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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Brain Res Bull. 2021 Feb 2;170:22–28. doi: 10.1016/j.brainresbull.2021.01.020

Detection of Endothelial Cell-Associated Human DNA Reveals Transplanted Human Bone Marrow Stem Cell Engraftment into CNS Capillaries of ALS Mice

Svitlana Garbuzova-Davis 1,2,3,4,*, Kayla J Boccio 1, Jared Ehrhart 1, Paul R Sanberg 1,2,4,5, Stanley H Appel 6, Cesario V Borlongan 1,2
PMCID: PMC7990719  NIHMSID: NIHMS1672392  PMID: 33545308

Abstract

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 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 EC 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.

Keywords: ALS, G93A SOD1 mice, blood-CNS-barrier, human bone marrow-derived stem cells, repair, endothelial cells, real-time PCR, LIVE/DEAD viability/cytotoxicity assay

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron dysfunction throughout the neural axis (Cleveland and Rothstein, 2001; Kiernan et al., 2011; Lomen-Hoerth, 2008; Strong et al., 2005; Talbot, 2009). Rapid progression of debilitating symptoms leads to paralysis and fatality within 3-5 years of diagnosis. Motor neuron death with subsequent muscle weakness is a hallmark pathology of ALS. Despite intense scientific efforts, treatment development is complicated by diffuse motor neuron death and by a complex of factors underlying disease pathogenesis (Garbuzova-Davis et al., 2016; Martin et al., 2000; Rothstein, 2009; Wijesekera and Leigh, 2009).

Numerous reports have demonstrated impairment of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in ALS, potentially representing an additional pathogenic disease mechanism. We (Garbuzova-Davis et al., 2012, 2007a, 2007b; Garbuzova-Davis and Sanberg, 2014) and others (Henkel et al., 2009; Kakaroubas et al., 2019; Miyazaki et al., 2011; Nicaise et al., 2009; Sasaki, 2015; Winkler et al., 2013; Zhong et al., 2008) showed structural and functional BBB/BSCB alterations in animal models of disease and in ALS patients. Degeneration of endothelial cells (ECs) and astrocyte end-feet processes, reduced pericyte capillary coverage, impairment of endothelial transport system, and dysfunction of tight junction proteins have been shown to compromise barrier integrity. Capillary leakage and microhemorrhages were also noted. The dysfunctional BBB/BSCB may allow detrimental factors from the systemic circulation to penetrate the central nervous system (CNS) and foster motor neuron degeneration. Since CNS vascular homeostasis is not effectively sustained in ALS due to the damaged capillary endothelium, restoring the altered barrier via cell administration may be a new therapeutic approach for this devastating neurovascular disease (Garbuzova-Davis et al., 2011; Rodrigues et al., 2012).

Our research has focused on repair of the altered endothelium in ALS by intravenous (iv) stem cell delivery into symptomatic G93A SOD1 mutant mice, mimicking a clinical approach. In initial studies, different doses of human bone marrow-derived CD34+ cells (hBM34+) were iv administered into ALS mice at 13 weeks of age. Results showed benefits of transplanted cells (primarily from highest dose of 1×106 cells/mouse) on restoration of CNS barrier competence and decreases in disease-related effectors, leading to enhanced motor neuron survival (Eve et al., 2018; Garbuzova-Davis et al., 2017, 2018b). However, a delayed effect on motor function and still severely damaged capillaries were observed in post-transplanted ALS mice. Addressing these limitations, administration of restricted-endothelial cell lineage may provide more benefits in barrier restoration. In our follow-up study, human bone marrow-derived endothelial progenitor cells (hBM-EPCs, 1×106 cells/mouse) were iv transplanted into symptomatic G93A SOD1 mutants. Transplanted cells significantly improved behavioral disease outcomes leading to delayed disease progression (Garbuzova-Davis et al., 2019b). This benefit likely occurred due to widespread distribution of transplanted cells and their engraftment into capillary lumen within the spinal cord and brain, resulting in substantially restored capillary ultrastructure, decreased capillary permeability, and enhanced motor neuron survival. These results demonstrated that systemic administration of hBM-EPCs vs. hBM34+ cells effectively restores the compromised barrier integrity in the CNS, potentially by replacement of damaged ECs, leading to microvascular repair in ALS.

