Impact of the Secretome of Human Mesenchymal Stem Cells on Brain Structure and Animal Behavior in a Rat Model of Parkinson's Disease

Abstract

Research in the last decade strongly suggests that mesenchymal stem cell (MSC)‐mediated therapeutic benefits are mainly due to their secretome, which has been proposed as a possible therapeutic tool for the treatment of Parkinson's disease (PD). Indeed, it has been shown that the MSC secretome increases neurogenesis and cell survival, and has numerous neuroprotective actions under different conditions. Additionally, using dynamic culturing conditions (through computer‐controlled bioreactors) can further modulate the MSC secretome, thereby generating a more potent neurotrophic factor cocktail (i.e., conditioned medium). In this study, we have characterized the MSC secretome by proteomic‐based analysis, investigating its therapeutic effects on the physiological recovery of a 6‐hydroxidopamine (6‐OHDA) PD rat model. For this purpose, we injected MSC secretome into the substantia nigra (SNc) and striatum (STR), characterizing the behavioral performance and determining histological parameters for injected animals versus untreated groups. We observed that the secretome potentiated the increase of dopaminergic neurons (i.e., tyrosine hydroxylase‐positive cells) and neuronal terminals in the SNc and STR, respectively, thereby supporting the recovery observed in the Parkinsonian rats’ motor performance outcomes (assessed by rotarod and staircase tests). Finally, proteomic characterization of the MSC secretome (through combined mass spectrometry analysis and Bioplex assays) revealed the presence of important neuroregulatory molecules, namely cystatin C, glia‐derived nexin, galectin‐1, pigment epithelium‐derived factor, vascular endothelial growth factor, brain‐derived neurotrophic factor, interleukin‐6, and glial cell line‐derived neurotrophic factor. Overall, we concluded that the use of human MSC secretome alone was able to partially revert the motor phenotype and the neuronal structure of 6‐OHDA PD animals. This indicates that the human MSC secretome could represent a novel therapeutic for the treatment of PD. Stem Cells Translational Medicine 2017;6:634–646

Secretome of Undifferentiated Neural Progenitor Cells Induces Histological and Motor Improvements in a Rat Model of Parkinson's Disease

“…For surgical procedures, animals were anesthetized intraperitoneally (i.p.) with ketamine (75 mg/kg) plus medetomidine (0.5 mg/kg), placed on a stereotaxic frame (Stoelting, Wood Dale, IL, https://www.stoeltingco.com/), and unilaterally injected using a 30‐gauge needle Hamilton syringe (Hamilton, Bonaduz, Switzerland, https://www.hamiltoncompany.com/), with either vehicle (0.2 mg/ml of ascorbic acid in 0.9% NaCl; sham group, n = 9) or 6‐OHDA hydrochloride (Sigma, H4381, St. Louis, MO, http://www.sigmaaldrich.com/portugal.html; 6‐OHDA group, n = 15) directly into the medial forebrain bundle (MFB; coordinates related to Bregma: AP = −4.4 mm, ML = − 1.0 mm, DV = −7.8 mm ) at a rate of 0.5 μl/min. Sham animals received 2 μl of 0.2 mg/ml of ascorbic acid (Sigma, A1968) in 0.9% NaCl and the 6‐OHDA animals were injected with 2 μl of 6‐OHDA hydrochloride (4 μg/μL) with 0.2 mg/ml of ascorbic acid in 0.9% NaCl.…”

“…Five weeks after 6‐OHDA injections, the animals received hNPCs transplants or hNPCs secretome. After anesthesia administration, animals were unilaterally injected, as described in previous section, with either vehicle (Neurobasal A medium; 6‐OHDA control group, n = 5), sterile saline (sham group, n = 9), hNPCs ( n = 5), or hNPCs CM ( n = 5) directly in the SNpc (coordinates related to Bregma: AP = − 5.3 mm, ML = −1.8 mm, DV = −7.4 mm) and into four striatum coordinates (coordinates related to Bregma: AP = −1.3 mm, ML = 4.7 mm, DV = −4.5 mm, and − 4.0 mm; AP = −0.4 mm, ML = 4.3 mm, DV = −4.5 mm, and 4.0 mm; AP = 0.4 mm, ML = 3.1 mm, DV = −4.5 mm, and − 4.0 mm; AP = 1.3 mm, ML = 2.7 mm; DV = −4.5 mm, and − 4.0 mm ). 6‐OHDA‐control group received 4 μl of Neurobasal A medium in the SNpc and 2 μl in each coordinate of striatum at a rate of 0.5 μl/min.…”

“…Therefore, this test was only used to select the animals that were truly injured upon 6‐OHDA lesions . All behavioral tests were performed as previously described .…”

“…All image analysis was completed using the ImageJ software (National Institute of Health, v1.48, Bethesda, MD, https://imagej.nih.gov/ij/). The optical density of TH‐positive fibers was measured by densitometry, as previously described . The data are presented as percentages of the contralateral striatum (intact side).…”

Strategies to improve the migration of mesenchymal stromal cells in cell therapy

“…MSCs have been used to treat various CNS disorders in preclinical and clinical studies, such as spinal cord and traumatic brain injury [116][117][118] , ischemic stroke [31,40,56,62,69,97,[119][120][121][122][123] , intracranial hemorrhage (ICH) [124] , Parkinson's disease [125,126] , multiple sclerosis [127] , experimental autoimmune encephalomyelitis (EAE) [47,128,129] , amyotrophic lateral sclerosis [130,131] , Alzheimer's disease [132] , Huntington's disease [133][134][135] , and so on. Potential repair mechanisms involve transdifferentiation to replace damaged neural cells and production of growth factors by MSCs.…”