JY was a major contributor to the work and designed the details of the experimental plan, performed all animal surgeries and most immunocytochemical staining, as well as writing the manuscript. LX performed revision experiments and some of the stereological and ICC work, did statistics analysis, and helped with the final preparation of the manuscript. AMW was responsible for most of the semiquantitative cell counts on ICC preparations and helped with stereology and the preparation of figures. GH designed and performed real-time PCR and wrote the corresponding portions of the manuscript. TJ and KJ established, maintained, and supplied human NSCs for transplantation. VEK, principal investigator, conceived and designed the project, established surgery procedures, troubleshot the experiments, and prepared the final manuscript.
Jun Yan, Leyan Xu, Annie M. Welsh, Glen Hatfield, and Vassilis E. Koliatsos have no financial interest in Neuralstem or in a product that might be developed as the result of this study. Karl Johe and Thomas Haze have financial interest in Neuralstem.
Effective treatments for degenerative and traumatic diseases of the nervous system are not currently available. The support or replacement of injured neurons with neural grafts, already an established approach in experimental therapeutics, has been recently invigorated with the addition of neural and embryonic stem-derived precursors as inexhaustible, self-propagating alternatives to fetal tissues. The adult spinal cord, i.e., the site of common devastating injuries and motor neuron disease, has been an especially challenging target for stem cell therapies. In most cases, neural stem cell (NSC) transplants have shown either poor differentiation or a preferential choice of glial lineages.
In the present investigation, we grafted NSCs from human fetal spinal cord grown in monolayer into the lumbar cord of normal or injured adult nude rats and observed large-scale differentiation of these cells into neurons that formed axons and synapses and established extensive contacts with host motor neurons. Spinal cord microenvironment appeared to influence fate choice, with centrally located cells taking on a predominant neuronal path, and cells located under the pia membrane persisting as NSCs or presenting with astrocytic phenotypes. Slightly fewer than one-tenth of grafted neurons differentiated into oligodendrocytes. The presence of lesions increased the frequency of astrocytic phenotypes in the white matter.
NSC grafts can show substantial neuronal differentiation in the normal and injured adult spinal cord with good potential of integration into host neural circuits. In view of recent similar findings from other laboratories, the extent of neuronal differentiation observed here disputes the notion of a spinal cord that is constitutively unfavorable to neuronal repair. Restoration of spinal cord circuitry in traumatic and degenerative diseases may be more realistic than previously thought, although major challenges remain, especially with respect to the establishment of neuromuscular connections.
When neural stem cells from human fetal spinal cord were grafted into the lumbar cord of normal or injured adult nude rats, substantial neuronal differentiation was found.
Every year, spinal cord injuries, many caused by road traffic accidents, paralyze about 11,000 people in the US. This paralysis occurs because the spinal cord is the main communication highway between the body and the brain. Information from the skin and other sensory organs is transmitted to the brain along the spinal cord by bundles of neurons, nervous system cells that transmit and receive messages. The brain then sends information back down the spinal cord to control movement, breathing, and other bodily functions. The bones of the spine normally protect the spinal cord but, if these are broken or dislocated, the spinal cord can be cut or compressed, which interrupts the information flow. Damage near the top of the spinal cord can paralyze the arms and legs (tetraplegia); damage lower down paralyzes the legs only (paraplegia). Spinal cord injuries also cause many other medical problems, including the loss of bowel and bladder control. Although the deleterious effects of spinal cord injuries can be minimized by quickly immobilizing the patient and using drugs to reduce inflammation, the damaged nerve fibers never regrow. Consequently, spinal cord injury is permanent.
Scientists are currently searching for ways to reverse spinal cord damage. One potential approach is to replace the damaged neurons using neural stem cells (NSCs). These cells, which can be isolated from embryos and from some areas of the adult nervous system, are able to develop into all the specialized cells types of the nervous system. However, because most attempts to repair spinal cord damage with NSC transplants have been unsuccessful, many scientists believe that the environment of the spinal cord is unsuitable for nerve regeneration. In this study, the researchers have investigated what happens to NSCs derived from the spinal cord of a human fetus after transplantation into the spinal cord of adult rats.
