Objective To investigate the possibility of theadipose tissue-derived stromal cells(ADSCs) to differentiate into the neuron-like cells and to explore a new cell source for the transplantation related to the central nervous system. Methods Adipose was digested by collagenase, cultured in the fetal bovine serum containing a medium. Trypse was used to digest the cells and the cell passage was performed. The 3rd to the 9th passage ADSCs were used to make an induction. Isobutylmethylxanthine, indomethacin, insulin, and dexamethasone were used to induce the ADSCs to differentiate into the neuron-like cells and adipocytes. Sudan black B and immunocytochemistry were used to identify the cells. Results A population of the ADSCs could be isolated from the adult human adipose tissue, they were processed to obtain a fibroblast-like population of the cells and could be maintained in vitro for an extendedperiod with the stable population doubling, and they were expanded as the undifferentiated cells in culture for more than 20 passages, which indicated their proliferative capacity. They expressed vimentin and nestin, and characteristics of the neuron precursor stem cells at an early stage of differentiation. And the majority of the ADSCs also expressed the neuron-specific enolase and βⅢ-tubulin, characteristics of the neurons. Isobutyl-methyxanthine, indomethacin, insulin, and dexamethasone induced 40%-50% of ADSCs to differentiate into adipocytes and 0.1%0.2% of ADSCs into neuron-like cells. The neuron-like cells had a complicated morphology of the neurons, and they exhibited a neuron phenotype, expressed nestin, vimentin, neuron-specific enolase and βⅢ-tubulin, but some neuron-like cells also expressed thesmooth muscle actin (SMA), and the characteristics of the smooth muscle cells; however, the neurons from the central nervous system were never reported to express this kind of protein. Therefore, the neuron-like cells from the ADSCs could be regarded as functional neurons. Conclusion Ourresults support the hypothesis that the adult adipose tissue contains the stem cells capable of differentiating into the neuron-like cells, and they can overcome their mesenchymal commitment, which represents an alternative autologous stemcell source for transplantation related to the central nervous system.
Objective To observe the chondrogenic differentiation of adipose-derived stem cells (ADSCs) by co-culturing chondrocytes and ADSCs. Methods ADSCs and chondrocytes were isolated and cultured from 8 healthy 4-month-old New Zealand rabbits (male or female, weighing 2.2-2.7 kg). ADSCs and chondrocytes at passage 2 were used. The 1 mL chondrocytes at concentration 2 × 104/mL and 1 mL ADSCs at concentration 2 × 104/mL were seeded on the upper layer and lower layer of Transwell 6-well plates separately in the experimental group, while ADSCs were cultured alone in the control group. The morphology changes of the induced ADSCs were observed by inverted phase contrast microscope. The glycosaminoglycan and collagen type II synthesized by the induced ADSCs were detected with toluidine blue staining and immunohistochemistry staining. The mRNA expressions of collagen type II, aggrecan, and SOX9 were detected with real-time fluorescent quantitative PCR. Results ADSCs in the experimental group gradually became chondrocytes-like in morphology and manifested as round; while ADSCs in the control group manifested as long spindle in morphology with whirlool growth pattern. At 14 days after co-culturing, the results of toluidine blue staining and immunohistochemistry staining were positive in the experimental group, while the results were negative in the control group. The results of real-time fluorescent quantitative PCR indicated that the expression levels of collagen type II, aggrecan, and SOX9 mRNA in the experimental group (1.43 ± 0.07, 2.13 ± 0.08, and 1.08 ± 0.08) were significantly higher than those in the control group (0.04 ± 0.03, 0.13 ± 0.04, and 0.10 ± 0.02) (P lt; 0.05). Conclusion ADSCs can differentiate into chondrocytes-like after co-culturing with chondrocytes.