However, transplanted hBM34+ cells or hBM-EPCs engraftment into vascular wall in ALS mouse brains and spinal cords was evaluated via immunohistochemistry using anti-human Von Willebrand Factor (vWF) (Garbuzova-Davis et al., 2017, 2019b). These data provided imperative information regarding potential replacement of injured ECs by “heathy” introduced human cells. Though, additional study is needed to confirm transplanted human cell engraftment within capillaries in mouse CNS tissues.

The aim of this study was to determine the presence of human DNA within microvascular endothelial cells isolated from the CNS tissues of G93A SOD1 mutant mice treated with human bone marrow stem cells. A specific focus was examining the viability of isolated ECs in cell-treated animals.

MATERIALS AND METHODS

Animals

All animals were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and used under standardized ethic procedures approved by the Institutional Animal Care and Use Committee at USF and conducted in compliance with the Guide for the Care and Use of Laboratory Animals as described (Garbuzova-Davis et al., 2017, 2018b, 2019b). In this study, fifteen transgenic male B6SJL-Tg(SOD1*G93A)1Gur/J mice, over-expressing human SOD1 carrying the Gly93⟶Ala mutation (G93A SOD1), were randomly selected from previous studies (Garbuzova-Davis et al., 2018b, 2019b) receiving human bone marrow stem cells or media at 13 weeks of age: Group 1 - hBM34+ cells (1×106 cells/mouse, n=5), Group 2 - hBM-EPCs (1×106 cells/mouse, n=5), and Group 3 - Media (n=5). G93A SOD1 mutant mice intravenously (iv, jugular vein) transplanted with either hBM34+ cells, hBM-EPCs, or an equal volume of media. A non-transplant control group (Group 4), consisted of mice from the background strain that did not carry the mutant SOD1 gene (control, n=5).

Cell preparation, transplant procedure, and perfusion

Cryopreserved human bone marrow CD34+ cells (hBM34+) and human bone marrow-derived endothelial progenitor cells (hBM-EPCs) were purchased from AllCells (Alameda, CA, USA) and CELPROGEN (Torrance, CA, USA), respectively. According to the company reports, cells were obtained from healthy adult donors and tested negative for various viruses and microbial growth using an infectious disease panel.

We previously detailed preparation of hBM34+ cells or hBM-EPCs for transplantation (Garbuzova-Davis et al., 2017, 2018b, 2019b). Briefly, cells were thawed rapidly at 37°C and then transferred into a centrifuge tube containing 12 mL of Dulbecco’s Phosphate Buffered Saline 1X (DPBS), pH 7.4 (Mediatech, Inc., Manassas, VA, USA). The cells were centrifuged (hBM34+: 200 g/10 min, hBM-EPCs: 100 g/7 min) at room temperature (RT), the supernatant discarded, and the process repeated. After the final wash, cell viability was assessed using the 0.4% trypan blue dye exclusion method before transplantation. Cell concentration was adjusted to 5,000 cells/μ1 (1×106 cells/200 μL/injection).

The hBM34+ cells or hBM-EPCs were delivered intravenously via the jugular vein of mice under anesthesia with isofluorane (2-5% at 2L O2/min) as we previously described (Garbuzova-Davis et al., 2017, 2018b, 2019b). The Media mice in Group 3 received 200 μL of DPBS, the same volume administered to the cell-transplanted mice. Animals in Groups 1-3 received cyclosporine A (CsA, 10 mg/kg ip) daily for the entire post-transplant period.

All cell-treated, media-treated, and control mice were sacrificed under Euthasol® (0.22 ml/kg body weight) and perfused transcardially with 0.1 M phosphate buffer (PB, pH 7.2) at 17 weeks of age. The brains and spinal cords were removed for further isolation of microvascular endothelial cells followed by detection of human DNA, immunohistochemical, and isolated EC viability analyses.