The researchers injected human NSCs that they had grown in dishes into the spinal cord of intact nude rats (animals that lack a functioning immune system and so do not destroy human cells) and into nude rats whose spinal cord had been damaged at the transplantation site. The survival and fate of the transplanted cells was assessed by staining thin slices of spinal cord with an antibody that binds to a human-specific protein and with antibodies that recognize proteins specific to NSCs, neurons, or other nervous system cells. The researchers report that the human cells survived well in the adult spinal cord of the injured and normal rats and migrated into the gray matter of the spinal cord (which contains neuronal cell bodies) and into the white matter (which contains the long extensions of nerve cells that carry nerve impulses). 75% and 60% of the human cells in the gray and white matter, respectively, contained a neuron-specific protein six months after transplantation but only 10% of those in the membrane surrounding the spinal cord became neurons; the rest developed into astrocytes (another nervous system cell type) or remained as stem cells. Finally, many of the human-derived neurons made the neurotransmitter GABA (one of the chemicals that transfers messages between neurons) and made contacts with host spinal cord neurons.
These findings suggest that human NSC grafts can, after all, develop into neurons (predominantly GABA-producing neurons) in normal and injured adult spinal cord and integrate into the existing spinal cord if the conditions are right. Although these animal experiments suggest that NSC transplants might help people with spinal injuries, they have some important limitations. For example, the spinal cord lesions used here are mild and unlike those seen in human patients. This and the use of nude rats might have reduced the scarring in the damaged spinal cord that is often a major barrier to nerve regeneration. Furthermore, the researchers did not test whether NSC transplants provide functional improvements after spinal cord injury. However, since other researchers have also recently reported that NSCs can grow and develop into neurons in injured adult spinal cord, these new results further strengthen hopes it might eventually be possible to use human NSCs to repair damaged spinal cords.
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Degenerative and traumatic diseases of the nervous system are characterized by loss of neurons and their connections. Effective treatments for these conditions are presently unavailable. In the field of experimental therapeutics two major approaches have been taken: prevention of cell death with compounds that interfere with decision-making steps in cell death pathways and the replacement or support of degenerating neurons with neural grafts [
The adult spinal cord represents an especially challenging environment for the survival and differentiation of NSCs because of the apparent lack of cells and/or signals promoting regeneration [
Human NSCs were prepared from the cervical and upper thoracic spinal cord of a single eight-week human fetus after an elective abortion. The tissue was donated by the mother in a manner fully compliant with the guidelines of the National Institutes of Health (NIH) and the Food and Drug Administration (FDA) and approved by an outside independent review board. Spinal cord tissue was cleared of meninges and dorsal root ganglia and dissociated into a single-cell suspension by mechanical trituration in serum-free, modified N2 medium composed of 100 mg/l human plasma apo-transferrin, 25 mg/l recombinant human insulin, 1.56 g/l glucose, 20 nM progesterone, 100 μM putrescine, and 30 nM sodium selenite in DMEM/F12, to which basic fibroblast growth factor (bFGF) (10 ng/ml) was added. The initial culture was serially expanded as monolayers in precoated flasks or plates as described [
The first passage was conducted at 16 d postplating. At this time point, the culture was composed mostly of dividing NSCs and postmitotic neurons. Dividing cells were harvested by brief treatment with trypsin (0.05% + 0.53 mM EDTA) followed by mechanical dissociation. A single-cell suspension was thus derived that was centrifuged at 1,400 rpm for five minutes. The cell pellet was resuspended in the growth medium, and cells were replated in new precoated plates at 1.2 × 106 cells in 20 ml of medium per 150 mm plate. Cells were harvested at approximately 75% confluence, which occurred within five or six days. This process was repeated for 20 passages. Cells from various passages were frozen in the growth medium plus 10% DMSO and stored in liquid nitrogen. Upon thawing, overall rate of recovery was 80%–95%. The resulting cell line, produced by epigenetic means only and by using bFGF as the sole mitogen, was coded “566RSC.”
Passage 10–12 cells were used in this study. One cryopreserved vial of the appropriate passage was thawed, washed, and cultured again as described above, 5–7 d prior to surgery. For multiple days of surgeries, cultures were seeded at varying densities, so that each flask reached confluence on the designated day of surgery. Cells were subsequently harvested by brief enzymatic treatment as described above, washed in a buffered saline solution, couriered to the surgery site on wet ice, and used within 24 h. Viability of cells on ice was typically greater than 80% within this 24-h period.