Objective To study the transfection and expression of pleiotrophin (Ptn) gene in mice adipose-derived stem cells (ADSCs) so as to provide a new approach for the treatment of ischemic injury. Methods ADSCs from clean inbred C57BL/6W mice (weighing, 15-20 g) were isolated and cultured in vitro. The cell surface markers (CD29 and CD44) of ADSCs were identified by flow cytometry. The ADSCs were transfected with plasmid pIRES2-LEGFPN1 (containing Ptn gene coding sequence) as experimental group (group A) and with plasmid pLEGFP-N1 (containing GFP gene coding sequence) as control group (group B). After ADSCs were transfected by different plasmids respectively, the cells containing Ptn gene were selected by G418 (the best selected concentration was 200 μg/mL), and the immunophenotype of the cells was identified by flow cytometry after transfection. Meanwhile, real-time fluorescence quantitative PCR and Western blot were used to analyse the expression levels of Ptn mRNA and PTN protein in selected cells. Results The mice ADSCs were isolated and cultured successfully in vitro. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.5% and 95.8%, respectively; the double positive rate of CD44 and CD29 was 93.6%. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.1% and 95.6%, respectively after transfection of Ptn gene; the double positive rate of CD44 and CD29 was 93.4%. The expression levels of Ptn gene and PTN protein in group A were significantly higher than those in group B (P lt; 0.05). Conclusion The ADSCs can be stablely transfected by Ptn gene, the transfected ADSCs can express PTN protein highly, which is a new idea for tissue engineering of vascular reconstruction.
Objective To introduce types and differentiation potentials of stem cells from adipose tissue, and its applications on regenerative medicine and advantages. Methods The literature of original experimental study and clinical research about bone marrow mesenchymal stem cells (BMSCs), adipose-derived stem cells (ADSCs), and dedifferentiated fat (DFAT) cells was extensively reviewed and analyzed. Results ADSCs can be isolated from stromal vascular fraction. As ADSCs have multi-lineage potentials, such as adipogenesis, osteogenesis, chondrogenesis, angiogenesis, myogenesis, and neurogenesis, they have already been successfully used in regenerative medicine areas. Dramatically, mature fat cells can be dedifferentiated and changed into fibroblast-like cells, named DFAT cells, via ceiling culture method. DFAT cells also had the same multi-lineage potentials as ADSCs, differentiating into adipocytes, osteocytes, chondrocytes, endothelial cells, muscle cells, and nerve cells. Compared with BMSCs which are commonly used as adult stem cells, ADSCs and DFAT cells have extensive sources and can be easily acquired. While compared with ADSCs, DFAT cells have good homogeneity and b proliferation capacity. Conclusion As a potential source of stem cells, adipose tissue will provide a new promising for regenerative medicine.
Objective To find a kind of simple and effective method for purifying and label ing stromal vascular fraction cells (SVFs) so as to provide a theoretical basis for cl inical application of SVFs. Methods The subcutaneous adi pose tissue were harvested form volunteers. The adi pose tissue was digested with 0.065%, 0.125%, and 0.185% type I collagenase,respectively. SVFs were harvested after digestion and counted. After trypan blue staining, the rate of viable cells was observed. SVFs was labeled by 1, 1’-dioctadecyl-3, 3, 3’, 3’-2-tetramethy-lindocyanine perchlorate (DiI). The fluorescent label ing and growth was observed under an inverted fluorescence microscope. MTT assay was used to detect cell proliferation. Results The number of SVFs was (138.68 ± 11.64) × 104, (183.80 ± 10.16) × 104, and (293.07 ± 8.31) × 104 in 0.065% group, 0.125% group, and 0.185% group, respectively, showing significant differences among 3 groups (P lt; 0.01). The rates of viable cells were 91% ± 2%, 90% ± 2%, and 81% ± 2% in 0.065% group, 0.125% group, and 0.185% group, respectively, and it was significantly higher in 0.065% group and 0.125% group than in 0.185% group (P lt; 0.01), but no significant difference was found between 0.065% group and 0.125% group (P=0.881). Inverted fluorescence microscope showed that the cell membranes could be labeled by DiI with intact cell membrane, abundant cytoplasm, and good shape, but nucleus could not labeled. SVFs labeled by DiI could be cultured successfully and maintained a normal form. MTT assay showed that similar curves of the cell growth were observed before and after DiI labeled to SVFs. Conclusion The optimal collagenase concentration for purifying SVFs is 0.125%. DiI is a kind of ideal fluorescent dye for SVFs.
