophysin was observed in the marginal layer, except for cuneate fasciculi where the relatively weak expression has been established, which testifies to the insufficient myelination of the conduction tracts fibers.Conclusions. Macro- and morphometric parameters of the spinal cord of human fetuses with 17–18 week anencephaly point out to the significant retardation in development. Numerous disorders in the structural organization of both grey matter and white matter have been found out. The proliferation of neural stem cells in neuroepithelium is approaching 0 %.

Cadmium induces alterations in the human spinal cord morphogenesis

The effects of cadmium on the central nervous system are still relatively poorly understood and its role in neurodegenerative diseases has been debated. In our research, cultured explants from 25 human foetal spinal cords (10-11 weeks gestational age) were incubated with 10 and 100μM cadmium chloride (CdCl(2)) for 24h. After treatment, an immunohistochemical study [for Sglial fibrillary acidic protein (GFAP) and choline acetyltransferase (ChAT)], a Western blot analysis (for GFAP, β-Tubulin III, nerve growth factor receptor, Caspase 8 and poly (ADP-ribose) polymerase), and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) assay (for detection of apoptotic bodies) were performed. The treatment with CdCl(2) induced a significant and dose-dependent change in the ratio motor neurons/glial cells in the ventral horns of human foetal spinal cord. The decrease of the choline acetyltransferase-positive cells (motor neurons) and the reduction of β Tubulin III indicate that CdCl(2) specifically affects motor neurons of the ventral horns. While the number of motor neurons decreased for the activation of apoptotic pathways (as shown by the increased expression of Caspase 8, nerve growth factor receptor, and poly (ADP-ribose) polymerase), glial cells, both in the subependymal zone and in the gray matter of the ventral horns, increased (as shown by the increase of GFAP expression). These results provide the evidence that during human spinal cord development, CdCl(2) may affect the fate of neural and glial cells thus, being potentially involved in the etiopathogenesis of neurodegenerative diseases.

Indicative oligodendrocyte dysfunction in spinal cords of human fetuses suffering from a lethal motoneuron disease

Human spinal cord development is still poorly understood and detailed molecular analyses of human motoneuron diseases could improve our understanding of the normal developmental processes of the spinal cord. Lethal Congenital Contracture Syndrome (LCCS, MIM 253310) provides a human model to study the early motoneuronal development. A typical phenotype of LCCS fetuses consists of multiple joint contractures, distinct facial features, and hydrops. Tissue pathology is characterized by severe muscle atrophy, lung hypoplasia, and degeneration of the anterior horn of the spinal cord as the hallmark of the syndrome. In this study we performed a global transcript analysis of LCCS spinal cords. The RNA expression profiles of these spinal cords were compared to age-matched healthy control fetuses, aborted for nonrelated causes. In addition, we applied phylogenetic footprinting methods to decipher the mechanisms of transcriptional regulation in the affected transcripts. Changes in transcripts involved with the development of the CNS and oligodendrocytes were obvious and the transcription factor PAX6 was identified as a key regulator during spinal cord development. In addition, transcript pathway analysis clearly indicated genes belonging to groups with neuronal functions to be affected. Our findings support the hypothesis that human motoneurons and oligodendrocytes are dependent on each other during their development and are influenced by distinct transcription factors previously known to act during murine and chick motoneuron development. These data provide valuable information about the molecular pathways putatively active in motoneuron diseases.

Development of the Human Spinal Cord: An Interpretation Based on Experimental Studies in Animals

Development of the Human Spinal Cord: An Interpretation Based on Experimental Studies in Animals. Edited by Joseph Altman, Shirley A. Bayer. Oxford University Press, New York, New York, 2001. 542 pages. This elegant, handsomely bound, beautifully illustrated treatise on human spinal cord development fills an important gap in the literature by analyzing and summarizing the descriptive morphology of the embryological development of what these world-renowned investigators have characterized as the structure that, with the exception of the cranial nerves, “is responsible for …

Neurons expressing NADPH-diaphorase in the developing human spinal cord.

The present study used nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry to identify populations of neurons containing nitric oxide synthase and to describe their putative migration during development of the human spinal cord. As early as week 6 (W6) of gestation, diaphorase expression was observed in sympathetic preganglionic neurons (SPNs) and interneurons of the ventral horn. As development proceeded, the SPNs translocated dorsally to form the intermediolateral nucleus, and the interneurons remained scattered throughout the ventral horn. In addition to the dorsal translocation of SPNs, a unique dorsomedially directed migratory pathway was observed. At later stages of development, other groups of SPNs were identified laterally in the lateral funiculus and medially in the intercalated and central autonomic regions. In addition, two "U-shaped" groups of diaphorase-labeled cells were identified around the ventral ventricular zone at W7. Cells of these groups appeared to translocate dorsally over the next weeks and presumably give rise to interneurons within the deep dorsal horn and surrounding the central canal. Furthermore, during W7-14 of gestation, the deep dorsal horn contained a number of diaphorase-positive cells, whereas the superficial dorsal horn was relatively free of staining. These data demonstrate that nitric oxide is present very early in human spinal cord development and that two unique cell migrations initially observed in rodents have now been identified in humans. Furthermore, nitric oxide may be expressed in some populations of neurons as they migrate to their final positions, suggesting that this molecule may play a role in neuronal development.

Mu opioid receptors in developing human spinal cord.