Nude rats (160–180 g, strain CR: NIH-RNU,
Rhizotomies involved transections of L4 and L5 roots with extraspinal avulsion of the corresponding spinal nerves as described [
Animals were subjected to dorsal laminectomy at the lower thoracic level and received four injections of 105 NSCs each in 0.5 μl suspension into ventral L4 and L5 on the left side 2 wk after lesion. Injections were made stereotaxically on a Kopf spinal unit 1 mm lateral to the midline using pulled-beveled glass micropipettes connected, via silastic tubing, to 10 μl Hamilton microsyringes. Animals were allowed to survive for 3 wk, 3 mo, or 6 mo (avulsion lesions), 3 or 6 mo (excitotoxic lesions), or 6 mo (sham lesions) (
The survival and phenotypic fate of NSCs were assessed with immunocytochemistry (ICC), including ABC-peroxidase ICC and dual-label immunofluorescence. Tissues were prepared from animals perfused with 4% freshly depolymerized, neutral-buffered paraformaldehyde. The thoracolumbar spinal cord segments with attached roots and lumbar nerves were further fixed by immersion in the same fixative for an additional 4 h after removal of the dura. Blocks containing the entire grafted area plus 1 mm border above and below were subdissected, equilibrated in 30% neutral-buffered sucrose, and frozen for further processing. L3–S1 roots were processed separately as whole-mount preparations or after teasing the rootlets with heat-coagulated tips of glass pipettes. Blocks were sectioned transversely (30 μm) on a freezing microtome; sections were kept in an antifreeze solution until processed for NSC survival or phenotypic studies. Survival studies utilized human nuclear antigen (HNu) immunoperoxidase-stained sections (~ 15 per animal, i.e. every 24th section through the L3–S1 block) with random sampling of the first section. HNu is a selective nuclear marker of cells of human origin [
After permeabilization with 0.1% Triton X-100 and nonspecific site blocking with 5% normal serum, slide-mounted sections were incubated in primary antibodies in 1 mg/ml BSA with 0.1% Triton X-100 (4 °C, overnight). Primary antibodies were used to address human (graft) versus rat (host) cell identity, mitotic activity, and neuronal, astrocytic, and oligodendrocytic phenotype specification and included a number of monoclonal antibodies and antisera as laid out in
Primary Antibodies
To assess NSC graft survival, we counted numbers of HNu (+) profiles through L4–L5 using stereological assumptions based on the optical fractionator concept [
To study NSC differentiation, we used a nonstereological method of counting total number of HNu (+) cells, as well as cells dually labeled with HNu and a phenotypic marker on randomly selected 100× fields through the ventral horn from immunofluorescent preparations. One field in each one of four sections, spaced 1 mm apart through the grafting area, was used from each animal. Numbers of HNu (+) and double-labeled profiles were pooled from all four fields counted from each case and grouped per experimental protocol. NSCs located at or near the meninges were counted separately. Percentages of double-labeled cells were generated per parenchymal or meningeal sites at different time points for each treatment group (
Variation in survival and differentiation, as function of experimental protocol, graft location, and time point postsurgery, was studied with one-way ANOVA followed by Tukey's multiple comparison post hoc test. Results are expressed as mean ± standard deviation.
RNA samples were extracted from 107 NSCs at various time points of differentiation in vitro (0, 14, 29, and 42 d after withdrawal of bFGF) using Trizol (Invitrogen [
Primer Sequences Used for Real-Time PCR
NSCs prepared for grafting were propagated as a monolayer culture in the presence of bFGF and delivered to animals within 24 h post-bFGF withdrawal. At that time, all cells expressed the NSC marker nestin (
(A) The vast majority of cells express the NSC-specific marker nestin (red) immediately before grafting. The DNA dye DAPI (blue) was used to reveal all cells in culture.
(B and C) At 14 days within the differentiation phase (i.e., after bFGF removal), ~ 50% of cells acquire MAP2 immunoreactivity and neuronal cytology, with characteristic processes (red, B). A smaller number of cells differentiate into GFAP (+) astrocytes (green, C).
(D) Real-time RT-PCR data showing increased neurotrophic factor and NRG expression in the course of NSC differentiation in vitro. The number of days on top of the columns is the days NSCs have been in a phase of differentiation (after withdrawal of fibroblast growth factor). Results are expressed as fold increases compared to levels expressed at the proliferation phase (day 0), the latter values designated as 1. Data represent average ± standard deviation of triplicate measurements of a representative cell culture sample at a given time point. The experiment was repeated twice with different sets of cell samples and yielded very similar results.
Scale bars: 50 μm.
