Abstract
Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset motor neuron disease causing a progressive, rapid and irreversible degeneration of motor neurons in the cortex, brain stem and spinal cord. No effective treatment is available and cell therapy clinical trials are currently being tested in ALS affected patients. It is well known that in ALS patients, approximately 50% of pericytes from the spinal cord barrier are lost. In the central nervous system, pericytes act in the formation and maintenance of the blood-brain barrier, a natural defense that slows the progression of symptoms in neurodegenerative diseases. Here we evaluated, for the first time, the therapeutic effect of human pericytes in vivo in SOD1 mice and in vitro in motor neurons and other neuronal cells derived from one ALS patient. Pericytes and mesenchymal stromal cells (MSCs) were derived from the same adipose tissue sample and were administered to SOD1 mice intraperitoneally. The effect of the two treatments was compared. Treatment with pericytes extended significantly animals survival in SOD1 males, but not in females that usually have a milder phenotype with higher survival rates. No significant differences were observed in the survival of mice treated with MSCs. Gene expression analysis in brain and spinal cord of end-stage animals showed that treatment with pericytes can stimulate the host antioxidant system. Additionally, pericytes induced the expression of SOD1 and CAT in motor neurons and other neuronal cells derived from one ALS patient carrying a mutation in FUS. Overall, treatment with pericytes was more effective than treatment with MSCs. Our results encourage further investigations and suggest that pericytes may be a good option for ALS treatment in the future.
Abbreviations
- ACTB:
-
Beta Actin
- ALS:
-
Amyotrophic Lateral Sclerosis
- basic FGF:
-
Basic fibroblast; growth factor
- BBB:
-
Blood Brain Barrier
- BNDF:
-
Brain-derived Neurotrophic Factor
- BSA:
-
Bovine serum albumin
- CAT:
-
catalase
- CD11b:
-
cluster of differentiation; molecule 11B
- CFDA-SE:
-
carboxyfluorescein diacetate succinimidyl ester
- CHAT:
-
Choline acetyltransferase
- c-Myc:
-
Proto-oncogene c-Myc
- DAPI:
-
4′,6-Diamidino-2-phenylindole dihydrochloride
- DMEM:
-
Dulbecco’s Modified Eagle Medium
- EB:
-
Embryoid bodies
- EBM:
-
Endothelial Cell Growth Medium
- EGF:
-
Epidermal growth factor
- FBS:
-
Fetal Bovine Serum
- FDA:
-
Food and Drug Administration
- FGF:
-
Fibroblast growth factors
- GAPDH:
-
Glyceraldehyde 3-phosphate dehydrogenase
- G-CSF:
-
Granulocyte colony-stimulating factor
- GDNF:
-
glial cell derived neurotrophic factor
- GFAP:
-
Glial fibrillary acidic protein
- GLP-1:
-
Glucagon-like Peptide 1
- GM-CSF:
-
Granulocyte-macrophage colony-stimulating factor
- GPX:
-
glutathione peroxidase
- GSR:
-
Glutathione reductase
- HBSS:
-
Hanks’ Balanced Salt Solution
- IFN-g:
-
Interferon gamma
- IGF-1:
-
Insulin-like growth factor 1
- IL-10:
-
Interleukin 10
- IL-12p70:
-
Interleukin 12p70
- IL-13:
-
Interleukin 13
- IL-15:
-
Interleukin 15
- IL-17A:
-
Interleukin 17A
- IL-1ra:
-
Interleukin 1ra
- IL-1β:
-
Interleukin 1β
- IL-2:
-
Interleukin
- IL-4:
-
Interleukin 4
- IL-5:
-
Interleukin 5
- IL-6:
-
Interleukin 6
- IL-7:
-
Interleukin 7
- IL-8:
-
Interleukin 8
- IL-9:
-
Interleukin 9
- IP-10:
-
C-X-C motif chemokine 10
- IPSC:
-
Induced Pluripotent Stem Cells
- Klf4:
-
Kruppel-like factor 4
- MCP-1:
-
Monocyte chemoattractant protein-1
- MIP-1α:
-
Macrophage Inflammatory Proteins −1α
- MIP-1β:
-
Macrophage Inflammatory Proteins -1 β
- MND:
-
Motor neuron diseases
- MNX1/HB9:
-
Motor neuron and pancreas homeobox 1/Homeobox HB9
- MSCs:
-
Mesenchymal stromal cells
- NB media:
-
Neuro Basal Media
- NF-kB:
-
factor nuclear kappa B
- NGF:
-
Nerve Growth Factor
- NPCs:
-
neural progenitor cells
- NSCs:
-
Neural Stem Cells
- Oct4:
-
octamer-binding transcription factor 4
- PaGE:
-
paw grip endurance
- PBS:
-
Phosphate-buffered saline
- PDGF –BB:
-
Platelet-derived growth factor C
- RANTES:
-
Regulated on Activation, Normal T Cell Expressed and Secreted
- Ri:
-
ROCK inhibitor
- RT-qPCR:
-
Real Time Quantitative Polymerase Chain Reaction
- SHH:
-
Sonic Hedgehog
- SOD1:
-
superoxide dismutase 1
- Sox2:
-
SRY (sex determining region Y)-box 2
- TNF-α:
-
Tumor necrosis fator α
- VEGF:
-
Vascular endothelial growth factor
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This work was supported by CEPID/FAPESP, INCT and CNPq.
