and the Miami Project to Cure Paralysis funding to Dr. Bullock. ... overlay to GFP signal in transplant with synaptophysin) and is pointed out by an orange arrow.
Durable engraftment, neuronal differentiation of human fetal neural stem cell transplants in penetrating ballistic-like brain injury accompanied by amelioration of cognitive deficits.
321.08 / X8
*S. GAJAVELLI1, M. S. SPURLOCK1, K. N. RIVERA1, A. I. AHMED1, S. W. LEE1, S. YOKOBORI1, PINGDEWINDE N SAM1, M. P. HEFFERAN2, K. JOHE2, T. G. HAZEL2, F. C. TORTELLA3, D. A. SHEAR3, R. M. BULLOCK1 1Miami
Project to Cure Paralysis, Dept. of Neurosurgery, University of Miami, Miami, FL; 2Neuralstem Inc., Germantown, MD; 3Brain Trauma Neuroprotection and Neurorestoration Branch, Walter Reed Army Inst. of Research, Silver Spring, MD
Introduction
1. Survival/Engraftment of hNSCs in PBBI rats
Traumatic Brain Injury (TBI) is a public health concern worldwide as evident from incidence data covering half the world population (A). One of the causes of TBI: nonfatal gunshot wounds is on the rise (inset A). Penetrating high velocity or low velocity projectiles that breach the cranium (e.g., gunshot wound to head) cause PTBI in both military (B) and civilians. PTBI survivors often live with permanent disabilities due to neuronal loss. There are few effective restorative therapies beyond physical therapy. Preclinical studies with rodent neural stem cells (NSCs) show beneficial effects. However the enthusiasm for clinical trials is lacking despite 2 decades of strong preclinical data in athymic rats. This could in part be due to absence of quantitative data on durable (>28 days) engraftment of human NSCs in animals models. Further, despite xenotransplantation, there is no consensus on duration and agent(s) of choice for immunosuppression. Poor engraftment of the human cells in rat TBI models impedes exploration of efficacy and safety issues. United States Food and Drug Administration (US FDA) nonbinding recommendations for cell therapy include five levels of evidence that together could demonstrate efficacy and safety of the intervention (D). Here, engraftment of human NSCs in immunosuppressed penetrating ballistic-like brain injury (PBBI) rats, a PTBI model. A
B
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Materials and Methods A
B
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D
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Confocal image of PBBI brain sections with GFP+ human NSCs (green fluorescence) at week 5,8 and16 weeks post transplantation suggesting robust and durable engraftment. Scale bar is 1 mm. Following transplantation green fluorescent human NSCs undergo morphological changes with time in vivo. At one-week post transplantation cells exhibit round undifferentiated neural stem cell morphology devoid of processes (A). Nuclear HuNu immunoreactivity (red) confirms human origin of cells, note absence of red cytoplasmic processes (B). Fluorescence overlay of GFP (green) with HuNu (red) renders hNSC yellow, which is absent in the host cells (on top right corner of C). By week 8 GFP cells have differentiated with numerous cellular processes (D-F). A cell in white square (D-F) is shown at higher magnification in (insets) to show an emanating cytoplasmic process. The differentiated cellular morphology persists at week 16 (G-I). Overlay of HuNu and DAPI fluorescence renders transplanted cells pink (F and I) in contrast to blue HuNu negative host cells(arrow). Scale bar 10mm.
2. Distribution, decreased stemness and absence of 3.Neuronal differentiation of transplant, amelioration glial differentiation of engrafted hNSCs of PBBI-induced cognitive deficit Distribution of GFP+ human NSCs at 8-weeks post transplantation. The image at center with is marked with white boxes that are shown at higher magnification (counter clockwise).Processes from transplant (A) cross the posterior commissure (B) contralateral thalamic surface (Contralateral thalamus; C), ventrally by the internal capsule (D). GFP cells and processes in the periaqueductal grey (E) and ipsilateral internal capsule (F). Transplant processes thus wrap thalamus bilaterally. Micron bar is 30mm.
Visual cues
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Probe Balloon expansion
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-3.0mm +3.0mm
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The experimental groups, sequence of events and outcomes measured is shown in (A). In isofluorane anesthetized male Sprague Dawley rats (~325 g) unilateral penetrating ballistic-like brain injury (PBBI) was produced by rapidly inflating a balloon as shown (B) resulting in a ~6mm wide lesion (red oval in C) centered at Bregma (upward pointing arrow in C). Immunosuppression was established two days before transplantation and maintained until end of the study. Two drops (200,000 cells/drop) of Ubiquitin promoter driven GFP expressing human NSCs (NSI-566RSC Neuralstem Inc., green circles in C) were placed around the PBBI lesion via intraparenchymal microinjection (4ml volume) 710 days post injury (C). Animals underwent the Morris Water Maze (MWM) learning and memory tasks at 8 weeks post-transplantation, Release points (N=North,E=East,W=West, S=South) and visual cues used are shown in D. The latency to platform was recorded using a manual timer, Path length and swim speed were determined using the EthoVision Video Tracking Software (Noldus EthoVision XT). Statistical analysis with GraphPad. Animals were sacrificed at 8 or 16 weeks post transplantation, brain section immunohistochemistry was used to asses engraftment, distribution and differentiation. Immunostained sections were imaged on a confocal microscope (Olympus FV1000). Acknowledgements: Melissa Carballosa-Gautam for imaging. This work was supported in part by DOD Grant W81XWH-16-2-0008 and the Miami Project to Cure Paralysis funding to Dr. Bullock.
