A Huntingtin Knockin Pig Model Recapitulates Features of Selective Neurodegeneration in Huntington's Disease.

Huntington's disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.

Digestion and utilization of plant-based diets by transgenic pigs secreting ß-glucanase, xylanase, and phytase in their salivary glands.

Novel transgenic (TG) pigs co-expressing three microbial enzymes, ß-glucanase, xylanase, and phytase, in their salivary glands were previously generated, which exhibited reduced phosphorus and nitrogen emissions and improved growth performances. In the present study, we attempted to explore the age-related change of the TG enzymic activity, the residual activity of the enzymes in the simulated gastrointestinal tract, and the effect of the transgenes on the digestion of nitrogen and phosphorus content in the fiber-rich, plant-based diets. Results showed that all the three enzymes were stably expressed over the growing and finishing periods in the F2 generation TG pigs. In simulated gastric juice, all the three enzymes exhibited excellent gastrointestinal environment adaptability. The apparent total tract digestibility of phosphorus was increased by 69.05% and 499.64%, while fecal phosphate outputs were decreased by 56.66% and 37.32%, in the TG pigs compared with the wild-type littermates fed with low non-starch polysaccharides diets and high fiber diets, respectively. Over half of available phosphorus and water-soluble phosphorus in fecal phosphorus were reduced. We also found the performance of phosphorus, calcium, and nitrogen retention rates were significantly improved, resulting in faster growth performance in TG pigs. The results indicate that TG pigs can effectively digest the high-fiber diets and exhibit good growth performance compared with wild type pigs.

Uterine luminal-derived extracellular vesicles: potential nanomaterials to improve embryo implantation.

Most pregnancy losses worldwide are caused by implantation failure for which there is a lack of effective therapeutics. Extracellular vesicles are considered potential endogenous nanomedicines because of their unique biological functions. However, the limited supply of ULF-EVs prevents their development and application in infertility diseases such as implantation failure. In this study, pigs were used as a human biomedical model, and ULF-EVs were isolated from the uterine luminal. We comprehensively characterized the proteins enriched in ULF-EVs and revealed their biological functions in promoting embryo implantation. By exogenously supplying ULF-EVs, we demonstrated that ULF-EVs improve embryo implantation, suggesting that ULF-EVs are a potential nanomaterial to treat implantation failure. Furthermore, we identified that MEP1B is important in improving embryo implantation by promoting trophoblast cell proliferation and migration. These results indicated that ULF-EVs can be a potential nanomaterial to improve embryo implantation.

Reflection-based vision guide method for coarse alignment of vector measurement during the satellite assembly, integration, and test process.

Vector measurement is a vital measurement item during the satellite assembly, integration, and test (AIT) process. With the increasing popularity of commercial spaceflight, the development cycle of a satellite is shorter, and the number of satellites has been growing rapidly. The traditional on-site vector measurement method is inefficient and significantly affects the development cycle of the satellite. Therefore, it is of utter importance to propose an online high-precision automatic vector measurement system. The most challenging step of the online automatic vector measurement is coarse alignment because a cubic prism must be identified, and the normal direction of its surface must be calculated at a certain precision in the unstructured environment during the coarse alignment step. A reflection-based vision guide method was proposed to identify and calculate the normal direction of the cubic prism. The working principle and advantage of the proposed vision guide system were described in detail. What is more, the calibration and calculation methods of the proposed vision guide system were also presented. Finally, experiments were conducted to verify the effectiveness of the proposed method.

A Cas9-transcription factor fusion protein enhances homology-directed repair efficiency.

Precise gene insertion or replacement in cells and animals that requires incorporation of a foreign DNA template into the genome target site by homology-directed repair (HDR) remains an inefficient process. One of the limiting factors for the inefficiency of HDR lies in the limited chance for colocalization of the donor template and target in the huge genome space. We here present a strategy to enhance HDR efficiency in animal cells by spatial and temporal colocalization of the donor and Cas9 by coupling the CRISPR system with a transcription factor (TF). We first identified that THAP domain-containing 11 (THAP11) can coordinate with CRISPR/Cas9 to increase HDR stably through screening multiple TFs from different species. We next designed donor structures with different fusion patterns with TF-specific DNA-binding motifs and found that appending two copies of THAP11-specific DNA binding motifs to both ends of the double-stranded donor DNA has an optimal effect to promote HDR. The THAP11-fused CRISPR system achieved more than twofold increase in HDR-mediated knock-in efficiency for enhanced green fluorescent protein (EGFP) tagging of endogenous genes in 293T cells. We also demonstrated up to 6-fold increases of knock-in through the combinational use of the TF-fused CRISPR and valnemulin, a recently discovered small-molecule HDR enhancer. This modified CRISPR system provides a simple but highly efficient platform to facilitate CRISPR-mediated KI manipulations.

