In Vitro Differentiation and Trophic/Tropic Profile of Human NSCs
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), approximately 5% were immunoreactive for the neuronal precursor-specific marker PSA-NCAM, and less than 1% expressed the neuronal markers TUJ1 and MAP2 or the astroglial marker glial fibrillary acidic protein (GFAP). With continued culture in the absence of bFGF, about 50% of cells acquired neuronal phenotypes as shown by MAP2 immunoreactivity and cytological profile (B), and some differentiated into GFAP (+) astrocytic profiles (C) within 2 wk. Very few cells expressed oligodendrocyte/Schwann cell markers within this time frame.
In Vitro Differentiation of Human NSCs Used for Transplantation
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 (D). Among neurotrophic factors, glial cell line-derived neurotrophic factor and BDNF transcripts increased 3- and 7-fold respectively, whereas VEGF increased 15-fold by day 29; significant sustained increases were also noted for IGF-1 (D). Among NRGs, NRG1 and NRG3 transcripts, particularly the latter, were also found to have significantly increased. The expression of all these factors declined after 4 wk, a pattern that may reflect some cellular deterioration that is common in long-term culturing of mature neurons.
Survival and Migration of Human NSCs in Rat Spinal Cord
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 (A and B). Stereological estimates of surviving HNu (+) cells show an average of 1.5 × 106 cells in the L4–L5 cord 6 mo postgrafting. This value represents a 3- to 4-fold increase of the cell population present in the initial graft (C) and implies that the initial graft survived well and underwent, on average, two mitotic divisions. This estimate is consistent with a low frequency (3%–5%) of HNu (+) cells that also expressed the nuclear antigen Ki67 at 3 and 6 mo postgrafting in all experimental conditions studied in this paper (F). Ki67 is a marker of all phases of cell cycle minus G0. and Ki67-positive cells were found randomly dispersed across the graft area without evidence of clustering in specific sites.
Survival and Migration of Human NSCs in Rat Spinal Cord
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 (C, far right graph). The migratory disposition of NSCs was further confirmed with the expression of doublecortin (Dcx), a microtubule-associated protein that serves as a specific marker for migrating neuronal precursors [25
], by about 80% of grafted cells three weeks postgrafting (D and E). Dcx expression is reduced to 10%–15% of HNu (+) cells at the grafting sites at 3 and 6 mo but remains very high (~ 80%) in HNu (+) cells on the contralateral side at 6 mo postgrafting, evidence for continuous migration (D).
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.
Differentiation of Human NSCs into Neuronal and Non-Neuronal Cells: Parenchyma versus Meninges, Gray versus White Matter
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 (A). In this location, the majority of grafted cells entered a neuronal lineage, as evidenced by a ≥ 75% rate of TUJ1 immunoreactivity at 3 wk and at 3 and 6 mo postgrafting (A, B, and 3G). These TUJ1 (+) cells have round or bipolar cell bodies and an average diameter of 10 μm (A and B). Rates of TUJ1 differentiation did not differ significantly among treatment groups and were consistent with results from dual ICC for HNu and the neuronal nuclear epitope NeuN. In addition, there were no significant differences in rates of TUJ1 (+) NSC-derived cells among various time points, evidence that the establishment of a neuronal lineage occurs very early in the life of these grafts. Compared to neuronal markers, the appearance of astrocytic phenotypes was slower. HNu (+) cells do not show significant GFAP immunoreactivity at 3 wk postgrafting. By 3–6 mo, ~ 5% of grafted HNu (+) cells in the avulsion cases and a slightly higher proportion of cells in HCA (−) and sham animals stained for GFAP (G). The astrocytic cytology of these GFAP (+) cells is confirmed by confocal microscopy (D). Nestin expression in grafted cells declined over the time course studied here and the greatest reduction was noted between 3 wk and 3 mo. A percentage of 11%–14% of HNu (+) cells remained nestin (+) even at 6 mo postgrafting (G).
Differentiation of Grafted Human NSCs into Neurons and Glial Cells
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 (H, far left graph). Because of this similarity, fate choices in ventral horn were taken as representative of the entire spinal gray matter and were entered as such in all subsequent comparisons with fate choices in the meninges and white matter (see below).
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%) (C, D, and 3G). At 6 mo, these rates were higher than those in ventral horn in all experimental groups (p < 0.05) (H, center and far right graph). In sections that were dually labeled for human nestin and GFAP, the two phenotypes were expressed by distinct populations of NSC-derived cells, i.e. the vast majority of GFAP (+) cells were nestin (−) and nestin (+) cells were GFAP (−). Only 6%–11% of HNu (+) cells colocalized TUJ1 immunoreactivity at pial sites (G). These rates are significantly lower compared to ventral horn and white matter (p < 0.05) (H, far left graph).
