Transplantation of Adipose-Derived Stem Cells Alleviates Striatal Degeneration in a Transgenic Mouse Model for Multiple System Atrophy.

Patients with multiple system atrophy (MSA), a progressive neurodegenerative disorder of adult onset, were found less than 9 years of life expectancy after onset. The disorders include bradykinesia and rigidity commonly seen in Parkinsonism disease and additional signs such as autonomic dysfunction, ataxia, or dementia. In clinical treatments, MSA poorly responds to levodopa, the drug used to remedy Parkinsonism disease. The exact cause of MSA is still unknown, and exploring a therapeutic solution to MSA remains critical. A transgenic mouse model was established to study the feasibility of human adipose-derived stem cell (ADSC) therapy in vivo. The human ADSCs were transplanted into the striatum of transgenic mice via intracerebral injection. As compared with sham control, we reported significantly enhanced rotarod performance of transgenic mice treated with ADSC at an effective dose, 2 × 105 ADSCs/mouse. Our ex vivo feasibility study supported that intracerebral transplantation of ADSC might alleviate striatal degeneration in MSA transgenic mouse model by improving the nigrostriatal pathway for dopamine, activating autophagy for α-synuclein clearance, decreasing inflammatory signal, and further cell apoptosis, improving myelination and cell survival at caudate-putamen.

Adipose-derived Stem Cells Stimulated with n-Butylidenephthalide Exhibit Therapeutic Effects in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) causes motor dysfunction and dopaminergic cell death. Drug treatments can effectively reduce symptoms but often cause unwanted side effects. Stem cell therapies using cell replacement or indirect beneficial secretomes have recently emerged as potential therapeutic strategies. Although various types of stem cells have been proposed as possible candidates, adipose-derived stem cells (ADSCs) are easily obtainable, more abundant, less ethically disputed, and able to differentiate into multiple cell lineages. However, treatment of PD using adult stem cells is known to be less efficacious than neuron or embryonic stem cell transplantation. Therefore, improved therapies are urgently needed. n-Butylidenephthalide (BP), which is extracted from Angelica sinensis, has been shown to have anti-inflammatory and neuroprotective effects. Indeed, we previously demonstrated that BP treatment of ADSCs enhances the expression of neurogenesis and homing factors such as nuclear receptor related 1 protein, stromal-derived factor 1, and brain-derived neurotrophic factor. In the present study, we examined the ability of BP-pretreated ADSC transplantation to improve PD motor symptoms and protect dopamine neurons in a mouse model of PD. We evaluated the results using neuronal behavior tests such as beam walking, rotarod, and locomotor activity tests. ADSCs with or without BP pretreatment were transplanted into the striatum. Our findings demonstrated that ADSC transplantation improved motor abilities with varied efficacies and that BP stimulation improved the therapeutic effects of transplantation. Dopaminergic cell numbers returned to normal in ADSC-transplanted mice after 22 d. In summary, stimulating ADSCs with BP improved PD recovery efficiency. Thus, our results provide important new strategies to improve stem cell therapies for neurodegenerative diseases in future studies.

Antifibrotic Activity of Human Placental Amnion Membrane-Derived CD34+ Mesenchymal Stem/Progenitor Cell Transplantation in Mice With Thioacetamide-Induced Liver Injury.

