Detection of endothelial cell-associated human DNA reveals transplanted human bone marrow stem cell engraftment into CNS capillaries of ALS mice.

Repairing the altered blood-CNS-barrier in amyotrophic lateral sclerosis (ALS) is imperative to prevent entry of detrimental blood-borne substances into the CNS. Cell transplantation with the goal of replacing damaged endothelial cells (ECs) may be a new therapeutic approach for barrier restoration. We showed positive effects of human bone marrow-derived CD34+ cells (hBM34+) and endothelial progenitor cells (hBM-EPCs) intravenous transplantation into symptomatic G93A SOD1 mutant mice on barrier reparative processes. These benefits mainly occurred by administered cells engraftment into vascular walls in ALS mice; however, additional studies are needed to confirm cell engraftment within capillaries. The aim of this investigation was to determine the presence of human DNA within microvascular ECs isolated from the CNS tissues of G93A SOD1 mutant mice treated with human bone marrow-derived stem cells. The CNS tissues were obtained from previously cell-treated and media-treated G93A mice at 17 weeks of age. Real-time PCR (RT-PCR) assay for detection of human DNA was performed in ECs isolated from mouse CNS tissue. Viability of these ECs was determined using the LIVE/DEAD viability/cytotoxicity assay. Results showed appropriate EC isolation as verified by immunoexpression of endothelial cell marker. Human DNA was detected in isolated ECs from cell-treated mice with greater concentrations in mice receiving hBM-EPCs vs. hBM34+ cells. Also, higher numbers of live ECs were determined in mice treated with hBM-EPCs vs. hBM34+ cells or media-injection. Results revealed that transplanted human cells engrafted into mouse capillary walls and efficaciously maintained endothelium function. These study results support our previous findings showing that intravenous administration of hBM-EPCs into symptomatic ALS mice was more beneficial than hBM34+ cell treatment in repair of barrier integrity, likely due to replacement of damaged ECs in mouse CNS vessels. Based on this evidence, hBM-EPCs may be advanced as a cell-specific approach for ALS therapy through restored CNS barrier integrity.

Beneficial Effects of Transplanted Human Bone Marrow Endothelial Progenitors on Functional and Cellular Components of Blood-Spinal Cord Barrier in ALS Mice.

Convincing evidence of blood-spinal cord barrier (BSCB) alterations has been demonstrated in amyotrophic lateral sclerosis (ALS) and barrier repair is imperative to prevent motor neuron dysfunction. We showed benefits of human bone marrow-derived CD34+ cells (hBM34+) and endothelial progenitor cells (hBM-EPCs) intravenous transplantation into symptomatic G93A SOD1 mutant mice on barrier reparative processes. These gains likely occurred by replacement of damaged endothelial cells, prolonging motor neuron survival. However, additional investigations are needed to confirm the effects of administered cells on integrity of the microvascular endothelium. The aim of this study was to determine tight junction protein levels, capillary pericyte coverage, microvascular basement membrane, and endothelial filamentous actin (F-actin) status in spinal cord capillaries of G93A SOD1 mutant mice treated with human bone marrow-derived stem cells. Tight junction proteins were detected in the spinal cords of cell-treated versus non-treated mice via Western blotting at four weeks after transplant. Capillary pericyte, basement membrane laminin, and endothelial F-actin magnitudes were determined in cervical/lumbar spinal cord tissues in ALS mice, including controls, by immunohistochemistry and fluorescent staining. Results showed that cell-treated versus media-treated ALS mice substantially increased tight junction protein levels, capillary pericyte coverage, basement membrane laminin immunoexpressions, and endothelial cytoskeletal F-actin fluorescent expressions. The greatest benefits were detected in mice receiving hBM-EPCs versus hBM34+ cells. These study results support treatment with a specific cell type derived from human bone marrow toward BSCB repair in ALS. Thus, hBM-EPCs may be advanced for clinical applications as a cell-specific approach for ALS therapy through restored barrier integrity.

Phenotypic characteristics of human bone marrow-derived endothelial progenitor cells in vitro support cell effectiveness for repair of the blood-spinal cord barrier in ALS.