Increased neuronal differentiation of GFP hNSCs with time in vivo is evident from 8 week images (AH) and corresponding 16 week images (A’-H’), in A, image of whole hemisphere at 8 weeks post transplantation shows overlap of DAPI, GFP, DCX and NeuN fluorescence. The black square in A is shown at higher magnification in (B-H). The DAPI stained nucleus indicated by white arrow in B is GFP (C), strongly DCX+ (D) with weak NeuN immunoreactivity (E). Overlay of GFP with DCX (F), DCX with NeuN (G) and GFP with NeuN (H) confirms neuronal differentiation of transplanted hNSC. Corresponding 16 week images (A’-H’) shows weak, diffuse DCX (D’) and stronger NeuN (E’) that is confirmed by fluorescence overlay (F’-H’). Scale bar 10mm. Transplant derived neurons show immunoreactivity to mature neuronal marker, calbindin (I-K). DAPI stained whole hemisphere image shows GFP transplant (I) that is calbindin positive (Cal+) (J), overlay renders transplant yellow (K). The white square (in I-K) near corpus callosum is shown at higher magnification to highlight a single GFP cell bearing neuronal morphology that is Cal+ (I’-K’). Scale bar 10mm. DAPI and anti-Synaptophysin antibody stained whole hemisphere with 16-week transplant shows synpatophysin immunoreactivity in gray matter and transplant but not white matter (L). Quantitation of synpatophysin immunoreactivity (red puncta in L) revealed quantitatively greater amount of synaptophysin puncta in transplant (green) than host (red)(M). The region in white box is shown at higher magnification as an orthogonal view (to resolve fluorescence overlap) with two representative synaptophysin signals, one each for transplant (orange arrow) and host (red arrow) (N). A zoomed in orthogonal view shows presence of orange signal (due to overlay to GFP signal in transplant with synaptophysin) and is pointed out by an orange arrow. Such overlap is absent in host synaptophysin signal (red arrow). Quantitation of fluorescence is shown in P. Scale bar 10mm. The scale bar for A, A’, I-L is 1000 mm.
During weeks 8 and 16 post transplantation the distribution of GFP cells and processes shows the E Examples of transplant derived cells (green extent of interaction between host and transplant. fluorescence) and processes in host parenchyma. Triangular shaped transplant derived cell in cortex is reminiscent of cortical pyramidal shaped cells (A). The rat cortex revealed by DAPI staining shows no such green fluorescence. Two transplant derived cells in the hippocampus cornus ammonis 1 (CA1) bear similarity to host neurons (blue nuclei within dotted lines). Extensive processes emanate from these cells in the same direction as the host processes (B). Transplant derived cells in thalamus (C) revealed by GFP and DAPI (blue) fluorescence. Cells in thalamus appear to extend multiple long processes along white matter tracts of the internal capsule (IC) dotted outline in D. A sagittal section shows brain stem and cerebellum in (E), higher magnification of the boxes F and G show presence of GFP fibers in corresponding higher resolution image on right. Numbers of GFP fibers diminish in the distal area (G) compared to same area proximal to transplant (F). The tip of a GFP process with growth-cone morphology is shown in (H), suggesting normal development of the fibers. Micron bar is 10mm for A-C, 1500mm for D, 1000mm for E, and 10mm for F-H.
Immunoreactivity of transplant to neural stemness marker, nestin, and cell proliferation marker, nuclear Ki67, diminished over time. Confocal images at 2-weeks post transplantation (A-D) show a DAPI (A) stained field containing transplanted GFP cells (B) is strongly immunoreactive to anti-nestin antibody (C), the overlay of three channels (D) shows GFP fluorescence cells with nestin-positive (red) processes. The reaction to anti-nestin antibody diminished by 8-weeks post transplantation (E-H) and mostly absent by 16-week post transplantation (I-L). Nuclear Ki-67 at 2-week post transplantation (MP) is diminished by 16-weeks post transplantation (Q-T). Scale bar 10mm. Astrocytic marker, GFAP (A) , Olig 2, oligodendrocytic precursor marker (B), mature oligodendrocyte marker myelin basic protein (MBP) (C) could not be detected in GFP cells at 16 weeks. Scale bar 20mm.
B
Morris Water Maze (MWM) behavioral outcome 8 weeks post transplantation of hNSCs. Paths taken by each group representative on day 4 from various release points is shown in A. Sham (no PBBI, no cells), vehicle (PBBI, no cells), and transplant (PBBI, cell transplant) are compared. Graphical representation of day 4 data is shown in B. Across four test days (C), latency to reach platform was significantly lower in sham group than in C D vehicle group (p