Isolation of microvascular endothelial cells from mouse CNS tissues

The isolation of mouse brain and spinal cord microvascular endothelial cells (ECs) procedure was performed as described (Ge and Pachter, 2006; Song and Pachter, 2003). Briefly, immediately after mouse perfusion, the brain and spinal cord tissues were immersed in ice-cold isolated buffer Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12, Cat. No. D8437, Millipore Sigma, Burlington, MA, USA) (Gibco™, Cat. No. 10-565-042, Fisher Scientific, Waltham, MA, USA) with 1% antibiotic-antimycotic (Gibco™, Cat. No. 15-240-062, Fisher Scientific, Waltham, MA, USA) solution and diced into small pieces (~ 1 mm). After rinsing mixed tissues with fresh isolated buffer, tissues were homogenized in a 7 mL Dounce tissue grinder (DWK Life Sciences Kimble™ Kontes™, Cat. No. K885300-0007, Fisher Scientific, Waltham, MA, USA) in isolation buffer with 20 strokes of larger clearance pestle (0.071 – 0.119 mm) and then with 20 strokes of smaller clearance pestle (0.02 – 0.056 mm). Homogenates were collected in a 15-mL tube and centrifuged at 200 × g for 5 min at RT. Supernatant was removed and pellet was resuspended in 14 mL of 18% dextran solution (Alfa Aesar™, MW 75,000, Cat. No. AAJ6098922, Fisher Scientific, Waltham, MA, USA) in DMEM-F-12 followed by centrifugation at 15,000 × g for 15 min at 4°C. After removing foamy myelin layer and dextran supernatant, collected pellet was resuspended in Hanks Balanced Salt Solution (HBSS, Alfa Aesar™, Cat. No. AAJ67807K2, Fisher Scientific, Waltham, MA, USA) and centrifuged again. The centrifugation steps were repeated two times and collected pellet then transferred in a 15-mL tube containing HBSS to wash out remaining dextran. After centrifugation at 1000 × g for 4 min at RT, pellet was filtered with a 250 μm cell strainer into a 15-mL tube and cell strainer rinsed 3 times with HBSS. Microvessel pellet was resuspended in HBSS and finally centrifuged at the same speed/duration at RT. Supernatant was removed and pellet was collected.

Next, collected pellet was resuspended with 5 mL of collagenase/dispase solution (Cat. No. NC0953723, Fisher Scientific, Waltham, MA, USA) for digesting microvessels. Suspension was placed in a 6-well plate and incubated for 60 min at 37°C. ECs were frequently observed being released from the large majority of vessel fragments during digestion and images were obtained using an inverted Olympus IX71 microscope. Microvessel suspension was then collected in a 15-mL tube and centrifuged at 1000 × g for 5 min at RT. After removing supernatant, ECs pellet was resuspended in pre-cooled MACS separation buffer solution (Cat. No. 130-091-376, Miltenyi Biotec, Auburn, CA, USA) and centrifuged again at the same speed/duration. Supernatant was removed and resuspended pellet was placed in 1.5 mL microcentrifuge tube with 90 ×L of pre-cooled MACS buffer. Then, cell pellet was incubated with rabbit anti-CD31 primary antibody (1:10, Cat. No. ab28364, Abcam, Cambridge, MA, USA) for 30 min at RT. Next, cells were washed in pre-cooled MACS buffer and centrifuged again at 1000 × g for 5 min at RT. Washing and centrifugation steps were repeated. Collected cell pellet was incubated in pre-cooled MACS buffer with anti-rabbit IgG microbeads (1:5, Cat. No. 130-048-602, Miltenyi Biotec, Auburn, CA, USA) for 15 min at 4°C using constant rotation. Pellet samples were prepared for EC isolation.