Objective To review research progress of adipose tissuederived stromal cells (ADSCs).Methods The recent articles on ADSCs were extensively reviewed, and the culture and differentiation ability of ADSCs were investigated.Results A population of stem cells could be isolated from adult adipose tissue, they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling. The majority of the isolated cells were mesenchymal origin, with a few pericytes,endothelial cells and smooth muscle cells. ADSCs could be induced to differentiate intomultiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic and adipogenic cells, they could also differentiate into nerve cells.Conclusion ADSCs can substitute mesenchymal stem cells and become an alternative stem cells source for tissue engineering.
ObjectiveTo review the research progress of adipose-derived stem cells (ADSCs) in skin scar prevention and treatment.MethodsThe related literature was extensively reviewed and analyzed. The recent in vitro and in vivo experiments and clinical studies on the role of ADSCs in skin scar prevention and treatment, and the possible mechanisms and biomaterials to optimize the effect of ADSCs were summarized.ResultsAs demonstrated by in vitro and in vivo experiments and clinical studies, ADSCs participate in the whole process of skin wound healing and may prevent and treat skin scars by reducing inflammation, promoting angiogenesis, or inhibiting (muscle) fibroblasts activity to reduce collagen deposition through the p38/mitogen-activated protein kinase, peroxisome proliferator activated receptor γ, transforming growth factor β1/Smads pathways. Moreover, bioengineered materials such as hydrogel from acellular porcine adipose tissue, porcine small-intestine submucosa, and poly (3-hydroxybutyrate-co-hydroxyvalerate) scaffold may further enhance the efficacy of ADSCs in preventing and treating skin scars.ConclusionRemarkable progress has been made in the application of ADSCs in skin scar prevention and treatment. While, further studies are still needed to explore the application methods of ADSCs in the clinic.
Objective To review the study on adi pose derived stem cells (ADSCs) in the therapy of urological diseases. Methods The recent l iterature concerning ADSCs in bladder repair, urethral reconstruction, incontinence treatment, and erectile dysfunction treatment was reviewed. Results The appl ication of tissue engineering using ADSCs has made significant achievements in the treatment of urological diseases and in animal studies, and has been initially used in cl inicaland has achieved a good therapeutic effect. Conclusion Tissue engineering using ADSCs has good prospects in the study on urological diseases, and is expected to widely used in the treatment of urological diseases.