Isolation and in vitro expansion of porcine spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genetic information to offspring through their ability to both self-renew and differentiate into mature spermatozoa. SSCs can be transplanted to establish donor-derived spermatogenesis in recipient animals, thus offering a novel reproductive tool for multiplication of elite individual animals to benefit livestock production. An optimal SSC culture in vitro can benefit various SSC-based studies and applications, such as mechanistic study of SSC biology, SSC transplantation process and SSC-based transgenesis technique. However, except for some model rodent animals, SSC culture remains an inefficient and unstable process. We here studied a workflow to isolate, purify and in vitro culture porcine SSCs from neonatal pig testes. Pig testicular cells were dissociated by two-step enzymatic digestion with collagenase type IV and trypsin. We enriched the spermatogonia from the testicular cell mix by differential plating for at least 3 times to remove firmly attached non-SSCs. We then tested the optimal culture medium formula by supplementation of different growth factors to the basic medium (DMEM/F12 + 1% FBS) and found that a combination of 20 ng/ml GDNF, 10 ng/ml LIF, 20 ng/ml FGF2 and 20 ng/ml IGF1 had the best effect on SSC growth in our defined experimental system. In the presence of 4 growth factors without specific feeders, the purified SSCs can be cultured in poly-L-lysine- and laminin-coated dishes for 28 days and remain preserving a continuous proliferation without losing the undifferentiated spermatogonial phenotype.

Non-Coding RNAs Regulate Spontaneous Abortion: A Global Network and System Perspective.

Spontaneous abortion is a common pregnancy complication that negatively impacts women's health and commercial pig production. It has been demonstrated that non-coding RNA (ncRNA) is involved in SA by affecting cell proliferation, invasion, apoptosis, epithelial-mesenchymal transformation (EMT), migration, and immune response. Over the last decade, research on ncRNAs in SA has primarily concentrated on micro RNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). In this review, we discuss recent ncRNA studies focused on the function and mechanism of miRNAs, lncRNAs, and circRNAs in regulating SA. Meanwhile, we suggest that a ceRNA regulatory network exists in the onset and development of SA. A deeper understanding of this network will accelerate the process of the quest for potential RNA markers for SA diagnosis and treatment.

Pig Coat Color Manipulation by MC1R Gene Editing.

Black coat color in pigs is determined by the dominant E allele at the MC1R locus. Through comparing MC1R gene sequences between recessive e and dominant ED1 alleles, we identified four missense mutations that could affect MC1R protein function for eumelanin synthesis. With the aim of devising a genetic modification method for pig coat color manipulation, we mutated the e allele in the Duroc breed to the dominant ED1 allele using CRISPR-mediated homologous recombination for the four mutation substitutions at the MC1R locus. The MC1R-modified Duroc pigs generated using the allele replacement strategy displayed uniform black coat color across the body. A genotyping assay showed that the MC1R-modified Duroc pigs had a heterozygous ED1/e allele at the MC1R locus; in addition, the pigs remained in the Duroc genetic background. Our work offers a gene editing method for pig coat color manipulation, which could value the culture of new pig varieties meeting the needs of diversified market.

Knockdown of RLIM inhibits XIST expression and improves developmental competence of cloned male pig embryos.

