Cell Tissue Res DOI 10.1007/s00441-014-1903-z

REGULAR ARTICLE

Multiple systemic transplantations of human amniotic mesenchymal stem cells exert therapeutic effects in an ALS mouse model Haitao Sun & Zongliu Hou & Huaqiang Yang & Mingyao Meng & Peng Li & Qingjian Zou & Lujun Yang & Yuxin Chen & Huihui Chai & Huilin Zhong & Zara Zhuyun Yang & Jing Zhao & Liangxue Lai & Xiaodan Jiang & Zhicheng Xiao

Received: 16 October 2013 / Accepted: 28 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstracts Amyotrophic lateral sclerosis (ALS) is an adultonset progressive neurodegenerative disease involving degeneration of motor neurons in the central nervous system. Stem cell treatment is a potential therapy for this fatal disorder. The human amniotic membrane (HAM), an extremely rich and easily accessible tissue, has been proposed as an attractive material in cellular therapy and regenerative medicine because of its advantageous characteristics. In the present study, we evaluate the long-term effects of a cellular treatment by intravenous administration of human amniotic mesenchymal stem cells (hAMSCs) derived from HAM into a hSOD1G93A mouse model. The mice received systemic administration of hAMSCs or phosphate-buffered saline (PBS) at the onset, progression and symptomatic stages of the disease. hAMSCs

were detected in the spinal cord at the final stage of the disease, in the form of isolates or clusters and were negative for β-tubulin III and GFAP. Compared with the treatment with PBS, multiple hAMSC transplantations significantly retarded disease progression, extended survival, improved motor function, prevented motor neuron loss and decreased neuroinflammation in mice. These findings demonstrate that hAMSC transplantation is a promising cellular treatment for ALS.

Keywords Human amniotic mesenchymal stem cells . Amyotrophic lateral sclerosis . SOD1 . Transplantation . Treatment

Haitao Sun and Zongliu Hou contributed equally to this work. H. Sun : P. Li : L. Yang : Y. Chen : H. Chai : X. Jiang : Z. Xiao Department of Neurosurgery, The National Key Clinic Specialty, The Neurosurgery Institute of Guangdong Province, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China Z. Hou : M. Meng Research Laboratory Center, Yan’an Hospital, Kunming Medical University, Kunming 650051, China

Z. Z. Yang : Z. Xiao Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia

J. Zhao Model Animal Research Center, Nanjing University, Nanjing 210061, China

H. Yang : Q. Zou : H. Zhong : L. Lai Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

X. Jiang (*) Laboratory of Neurosurgery Institute, Zhujiang Hospital, Southern Medical University, 253# Gongye Road, Guangzhou 510282, China e-mail: [email protected]

Z. Z. Yang : Z. Xiao The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228, China

Z. Xiao (*) Monash Immunology and Stem Cell Labor, Monash University, level 3, Blg 75, Clayton, Victoria 3800, Australia e-mail: [email protected]

