Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

Background

There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.

Results

Goat MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency (CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection. BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture, exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.

Conclusions

Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.

Electronic supplementary material

The online version of this article (doi:10.1186/2049-1891-6-1) contains supplementary material, which is available to authorized users.     

Ultrastructural and Extracellular Protein Changes in Cell Suspension Cultures of Populus euphratica Associated with Low Temperature-induced Cold Acclimation

Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of-17.5 ℃) in cell suspension at 4-5 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of-12.5 ℃ in nonacclimated cells to LT50 of-17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.     

油樟油细胞和粘液细胞发育的超微结构

利用超薄切片法和透射电镜研究油樟油细胞和粘液细胞发育过程。依据油细胞3层细胞壁的发育将其分为4个阶段。阶段1：仅有初生纤维素壁层，又可分为原始细胞和细胞液泡化二时期。质体内具白色小泡和黑色嗜饿滴，细胞质中有黑色或灰色的嗜饿物质，以及嗜饿物质与液泡的融合。阶段2：栓质化壁层的形成。片层状的栓质叠加在初生纤维素壁内侧。阶段3：内纤维素壁层的形成。较厚而结构松散的内纤维素壁层逐步形成，并叠加在栓质化壁层的内侧，大液泡成为充满嗜饿油脂的油囊。阶段4：油细胞成熟及细胞质解体。杯形构造由内纤维素壁层向细胞腔内突起形成，油囊由液泡包被连接到杯形构造中。解体的细胞质变得电子不透明或呈杂乱状态。粘液细胞发育方式有两种：一种是由内纤维素形成以后的油细胞发育而来，其细胞质中不断产生以同心圆或螺旋线方式排列的多膜结构，并充满整个细胞腔，最后多膜结构解体而成为丝状或颗粒状的粘液；另一种是由已完全成熟的油细胞发育而来，其油囊中的油呈不均匀的状态，并产生局部降解点，逐步扩大，最后油完全降解成颗粒成或丝状的粘液。     

Effect of atorvastatin on TRPC5 expression in atherosclerosis of apolipoprotein E-knockout mice

AIM: To observe the changes of transient receptor potential channel 5 (TRPC5) in vascular smooth muscle cells (VSMCs) of apolipoprotein E-knockout (ApoE^-/-) mice and the effect of atorvastatin interference, and to investigate the mechanism of atorvastatin therapy. METHODS: Male ApoE^-/- mice at 6 weeks of age were used to establish the atherosclerosis model by feeding with hyperlipidic diet. The mice were randomly divided into model group and atorvastatin group. The mice in atorvastatin group were lavaged with atorvastatin at 20 mg·kg^-1·d^-1, while the mice in model group received normal saline. The healthy C57BL/6J mice with the same age and the same genetic background, feeding with ordinary food, served as control group. At the time points of 14 and 24 weeks, the mice were sacrificed. The serum was collected for detecting the lipid levels. The aortic roots of the heart were taken to make paraffin sections with HE staining for measuring and comparing the relative atherosclerotic plaque area in each section. The expression of TRPC5 in VSMCs was examined with immunohistochemical staining. The mRNA levels of TRPC5 in the serum and the thoracoabdominal aorta were measured by real-time PCR. RESULTS: Compared with model group, blood lipids in atorvastatin group were significantly decreased, and the formation of plaque under aorta intima also decreased. The protein expression of TRPC5 in atorvastatin group decreased significantly compared with model group. Compared with 20-week model group, TRPC5 in 30-week model group showed increasing tendency, but has no statistical significance. Compared with 20-week atorvastatin group, TRPC5 of 30-week atorvastatin group declined. CONCLUSION: Atorvastatin suppresses TRPC5 expression, thus attenuating atherosclerotic development in ApoE^-/- mice.     

