Human Amniotic Epithelial Cells Recover Mouse Model of Parkinson’S Disease Mainly by Neuroprotective，Anti-Oxidative and Anti- Infammatory Factors
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Human Amniotic Epithelial Cells Recover Mouse model of Parkinson’s Disease mainly by NeuroprotectiveAnti-oxidative and Anti- inammatory factors Jiaofei Zhang Shanghai Jiao Tong University School of Medicine Aliated Renji Hospital Hui Li Shanghai Jiao Tong University Hao Yang Xi'an Jiaotong University Jianhua Lin Shanghai Jiao Tong University School of Medicine You Wang Shanghai Jiao Tong University School of Medicine Qianjun Zhang ( [email protected] ) Central South University Xiangya School of Medicine Wei-Qiang Gao ( [email protected] ) Shanghai Jiao Tong University School of Medicine Huiming Xu ( [email protected] ) Shanghai Jiao Tong University School of Medicine Aliated Renji Hospital https://orcid.org/0000- 0003-4823-6920 Research Keywords: Parkinson’s disease. human amniotic epithelial cells. neuroprotection. dopaminergic neurons. anti-inammatory factors. anti-oxidative stress Posted Date: April 1st, 2020 DOI: https://doi.org/10.21203/rs.3.rs-20116/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/21 Abstract Objectives: Human amniotic epithelial cells (hAECs) have been reported to have neuroprotective roles in neurological diseases including Parkinson’s Disease (PD) in animal models. However, the mechanism is not fully understood. Materials and method : Firstly, hAECs were transplanted into the striatum of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine ( MPTP )-induced PD mice. And the motor decits were tested by Rotarod test. Secondly, injury severity of nigral dopaminergic neurons in substantia nigra and striatal axon bers in PD mice was estimated by immunochemistry with tyrosine hydroxylase antibody. Thirdly, neuroinammation was measured by microglial activation and the level of inammatory factor levels. Lastly, the oxidative stress was tested by the level of reactive oxidative species (ROS) levels. In vitro, we examined a full spectrum of soluble proteins of hAECs by Raybiotech human Antibody Array 507. Besides, antibody neutralization experiments were used to determine the role of many factors on dopaminergic neurons. Results: hAECs signicantly attenuated the motor decits of PD mice. Moreover, the grafts prevented the loss of nigral dopaminergic neurons, promoted the outgrowth of their neurites and striatal axon bers in PD mice. More importantly, decreased microglial activation, inammatory factor levels and MPTP- induced excessive ROS levels were also observed in hAEC-treated PD mice. In vitro, analysis of an antibody array of 507 soluble target proteins using hAEC-CM revealed that the levels of many neurotrophic factors, growth factors neuronal cell adhesion molecule ( NrCAM )and anti-inammatory factors were evidently high. In addition, antibody neutralization experiments showed that many of them contributed to the survival and growth of dopaminergic neurons and the outgrowth of their neurites. Conclusion: Our study indicates that the neuroprotection effects by hAECs grafts are achieved not only by neuroprotection and neurite outgrowth-promoting activities, but also by anti-inammation and anti- oxidation functions in the microenvironment of the damaged dopaminergic neurons, so to facilitate the restoration of neurological functions. Introduction Parkinson’s disease (PD) is an age-related neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and appearance of cytoplasmic aggregated lewy bodies. Accumulating evidence indicates that aging, oxidative stress, and neuroinammation play a crucial role in the pathogenesis of PD [1–4]. Although pharmaceutics and neurosurgery can relieve the symptoms, it is still dicult to halt the progress of the neurodegeneration. Therefore, seeking an effective therapeutic strategy is crucial. Currently, increasing evidence has shown that stem cells are a promising strategy in the treatment of neurodegeneration disorders including PD [5]. Stem cells including embryonic stem cells, induced pluripotent stem cells, neural stem cells, bone-marrow mesenchymal stem cells and adipose-derived stromal cells have been reported to ameliorate behavioral decits and to promote the survival of dopaminergic neurons in PD animal models [6–11]. However, their Page 2/21 clinical application is limited due to some problems such as oncogenicity, immunological rejection, ethical issue, scarcity and quantity of stem cell resources [12–14]. In contrast, human amniotic epithelial cells (hAECs), a type of cells derived from human term amnion, have some unique advantages including non-tumorigenic, low immunogenic potential, less ethical dispute and an adequate resource [15, 16], and have attracted much