The expression of representative neurotrophic factors and NRGs was studied by real-time PCR at 0, 14, 29, and 42 days post-bFGF withdrawal, i.e. during the differentiation phase (
The examination of all stained sections from lesioned and control animals shows a robust engraftment and excellent long-term survival of NSCs in the adult spinal cord environment (
Photomicrographs and graphs in (A–C) illustrate the localization and numbers of HNu (+) cells at different time points postgrafting, whereas (D) and (E) support the migratory phenotype of grafted HNu (+) cells, and (F) confirms their low mitotic activity.
(A and B) At 3 mo postgrafting most HNu (+) cells, indicated as red profiles with an arrow in (A), are located around the injection sites and along needle tracks. By six months (B), HNu (+) cells show widespread migration away from the injection site in both the gray and white matter, and many are seen in the white matter and a few in the gray matter of the contralateral side. (B) is a composite of several fields to show the extent of migration. Arrow in (B) shows the colonization, by NSC-derived cells, of the central nervous system portion of the dorsal root (note the central nervous system–peripheral nervous system transition zone).
(C) Bar graphs showing HNu (+) cell numbers at the time of grafting (0) and at three weeks (3w), three months (3m), and six months (6m) postgrafting in the different treatment groups (avulsion, red; HCA treatment, blue; sham, green). Far left graph shows numbers of HNu (+) cells ipsilateral to the grafting site (Ipsi), and far right graph shows numbers on the contralateral gray matter (Contra). Brackets show the results of post hoc testing when ANOVA was significant in the avulsion and HCA groups ipsilateral to grafting; in all other cases, significance was established with a Student's t-test. Asterisk indicates statistical significance at
(D) Dcx, a marker for migrating neuronal precursors, was expressed by about 80% of grafted cells 3 wk postgrafting. Dcx expression is reduced to 10%–15% of HNu (+) cells surrounding the grafting sites at 3 and 6 mo but remains very high (~ 80%) in HNu (+) cells on the contralateral gray matter up to 6 mo postgrafting.
(E) A confocal image of HNu (+) (red) cells also labeled with Dcx (green) at 3 wk postgrafting.
(F) The three images illustrate, on a section that was dually stained for HNu (red nuclear marker on the left) and Ki67 (green nuclear marker in the center), the very low rate of mitotic activity (double-stained nuclei on the right) in NSC grafts. The single double-stained nuclear profile is indicated with an arrow.
Scale bars: (A) 200 μm; (B) 600 μm; (E) 10 μm; (F) 20 μm.
A comparison among animals surviving for 3 wk, 3 mo, and 6 mo shows a tendency of NSCs to migrate away from the initial grafting sites and populate both gray and white matter, as well as the proximal end of ventral and dorsal roots in the avulsion cases. Approximately 3% of NSC-derived cells were found in the contralateral side. There were more cells in the contralateral side in avulsion-injured than in HCA-injured animals three months postgrafting (
In summary, human NSCs survive very well in the spinal cords of nude rats with minimal further mitotic activity irrespective of the presence or absence of lesions, and migrate extensively into the ipsilateral and contralateral spinal cord.
Based on the aiming of the grafting pipettes, the main portion of NSC grafts reviewed here was confined within the ventral horn of L4–L5 segments (
Photomicrographs (A–F) illustrate cases of neuronal (A and B), astrocytic (C and D), and oligodendrocytic (E and F) differentiation of HNu (+) cells by epifluorescence (A, C, and E) or confocal (B, D, and F) microscopy. (G) is a composite of bar graphs illustrating the general differences in fate choice between parenchymal (upper level) and meningeal (lower level) sites of NSC grafts. (H) provides further detail in differential fate choice among three parenchymal sites and the pia compared side-by-side.
(A and B) These two sections are stained for HNu and TUJ1 and show the abundance of NSC-derived neurons within the parenchyma of the ventral horn by epifluorescence (A) and confocal microscopy (B). Both preparations are taken from animals killed three months postgrafting. Inset is a magnification of demarcated area in (A). Note the homogeneous appearance of TUJ1 (+) cells in the A inset. Confocal sections have been virtually resectioned at the
(C and D) These sections, dually stained for HNu and GFAP, illustrate the substantial astrocytic differentiation of NSCs located by the pia membrane by (Figure 3, continued) epifluorescence (C) and confocal microscopy (D). Inset is a magnification of demarcated area in (C), and representative astrocytes are indicated with arrows. Confocal sections have been processed as in (B).