Isolation of ECs was performed by immunomagnetic cell sorting using a Mini-MACS kit (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s protocol. Briefly, MS column (Cat. No. 130-042-201, Miltenyi Biotec, Auburn, CA, USA) was inserted in MiniMACS Separator (Cat. No. 130-042-102, Miltenyi Biotec, Auburn, CA, USA), attached to MACS MiltiStand (Cat. No. 130-042-303, Miltenyi Biotec, Auburn, CA), and a collection tube was placed under column. After rinsing MS column with 500 μL of MACS rinsing solution (Cat. No. 130-091-222, Miltenyi Biotec, Auburn, CA), cells were resuspended in 500 μL of MACS buffer solution and processed through 40 μm cell strainer into MS column. The unlabeled cell fraction was collected by washing MS column and cell strainer with 500 μL of MACS buffer solution 3 times and suspension was discarded. After the final wash, MS column was removed from the separator and placed into 1.5 mL tube. Magnetically labeled cells were flushed by adding MACS buffer solution onto MS column. Cell suspension was centrifuged at 1000 × g for 5 min at RT and supernatant removed. Purified ECs were washed in DMEM/F-12 and centrifuged again at the same speed/duration and cell pellet was resuspended in DMEM/F-12. The isolated ECs number was assessed using the 0.4% trypan blue dye exclusion method for each mouse.

Immunocytochemistry

To verify that isolated ECs from mouse CNS tissue displayed endothelial cell phenotype, immunocytochemical staining for CD105 (endoglin) was performed as we previously described (Garbuzova-Davis et al., 2019a). Briefly, randomly selected isolated ECs from each mouse group were cultured on Ibidi USA U-slide 18 well flat collagen pre-coated culture slide (Cat. No. 81826, Ibidi, Fitchburg, WI, USA) at concentration of 2,500 cell/well in 30 μL of DMEM/F-12/10% FBS/1% antibiotic-antimycotic. After 24 hours of incubation at 37°C, cells were fixed by 4% paraformaldehyde in DPBS solution. The rabbit polyclonal primary antibody CD105 (1:200, Cat No. ab107595, Abcam, Cambridge, MA, USA) was applied into culture slides after pre-incubation in blocking solution (10% normal goat serum/3% Triton 100X/DPBS) for 60 min at RT. After incubation overnight at 4°C, cells were rinsed in DPBS and incubated with goat anti-rabbit secondary antibody conjugated to fluorescein isothiocyanate (FITC, 1:500, Cat. No. A11008, Molecular Probes, Eugene, OR, USA) for 2 hours at RT. Then, cell slides were washed with DPBS followed by adding Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA). Immunoexpressions of CD105 in cell cultures were examined under an epifluorescent inverted Olympus IX71 microscope.