Ectopic expression of Xist on the putative active X chromosome is a primary cause of the low developmental efficiency of cloned mouse and pig embryos. Suppression of abnormal Xist expression via gene knockout or RNA interference (RNAi) can significantly enhance the developmental competence of cloned mouse and pig embryos. RLIM is a Xist expression activator, whereas REX1 is an Xist transcription inhibitor, as RLIM triggers Xist expression by mediating the proteasomal degradation of REX1 to induce imprinted and random X chromosome inactivation in mice. This study aimed to test whether the knockdown of RLIM and overexpression of REX1 can repress aberrant Xist expression and improve the developmental ability of cloned male pig embryos. Results showed that injection of anti-RLIM small interfering RNA significantly decreased Xist messenger RNA abundance, increased REX1 protein level, and enhanced the preimplantation development of cloned male porcine embryos. These positive effects were not observed in cloned male pig embryos injected with REX1 expression plasmid, which might be due to the low expression efficiency of injected REX1 plasmid and/or the short half-life of expressed REX1 protein. The findings from this study indicated that RLIM participated in the ectopic activation of Xist expression in cloned pig embryos by targeting REX1 degradation. Furthermore, this study provided a new method to improve cloned pig embryo development by the inhibition of Xist expression via RNAi of RLIM.

Resistance to pseudorabies virus by knockout of nectin1/2 in pig cells.

Pseudorabies virus (PRV) is a pig pathogen that causes substantial economic losses to the pig industry. Infection of host cells by PRV is mediated by the membrane proteins nectin1 and nectin2, which are presumed to be receptors for PRV infection. Here, we generated nectin1/2 knockout (KO) cells with the aim of establishing a PRV-resistant cell model. Nectin1 and 2 were ablated in PK15 cells by CRISPR/Cas9-mediated gene targeting. PRV infection in either nectin1 or nectin2 KO cells showed a significant reduction in viral growth compared with wild-type (WT) cells. We further simultaneously deleted nectin1 and nectin2 in PK15 cells and found that double KO cells showed no further increase in resistance to PRV compared with single gene-KO cells, despite being more resistant than WT. By investigating the cell entry steps of PRV infection, we found that nectin1 or/and nectin2 KO did not greatly affect virus attachment or internalization to cells but blocked cell-to-cell spread. Our results demonstrate that KO of either nectin1 or nectin2 confers PRV resistance to PK15 cells. This strategy could be applied to establish PRV-resistant pigs with nectin1/2 modifications to benefit the pig industry.

Transgenic pigs expressing ß-xylanase in the parotid gland improve nutrient utilization.

Xylan is one of the main anti-nutritional factors in pig's feed. Although supplementation of ß-xylanase in diet can improve the utilization of nutrients in animals, it is limited by feed cost, manufacturing process and storage stability. To determine whether the expression of endogenous ß-xylanase gene xynB in vivo can improve digestibility of dietary xylan and absorption of nutrients, we produced transgenic pigs which express the xynB from Aspergillus Niger CGMCC1067 in the parotid gland via nuclear transfer. In four live transgenic founders, ß-xylanase activities in the saliva were 0.74, 0.59, 0.37 and 0.24 U/mL, respectively. Compared with non-transgenic pigs, the content of crude protein (CP) in feces reduced by 15.5% (P < 0.05). Furthermore, in 100 of the 271 F1 pigs the xynB gene was detectable. The digestibility of gross energy and CP in F1 transgenic pigs were increased by 5% and 22%, respectively, with the CP content in feces decreased by 6.4%. Taken together, our study showed that the transgenic pigs producing ß-xylanase from parotid gland can reduce the anti-nutritional effect in animal diet and improve the utilization of nutrients.

Establishment of gene-edited pigs expressing human blood-coagulation factor VII and albumin for bioartificial liver use.

BACKGROUND AND AIM: Bioartificial livers (BALs) are considered as a solution to bridge patients with acute liver failure to liver transplantation or to assist in spontaneous recovery for patients with end-stage liver disease. Pig is the best donor of hepatocytes for BALs in clinical trials, because metabolic and detoxification function of its liver are close to human. However, using pig hepatocytes for BALs remains controversial for safety concern owing to nonhuman proteins secretion. Herein, we attempt to establish modified pigs expressing humanized liver proteins, blood-coagulation factor VII (F7), and albumin (ALB). These pigs should also be porcine endogenous retrovirus subtype C (PERV-C) free so that their ability of transmitting PERV to human could be diminished seriously. METHODS: We devised both homology-dependent and independent knock-in approaches to insert a fusion of hF7 and hALB gene downstream the site of pig endogenous F7 promoter in pig fetal fibroblasts negative for PERV-C. The modified pigs were then generated through somatic cell nuclear transfer. RESULTS: We obtained 14 and 10 cloned pigs by homology-dependent and independent approaches, respectively. Among them, 19 cloned pigs were with expected gene modification and 13 are alive to date. These modified pigs can successfully express hF7 and hALB in the liver and serum, and the expressed hF7 exhibits normal coagulation activity. CONCLUSIONS: The gene-edited pigs expressing hF7 and hALB in the liver were generated successfully. We anticipate that our pigs could provide an alternative cell source for BALs as a promising treatment for patients with acute liver failure.