(E and F) Oligodendrocyte differentiation in ventral white matter based on APC immunoreactivity in the cytoplasm of cells with HNu (+) nuclei as shown with epifluorescence (E) and confocal microscopy (F). Blue nuclei represent DAPI counterstain. Arrow depicts a double-labeled cell. Arrowheads point to host oligodendrocytes (APC [+], HNu [−] cells). Confocal sections have been processed as in (B).
(G) Bar graphs depicting the fate choices of NSC grafts in the parenchyma (including ventral and dorsal horn and ventral white matter, upper graphs) or the meninges (lower graphs) at three weeks (3w), three months (3m), and six months (6m) in different treatment groups (avulsion, red; HCA treatment, blue; sham, green). Neuronal fate is represented by numbers of TUJ1-labeled HNu (+) cells, and astrocytic fate is represented by numbers of GFAP-labeled HNu (+) cells. NSCs in a neural stem/precursor state are depicted here as nestin-and HNu double-labeled cells. Asterisks indicate critical post hoc differences between subgroups where ANOVA is significant (
(H) These graphs provide further detail into the role of spinal microenvironment in the fate choice of grafted NSCs by differentiating among three parenchymal sites and the pia. Cell fates are represented by the same markers as in (G) Asterisks on top of brackets indicate important post hoc differences where ANOVA is significant (
Scale bars: (A), (C), (E) 20 μm; (B), (D), (F) 10 μm.
The fate of grafted human NSCs located in dorsal horn exhibited similar patterns as in ventral horn. At 6 mo postgrafting, rates of TUJ1 (+) NSC-derived cells in dorsal horn for avulsion, HCA, and sham groups were 71.1% ± 8.3%, 68.4% ± 13.6%, and 68.8% ± 4.3%, respectively (
In all animals examined, the pia adjacent to the inoculation sites contained large numbers of grafted NSCs. At those sites, differentiation pattern was different from parenchymal sites. For example, the rates of GFAP (+), astrocyte-like, NSC-derived cells were 30%–50% in the various experimental groups, and there were more cells persisting in a nestin (+) state (40%–53%) (
The neuronal differentiation of NSCs that were dispersed in the ventral white matter, apparently by migration from the ventral horn inoculation sites, was not as prominent as in gray matter. At 6 mo postgrafting, percentages of dually labeled cell for TUJ1 and HNu in ventral white matter are 59.6% ± 6.3%, 57.8% ± 3.0%, and 59.9% ± 4.5% for avulsion, HCA, and sham treatments, respectively (
In both parenchymal and meningeal locations, we observed infrequent HNu (+) cells that also expressed A2B5, a ganglioside antigen present in the common glial precursor O-2A, at 3 mo as well as 6 mo postgrafting. In 6-mo grafts, a population of smaller HNu (+) cells in the ventral horn expressed the mature oligodendrocyte marker adenomatus polyposis coli (APC) in the ventral horn (8.8% ± 4.1%, 8.9% ± 3.0%, and 9.0% ± 1.8 % in avulsion, HCA, and sham groups respectively), white matter (13.8% ± 2.6%, 12.2% ± 5.3%, and 12.0% ± 7.4%), and pia (6.4% ± 4.6%, 7.2% ± 3.3%, and 8.5% ± 1.9%). On confocal imaging, these cells have a thin cytoplasm and multiple APC (+) radial processes consistent with oligodendrocytic cytology (
In concert, the fate of grafted NSCs depends on location, i.e., the parenchymal microenvironment overall promotes a neuronal differentiation, and there is further inductive influence in this direction in the gray matter. The meningeal environment appears to facilitate astrocytic differentiation or to induce NSCs to remain in a nestin (+) state. In both locations, about one-tenth of surviving NSCs differentiate in the oligodendrocytic lineage. As proof of the concept that the predominantly neuronal differentiation of NSCs is not dependent on the athymic state of nude rats, we performed a small study in which we grafted the lumbar cord of normal rats. These animals were treated with FK506 to prevent xenograft rejection. Two months postgrafting, animals were prepared exactly as the nude rats and the neuronal differentiation of NSCs was explored with dual immunofluorescence using NeuN and TUJ1 as neuronal markers. As in the case of nude rats, the vast majority of NSCs had differentiated into neuronal cells (
Outlined areas in (A) and (C) are enlarged in (B) and (D). All images illustrate the neuronal differentiation of NSCs two months postgrafting based on dual-label immunofluorescence for HNu (red) and a neuronal marker (green, representing TUJ1 and NeuN in [A and B], and [C and D], respectively). The predominance of double-labeled profiles in both (B) and (D) (indicated with asterisks) matches the avid neuronal differentiation of human NSCs in nude rats as illustrated in
Scale bars: (A and B) 20 μm; (C and D) 10 μm.