Real-time PCR for detection of human DNA in isolated ECs from mouse CNS tissue

Total genomic DNA was collected from isolated ECs in microvessels obtained from the CNS (brains and spinal cords) tissues of cell-treated, media-treated, and control mice. The DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Cat. No. 69504, Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. Briefly, cells pellets were digested with proteinase K and the DNA was isolated by centrifugation at 6000 × g for 1 min at RT using the DNeasy Mini spin column. Following the final elution, the total DNA concentration was determined using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) at an absorbance of A260/A280 to assess quality and purity of the isolated DNA. Samples were then diluted with nuclease-free water (Cat. No. FEREN0521, Thermo Scientific, Waltham MA, USA) to normalize the DNA concentration of each sample to 5 ng/μL in preparation for real-time PCR (RT-PCR) analysis. Detection of human DNA was determined in duplicate using a standard SYBR™ Green PCR Master Mix kit (Cat. No. 4385610, Thermo Scientific, Waltham, MA, USA) with primers specific for the human Arthrobacter luteus (Alu) repeat sequences using forward PCR primer (5’-CATGGTGAAACCCCGTCTCTA-3’) and reverse PCR primer (5’-GCCTCAGCCTCCCGAGTAG-3’). Additionally, mouse DNA was detected with primers specific for the murine Y chromosome using forward PCR primer (5’-TTTTGCCTCCCATAGTAGTATTTCCT-3’) and reverse primer (5’-TGTACCGCTCTGCCAACCA-3’) as described (McBride et al., 2003). All primers were purchased from Thermo Scientific (Cat. No. A15610, Custom Value DNA Oligos, Waltham MA, USA). Validation of primers was performed using pure hBM-EPCs and hBM34+ cells. The PCR was performed using the Applied Biosystems StepOnePlus RT-PCR system (Cat. No. 4376373, Thermo Scientific, Waltham MA, USA). RT-PCR assay for detection of the human Alu-sequences and murine Y chromosome DNA was performed in a MicroAmp Fast Optical 96-Well PCR Reaction Plate (Cat. No. 4346906, Thermo Scientific, Waltham MA, USA) in a 30 μL total volume consisting of 15 μL SYBR Green Mix, 1μl (150 nM final concentration) of each forward and reverse human Alu or murine Y chromosome primers, 5μL DNA template previously normalized to 5 ng/μL, and 8 μL nuclease-free water. All reagents were mixed in tubes on ice under a biosafety hood to prevent airborne contamination. Tubes were then vortexed for 30 sec to mix reagents and centrifuged at 6000 × g for 1 min at RT to remove air bubbles. Mixture collected from the bottom of tubes was added to a 96-well PCR plate and then the wells were sealed with optically transparent adhesive film (Cat. No. 4360954, Thermo Scientific, Waltham MA, USA). The plates were placed in the PCR system and the reaction was performed by denaturation at 95° for 3 min followed by 40 cycles of amplification at 95°C for 3 sec, and 60°C for 30 sec. Results were analyzed with the StepOnePlus™ software (version 2.3).

Isolated EC viability assay

Viability of isolated ECs from CNS tissue of cell-treated, non-treated G93A SOD1 mutant mice, and controls was determined using the LIVE/DEAD viability/cytotoxicity kit (Cat. No. L3224, Fisher Scientific, Waltham, MA, USA) as we previously described (Ehrhart et al., 2018). Briefly, cells were plated on Ibidi USA U-slide 18 well flat collagen pre-coated culture slide (Cat. No. 81826, Ibidi, Fitchburg, WI, USA) at concentration of 2,500 cell/well in 30 μL of DMEM/F-12/10% FBS/1% antibiotic-antimycotic in duplicate. Cell culture slides were incubated at 37°C for 24 hours. Next day, culture media was removed, and cells were washed with DPBS twice in each well. The combined LIVE/DEAD assay reagents (30 μL) were added to each well and incubated at RT for 30 min. After incubation, randomly selected images (n=5/well, totaling n=10/mouse) were obtained at 20X magnification for cell quantification using the epifluorescent inverted Olympus IX71 microscope. Live cells were labeled with green fluorescence through the conversion of non-fluorescent cell-permanent calcein acetoxymethyl to intensely fluorescent calcein by ubiquitous intracellular esterase enzyme activity. Dead cells were identified using ethidium homo dimer-1 (EthD-1), which enters cells through damaged membranes and produces a red fluorescence upon binding to nucleic acids. Cell counts of live (green) and dead (red) cells were determined using NIH ImageJ software (version 1.46).

Statistical analysis

Data are presented as means ± S.E.M. One-way ANOVA with post-hoc Tukey HSD (Honesty Significant Difference) multiple comparison test using online statistical software (astatsa.com, 2016 Navendu Vasavada) was performed for statistical analysis. Significance was defined as p < 0.05.

RESULTS

Characteristic of isolated endothelial cells from mouse CNS tissue

Microvascular endothelial cells (ECs) were isolated from the CNS (brain and spinal cord) tissues of G93A SOD1 mutant mice treated with hBM34+ cells or hBM-EPCs and compared to media-injected or control animals. Isolation of microvascular ECs was performed in mice at 17 weeks of age (4 weeks post-transplant). The multi-step process demonstrated appropriate degrading of microvessels by dextran (Figure 1A) and releasing of ECs from digested microvessels by collagenase/dispase enzyme (Figure 1B). Similar numbers of ECs were isolated from each animal group: controls - 60.76 × 103 ± 5.02 × 103, media-treated - 58.08 × 103 ± 8.46 × 103, hBM34+ - 56.08 × 103 ± 9.03 × 103, and hBM-EPCs - 59.76 × 103 ± 7.49 × 103 cells. Verification of isolated ECs was conducted via immunocytochemical staining of cultured cells for CD105 (endoglin). Results showed positive CD105 immunoexpressions in cells (Figure 1C, 1D), indicating proper isolation of microvascular ECs and endothelial cell phenotype.

