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. Author manuscript; available in PMC: 2017 Jul 17.
Published in final edited form as: Stroke. 2010 Jan 14;41(3):516–523. doi: 10.1161/STROKEAHA.109.573691

Human Neural Stem Cell Grafts Modify Microglial Response and Enhance Axonal Sprouting In Neonatal Hypoxic-Ischemic Brain Injury

Marcel M Daadi 1,*, Alexis Davis 1, Ahmet Arac 1, Zongjin Li 2, Anne-Lise Maag 1, Rishi Bhatnagar 1, Kewen Jiang 1, Guohua Sun 1, Joseph C Wu 2, Gary K Steinberg 1
PMCID: PMC5512869  NIHMSID: NIHMS190801  PMID: 20075340

Abstract

Background and Purpose

Hypoxic-Ischemic (HI) brain injury in newborn infants represents a major cause of cerebral palsy, development delay and epilepsy. Stem cell-based therapy has the potential to rescue and replace the ischemic tissue caused by HI and to restore function. However, the mechanisms by which stem cell transplants induce functional recovery are yet to be elucidated. In the present study, we sought to investigate the efficacy of human neural stem cells (hNSCs) derived from human embryonic stem cells (hESCs), in the rat model of neonatal HI and the mechanisms enhancing brain repair.

Methods

The hNSCs were genetically engineered for in vivo molecular imaging and for postmortem histological tracking. Twenty-four hours after the induction of HI, animals were grafted with hNSCs into the forebrain. Motor behavioral tests were performed the fourth week after transplantation. We used immunocytochemistry and neuroanatomical tracing to analyze neural differentiation, axonal sprouting and microglia response. Treatment-induced changes in gene expression were investigated by microarray and quantitative PCR.

Results

Bioluminescence imaging (BLI) permitted longitudinal tracking of grafted hNSCs in real time. HI transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the rotarod test. The grafts showed good survival, dispersion and differentiation. We observed an increase of uniformly distributed microglia cells in the grafted side. Anterograde neuronanatomical tracing demonstrated significant contralesional sprouting. Microarray analysis revealed upregulation of genes involved in neurogenesis, gliogenesis and neurotrophic support.

Conclusions

These results suggest that hNSC transplants enhance endogenous brain repair through multiple modalities in response to HI.

Keywords: neural stem cells, hypoxia ischemia, microglia, axonal sprouting, cell therapy

Introduction

HI brain injury causes brain damage in the fetus and newborn infants and represents a major cause of cerebral palsy, cognitive impairment, learning disability and epilepsy 1. While mild body hypothermia has been shown to improve the outcome following neonatal HI encephalopathy when initiated within 6 hours of birth 2, 3; there are no other effective interventions to improve the chronic sequelae of perinatal asphyxia. Neural stem cell-based therapy offers the prospect to rescue damaged tissue, to replace lost cells and to restore neurologic function after cerebral HI.

Early imaging studies in stroke patients 4–7 and microstimulation in experimental model of stroke 8–10 reported that in response to ischemic injury, the brain undergoes limited compensatory changes in an effort to recover from structural and functional loss 11. These changes or neuroplasticity are most prominent in early weeks and include axonal, dendritic and synaptic changes, inflammatory and immune adaptation, neurogenesis, gliogenesis and angiogenesis 12. Axonal sprouting and reorganization manifests by changes in dendritic arborization, spine remodeling, branching into the denervated areas and de novo formation of novel projections 13–17. Although limited, this spontaneous plasticity-mediated recovery is a promising target for drug and cell therapeutic interventions 17–21.

In parallel to endogenous CNS plasticity, inflammation and immune components are markedly activated de novo in neonatal brain and peripheral organs after HI 22, 23. Noteworthy, this inflammatory response to ischemic injury may exert neuroprotective and regenerative effects on the CNS 24, 25.

In the present study, we evaluated functional recovery after transplantation of multipotent hESC-derived hNSCs into the rat model of neonatal HI. We also investigated modalities by which grafted hNSCs provide therapeutic benefits to HI-damaged brain.