Because of the predominant neuronal fate of grafted NSCs excitatory, inhibitory (GABA) and cholinergic neurotransmitter markers were examined in order to ascertain the degree of differentiation of cells into the neuronal lineage (
Photomicrographs (A–J) illustrate evidence of glutamatergic (A and B, G and H), GABAergic (C–F), and cholinergic (I and J) neurotransmission in NSC grafts. As in previous figures, confocal microscopy is used primarily to confirm the colocalization of two markers in the same cellular compartment along three planes of sectioning.
(A and B) These sections, stained for HNu and the prevalent AMPA receptor epitope GluR2/3, show both cytoplasmic and synaptic staining by epifluorescence (A) or confocal (B) microscopy. Insets in (A) represent magnifications of indicated neurons in main image; top- and bottom-left insets show two medium-size HNu (+) cells with cytoplasmic immunoreactivity, whereas bottom-right inset illustrates a larger HNu (+) cell containing multiple GluR2/3 (+) boutons.
(C and D) These sections are stained for HNu and the GABA-synthesizing enzyme GAD and visualized with epifluorescence (C) or confocal microscopy (D). Arrows in (C) indicate multiple HNu (+) cells with cytoplasmic GAD immunoreactivity.
(E and F) Confocal microscopy of a field stained with both human Syn (red in single-channel image on top left, to label graft-derived terminals) and GAD (green in single-channel image on bottom left, to label GABAergic terminals) shows colocalization of the two proteins (yellow color in merged images in F) in multiple synaptic boutons. Nearly all graft-derived boutons are inhibitory (F).
(G and H) These sections (G, epifluorescence; H, confocal) are stained for human Syn to label graft-derived terminals (red) and mixed VGLUT1/ VGLUT2 antibodies to label glutamatergic terminals in the field (green). Despite significant overlap and apposition of graft-derived and VGLUT1/2 (+) terminals (G), the two groups of terminals are separate (H).
(I and J) These two sections were dually stained for: HNu and choline acetyltransferase (I and insert) epifluorescence; confocal microscopy (J); and show that some of the largest NSC-derived neurons express cholinergic phenotypes. These cells elaborate multiple primary dendrites (I and insert). (J) is the confocal image of the neuron in the inset.
Scale bars: (A), (C), (G), (I) 20 μm; (B), (D–F), (H), (J) 10 μm.
Examination of sections stained for HNu, GAD, and GluR2/3 showed a large number of NSC-derived neurons colocalizing both GAD and GluR2/3. To distinguish between GABAergic and glutamatergic phenotypes in the nerve terminals, we performed dual ICC for human-specific synaptophysin (Syn, to mark graft-derived terminals) and GAD or vesicular glutamate transporter type 1 and 2 (VGLUT1/2). GAD and VGLUT1/2 are sensitive and selective markers for GABAergic and glutamatergic neurons respectively [
Using GAD immunoreactivity as a marker of GABAergic neurons, we counted HNu and GAD (+) profiles and calculated rates of dually labeled cells in the total population of HNu (+) cells at 6 mo, i.e. the longest survival time examined. At that time point, a significant percentage of HNu (+) cells in all three experimental groups (avulsion: 60.5% ± 0.47%; HCA lesion: 56.4% ± 3.19%; sham: 49.57% ± 4.04%) were also GAD immunoreactive. Frequency of differentiation did not vary significantly by type of treatment.
A very small percentage of HNu (+) cells (less than 1%), first appearing at 3 mo and consistently seen at 6 mo postgrafting, colocalized choline acetyltransferase immunoreactivity. Choline acetyltransferase (+) neurons were larger than other neuronal HNu (+) cells (15–25 μm in diameter) and displayed multipolar cytologies (
These findings indicate that a majority of NSC-derived neurons develop and sustain stable bipolar cytologies and GABAergic phenotypes for at least six months after grafting. These cells are contacted by GABAergic terminals from other graft and host neurons and glutamatergic terminals from the host. A small, but consistent, percentage of graft-derived neurons evolve into larger multipolar neurons with cholinergic phenotypes.
By 3 mo postgrafting, HNu (+) neurons elaborate axons (
(A) This photograph was taken through the ventral horn of a HNu/70 kDa neurofilament protein stained section 3 mo postgrafting and shows bundles of human 70 kDa neurofilament protein (+) axons (indicated with white arrows) originating in HNu (+) grafts (one indicated with an asterisk on top right) and coursing together (red arrows on bottom left) toward the ventral white matter.