Figure 1. Microvascular ECs isolated from mouse CNS tissue.

Figure 1.

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(A) Phase-contrast image shows freshly isolated microvessel (arrows) from tissue after incubation with dextran solution. (B) ECs (arrows) released from microvessel were apparent after treatment with collagenase/dispase enzyme. (C) Phase-contrast image shows fixed ECs (arrow) isolated from microvessels after culture for 24 hrs. (D) Cultured ECs immunoexpressed CD105 (green, arrow), indicating EC phenotype. Cell nucleus is indicated by DAPI (blue) staining. Scale bar in A-D is 50 μm.

Real-time PCR analysis

Real-time PCR (RT-PCR) was used to detect human DNA in ECs isolated from the CNS tissues of mice treated with hBM34+ cells or hBM-EPCs. RT-PCR reactions were based on amplification of human-specific Alu sequences. Total DNA from isolated ECs was extracted, quantified, and the concentration normalized between all samples. Validation of the primers was initially performed by using human DNA isolated from pure hBM34+ cells and hBM-EPCs. Results showed positive amplification curves of human DNA in hBM34+ cell (orange trace) and hBM-EPC (blue trace) samples indicating values above cycle threshold (Ct) and are referred to as positive controls for the human Alu primer pair (Supplemental Figure 1). Below Ct, samples containing only mouse DNA (green trace) did not result in amplification and are referred to as negative controls. No RT-PCR products were observed from human DNA samples using the murine Y chromosome primer pair. Also, using mouse DNA resulted in no amplification products from the human Alu primer pair, showing specificity of primers to human DNA.

After validation of the primers, following extraction of genomic DNA from isolated ECs of CNS microvessels in mice treated with either 1 × 106 hBM34+ cells or hBM-EPCs, RT-PCR was performed to detect the presence of human DNA. Results demonstrated positive amplification of human DNA present in samples crossing above Ct, while samples negative for human DNA from media and control mice consistently remained below Ct (Figure 2A). Human DNA was detected in PCR products with Ct values between 15 and 30 amplification cycles, indicating possible variability of the transplanted human cells engraftment into the capillary wall of cell-treated mice. A potentially greater presence of human DNA validated by amplification cycle was observed in ECs isolated from microvasculature of mice receiving hBM-EPCs (15-18 amplification cycles) vs. hBM34+ cells (22-29 amplification cycles). Analysis of amplification data showed significantly (p < 0.01) lower average Ct value in ECs isolated from hBM-EPCs (16.20 ± 0.73) vs. hBM34+ cell (25.80 ± 1.53) treated ALS mice, indicating greater presence of human DNA in microvascular cells of mice treated with hBM-EPCs (Figure 2B, 2C).

Figure 2. Detection of human DNA in isolated microvascular endothelial cells.

Figure 2.

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(A) RT-PCR amplification plot shows positive reactions (above Ct), indicating the presence of human DNA from isolated ECs of mice treated with hBM34+ cells or hBM-EPCs. A greater concentration of human DNA was observed in isolated ECs from microvasculature of mice receiving hBM-EPCs (between 15 and 18 amplification cycles) vs. hBM34+ cells (between 22 and 29 amplification cycles). Samples negative for human DNA, containing primarily mouse DNA, were shown below Ct. (B) Significantly lower Ct value was detected in ECs isolated from hBM-EPCs vs. hBM34+ cell treated ALS mice. (C) Ct data on a Log scale showed the same significant differences between cell-treated mice. ** p < 0.01.