Amyotrophic lateral sclerosis (ALS) was recently recognized as a neurovascular disease. Accumulating evidence demonstrated blood-spinal-cord barrier (BSCB) impairment mainly via endothelial cell (EC) degeneration in ALS patients and animal models. BSCB repair may be a therapeutic approach for ALS. We showed benefits of human bone marrow endothelial progenitor cell (hBMEPC) transplantation into symptomatic ALS mice on barrier restoration; however, cellular mechanisms remain unclear. The study aimed to characterize hBMEPCs in vitro under normogenic conditions. hBMEPCs were cultured at different time points. Enzyme-linked immunosorbent assay (ELISA) was used to detect concentrations of angiogenic factors (VEGF-A, angiogenin-1, and endoglin) and angiogenic inhibitor endostatin in conditioned media. Double immunocytochemical staining for CD105, ZO-1, and occludin with F-actin was performed. Results showed predominantly gradual significant post-culture increases of VEGF-A and angiogenin-1 levels. Cultured cells displayed distinct rounded or elongated cellular morphologies and positively immunoexpressed for CD105, indicating EC phenotype. Cytoskeletal F-actin filaments were re-arranged according to cell morphologies. Immunopositive expressions for ZO-1 were detected near inner cell membrane and for occludin on cell membrane surface of adjacent hBMEPCs. Together, secretion of angiogenic factors by cultured cells provides evidence for a potential mechanism underlying endogenous EC repair in ALS through hBMEPC transplantation, leading to restored barrier integrity. Also, ZO-1 and occludin immunoexpressions, confirming hBMEPC interactions in vitro, may reflect post-transplant cell actions in vivo.

Human Bone Marrow Endothelial Progenitor Cell Transplantation into Symptomatic ALS Mice Delays Disease Progression and Increases Motor Neuron Survival by Repairing Blood-Spinal Cord Barrier.

Convincing evidence demonstrated impairment of the blood-spinal cord barrier (BSCB) in Amyotrophic Lateral Sclerosis (ALS), mainly by endothelial cell (EC) alterations. Replacing damaged ECs by cell transplantation is a potential barrier repair strategy. Recently, we showed that intravenous (iv) administration of human bone marrow CD34+ (hBM34+) cells into symptomatic ALS mice benefits BSCB restoration and postpones disease progression. However, delayed effect on motor function and some severely damaged capillaries were noted. We hypothesized that hematopoietic cells from a restricted lineage would be more effective. This study aimed to establish the effects of human bone marrow-derived endothelial progenitor cells (hBMEPCs) systemically transplanted into G93A mice at symptomatic disease stage. Results showed that transplanted hBMEPCs significantly improved behavioral disease outcomes, engrafted widely into capillaries of the gray/white matter spinal cord and brain motor cortex/brainstem, substantially restored capillary ultrastructure, significantly decreased EB extravasation into spinal cord parenchyma, meaningfully re-established perivascular astrocyte end-feet, and enhanced spinal cord motor neuron survival. These results provide novel evidence that transplantation of hBMEPCs effectively repairs the BSCB, potentially preventing entry of detrimental peripheral factors, including immune/inflammatory cells, which contribute to motor neuron dysfunction. Transplanting EC progenitor cells may be a promising strategy for barrier repair therapy in this disease.

Transplantation of human bone marrow stem cells into symptomatic ALS mice enhances structural and functional blood-spinal cord barrier repair.

Accumulating evidence shows alterations in the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in ALS patients and in animal models of disease, mainly by endothelial cell (EC) damage. Repair of the altered barrier in the CNS by replacement of ECs via cell transplantation may be a new therapeutic approach for ALS. Recently, we demonstrated positive effects towards BSCB repair by intravenous administration of unmodified human bone marrow CD34+ (hBM34+) cells at different doses into symptomatic ALS mice. However, particular benefits of these transplanted cells on microvascular integrity in symptomatic ALS mice are still unclear. The aim of the present study was to determine the structural and functional spinal cord capillary integrity in symptomatic ALS mice after intravenous administration of hBM34+ cells. The G93A mice at 13 weeks of age intravenously received one of three different cell doses (5 × 104, 5 × 105, or 1 × 106) and were euthanized at 17 weeks of age (4 weeks post-transplant). Control groups were media-treated and non-carrier mutant SOD1 gene mice. Capillary ultrastructural (electron microscopy), immunohistochemical (laminin and HuNu), and histological (myelin and capillary density) analyses were performed in the cervical and lumbar spinal cords. Capillary permeability in the spinal cords was determined by Evans Blue (EB) injection. Results showed significant restoration of ultrastructural capillary morphology, improvement of basement membrane integrity, enhancement of axonal myelin coherence, and stabilization of capillary density in the spinal cords primarily of ALS mice receiving the high dose of 1 × 106 cells. Moreover, substantial reduction of parenchymal EB levels was determined in these mice, confirming our previous results on capillary permeability. Additionally, transplanted cells were detected in blood smears of sacrificed late symptomatic mice by HuNu marker. Altogether, these results provide novel evidence that unmodified bone marrow hematopoietic stem cell treatment at optimal dose might be beneficial for structural and functional repair of the damaged BSCB in advanced stage of ALS, potentially resulting in delayed disease progression by increased motor neuron survival.