(B) This photograph shows an NSC graft in the ventral horn of a human Syn-stained section three months postgrafting. The sharp colocalization of Syn (+) puncta with the graft region (boundaries demarcated with arrows) is due to the selectivity of the antibody for human, but not rat, Syn protein.
(C and D) These images (C, epifluorescence; D, confocal) were taken from triple-stained sections with HNu (red), TUJ1 (blue), and the presynaptic marker Bsn (green). The Bsn antibody used here recognizes rat and mouse, but not human, protein. (C) depicts a dense field of rat Bsn (+) terminals in proximity to HNu and TUJ1 (+) profiles. Examples of contacts between rat terminals and NSC-derived neurons are shown with arrowheads in the inset, which is a magnification of the profile at the center of the main image. The very large number of such terminals on NSC-derived cell bodies is best illustrated with confocal microscopy (D).
(E and F) These photographs (E, epifluorescence; F, confocal) were taken from sections stained with HNu (red), TUJ1 (blue), and mixed VGLUT1/VGLUT2 antibodies (green) and show the innervation of HNu and TUJ1 (+) cells by glutamatergic terminals putatively originating in the host.
Scale bars: (A) 80 μm; (B) 20 μm; (C–F) 10 μm.
Conversely, preparations stained with human-specific Syn and TUJ1 or choline acetyltransferase revealed dense terminal fields of boutons apposed to host neurons, including large and small motor neurons both on the side of grafting as well as the contralateral side. Large numbers of host motor neurons were seen to be contacted in such a fashion by graft-specific terminals (
Host motor neurons are depicted as large TUJ1 (+) cell bodies, and NSC-derived terminals are labeled with human Syn antibodies.
(A) This epifluorescence image shows the site of the original graft (arrow in lower left) and two synaptic fields with host motor neuron pools marked as (1) and (2), with respectively higher and lower density of synaptic appositions. The low-density field (2) is further enlarged in the inset.
(B) This confocal image shows, in great detail, a large number of somatic and dendritic terminals from graft-derived nerve cells on a host motor neuron.
Scale bars: (A) 200 μm; (B) 20 μm.
In summary, NSC-derived neurons do not only develop differentiated neurotransmitter phenotypes in the adult spinal cord, but also elaborate axons and synaptic specializations and form synaptic contacts with host spinal cord neurons.
Our findings indicate a large-scale differentiation and some structural integration of human NSCs grafted into the normal and injured spinal cord of T cell deficient (nude) rats. Under the present experimental conditions, human NSCs survive well with limited further mitotic activity and migrate extensively for at least six months postgrafting. Although the vast majority of NSCs in the spinal cord parenchyma take on a neuronal fate, the meningeal environment is associated with astrocytic phenotypes or the persistence of grafted NSCs in a perpetual nestin (+) state.
A majority of NSC-derived neurons in spinal cord parenchyma have bipolar cytologies and GABAergic phenotypes for at least six months after grafting and receive GABAergic innervation from other graft and host neurons, in addition to glutamatergic innervation from the host. A small percentage of graft-derived neurons evolve into larger multipolar neurons with cholinergic phenotypes. NSC-derived neurons elaborate axons and synaptic specializations and appear to engage in reciprocal innervation with host spinal cord neurons. Importantly, when immunological obstacles are overcome, rodent models are quite suitable for the preclinical evaluation of human NSCs as tools for repair of the injured spinal cord.
The 3- to 4-fold increase in NSC numbers postgrafting, although not of the magnitude or rate to cause tumors, persisted during the period of active neuronal differentiation. Because it is implausible that the differentiation and dividing pools of NSCs are the same cell population, we hypothesize that the subset of grafted NSCs that persists in a nestin (+) state, for example NSC-derived cells located near the pial surface, is the one that gives rise to additional neuronal lineage, possibly on an ongoing basis. These cells may persist in a stem cell state either because of accidental initial placement near the pial environment or via active migration and tropism to pial sites. These are complex phenomena that require NSC-host signaling and require further investigation.