Viability of isolated endothelial cells from mouse CNS tissue

Viability of isolated ECs was determined in a separate set of cultured cells using the LIVE/DEAD viability/cytotoxicity assay. Results showed numerous viable ECs (85.81 ± 1.88 cells) obtained from control mice (Figure 3Aa, 3B). Significant (p < 0.01) reduction of live cells (59.33 ± 0.87 cells) was detected in media-injected animals vs. controls (Figure 3Ab, 3B). ECs isolated from ALS mice treated with hBM34+ cells or hBM-EPCs demonstrated significant (p < 0.01) increases of viable cells (72.61 ± 1.01 and 79.92 ± 2.63 cells, respectively) compared to media-injected animals (Figure 3Ac, 3Ad, 3B). Importantly, significantly (p < 0.05) higher numbers of live cells were determined in mice with hBM-EPC vs. hBM34+ cell treatment. Specifically, ECs isolated from hBM-EPC-treated mice had a greater ratio of live to dead cells (4.60:1) compared to cells obtained from hBM34+ cell treatment (2.67:1, p < 0.05) or media-injected animals (1.46:1, p < 0.01) (Figure 3C).

Figure 3. Viability of isolated endothelial cells in vitro.

Figure 3.

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(A) Fluorescent microscopy images demonstrate numerous viable (green, asterisks) ECs isolated from control (Aa) vs. media (Ab) mice. More dead ECs (red, arrowheads) were detected in media-treated mice. Isolated ECs from mice treated with hBM34+ cells (Ac) or hBM-EPCs (Ad) showed substantial increases of live cells (green, asterisks) and reduced numbers of dead (red, arrowheads). Scale bar in Aa-Ad is 50 μm. (B) Percentage of live ECs was significantly higher in hBM-EPC-treated mice vs. hBM34+ cell treatment or media-injected animals. (C) ECs isolated from hBM-EPC-treated mice had a greater live (green)/dead (red) cell ratio compared to media-treated or hBM34+ cell-treated mice. *p < 0.05, **p < 0.01.

DISCUSSION

The current study evaluated the presence of human DNA within isolated microvascular ECs from the CNS tissues of G93A SOD1 mutant mice treated with hBM34+ cells or hBM-EPCs at the symptomatic disease stage. Additionally, viability of isolated ECs from cell-treated and non-treated animals was examined. The major study findings revealed that isolated microvascular ECs: (1) were appropriately obtained from mouse CNS tissue and immunoexpressed endothelial cell marker; (2) exhibited human DNA in cell-treated mice; (3) demonstrated greater human DNA concentrations in mice receiving hBM-EPCs vs. hBM34+ cells; and (4) showed higher numbers of live cells in mice treated with hBM-EPCs vs. hBM34+ cells or media-injection. These study results reveal that iv transplanted human cells engrafted into capillary walls within mouse CNS tissue with superior grafting ability of hBM-EPCs transplanted into symptomatic ALS mice.

However, the discrepancy between the presence of human DNA in ECs isolated from CNS tissues of mice receiving hBM-EPCs or hBM34+ cells at the same cell dose needs further discussion. It is our suggestion that the two types of human bone marrow-derived cells may have different engrafting potentials towards replacing damaged ECs with “heathy” transplanted human cells. Although transplanted hBM34+ cells were found in vivo to differentiate into ECs and engraft within the capillary lumen in the spinal cord of ALS mice (Garbuzova-Davis et al., 2017), some administered cells were observed at a distance from blood vessels. In contrast, transplanted hBM-EPCs predominantly engrafted into CNS capillaries in ALS mice; no cells were identified outside of microvessels (Garbuzova-Davis et al., 2019b). Thus, hBM-EPCs provided superior re-establishment of barrier integrity in ALS vs. hBM34+ cell transplants likely due to these cells’ rapid differentiation and engraftment capability, leading to replacement of damaged ECs in mouse CNS vessels. This microvascular “homing” of transplanted hBM-EPCs on the luminal capillary surface may have resulted in greater human DNA concentrations (indicating more cells of human origin) in ECs isolated from CNS tissues of mice receiving hBM-EPCs vs. hBM34+ cells. However, one limitation of this study is that ECs were isolated from combined brain and spinal cord tissues to yield sufficient cell numbers for multiple tests. Determining human DNA in microvascular ECs isolated separately from the brains and spinal cords of mice may more accurately characterize transplanted cell engraftment and biodistribution in regard to repair of damaged BBB and/or BSCB in ALS. This study will be addressed in a future investigation.