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 (H, far left graph). Differences in TUJ1 (+) ratios between NSC-derived cells located in ventral white matter and in ventral horn are significant within the same treatment group (p < 0.05 for each one of the three groups). At the ventral white matter, percentages of GFAP (+) cells are 18.5% ± 5.6%, 20.0% ± 5.1%, and 16.1% ± 6.1% in the avulsion, HCA, and sham groups; in the avulsion group, GFAP differentiation rate is significantly higher than in ventral horn (p < 0.05) (H, center graph). Nestin expression by HNu (+) cells is similar between ventral white matter and ventral horn (H, far right graph).
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 (E and F).
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 ().
Differentiation of Human NSCs into Neurons after Transplantation into the Lumbar Spinal Cord of Normal Adult Sprague-Dawley Rats
GABAergic and Cholinergic Neurotransmitter Phenotypes Expressed by Grafted NSCs
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 (). Almost without exception, grafting sites were markedly enriched in metabotropic glutamatergic (A and B) and GABAergic (C and D) neurotransmitter markers. Individual bipolar NSC-derived cells were seen to express strong immunoreactivity for glutamate receptor subunit 2 and 3 (GluR2/3) and glutamate decarboxylase (GAD) and to be contacted by terminals enriched in these two neurotransmitter markers. Both GluR2/3 and GAD immunoreactivity first became apparent in grafting sites at 3 mo and persisted without appreciable changes at 6 mo postgrafting.
Neurotransmitter Differentiation of Grafted Human NSCs
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 [26
], and terminal staining is especially robust in the spinal cord (E and G). To label a maximal number of glutamatergic terminals, antibodies against VGLUT1 and VGLUT2 were used in combination. Large numbers of GAD and VGLUT1/2 (+) terminals were seen at the grafting sites, but fields of VGLUT1/2 (+) puncta only partially overlapped the graft (G). In multiple fields in sections dually stained for human-specific Syn and GAD, we found that a majority of Syn (+) terminals colocalized GAD immunoreactivity by confocal microscopy (F). In contrast, we observed no synaptic colocalization of human Syn with VGLUT1/2 immunoreactivity, despite dense apposition of individually labeled terminals (H).
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 (I and J).
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.
Evidence for Structural Integration of Human NSCs in Rat Spinal Cord: Axons and Synapses
By 3 mo postgrafting, HNu (+) neurons elaborate axons (A) and synapses (B) that can be specifically linked to graft origin with antibodies selective for human Syn and neurofilament proteins. Axons bundle in groups that traverse across the white matter (A). Axon projections into the ventral root were rare or inconsistent. To evaluate the ability of HNu (+) neurons to integrate within the host circuitry, we labeled neural structures with graft- and host-selective markers. For example, to separate host from graft terminals, we used a monoclonal antibody for the presynaptic protein Bsn that selectively recognizes rat and mouse, but not human, epitopes. In sections stained for HNu (to establish graft origin), TUJ1 (to establish neuronal differentiation), and Bsn (to detect terminals from host rat axons), we found that HNu (+) and TUJ1 (+) cells in parenchymal locations were contacted by synaptic boutons of rat origin (C and D), i.e., evidence that the host species (rat) innervates graft-derived (human) neuronal cells. Because graft-derived terminals are negative for markers of glutamatergic neurotransmission, VGLUT1/2 immunoreactivity can be used to differentiate between graft and host terminals. By combining ICC staining for HNu and TUJ1 (to mark graft-derived neurons) with ICC staining for VGLUT1/2 (to label most host-derived glutamatergic terminals), we found dense appositions between graft-derived neurons and host glutamatergic terminals (E and F). In preparations stained for VGLUT1/2, GFAP, and HNu, we never observed such contacts between host terminals and graft-derived astrocytic profiles.
Maturation of Human NSC-Derived Neurons Based on the Elaboration of Axons, Synapses, and Innervation by Host Neurons
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 (A and B). Treatment variance (avulsion, HCA lesion, or sham treatment) did not seem to affect the presence or absence of such dense contacts. In sections stained for human Syn and GFAP, we did not observe any appositions between graft-derived terminals and astrocytes.
Innervation of Host Motor Neurons by Graft-Derived Nerve Cells as Shown on Sections Stained with Human Syn (Red) and TUJ1 (Green) and Studied with Epifluorescence or Confocal Microscopy
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.