ObjectiveTo investigate the effect of burn on the fat metabolism by observing the effect of burn serum on the proliferation and adipose differentiation of 3T3-L1 preadipocytes. MethodsForty-eight male Sprague Dawley rats were randomly divided into sham burn group and burn at 1, 4, 7, 14, and 21 days groups, 8 rats in each group. The rats in burn groups were made the full-thickness thermal burns comprising 30% total body surface area. At 1, 4, 7, 14, and 21 days after burn, the serum of burn rats was collected. The rats in sham burn group were not treated as normal control. The proliferation activity of 3T3-Ll cells was detected using MTT method after treated by normal and burn serum. The burn serum having the highest proliferation inhibitory effect was chosen for subsequent study. The growth of 3T3-L1 cells in normal serum group (group A), burn serum group (group B), normal serum and adipogenic induction group (group C), burn serum and adipogenic induction group (group D) was observed using inverted microscope. After 7 days of treatment, the adipocytes was stained by oil red O and the absorbance (A) value was measured. The mRNA and protein levels of preoxisome proliferator-activated receptor γ (PPAR-γ) and lipoprotein lipase (LPL) were detected by real-time quantitative PCR and Western blot. ResultsThe proliferation ability of 3T3-L1 cells was significantly reduced in the group treated by 4-or 7-day burn serum (P<0.05), especially 7-day burn serum treatment group (P<0.05). Under inverted microscope, the cell morphology in group A and group B had no obvious change, but a large number of fat cells were observed in group C and a few were observed in group D. The positive or weak positive oil red O staining was observed in group C or group D, respectively. The cell counting and A value were significantly higher in group A than in group B, and in group C than in group D (P<0.05). The mRNA level of PPAR-γ in group B was significantly reduced when compared with that in group A (P<0.05). No significant difference was found in LPL mRNA levels and protein levels of PPAR-γ and LPL between group A and group B (P>0.05). The mRNA and protein levels of PPAR-γ and LPL were significantly attenuated in group D when compared with those in group C (P<0.05). ConclusionThe adipose differentiation of 3T3-L1 preadipocytes can be significantly reduced after treated by 7-day burn serum of rat.
Objective To construct chemically extracted acellular nerve allograft (CEANA) with Schwann cells (SCs) from different tissues and to compare the effect of repairing peripheral nerve defect. Methods Bone marrow mesenchymal stem cells (BMSCs) and adi pose-derived stem cells (ADSCs) were isolated and cultured from 3 4-week-old SD mice with weighing 80-120 g. BMSCs and ADSCs were induced to differentiated MSC (dMSC) and differentiated ADSC (dADSC) in vitro.dMSC and dADSC were identified by p75 protein and gl ial fibrillary acidic protein (GFAP). SCs were isolated and culturedfrom 10 3-day-old SD mice with weighing 6-8 g. CEANA were made from bilateral sciatic nerves of 20 adult Wistar mice with weighing 200-250 g. Forty adult SD mice were made the model of left sciatic nerve defect (15 mm) and divided into 5 groups (n=8 per group) according to CEANA with different sources of SCs: autografting (group A), acellular grafting with SCs (5 × 105) (group B), acellular grafting with dMSCs (5 × 105) (group C), acellular grafting with dADSCs (5 × 105) (group D), and acellular grafting alone (group E). Motor and sensory nerve recovery was assessed by Von Frey and tension of the triceps surae muscle testing 12 weeks after operation. Then wet weight recovery ratio of triceps surae muscles was measured and histomorphometric assessment of nerve grafts was evaluated. Results BMSCs and ADSCs did not express antigens CD34 and CD45, and expressed antigen CD90. BMSCs and ADSC were differentiated into similar morphous of SCs and confirmed by the detection of SCs-specific cellsurface markers. The mean 50% withdrawal threshold in groups A, B, C, D, and E was (13.8 ± 2.3), (15.4 ± 6.5), (16.9 ± 5.3), (16.3 ± 3.5), and (20.0 ± 5.3) g, showing significant difference between group A and group E (P lt; 0.01). The recovery of tension of the triceps surae muscle in groups A, B, C, D, and E was 87.0% ± 9.7%, 70.0% ± 6.6%, 69.0% ± 6.7%, 65.0% ± 9.8%, and 45.0%± 12.1%, showing significant differences between groups A, B, C, D, and group E (P lt; 0.05). No inflammatory reactionexisted around nerve graft. The histological observation indicated that the number of myel inated nerve fiber and the myel in sheath thickness in group E were significantly smaller than that in groups B, C, and D (P lt; 0.01). The fiber diameter of group B was significantly bigger than that of groups C and D (P lt; 0.05) Conclusion CEANA supplementing with dADSC has similar repair effect in peripheral nerve defect to supplementing with dMSC or SCs. dADSC, as an ideal seeding cell in nerve tissue engineering, can be benefit for treatment of peripheral nerve injuries.