more attention for treating neurological diseases including PD. hAECs are derived from the human amniotic membrane that is generated from the epiblast prior to gastrulation [17]. Thus, hAECs possess some pluripotent characteristics and have the potential ability to differentiate into neural cells [18, 19]. In addition, hAECs also synthesize and release some neurotrophic factors and growth factors [20, 21], which have been demonstrated to play an important role in improving the microenvironment benecial for the repair/regeneration of damaged/degenerated neurons and restoration of the neurological functions [22–24]. Interestingly, some anti-inammatory factors are also secreted by hAECs [25], which could contribute to the attenuation of neuroinammatory response in PD. Thus, hAECs may be a desirable cell resource for cell therapy of PD. Kakishita et al rstly reported that hAECs transplanted into the striatum of PD rats could partially differentiated tyrosine hydroxylase (TH) positive cells and the grafts ameliorated apomorphin-induced rotational asymmetry [26]. Moreover, hAECs could prevent the degeneration of nigral dopamine neurons in PD rats [27]. These studies suggested that hAECs play a neuroprotective effect on PD animal model. However, the underlying molecular mechanism remains unclear. In the present study, we not only transplanted hAECs into the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice to conrm that hAECs has neuroprotective effects and improve motor behavior in PD mice, we also focused on the molecules that are responsible for the underlying repair mechanism. In particular, we performed protein chip assay to get a full spectrum of a large number of soluble factors produced by hAECs, and further analyzed what rich paracrine factors contribute to the survival and outgrowth of lesioned dopaminergic neurons by antibody neutralization experiment. In addition, we investigated for the rst time the anti-inammatory and anti-oxidation roles of hAECs on MPTP-induced PD mice. Materials And Methods Isolation and Culture of hAECs hAECs were isolated from human amnion according to the protocol described previously with minor modication [28]. In brief, the placenta was obtained from women undergoing caesarean sections. The amniotic membrane was stripped from adjacent chorion and rinsed in Hank’s Balanced Salt Solution (HBSS, Life Technology, Grand island, NY). Then the epithelium layer was thoroughly scraped out from the underlying tissues and was further cut into small pieces. Then the small pieces were digested with 0.25% trypsin (Sigma-Aldrich, Darmstadt, Germany) for 30 min at 37℃, and collagenase (0.1mg/ml, Sigma-Aldrich) for 1h at 37℃, and then with DNase I (0.1mg/ml, Roche, Basel, Switzerland) for 10 min. Lastly, the cell pellet was resuspended in DMEM/F12 containing 10% FBS and 1% penicillin/streptomycin (P/S, all from Life Technology). The cells were passaged at 90% conuence. Page 3/21 Flow Cytometry The cells were dissociated with trypsin and washed with cold PBS, then they were separately stained with IgG or monoclonal antibodies conjugated to PE or Percp, or APC or FITC: Epcam-PE, CD29-FITC, CD49f- FITC, CD73-FITC, CD105-APC, CD90-FITC, CD34-Percp, CD31-FITC, CD45-FITC, HLA-DR-FITC (all from eBioscience, San Diego, USA). Upon completion of washing with PBS, labeled cells were re-suspended and at least 105 events were acquired by using a BD AccuriTM C6 Flow Cytometer (BD, NJ, USA). Transplantation of hAECs into PD Mice Induced by MPTP A repeated MPTP (30mg/kg, intraperitoneal injection for six consecutive days) were administered to C57BL/6 mice to induce PD mouse model. Mice treated with PBS were used as a control (CON). Recipient mice were divided into three groups : “CON” group (n≥8), “MPTP+PBS” group (n≥8) and “MPTP+hAECs” group (n≥8). In the “MPTP+hAECs” group, mice treated with MPTP received unilateral transplantation of hAECs at day 7. hAECs were digested and prelabeled with PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma-Aldrich). Cell suspension (3ml, 1×106 cells) in PBS was stereotaxically injected into the unilateral striatum of recipient mice according to the following coordinates: 0.8 mm posterior to bregma; 2.0mm lateral to bregma; 3.0 mm ventral from the dural surface with the tooth bar set at zero. PBS was injected in a manner as above. Rotarod test The rotarod behavior test was measured using a rotarod device (RWD life Science, Shenzhen, China). The parameters of the rotarod system include starting speed, acceleration and the highest speed (2rpm, 1.5rpm/s, 50rpm). Every mouse underwent three consecutive trials and the longest latency time for each mouse to fall off the rotating rod for the last two trials was measured at week 1 and week 2 after transplantation of hAECs. The rest period