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Figure S1
Cell lines characterization performed in the 8th passage, the same used for in vivo experiments. (A) In vitro differentiation potential of MSCs and pericytes into adipocytes, osteoblasts and chondroblasts. (B) Immunophenotypic profile of MSCs and pericytes after 8 passages in culture, assessed by flow cytometry. (C) Multiplex ligation-dependent probe amplification (MLPA) analysis using SALSA P070 and SALSA P036 subtelomeric probes, evaluated in MSCs and pericytes. (PDF 3905 kb)
Figure S2
Macrophage phagocytosis assay. (A, C) Anti-human lamin A/C (green) immunostaining of human cells (MSCs, or pericytes, respectively) co-cultured with macrophages (unlabeled, obtained from one SOD1 female). (B, D) Anti-human lamin A/C (green) immunostaining of human cells (MSCs, or pericytes, respectively) without macrophage co-culture. (E) DAPI nuclei staining in macrophages cultured alone. Magnification 200X. (F) positive control showing India Ink internalization by macrophages, 400X magnification. (H) Graphical representation of human MSCs and pericytes quantification with or without co-culture with macrophages (p = 0.3024 and p = 0.0906, respectively). The experiment was carried out in triplicate. (PDF 19208 kb)
Figure S3
Dose-dependent immunosuppressive potential of MSCs and pericytes in CFDA-labeled SOD1 mice PBMCs and lymphocytes, after 5 days of co-culture with increasing doses of MSCs and pericytes. Bars represent the mean fluorescence of peak area of CFDA-labeled activated PBMCs and lymphocytes (obtained from one SOD1 female), evaluated by flow cytometry after the co-culture period. Increasing concentrations of human cells and a fixed amount of 1.104 activated PBMCs and lymphocytes were used. Activated PBMCs and lymphocytes cultured alone were used as a control. No differences were found between MSCs and pericytes. ***p < 0.001. The experiment was carried out in triplicate. (PDF 475 kb)
Figure S4
Individual and average smoothed profiles (bold curve) of the longitudinal data relating to weight (A), motor score (B), rotarod (C) e PaGE (D). n = 22 mice per group, being 11 males and 11 females. (PDF 192 kb)
Figure S5
Cytokine quantification by the 27-human cytokine plex (luminex) in culture media of co-cultured ALS patient derived motor neurons and other neuronal cells (NMs) with MSCs or pericytes. Only differentially secreted cytokines were represented. IL-2 IL-4 IL-5, IL-7 IL-9 IL-10, MIP-1a, were not detected in this assay and levels of basic-FGF, Eotaxin, GM-CSF, IFN-γ, IL-1β, IL-12p70, IL-13, IL-17a, MP-1β and PDGF-bb were not altered in comparison to the control (MNNs alone). *p < 0.05 **p < 0.01. For neuronal differentiation, iPSCs from one ALS patient was used. The experiment was carried out in four replicas. (PDF 736 kb)
Figure S6
Validation of iPSC derived motor neurons and other neuronal cells. Amplification curves obtained after RT-qPCR, evaluating the expression of neuronal markers (ISL1, CHAT and MNX1) and the expression of the endogenous gene (BACT), with taqman probes. (PDF 1356 kb)
Table S1
Primers for qRT-PCR. (PDF 492 kb)
Table S2
Results of the mixed model analysis for physical performance tests assessed weekly in SOD1 treated mice (n = 22 mice per group, being 11 males and 11 females), for weight, PaGE, rotarod and motor score evaluations. (PDF 615 kb)
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Coatti, G.C., Frangini, M., Valadares, M.C. et al. Pericytes Extend Survival of ALS SOD1 Mice and Induce the Expression of Antioxidant Enzymes in the Murine Model and in IPSCs Derived Neuronal Cells from an ALS Patient. Stem Cell Rev and Rep 13, 686–698 (2017). https://doi.org/10.1007/s12015-017-9752-2
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DOI: https://doi.org/10.1007/s12015-017-9752-2