Cell Tissue Res

Introduction Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease involving the loss of the upper and lower motor neurons in the brain and spinal cord. Patients with ALS develop progressive muscular atrophy that results in eventual paralysis and they usually die because of respiratory failure within 3–5 years after symptoms begin (Boillée et al. 2006a; Dion et al. 2009). The most common form of ALS is sporadic ALS, which is associated with neurofilament disorganization, oxidative stress, mitochondrial dysfunction, astrogliosis and excitotoxicity (Bungener et al. 2005; Choi et al. 2013). Only approximately 10 % ALS cases are diagnosed as familial ALS (fALS). Approximately 20 % of fALS cases are attributed to mutations in the gene that encodes copper/zinc superoxide dismutase 1 (SOD1) (Rosen et al. 1993). Although research on this devastating disease has shown progress, the precise etiopathogenetic mechanisms of the disease remain unclear and no effective therapies are currently available. Stem cell therapy has recently emerged as a promising approach for neurological disease treatment. Several stem cell types, such as embryonic stem cells (ESCs), neural stem/progenitor cells, glial-restricted progenitor cells, umbilical cord blood mesenchymal stem cells and bone marrow mesenchymal stem cells, have exerted positive effects in ALS rodent models (Boulis et al. 2011; Lepore et al. 2008; Lunn et al. 2011; Silani et al. 2010; Thonhoff et al. 2009). However, the moderate improvement in motor behavior and the slight retardation in disease progression by these stem cells are likely related to indirect effects, such as induction of neurogenesis, release of trophic factors and decrease of inflammation, rather than the actual substitution of degenerating neurons. Neuroinflammation, which mainly involves microglial activation and reactive astrogliosis, has an important function in ALS and directly results in motor neuron death (Boillée et al. 2006b; Hall et al. 1998; Henkel et al. 2004; McGeer and McGeer 2002; Philips and Robberecht 2011; Zhao et al. 2013). Thus, a therapeutic strategy that combines inflammation modulation with cell therapy or neuroprotective processes may be a feasible clinical approach for ALS (Lindvall et al. 2004). The human amniotic membrane (HAM), an extremely rich and easily accessible tissue, has been proposed as an attractive material in cellular therapy and regenerative medicine because of its advantageous characteristics (Díaz-Prado et al. 2011; Insausti et al. 2010). HAM-derived stem cells have already been applied in various preclinical and clinical studies for repair of tissues, including corneal tissue, spinal cord injury, brain infarction and Parkinson’s disease (Díaz-Prado et al. 2011). Amniotic mesenchymal stem cells (hAMSCs) derived from HAM can be easily acquired noninvasively without pain and risk of morbidity. Human embryos need not be sacrificed

when isolating hAMSCs, thereby avoiding ethical concerns. In addition, hAMSCs have a high proliferation rate, without the limitation that the proliferative potential and differentiation abilities decrease as the donor’s age increases in the traditionally used bone marrow mesenchymal stem cells (BMSCs) (Díaz-Prado et al. 2011; In ’t Anker et al. 2004). hAMSCs also display ESC properties. For example, hAMSCs can express surface markers and transcription factors (e.g., OCT-4, octamer-binding transcription factor 4) and differentiate into all three germinal cells lines (Bilic et al. 2008; Díaz-Prado et al. 2011; In ’t Anker et al. 2004; Parolini et al. 2008). Importantly, hAMSCs are immune privileged and are less likely to be rejected after transplantation, which results from the low expression levels of HLA-ABC antigens but do not express HLA-DR and HLA-G antigens on the surface (Chang et al. 2010; Kim et al. 2013; Parolini et al. 2008; Portmann-Lanz et al. 2010). Morever, hAMSCs have anti-inflammatory properties and induce the production of the anti-inflammatory cytokine IL-10 (Boillée et al. 2006a; Chang et al. 2006; Kang et al. 2012; Kim et al. 2013). These cells can also modulate T-cell proliferation by actively suppressing the proliferation of T lymphocytes and inhibiting the differentiation of monocytes, suggesting that transplanted hAMSCs can modulate host immune-related inflammatory responses (Chang et al. 2006; Ilancheran et al. 2007; Kim et al. 2013; Wolbank et al. 2007). Thus, this preclinical study aims to evaluate the therapeutic effects of multiple intravenous transplantations of hAMSCs upon disease progression in an ALS mouse model.

Materials and methods Preparation of hAMSCs Amnion membranes were obtained from placentas (two male and three female fetuses) delivered by healthy donor mothers with normal pregnancy undergoing elective caesarean section (37–40 weeks gestation). Informed consent was obtained from all mothers (n=5) who participated in this study. The membranes were rinsed thoroughly using phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA, USA) with antibiotics (100 U/ml ampicillin and 100 μg/ml streptomycin) (Hyclone, Logan, UT, USA). The membranes were cut into pieces of approximately 1 mm3 and then cultured in α-modified Eagle’s medium (α-MEM; Hyclone, Logan, UT) supplemented with 20 % (v/v) fetal bovine serum (FBS; Hyclone). The culture medium was replaced twice a week. Adherent cells at 60– 80 % confluence were dissociated using 0.25 % trypsin (Invitrogen) for subculture. hAMSCs at the fourth passage were infected with lentiviral FUGW encoding the enhanced green fluorescent protein (EGFP) gene. The 293T cells were seeded at 4×106 cells per 100 mm dish (Corning, NY, USA). After 1 day, lentiviral vectors were packaged by co-