Fumarylacetoacetate Hydrolase Knock-out Rabbit Model for Hereditary Tyrosinemia Type 1.

Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH-/- rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH-/- rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH-/- rabbits are an attractive alternative for modeling the consequences of HT1.

DZNep and UNC0642 enhance in vitro developmental competence of cloned pig embryos.

Somatic cell nuclear transfer in mammalian cloning suffers from a faulty epigenetic reprogramming, which is believed to cause developmental failures in cloned embryos. Regulating the epigenetic-modifying enzymes can rescue the chromatin of cloned embryos from aberrant epigenetic status, thereby potentially promoting cloning efficiency. In this study, we investigated the effect of two histone methyltransferase inhibitors, namely, DZNep and UNC0642, on the in vitro developmental competence of cloned pig embryos. We found that (1) treatment with 10 nM DZNep or 5 nM UNC0642 for 24 h after activation had the best promoting effect on the development of cloned embryos (blastocyst rate 10.32% vs 18.08% for DZNep, and 10.44% vs 18.14% for UNC0642); (2) 10 nM DZNep and 5 nM UNC0642 significantly decreased the levels of H3K27me3 and H3K9me2, respectively, at the 2-cell, 4-cell and blastocyst stages; (3) the apoptosis level was lower in the treatment groups than in untreated control; and (4) the transcriptional expression of epigenetic genes (EZH2, GLP, G9a, Setdb1, Setdb2, Suv39h1 and Suv39h2) was decreased and pluripotency genes (Nanog, Pou5f1, Sox2 and Bmp4) was increased in treatment groups compared with control. These results indicated that treatment with DZNep and UNC0642 improves the epigenetic reprogramming of cloned embryos, which could render beneficial effect on the embryo quality and aberrant gene expression, and finally improve the developmental competence of cloned pig embryos.

Advances in site-specific integration of transgene in animal genome.

The traditional transgenic technologies, such as embryo microinjection, transposon-mediated integration, or lentiviral transfection, usually result in random insertions of the foreign DNA into the host genome, which could have various disadvantages in the establishment of transgenic animals. Therefore, a strategy for site-specific integration of a transgene is needed to generate genetically modified animals with accurate and identical genotypes. However, the efficiency for site-specific integration of transgene is very low, which is mainly caused by two issues. The first one is the low efficiency of inducing double-strand break (DSB) at the target site of host genome in the initial process. The second one is the low efficiency of homologous recombination repair (HDR) between the target site and the donor plasmid carrying homologous arm and foreign genes. HDR is the most common mechanism for site-specific integration of a transgene. DSBs can stimulate DNA repair mainly by two competitive mechanisms, HDR and nonhomologous end joining (NHEJ). Hence, activation of HDR or inhibition of NHEJ can promote the HDR in the integration processes, thereby optimizing a specific targeting of the transgene. In this review, we summarize the recent advances in strategies for improving the site-specific integration of foreign transgene in transgenic technologies.

RAG1/2 knockout pigs with severe combined immunodeficiency.

Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.

Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer.

The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.

Establishment of porcine Xist knockout model using CRISPR/Cas9 system.

Somatic cell nuclear transfer technique has great applications in livestock breeding, production of genetically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this technique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that it may directly or indirectly affects cloning efficiency. In this study, multiple sgRNAs were designed based on the CRISPR/Cas system, and two sites (Target 3 and Target 4) whose mutation efficiency were 1% and 3% at the cellular level were selected. We successfully knocked out Xist with 100% efficiency by microinjecting sgRNAs for Target 3 and Target 4 in embryo. Finally, 6 cloning piglets were born including two Xist-fully-knockout piglets. The follow-up studies on increasing cloning efficiency can be carried out based on the Xist-knockout model.

Transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting.

Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.

Multiple systemic transplantations of human amniotic mesenchymal stem cells exert therapeutic effects in an ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is an adult-onset 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 hSOD1(G93A) 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 ß-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.