MATERIALS AND METHODS

Derivation of multipotent hNSCs

Human NSCs were derived from hESCs and perpetuated, as previously described 26, 27.

Induction of HI and cell transplantation

All animal experimentations were conducted according to the National Institute of Health guidelines and approved by the Institutional Animal Care and Use Committee. Seven-day-old Sprague Dawley rats were subjected to permanent ligation of the left carotid artery followed by 90 min in a hypoxic chamber (8%O2 and 92%N2, at 37°C) 28. The newborns were divided into HI vehicle (total n=12) and HI transplant (total n=12) groups. Twenty-four hours after the induction of HI, animals were placed in a stereotaxic apparatus with neonatal rat adaptor (Kopf Instruments). The skull bregma was determined and single cell suspension (50,000 cell/μl) of hNSCs (passages P9-P15) was transplanted, using a Hamilton syringe, into 3 sites (2 μl/site) in the left ischemic hemisphere at the following stereotaxic coordinates in mm: AP: +0.0, ML: +3.0, DV: −4.0; AP: +0.5, ML: +2.5, DV: −3.5; AP: −1.0, ML: +2.0, DV: −4.0. The injection rate was 1 μl/min, and the cannula was left in place for 5 min before retraction.

BLI of grafted hNSCs in vivo

BLI was performed using the Xenogen in vivo imaging system. Due to space limitation, we refer the reader to our recent publication 27 where we described in detail the BLI technique.

Behavioral tests

The animals were evaluated 4 weeks after transplantation for their sensorimotor skills in the cylinder and in the rotarod tests, as we previously reported 26, 29.

Transcriptome analysis

Total RNA was isolated from the ipsilateral hemisphere of normal, HI and HI-transplant groups (n=3) using RNAeasy kit (Quiagen) according to the manufacturer’s instruction. The Oligo GEArray neurogenesis and neural stem cell microarrays were purchased from SuperArray (SA Biosciences Corporation). The protocol for the microarray analyses followed manufacturer’s recommendations. Array spot density and differential probe expression was calculated using SuperArray’s GEArray Expression Analysis Suite software. Spot density was normalized to select positive and negative controls spotted onto each array.

Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was extracted from tissue dissected out from the ipsilateral hemisphere of vehicle and hNSC-treated animals (n=3). Aliquots (1 μg) of total RNA from the cells were reverse transcribed (RT) as previously described 30. Real time PCR was performed using Maxima SYBR Green qPCR Master Mix (Fermentas) in the Stratagene thermocycler MX3000P (Stratagene) according to the supplier’s instructions. Data were expressed as mean ±SEM. The ΔCt for each candidate was calculated as ΔCt of [Ct (gene of interest) - Ct (18S)] and the ΔΔCt was the difference between the Ct of treated sample (transplanted animals) and the Ct of control vehicle sample. The relative expression was calculated as the 2ÙΔΔCt according to the methods 31 and plotted as relative levels of gene expression. The rat primers were designed using the Primer3 software and are available upon request.

Biotinylated dextran amine (BDA) injections

The last week before euthanasia of the animals, three randomly selected animals from both animal groups were anesthetized and placed in the stereotaxic apparatus. After craniotomy, 0.5 μl of BDA (10,000 molecular weight, Molecular Probes; 10% wt/vol solution in sterile PBS) was injected stereotaxically into the sensorimotor cortex opposite to the HI lesion site at the stereotaxic coordinates: AP: +0.5 m, ML: 2.5mm, and DV: −1.5mm. The scalp was then closed and the animal returned to its cage.

Histopathology, immunocytochemistry and microscopical analysis

Immunocytochemistry was performed, as we previously reported 26. The following primary antibodies were used: anti-human Nuclei (hNuc), anti-NeuN, anti-GAD65/67, anti-glial fibrillary acidic protein (GFAP), anti-galactocerebrocide (GC), anti-Nestin (Chemicon), anti-TuJ1 (Covance); anti-CNPase (Aves Labs); anti-doublecortin (DCX, SantaCruz Biotechnology). Secondary antibodies raised in the appropriate hosts and conjugated to FITC, RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoResearch) were used. Cells and sections were counterstained with the nuclear marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Fluorescence was detected, analyzed and photographed with a Zeiss LSM550 laser scanning confocal photomicroscope (Carl Zeiss).