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transfecting FUGW (Addgene, Cambridge, MA, USA) with auxiliary packaging vectors psPAX2 and pMD2-G. The 293T cells and auxiliary packaging vectors were all gifts from Dr Duanqing Pei’s laboratory at the Guangzhou Institute of Biomedicine and Health, Guangzhu, China 510530. Lentiviruses were harvested after 48 h and centrifuged at 80,000g for 2 h at 4 °C. After centrifugation, the supernatant was carefully aspirated and the pellet was suspended in 200 μl Opti-MEM® Reduced Serum Medium (GBICO). hAMSCs were seeded at 2×105 cells per well in 6-well plate (Corning) 1 day before infection. Viruses were added 50 μl per well and incubated with hAMSCs overnight. Then, the cells were recovered with fresh medium. hAMSCs at the sixth to eighth passages were used for transplantation. This study was approved by the Human Research Ethics Committees of Yan’an Hospital, Kunming Medical University and Zhujiang Hospital, Southern Medical University. FACS and immunofluorescence analysis of hAMSCs hAMSCs at the sixth passage were stained using fluorescein isothiocyannate (FITC)-, phycoetrythrin- , or prolylcarboxypeptidase-labeled mouse monoclonal antibodies and then analyzed using a fluorescence-activated cell sorter (FACS) (BD Biosciences) system equipped with Cell Quest software. Approximately 10,000 cells were measured per sample. Antibodies CD14, C29, CD34, CD44, CD45, CD73, CD90, CD123, CD166, HLA-DR, SSEA-4 and OCT-4 were purchased from BD Pharmingen (San Diego, CA, USA). Cells grown on coverslips (Thermo Fisher Scientific, Bremen, Germany) in 24-well tissue culture plates at a density of 8,000 cells/well were washed with PBS (Invitrogen), fixed with 4 % paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS (Invitrogen), permeabilized and then blocked with tris-buffered saline (TBS) containing 0.05 M Tris, 150 mM NaCl and 0.5 % Triton X-100 (all Sigma-Aldrich) supplemented with 5.0 % normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h. The cells were incubated with rabbit anti-Vimentin (1:200; Abcam, Cambridge, UK) overnight at 4 °C and then stained with FITC-conjugated goat anti-rabbit antibody (Sigma-Aldrich) for 1 h at 37 °C. Finally, the cells were washed with PBS (Invitrogen) and mounted with diamidino-2-phenylindole (DAPI; Vector Laboratories). Multi-differentiation of hAMSCs in vitro Osteogenic induction: Cells at 50 % confluence were cultured in α-MEM supplemented with 10 % FBS (Hyclone), 50 μg/L gentamicin sulfate (Sigma-Aldrich) and osteogenic medium (0.1 μM dexamethasone, 10 mM betaglycerophosphate and 50 μM ascorbic acid-2-phosphate) (Sigma-Aldrich) for