Another study finding was significant increase of cell viability in isolated ECs from ALS mice treated with hBM34+ cells or hBM-EPCs compared to media-injected animals using LIVE/DEAD viability/cytotoxicity assay. Moreover, higher live cell numbers and live/dead cell ratios were identified in ECs isolated from mice receiving hBM-EPC vs. hBM34+ cell transplant. These data supported the ability of transplanted cells, mainly of hBM-EPCs, to maintain “healthy” vascular homeostasis in ALS. However, significant reduction of isolated ECs viability in media-treated ALS mice vs. controls requires specific attention. We previously demonstrated ECs degeneration in the brain and spinal cord in both G93A SOD1 mutant mice and sporadic ALS patients (Garbuzova-Davis et al., 2012, 2007a, 2007b) leading to CNS barrier impairment. Although the mechanism(s) of ECs damage is still unknown, unfavorable humoral factors in ALS peripheral blood may be an impediment to these cells’ survival. Increased levels of pro-inflammatory (IL-1β, IL-1α, IL-12, and TNF-α) vs. anti-inflammatory cytokines (IL-4, IL-6, IL-10) in blood plasma from G93A SOD1 mutant mice at all disease stages have been reported (Jeyachandran et al., 2015; Moreno-Martínez et al., 2019). In ALS patients, significantly increased levels of peripheral blood inflammatory cytokines such as TNF-α, IL-6, and IL-1β were also noted (Ehrhart et al., 2015; Hu et al., 2017; Lam et al., 2016). Moreover, the potential role of humoral IL-6 cytokine in mediating EC inflammation via the trans-signaling pathway has been discussed (Garbuzova-Davis et al., 2018a). These up-regulated inflammatory cytokines, circulating in blood, may affect not only the integrity of endogenous ECs, but also the viability of systemically transplanted cells. Vulnerability of ECs to various harmful blood substances, however, needs to be determined. Although our data showed significant viability increase of isolated ECs from cell-treated vs. media-treated ALS mice, elucidating content of pro- and anti-inflammatory cytokines within these cells is imperative. This study is currently in progress.

In summary, our study results demonstrated the presence of human DNA in ECs isolated from the CNS tissues of G93A SOD1 mutant mice treated with hBM34+ cells or hBM-EPCs, revealing a greater engraftment capability of transplanted hBM-EPCs into capillary walls. Also, a significant increase of cell viability was determined in isolated ECs from cell-treated ALS mice compared to media-injected animals. However, a higher cell viability was identified in isolated ECs from mice receiving hBM-EPC vs. hBM34+ cell transplant. These study results confirm that intravenous administration of hBM-EPCs into symptomatic ALS mice was more beneficial than hBM34+ cell treatment in terms of cell engraftment to the CNS capillary wall. Based on these data, hBM-EPCs may be advanced into clinical applications as a cell-specific approach for ALS therapy towards restoration of CNS barrier integrity.

Supplementary Material

1

Highlights.

The major study findings revealed that isolated microvascular ECs from mouse CNS tissue:

  • • Were appropriately obtained and immunoexpressed endothelial cell marker

  • • Exhibited human DNA in cell-treated mice

  • • Demonstrated greater human DNA concentrations in mice receiving hBM-EPCs vs. hBM34+ cells

  • • Showed higher numbers of live cells in mice treated with hBM-EPCs vs. hBM34+ cells or media-injection

Acknowledgments

This study was supported by the NIH, NINDS (1R01NS090962) grant.

Footnotes

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Declarations of Interest: none

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