For the rat anti-Iba-1 (ionized calcium binding adapter molecule, Wako Bioproducts) and BDA immunohistochemistry was carried out, as we previously described 29. For each animal, quantitative estimates of the number of Iba-1+ cells were stereologically determined using the optical fractionator procedure 32, as we previously described in detail 29. The quantitative analysis of BDA labeled fibers was performed, as we previously reported 29. The infarct volume was estimated by summing up the infarcted areas in all animals of each group. To eliminate the effects of edema, infarct size was calculated as the contralateral hemisphere – ipsilateral nonischemic hemisphere / contralateral hemiphere and expressed as percentage (x 100%) of infarcted hemisphere.

Statistics

Outcome measurement for each experiment was reported as mean±SEM. Data were analyzed using SPSS 11 for Mac OS X (SPSS Inc.). Significance of inter-group differences was performed by applying Student’s t-test where appropriate. One-Way ANOVA analysis was used to compare groups. Differences between the groups were determined using Bonferroni’s post hoc test. A P-value of less than 0.05 was considered to be statistically significant.

Results

1. Real time imaging of hNSC transplants

The hNSCs grew as an adherent monolayer culture and all progeny expressed double fusion (DF) construct carrying fLuc and eGFP reporter genes (Fig. 1A). Image analysis (Fig. 1B) demonstrated a linear correlation between plating density and the fLuc activity (Fig. 1C). This stable and efficient expression of fLuc gene, enabled us to non-invasively image, in real time, the survival of the grafted hNSCs at different time points (Fig. 1D).

Figure 1. Imaging of hESC-derived hNSCs in HI neonates.

Figure 1

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(A) Neural stem cells were isolated through a multistep process previously described 26, 27. Flow cytometry analysis demonstrated the purity of the eGFP-expressing hNSCs. (B) In vitro imaging analysis of genetically engineered hNSCs show increasing fLuc activity with cell density and a linear correlation (R2=0.98) (C). Data are representative of three independent experiments performed in triplicate. Representative BLI imaging of a neonate rat transplanted with the hNSCs and monitored for 4 weeks (D). Color scale bar is in photon/sec/cm2/sr.

2. Characterization of the HI infarct size and the hNSC graft

Microscopic examination of cresyl violet stained sections showed significant loss of brain tissue in HI animals in the neocortex, the striatum and extending caudally to the hippocampus. This atrophy was accompanied sometimes by the formation of a porencephalic cyst. The estimation of the infracted region revealed 17.8±8.6% of the hemisphere (n=6) for the transplanted group and 20.66±12.5% (n=6) for the vehicle group. Although hNSC grafted animals showed smaller infarct volume, the difference to vehicle group was not significant (n=6). Grafted hNSCs identified with hNuc demonstrated good survival dispersion and integration into the HI-damaged tissue (Fig. 2A, B). Transplanted cells expressed the neuroepithelial stem cell marker nestin in 12.8±3.4% and differentiated into neurons expressing NeuN, TuJ1+ (42.9 ± 3.5%), DCX (4.8±1.1%) (Fig. 2C–E) and the GABAergic marker GAD (47.6±4.3%) (Fig. 2G). Grafted cells also differentiated into GFAP-expressing astrocytes (Fig. 2F).

Figure 2. Characterization of hNSC grafts.

Figure 2

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(A) Example of triple labeling of transplanted animal with the human nuclear specific marker hNuc (purple) and the neuronal markers DCX (red) and TuJ1 (green). (B) Higher magnification showing the dispersion of the grafted hNuc+ (purple) hNSCs in the striatum. Photomicrographs show co-expression of hNuc (purple) and the neuronal markers: TuJ1 (green) (C), DCX (green) (D), and NeuN (green) (E). (F) Co-localization of hNuc and the astroglial marker GFAP, (G), and example of grafted cell expressing the GABAergic neurotransmitter marker GAD. Bars: (A, B) 100 μm; (B-far right, E) 20 μm; (C, D, F, G) 10 μm.