Effect of gonadotropin-releasing hormone analogue on human breast cancer cell line in vitro

AIM: To investigate the effects of gonadotropin-releasing hormone (GnRH) analogue on the growth of breast cancer cell lines MCF-7 and MDA-MB-231 in vitro and to explore the related mechanisms with PI3K/Akt or ERK/MAPK pathways. METHODS: The proliferation of human breast cancer cell lines MCF-7 and MDA-MB-231 treatment with triptorelin was detected by MTT assay and the distribution of the cell cycle was determined by flow cytometry. The phosphorylation of the ERK1/2 and Akt was evaluated by Western blotting. RESULTS: Triptorelin inhibited the proliferation of MCF-7 cells at concentration of 10^-5 mol/L after treated for 192 h or at concentration of 10^-4 mol/L after treated for 168 h and 192 h. Triptorelin inhibited the proliferation of MDA-MB-231 cells at concentration of 10^-4 mol/L after treated for 192 h (P<0.05).Treatment with triptorelin for 192 h at concentration of 10^-4 mol/L had no statistical significance effect on phosphorylation of ERK1/2 and Akt(P>0.05).CONCLUSION: Inhibitory effect of GnRH analogue triptorelin on human breast cancer cells is not just the connection with the down-regulation of pituitary hormone, but also a direct inhibitory effect. The role may not be involved in the activation of ERK/MAPK and PI3K/Akt signaling pathways.     

Preparation of recombinant PTD-HSP27 and verification of its ability to penetrate the cell membrane of human lens epithelial cells and rabbit cornea

AIM: To construct the prokaryotic expression system containing protein transduction domain (PTD) with heat shock protein 27 (HSP27) in order to prepare and purify the recombinant protein, and to verify whether the recombinant protein PTD-HSP27 has the ability to penetrate the human lens epithelial cell (HLEC) membrane and the rabbit cornea. METHODS: The plasmid pKYB-PTD-HSPB1-6His was constructed by the technique of overlap extension PCR. The plasmid was transformed and PTD-HSP27 was purified through nickel affinity chromatography column and identified by Western blotting. PTD-HSP27-6His was labeled with the fluorescein isothiocyanate (FITC). The penetrating ability of PTD-HSP27 into HLECs and rabbit cornea was tested. RESULTS: The recombinant PTD-HSP27 plasmid was successfully cloned and effectively expressed. The correctness of the recombinant protein PTD-HSP27 was demonstrated. Fluorescence microscopic examination showed that PTD-HSP27-FITC was internalized by HLECs. Fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor. CONCLUSION: The recombined gene PTD-HSPB1 was constructed by overlap extension PCR technique and the PTD-HSP27 fusion protein was prepared and purified by nickel affinity chromatography column. Using the technique of PTD-fusion protein, HSP27 was transduced into HLECs and passed through the cornea.     

Expression of Gab2 in human osteosarcoma U2-OS cells

AIM: To investigate the expression of Grb2-associated binding protein 2 (Gab2) in human osteosarcoma cells and its relationship with the invasion and metastases of human osteosarcoma cells. METHODS: The technique of small RNA interference was used to transfect human osteosarcoma U2-OS cell lines. Western blotting and RT-PCR were used to detect the protein and mRNA expression of Gab2 in transfected U2-OS cells. After transfection, through chemotaxis and invasion assays in vitro, the cell migration and invasion abilities were detected. RESULTS: After transfection, the expression of Gab2 at mRNA and protein levels in Gab2 siRNA transfected cells (SiGab2/U2-OS) was lower than that in scrambled siRNA transfected cells (Scr/U2-OS) and U2-OS cells. After stimulation with epidermal growth factor (EGF) at concentration of 10 μg/L, the migration SiGab2/U2-OS cells was significantly less than Scr/U2-OS cells and U2-OS cells (P<0.01). The number of invasion cells of SiGab2/U2-OS group was significantly lower than the other 2 control groups (P<0.01). CONCLUSION: Inhibition of Gab2 expression obviously attenuates the migration and invasion abilities of human osteosarcoma U2-OS cell line.     