The migratory spread of human NSCs was especially evident at three and six months, but a migratory disposition was ascertained by Dcx immunoreactivity as early as three weeks postgrafting. Although most HNu (+) cells remained within the gray matter, there was also colonization of the white matter. A consistent phenomenon was the early establishment of linear pathways of migration from the initial graft into the white matter perpendicular to the spinal cord surface, which often appeared to extend all the way to the pia. In 4′,6-diamidino-2-phenylindole-counterstained preparations, it was apparent that these migratory pathways were associated with microvessels. The generality of this phenomenon invites further exploration of the role of microvessels as guiding structures for NSC migration and of the role of specific cells in the host vasculature as generators of tropic and other signals for grafted NSCs.
Neuronal differentiation of NSC grafts in the adult spinal cord has been the exception, rather than the rule, in the literature [
The extensive neuronal differentiation of the NSC preparation used in the present study may be due to several reasons, including species of NSC origin and culture method. For example, human NSCs may have a greater pluripotentiality compared to rodent NSCs [
The robust differentiation of NSCs in this study is inconsistent with the general notion that the adult cord environment is constitutively unfavorable to the survival and differentiation of NSCs [
Our findings also show that local factors can influence the fate choice of grafted NSCs, i.e., many more NSCs turn into astrocytes or remain nestin (+) when located close to the meninges (pia). The gray matter may exert additional inductive influences in the direction of neuronal differentiation and certain lesions, i.e., root avulsions, appear to facilitate an astrocytic fate choice. These patterns are welcome evidence of NSC plasticity and suggest that signals from the host microenvironment play significant roles in the differentiation choices of these cells in vivo. However, before various preparations of cells are directly compared in the same animal model, it is premature to draw generic conclusions about differentiation trends of human NSCs in the adult mammalian cord.
An unexpected finding in this paper was the measurable differentiation of human NSCs into mature oligodendrocytes six months after transplantation, especially given the low preference of these cells for oligodendrocyte fate choice in vitro. This is yet another indication of an important role of the mature spinal cord environment in inducing relatively immature precursors into diverse differentiation pathways. Oligodendrocyte differentiation was the predominant fate of a preparation of brain-derived human NSCs that was recently grafted into the contused spinal cord of NOD-
The predominant neurotransmitter phenotype of differentiated NSCs is GABAergic, although a sizeable minority of these cells acquires cholinergic phenotypes. It has been reported that “priming” of human NSCs prior to grafting may advance them more fully to cholinergic fates postgrafting [
A substantial degree of elongation of NSC-derived axons was observed within the spinal cord parenchyma. This observation suggests very little, if any, inhibitory effect from the host in the elaboration and elongation of these axons. The myelin-associated glycoprotein and the oligodendrocyte-myelin glycoproteins NOGO A, B, and C have been shown to inhibit axonal regeneration [
The partial anatomical integration of grafted cells with host neurons via the establishment of contacts observed here has important theoretical and practical implications. Although this is a novel phenomenon and the molecular mechanisms are unknown, a number of trophic and cell–cell recognition transcripts shown to be expressed by cultured human NSCs are likely to play significant roles in this process. These protein signals are expressed and released in amounts and gradients that allow highly conserved tropic and trophic interactions to occur irrespective of age of the host, or the fact that host and graft belong to different species. Based on our PCR data on human NSCs in culture, these proteins include: the prototypical glial-derived growth factor glial cell line-derived neurotrophic factor and the neurotrophin BDNF, i.e. potent trophic factors for motor neurons that can account for the marked ability of human NSCs to attract host motor axons [
In conclusion, NSCs from human spinal cord propagated in vitro can be grafted successfully in the adult rat spinal cord under a variety of experimental conditions and show marked differentiation into projection neurons that appear to engage in circuit formation with the host irrespective of species differences. These outcomes indicate that, with successful suppression of host immune rejection of the graft, rodent models can be used for the preclinical evaluation of human NSCs as cell support or replenishment tools to a much larger extent than previously anticipated. It appears that cellular and molecular factors that signal synaptic contact formation are remarkably conserved within the mammalian class, despite the fact that spatial and temporal patterning of developmental events may differ among species. Together, our findings add weight to the argument that restoration of spinal cord circuitry in traumatic and degenerative diseases may be a realistic goal that needs to be further refined at the preclinical stage prior to its eventual consideration for clinical applications.
adenomatus polyposis coli
basic fibroblast growth factor
Bassoon
doublecortin
gamma-aminobutyric acid
glutamate decarboxylase
glial fibrillary acidic protein
glutamate receptor subunit 2 and 3
L-homocysteic acid
human nuclear antigen
immunocytochemistry
insulin-like growth factor-1
immunoglobulin G
neuregulin
neural stem cell
synaptophysin
vascular endothelial growth factor
vesicular glutamate transporter