3. hNSC grafts enhance axonal sprouting

To investigate whether the transplanted hNSCs influenced the rewiring of the HI-damaged tissue, we used BDA to anterogradely label the sensorimotor cortical projection originating from the contralateral side. We measured the density of BDA+ crossing fibers in the corpus callosum and of BDA+ terminals in the ipsilateral sensory-motor cortex, the striatum and thalamic nuclei. The quantitative analysis of BDA labeled fibers and terminals, normalized to the total number of labeled somas at the injections site 29, revealed an increase in both the number of fibers crossing towards the ipsilateral hemisphere (Fig. 3B) and in the number of terminals in the ipsilateral sensorimotor cortex (Fig. 3A), the striatum (Fig. 3C) and thalamic nuclei (Fig. 3D).

Figure 3. Transplanted hNSCs enhanced axonal sprouting.

Figure 3

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Confocal image analysis and quantification of BDA-labeled axons and axon terminals in the sensorimotor cortex (A), corpus callosum (B), striatum (C) and in thalamus (D) show significant increase on the ipsilateral HI-lesioned and hNSC-transplanted side (n=3 per group). Data are expressed as mean ± SEM, *P < 0.05 versus vehicle group.

4. Transcriptome analysis

Gene microarray analysis revealed, in comparison to vehicle group, a significant increase in CNS rat endogenous genes involved: 1) in neurogenesis and cell migration (DCX, Chemokine (C-X-C motif) receptor 4 (CXCR4), Oligodendrocyte lineage transcription factor 2 (Olig2) and FGF2, 2) in myelination (myelin basic protein, MBP) and 3) in genes involved in cell survival and neurite outgrowth (Glial derived neurotrophic factor: GDNF, Neurturin: NTN and insulin growth factor-1: IGF1) (Fig. 4A) 33. Subsequently, we confirmed the microarray expression patterns of selected genes using real time quantitative RT-PCR (Fig. 4B).

Figure 4. Microarray analysis and quantitative real-time RT-PCR measurement of selected genes up-regulated in transplanted animals.

Figure 4

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Genes from the expression profile, with P-values below 0.01 were selected for analysis. (A) Fold of increase of genes involved in neurogenesis, gliogenesis and neurotrophic support in HI and HI-transplant groups, relative to naïve group. (B) Quantitative RT-PCR of the selected genes confirming the expression patterns seen in the microarray data.

5. Grafts of hNSCs modulate microglial presence in HI damaged area

Immunostaining with the pan-microglial marker Iba1 demonstrated that microglia were homogenously distributed throughout the brain in both vehicle and hNSC-grafted animals without adverse infiltration or reaction against the grafts (Fig. 5A, B). Stereological quantitative analysis of the Iba1 positive cells demonstrated a significant increase in the transplanted striatum (Fig. 5D).

Figure 5. Stereological analysis of Iba1-expressing microglia in vehicle and transplanted groups.

Figure 5

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Stereological analysis of Iba1-expressing microglia in transplanted animals (D) showed more abundant and homogenously distributed microglia. (E, F), with significant increase in the grafted striatum (F). (Tx: transplant) Bars: (C, D) 20 μm.

7. Improvement of motor behavioral in rats that received the hNSC grafts

To determine the ability of hNSCs to functionally engraft, one month after transplantation animals’ sensorimotor skills were evaluated using two neurobehavioral tests. Our results showed that during the fourth week, HI transplanted animals significantly improved in their use of the contralateral impeded forelimb (Fig. 6A,B). The hNSC grafts significantly ameliorated the locomotor deficits in the rotarod test (Fig. 6C).

Figure 6. Transplantation of hNSCs improves sensorimotor function.

Figure 6

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The independent use of the impaired contralateral forelimb significantly increased in the transplant group (B) (n=3) on days 28 and 30 post transplantation (*P<0.05 vs vehicle group: A). Bars represent percentages ± SEM of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. On days 29 and 30, transplant group (n=9) showed significant improvement (*P<0.05) on the rotarod test.