The distribution of mu opioid receptors was studied in human fetal spinal cords between 12-13 and 24-25 wk gestational ages. Autoradiographic localisation using [3H] DAMGO revealed the presence of mu receptors in the dorsal horn at all age groups with a higher density in the superficial laminae (I-II). A biphasic expression was noted. Receptor density increased in the dorsal horn, including the superficial laminae, between 12-13 and 16-17 wk. This could be associated with a spurt in neurogenesis. The density increased again at 24-25 wk in laminae I-II which resembled the adult pattern of distribution. A dramatic proliferation of cells was noted from the region of the ventricular zone between 16-17 and 24-25 wk. These were considered to be glial cells from their histological features. Mu receptor expression was noted over a large area of the spinal cord including the lateral funiculus at 24-25 wk. This may be due to receptor expression by glial cells. The study presents evidence of mu receptor expression by both neurons and glia during early development of human spinal cord.

Synchronized overproduction of AMPA, kainate, and NMDA glutamate receptors during human spinal cord development.

Quantitative receptor autoradiography was used to map the distribution in the developing human spinal cord of the three types of ionotropic glutamate receptors. N-methyl-D-Aspartate (NMDA) receptors were labeled with [3H]glutamate, kainic acid (KA) receptors were labeled with [3H]KA, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptors were labeled with [3H]AMPA. In the adult, labeling of all three receptor subtypes is largely restricted to the substantia gelatinosa (SG) in the dorsal horn, with very low level labeling elsewhere in the spinal gray matter. In marked distinction, in late fetal life, high level ligand binding is seen throughout the spinal gray matter. In early postnatal life, binding sites diminish in all regions, but least so in the SG, until the adult pattern emerges. Thus a coordinated transient high level of ionotropic glutamate receptor expression occurs within the developing spinal cord. Saturation analysis of ligand binding shows that the affinity of [3H]KA and [3H]AMPA binding is not developmentally regulated. In contrast, the affinity of [3H]glutamate binding to the NMDA receptor in the fetal ventral horn is three-fold greater than in the adult ventral horn. Thus, in addition to quantitative changes in glutamate receptor expression, qualitative changes occur in the expression of NMDA receptors during development. The distinct glutamate receptor phenotype of fetal and early postnatal spinal cord cells suggests that alterations in the excitable properties of these cells plays an important role in activity-dependent development and in susceptibility to excitotoxic injury.

Human fetal spinal cord xenografts survive in the eye of athymic nude rat hosts.

Human fetal spinal cord tissue was recovered from elective abortions and grafted to the anterior chamber of the eye of adult athymic nude rats. The transplants slowly became vascularized from the host iris during the first months. There was a clear cut stage-dependent survival and growth along a more "human" time-table. Fetal spinal cord tissue from embryos younger than gestational week 8 showed a much better survival and growth than tissue from older stages. Using laminin immunohistochemistry blood vessels could be visualized in the grafts. The pattern of vascularization was, however, clearly abnormal; there were fewer vessels which had abnormally thick walls as compared to those in the normal spinal cord. Similar to rat spinal cord allografts the human spinal cord xenografts displayed a relative gliosis and were surrounded by a glial layer visualized with antibodies against glial fibrillary acidic protein. Neurofilament-immunoreactive fibres were found inside the glial layer. A variety of neurons were found including large polygonal motoneuron-shaped cells, albeit with CGRP and AChE negative cell bodies. Both Substance P and enkephalin-immunoreactive cells and fibres were found. It is concluded that xenografted fetal human spinal cord survives, grows and may provide a useful model for experimental studies of human spinal cord development and connectivity.

Comparative study by S-100 and GFAP immunohistochemistry of glial cell populations in the early stages of human spinal cord development

The present study reports the presence and distribution of S-100-containing cells in the developing human spinal cord. From the earliest stages investigated, S-100-immunostained cells bordered the ventral third of the central canal or appeared dispersed in the basal lamina. On the other hand, radial glia processes were clearly depicted after GFAP immunolabelling. The presence and peculiar distribution of S-100-immunoreactive cells could be significant in the study of the early events of glial differentiation and migration.

Ultrastructure of marginal zone during prenatal development of human spinal cord.

Electron microscopic studies were conducted in the marginal zone (lamina I) in human fetuses ranging from 8-25 weeks of gestational age. At 8 weeks the neurons have indented nuclei and sparse organelles in the cytoplasm. The neuropil shows contacts between the axons and dendritic profiles. Some of them are well defined synapses with post synaptic thickening and agranular spherical vesicles in the presynaptic terminal. At 18 weeks compactly packed organelles with long cisternae of rough endoplasmic reticulum could be visualized in the neuronal cytoplasm. At 25 weeks the neurons have heterochromatin patches in the nuclei. Axosomatic, dendrodendritic, axoaxonic, symmetrical, asymmetrical and multisynaptic contacts with agranular and dense core vesicles are seen at different sequential age periods.

Fatty acid accretion in the development of human spinal cord

Lumbar and cervical segments of fetal and infant spinal cord were examined to determine the deposition rate of individual fatty acids in the spinal cord during the last trimester of fetal development and first 16 weeks of life. Fatty acid accretion rates were determined by regression analysis of spinal cord fatty acids at varying developmental ages. During the last trimester of fetal growth, accretion of major tissue fatty acids occurred earlier in cervical segments of spinal cord and at a greater rate than observed in lumbar regions. Postnatal accretion of fatty acids in cervical regions of spinal cord was minimal on a per gram of tissue basis with the exception of moderate increases in total ω-9 and saturated fatty acids, reflecting myelination. Significant postnatal accretion of fatty acids in lumbar regions of the spinal cord occurred some 4 weeks postnatally at rates significantly in excess of in utero rates. These developmental changes in fatty acid accretion are quantitatively relevant to estimates of fatty acid incorporation into spinal cord and parallel known processes of myelination and increasing neuro-motor activity in the developing infant.