Effects of myosin light chain kinase on regulation of endothelial barrier function

Myosin light chain kinase (MLCK) activates the regulatory light chain of myosin II, and the phosphorylated myosin light chain leads to actomyosin contractile activity, as well as the cell contraction and increasing intercellular gap, which finally results in endothelial barrier dysfunction. MLCK-dependent hyperpermeability occurs in response to multiple cell signaling molecules and signaling pathways, including Ca²⁺, Src, PKC, NO, cGMP and mitogen activated protein kinases (MAPK). In this review, different mechanisms of endothelial hyperpermeability mediated by MLCK are discussed.     

Role of 17-AAG in inducing apoptosis and cell cycle arrest of HCT-15 cells

AIM: To investigate the effects of 17-AAG on apoptosis and cell cycle of HCT-15 cells and to clarify the related mechanisms. METHODS: MTT method was employed to evaluate the inhibitory effects of 17-AAG with Aifferent time and different doses on the proliferation of HCT-15 cells. The cells were stained with Annexin V-FITC/propidiumiodide and measured by flow cytometry. The expression of STAT3, cyclin D1, Cyt C, caspase 9 and caspase 3 at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS: Treatment with 17-AAG at concentration of 1.25~20 mg/L for 24 h and 48 h significantly inhibited the activity of HCT-15 cells at both time-and concentration-dependent manners. Treatment with 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h significantly induced apoptosis and cell cycle arrest of HCT-15 cells. The exposure of 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h to the HCT-15 cells significantly down-regulated the expression of STAT3 and cyclin D1 at mRNA and protein levels, but up-regulated Cyt C, caspase 9 and caspase 3 mRNA and protein in a concentration-dependent manner. CONCLUSION: 17-AAG inhibits the cell activity, induces apoptosis and G₁ arrest by down-regulating the expression of cyclin D1, and promoting the mitochondria apoptosis through STAT3 pathway.     

Mesenchymal stem cell-induced CD8α+ Jagged2high CD11bhigh regulatory dendritic cells prevent and treat mouse aGVHD

AIM: To investigate the role of mesenchymal stem cell-induced regulatory dendritic cells (MSC-DCregs) in mouse acute graft-versus-host disease(aGVHD) model. METHODS: Bone marrow cells from BALB/c (H-2^d) mice were isolated and were induced to differentiate into DCs. The DCs were selected by flow cytometry, and after 10 d co-culture with MSCs, they were induced to be MSC-DCregs. Male 8-week-old C57BL/6 (H-2^b) mice were used as donor mice. The female 8-week-old BALB/c (H-2^d) mice, who had received 100 cm source-skin distance, 30 cm×30 cm radiation field, 700 cGy total body irradiation (TBI) pretreatment were used as recipient mice. The recipients were divided into 5 groups: control group, TBI group (injected with medium only), bone marrow transplantation group (injected with 1×10⁷ bone marrow cells), aGVHD group (injected with 1×10⁷ bone marrow cells and 1×10⁷ spleen cells), and MSC-DCregs group (injected with 1×10⁷ bone marrow cells, 1×10⁷ spleen cells and 1×10⁶ MSC-DCregs). The white blood cell count, recipients' chimerism, clinical evaluation of aGVHD, survival analysis and pathological changes were determined. RESULTS: Hematopoieic recovery was seen at 10 d after transplantation. The recipients' chimerism was parallel to the donors' at 30 d. The median survival time of the mice in aGVHD group and MSC-DCregs group was 27 d and 33 d, and the survival rates at 30 d were 20% and 100% (P<0.01), respectively. The clinical scores of the mice in MSC-DCregs group were lower than those in aGVHD group (P<0.01). Moreover, the pathological changes in the skin and liver of the mice in MSC-DCregs group were less serious than those in aGVHD group. CONCLUSION: The MSC-DCregs induce an aGVHD tolerance in vivo, and further research of its mechanism is still in great necessary.