2 weeks. The cells were then stained with Alizarin red S (Sigma-Aldrich). Adipogenic induction: Cells at 80 % confluence were cultured in α-MEM supplemented with 10 % FBS (Hyclone), 50 μg/L gentamicin sulfate (Sigma-Aldrich), and adipogenic supplements (0.5 μM dexamethasone, 0.5 mM 3isobutyl-1-methylxanthine, and 60 μM indomethacin) (Sigma-Aldrich) for 2 weeks. The cells were then stained with oil red O (Sigma-Aldrich). Neurogenic induction: Cells at 50 % confluence were cultured in α-MEM supplemented with 10 % FBS (Hyclone), 50 μg/L gentamicin sulfate (Sigma-Aldrich) and neurogenic supplements (0.1 μM dexamethasone, 0.5 μM linoleic acid, 10 ng/ml platelet-derived growth factor and 10 ng/ml basic fibroblast growth factor or bFGF) (SigmaAldrich) for 24 h and then processed without serum for 5 h. Immunocytochemical staining with anti-neurofilament M antibody (1:200; Millipore, Temecula, CA, USA) was performed to assess the capacity of neuronal differentiation. Animals All animal use protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Southern Medical University and carried out following the Guide for the Care and Use of Laboratory Animals. Transgenic mice harboring a high copy number of the hSOD1G93A [B6SJL-TgN (SOD1-G93A) 1Gur] transgene, as reported by Gurney et al. (1994), were used in this study. The breeding pairs were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Hemizygous transgenic progeny were maintained by mating the transgenic males with F1 hybrid wild-type (WT) females. The progeny were genotyped by polymerase chain reaction using genomic DNA isolated from mouse tail after birth as previously described (Gurney et al. 1994). The study included hSOD1G93A mice transplanted with hAMSCs (n= 12, 6 males, 6 females), PBS-injected transgenic mice (n=12, 6 males, 6 females) and normal WT mice (n=12, 6 males, 6 females). All mice were kept on a 12:12 h dark:light cycle at room temperature (23 °C). Food and water were provided ad libitum. Surgery The hAMSCs were administered intravenously in the mice under anesthesia as previously described (Garbuzova-Davis et al. 2003). Briefly, the jugular vein was exposed and then isolated with blunt dissection. A 250-μl Hamilton syringe attached with a 31-gauge needle (Hamilton, Reno, NV, USA) was laid into the lumen and fixed in place. The cells (1×106) in 200 μl PBS (Invitrogen) were delivered over 10 min. Afterward, the needle was withdrawn and the incision was closed. Animals received cells or PBS at 12, 14 and

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16 weeks. All animals were immunosuppressed with intraperitoneal injection of CsA in 0.1 ml at a dose of 10 mg/kg/d (Novartis, Basel, Switzerland), starting from 3 days before the transplantation until the end of the study.

consecutive weeks, the earliest time was retrospectively defined as onset.

Histology and immunofluorescence of mouse spinal cord Evaluation of motor functions and survival The disease onset and progression in the disease model were monitored using clinical observation, weight measurement, Rotarod performance test, PaGE test and CatWalk gait analysis, all of which are commonly performed to evaluate the hSOD1G93A mouse (Weydt et al. 2003). CatWalk gait analysis was performed to determine dynamic and voluntarily walking patterns in rodent models; this method is constant and reliable for quantitatively investigating multiple gait parameters (Mead et al. 2011). From 8 weeks of age, the animals (hAMSCs-transplanted, four males and four females; PBSinjected, four males and four females; WT mice, four males and four females) were evaluated weekly with behavioral tests and the investigator was blinded to the treatment. The first 2 weeks of tests were recognized as training. The animals were placed in a plastic bowl and then weighed using an electronic scale. Weighing was performed between 11:00 a.m. and 2:00 p.m. hours weekly. The motor coordination, balance and strength of each animal were assessed by a rotarod apparatus (Anhui Zhenghua Bio Equipment, China). Each mouse was provided three attempts and the longest latency for a mouse remaining on the rotating cylinder to fall was recorded, with a cut-off time of 180 s. For the PaGE test, the time for which a mouse could hold onto the inverted lid of a wire cage was measured. The duration until the mouse let go of the grip with both hind limbs was timed, with a cut-off time of 90 s. Each animal was given three tests and the longest latency was recorded. Gait analysis of each mouse was performed using the CatWalk gait analysis system v.9.0 (Noldus, Wageningen, The Netherlands). The animal was placed on the walkway in complete darkness and gait patterns were recorded in another room. Six runs crossed at the same speed, with three full consecutive step patterns per run recorded for each mouse and three were selected for analysis. Stride length was defined as the distance between consecutive foot trace in mm. The test was carried out weekly until the animal was not able to complete the performance because of hind limb paralysis. The signs of motor deficit in mice were assessed using the behavior scoring system described by Vercelli et al. (2008). The scoring system was as follows: 4 points, healthy without any sign of motor dysfunction; 3 points, signs of obvious hind limb tremors when suspended by the tail; 2 points, signs of gait abnormalities; 1 point, signs of at least one hind limb dragging; and 0 point, disabled to right themselves within 30 s and this time point was considered as the time of death. When the mice showed signs of a score