Discussion

Genetically engineered hNSCs were efficiently and non-invasively imaged in real time, after transplantation into HI model of newborn rats and up to five weeks of age. We report that hNSCs engrafted into the ischemic brain, enhanced axonal sprouting and the expression of genes involved in neurogenesis, gliogenesis and neurotrophic support, modulated microglial response and improved motor function of the animals.

It is generally believed that transplanted non-neural cells, such as those derived from bone marrow or cord blood, exert neurotrophic effects on ischemia-injured tissue and may not survive for long-term 34. Whereas neural stem cells are thought to provide cell replacement and neurotrophic support 29, 35, 36. This neurotrophic support may be responsible for the significant axonal sprouting that we measured in the cortex, the striatum and the thalamus. The number of BDA labeled axon terminals may vary with the size of the injection site and the number of BDA+ cell bodies. The BDA infusion led to small (3–5 mm2) and circumscribed injection sites in the sensorimotor cortex. There was no significant difference in the size of the injection site and the total number of labeled cells between vehicle and transplanted groups. In addition, the data were normalized to the total number of labeled cells. Thus, it is unlikely that the increase of axonal sprouting we observed is due to differences in the size or location of the injection site.

After ischemia, regions of the contralateral hemispheres become activated during the early phase of partial regain of function or spontaneous recovery in experimental models 37 and in stroke patients 38–40. In neonate HI rats contralesional sprouting could give rise to alternative motor descending pathways from the motor cortex relaying in sub-cortical structures, such as the red nucleus and the pontine formation, or direct cortico-spinal projections may be formed to compensate for functional loss 41. The newly formed pathways could be generated by multiple mechanisms, including sprouting from the surviving neurons, unmasking of existing pathways that are functionally inactive or the compensatory descending control channels through alternative functionally active but redundant pathways 42, 43.

The intracerebral or intravenous delivery of multipotent adult progenitor cells (MAPCs) provided protection to HI damaged tissue in neonate and improved motor and neurologic scores compared to vehicle group 44. Using retrograde and anterograte neuroanatomical tracing, Park et al. 36 demonstrated that neural stem cells grafted into HI-lesioned neonate, along with a polyglycolic acid-based biodegradable polymer scaffold reestablished long-distance cross-callosal neuronal connections. Recent genomic analysis studies demonstrated that in addition to these growth factors, axonal sprouting is activity-dependent in constraint-induced movement therapy 45. Together, these data suggest that grafted hNSCs could exert neurotrophic and/or activity-mediated effects on HI-damaged local network and enhance axonal sprouting. Based on these observations, it seems reasonable to propose that the newly innervated and recruited area of the contralesional hemisphere is becoming a part of a reorganized network promoting motor recovery.

Our data are in line with previous studies that show an increase in the resident microglia/macrophage cells (CD11b) in ischemic animals that received neurosphere-derived cells 46. Capone et al. suggest that microglia activation is required for neurosphere graft neuroprotective action through secretion of growth factors, including insulin growth factor-1, VEGF-A, transforming growth factor-β1 and BDNF 46. Indeed, BDNF treatment of HI neonates improves spatial memory 47 and selective ablation of microglia via mutant thymidine kinase gene driven by myeloid-specific CD11b promoter exacerbated ischemic injury 48. Thus, these findings support the notion that microglia play a dual role, pro-inflammatory or anti-inflammatory/neurotrophic depending on their state of activation and functional phenotype 24.

In conclusion, we provide evidence that growth factor-isolated and perpetuated hNSCs from hESCs are amenable to genetic modification for real time in vivo imaging and potentially for other therapeutic genes. We showed that these hNSCs are able to modify the host microenvironment and enhance neuroanatomical plasticity after HI in neonates and improve sensorimotor skills.

Acknowledgments

The authors thank Beth Hoyte for preparation of the figures. This work was supported in part by Russell and Elizabeth Siegelman, Bernard and Ronni Lacroute, the William Randolph Hearst Foundation, CIRM RS1-00322, Edward G. Hills Fund and NIH NINDS grants RO1 NS27292, P01 NS37520 and R01 NS058784.

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