Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord

Abstract

Background: 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.

Methods and findings: 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.

Conclusions: 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.

Figure 1. In Vitro Differentiation of Human NSCs Used for Transplantation

(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.

Figure 2. Survival and Migration of Human NSCs in Rat Spinal Cord

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 p ≤ 0.05. Method of section selection is illustrated on the extreme left. (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.

Figure 3. Differentiation of Grafted Human NSCs into Neurons and Glial Cells

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 x and y planes to confirm the identity of the double-stained structure. (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 (p ≤ 0.05). (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 (p ≤ 0.05). Scale bars: (A), (C), (E) 20 μm; (B), (D), (F) 10 μm.

Figure 4. Differentiation of Human NSCs into Neurons after Transplantation into the Lumbar Spinal Cord of Normal Adult Sprague-Dawley Rats

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 Figure 3. Single HNu-labeled profiles (D) are shown with arrowheads Scale bars: (A and B) 20 μm; (C and D) 10 μm.

Figure 5. Neurotransmitter Differentiation of Grafted Human NSCs

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.

Figure 6. Maturation of Human NSC-Derived Neurons Based on the Elaboration of Axons, Synapses, and Innervation by Host Neurons

(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.

Figure 7. 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

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.