: Liver fibrosis represents the end stage of chronic liver inflammatory diseases and is defined by the abnormal accumulation of extracellular matrix in the liver. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension. Liver transplantation has been the most effective treatment for these diseases, but the procedure is limited by the shortage of suitable donors. Mesenchymal stromal cells (MSCs) have shown great potential in the treatment of chronic inflammatory diseases associated with fibrosis. This study aimed to evaluate the therapeutic effect of MSC-based cell transplantation as an alternative treatment for liver fibrosis. A CD34-positive subpopulation of human placental amnion membrane-derived stem/progenitor cells (CD34+ AMSPCs) was isolated through the depletion of CD34-negative stromal fibroblasts (CD34- AMSFCs) facilitated by CD34 fluorescence-activated cell sorting, enriched and expanded ex vivo. These cells express pluripotency markers and demonstrate multidirectional differentiation potentials. Comparative analysis was made between CD34+ AMSPCs and CD34- AMSFCs in terms of the expressions of stemness surface markers, embryonic surface antigens, and multilineage differentiation potentials. A mouse model of liver fibrosis was established by thioacetamide (TAA) administration. When injected into the spleen of TAA-injured mice, human placental amnion membrane-derived MSCs (hAM-MSCs) can engraft into the injury site, ameliorate liver fibrosis, and restore liver function, as shown by pathological and blood biochemical analysis and downregulated gene expressions associated with liver damage. CD34+ AMSPCs represent a more primitive subset of hAM-MSCs and could be a suitable candidate with a potentially better safety profile for cell-based therapy in treatment of liver diseases associated with fibrosis. SIGNIFICANCE: In this study, a CD34+ subpopulation of stem/progenitor cells derived from neonatal placental amnion membrane, denoted as CD34+ AMSPCs, were identified, enriched, and characterized. These cells are highly proliferative, express mesenchymal stromal cells and pluripotent stem cell markers, and demonstrate multidirectional differentiation potentials, indicating their promising application in clinical regenerative therapies. CD34+ AMSPC transplantation ameliorated liver fibrosis in mice with drug-induced liver injury. These cells represent a potential therapeutic agent for treating liver diseases associated with fibrosis.

Zebrafish arl6ip1 is required for neural crest development during embryogenesis.

BACKGROUND: Although the embryonic expression pattern of ADP ribosylation factor-like 6 interacting protein 1 (Arl6ip1) has been reported, its function in neural crest development is unclear. METHODS/PRINCIPAL FINDINGS: We found that knockdown of Arl6ip1 caused defective embryonic neural crest derivatives that were particularly severe in craniofacial cartilages. Expressions of the ectodermal patterning factors msxb, dlx3b, and pax3 were normal, but the expressions of the neural crest specifier genes foxd3, snai1b, and sox10 were greatly reduced. These findings suggest that arl6ip1 is essential for specification of neural crest derivatives, but not neural crest induction. Furthermore, we revealed that the streams of crestin- and sox10-expressing neural crest cells, which migrate ventrally from neural tube into trunk, were disrupted in arl6ip1 morphants. This migration defect was not only in the trunk neural crest, but also in the enteric tract where the vagal-derived neural crest cells failed to populate the enteric nervous system. We found that this migration defect was induced by dampened Shh signaling, which may have resulted from defective cilia. These data further suggested that arl6ip1 is required for neural crest migration. Finally, by double-staining of TUNEL and crestin, we confirmed that the loss of neural crest cells could not be attributed to apoptosis. CONCLUSIONS/SIGNIFICANCE: Therefore, we concluded that arl6ip1 is required for neural crest migration and sublineage specification.

Nkx2.7 and Nkx2.5 function redundantly and are required for cardiac morphogenesis of zebrafish embryos.

BACKGROUND: Nkx2.7 is the tinman-related gene, as well as orthologs of Nkx2.5 and Nkx-2.3. Nkx2.7 and Nkx2.5 express in zebrafish heart fields of lateral plate mesoderm. The temporal and spatial expression patterns of Nkx2.7 are similar to those of Nkx2.5, but their functions during cardiogenesis remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here, Nkx2.7 is demonstrated to compensate for Nkx2.5 loss of function and play a predominant role in the lateral development of the heart, including normal cardiac looping and chamber formation. Knocking down Nkx2.5 showed that heart development was normal from 24 to 72 hpf. However, when knocking down either Nkx2.7 or Nkx2.5 together with Nkx2.7, it appeared that the heart failed to undergo looping and showed defective chambers, although embryos developed normally before the early heart tube stage. Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage. We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20. Although heart defects were induced by overexpression of an injection of 150-pg Nkx2.5 or 5-pg Nkx2.7 mRNA, either Nkx2.5 or Nkx2.7 mRNA rescued the defects induced by Nkx2.7-morpholino(MO) and Nkx2.5-MO with Nkx2.7-MO. CONCLUSIONS AND SIGNIFICANCE: Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage.

Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.

In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (-870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from -210 to 34 (-210/34) was sufficient to drive heart-specific expression, with a -210/-73 motif as a basal promoter and a -210/-174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at -119, evidence that A at -119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2.