Somatic cell nucleus transfer (SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer (NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts (P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells (85% to 91%) than those at 6 and 12 months (66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group (8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.
Animals ; Blastocyst ; physiology ; Cell Proliferation ; Cells, Cultured ; Ear ; embryology ; growth & development ; Fibroblasts ; cytology ; physiology ; transplantation ; Nuclear Transfer Techniques ; Swine ; Swine, Miniature ; anatomy & histology ; embryology ; growth & development

Soft tissue augmentation is a process of implanting tissues or materials to treat wrinkles or soft tissue defects in the body. Over the years, various materials have evolved to correct soft tissue defects, including a number of tissues and polymers. Autogenous dermis, autogenous fat, autogenous dermis-fat, allogenic dermis, synthetic implants, and fillers have been widely accepted for soft tissue augmentations. Tissue engineering technology has also been introduced and opened a new venue of opportunities in this field. In particular, a long-lasting filler consisting of hyaluronic acid filler and living human mesenchymal cells called "injectable tissue-engineered soft tissue" has been created and applied clinically, as this strategy has many advantages over conventional methods. Fibroblasts and adipose-derived stromal vascular fraction cells can be clinically used as injectable tissue-engineered soft tissue at present. In this review, information on the soft tissue augmentation method using the injectable tissue-engineered soft tissue is provided.
Adipocytes/transplantation ; Adipose Tissue/cytology ; Biocompatible Materials ; Connective Tissue/*surgery ; Dermatologic Surgical Procedures/*methods ; Face ; Fibroblasts/transplantation ; Humans ; Hyaluronic Acid/therapeutic use ; Injections, Intradermal ; Mesenchymal Stem Cell Transplantation/*methods ; Mesenchymal Stromal Cells ; Skin ; Skin Aging ; Tissue Engineering/*methods

In covering wounds, efforts should include utilization of the safest and least invasive methods with goals of achieving optimal functional and cosmetic outcome. The recent development of advanced wound healing technology has triggered the use of cells to improve wound healing conditions. The purpose of this review is to provide information on clinically available cell-based treatment options for healing of acute and chronic wounds. Compared with a variety of conventional methods, such as skin grafts and local flaps, the cell therapy technique is simple, less time-consuming, and reduces the surgical burden for patients in the repair of acute wounds. Cell therapy has also been developed for chronic wound healing. By transplanting cells with an excellent wound healing capacity profile to chronic wounds, in which wound healing cannot be achieved successfully, attempts are made to convert the wound bed into the environment where maximum wound healing can be achieved. Fibroblasts, keratinocytes, adipose-derived stromal vascular fraction cells, bone marrow stem cells, and platelets have been used for wound healing in clinical practice. Some formulations are commercially available. To establish the cell therapy as a standard treatment, however, further research is needed.
Blood Platelets/metabolism ; Cell- and Tissue-Based Therapy ; Diabetes Mellitus, Type 2/complications/pathology ; Fibroblasts/cytology/transplantation ; Humans ; Keratinocytes/cytology/transplantation ; Stromal Cells/cytology/transplantation ; Tissue Engineering ; Ulcer/etiology/therapy ; *Wound Healing

Animals ; Cellular Reprogramming ; Dentate Gyrus ; cytology ; Fibroblasts ; cytology ; Mice ; Neural Stem Cells ; cytology ; transplantation ; Swine

OBJECTIVETo observe the effect and safety of autologous cultured skin fibroblasts transplantation for treating depressed facial skin defects.

METHODSA total of 19 patients were treated from Jan, 2010 to Oct, 2010. Autologous skin fibroblasts were separated from postauricular skin biopsy or resected skin tissue in other surgeries such as blepharoplasty. They were cultured and expanded with exclusive method. Cells (2 x 10(7)/ml) within three passages were injected intradermally at the site of skin depression three times at one-month interval. Adverse events were observed and recorded. Clinical effects were evaluated and graded by two unrelated physicians before and 6 months after the first injection.

RESULTSCells from 16 patients were successfully cultured at the first time. The other 3 patients underwent a second harvest. A total amount of 6 x 10(8) cells could be reached within three passages in 45 days. 16 out of 19 patients accomplished the whole course of this study. Minor adverse events were observed in two patients including small ulcer caused by over injection in one patient and slightly redness and swelling in the other. The redness disappeared after a week without any treatment. No serious complications were observed. Significant difference was noticed between the scores obtained before and after the treatment.

CONCLUSIONSFrom this study, neither serious complications nor excessive cell proliferation or scar formation was found after cell injection. The effect of using autologous fibroblast transplantation was obvious and long-lasting, which provides a new choice for the treatment of depressed facial skin defects.

Adult ; Cells, Cultured ; Cicatrix ; therapy ; Face ; abnormalities ; Female ; Fibroblasts ; transplantation ; Humans ; Male ; Middle Aged ; Skin ; cytology ; Transplantation, Autologous ; Treatment Outcome ; Young Adult

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic beta-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.
Animals ; Biological Markers/metabolism ; *Cell Culture Techniques ; *Cell Differentiation ; Cell Proliferation/drug effects ; Cell Separation ; Cells, Cultured ; Dermis/*cytology/drug effects ; Diabetes Mellitus, Experimental/*surgery ; Female ; Fibroblasts/*cytology/drug effects ; Genitalia, Female/*cytology ; Glucose/metabolism ; Hepatocyte Nuclear Factor 3-beta/metabolism ; Homeodomain Proteins/metabolism ; Humans ; Insulin/pharmacology/secretion ; Insulin-Secreting Cells/*cytology/metabolism ; *Islets of Langerhans Transplantation ; Mesenchymal Stem Cells/*cytology/drug effects/metabolism ; Mice ; Mice, Nude ; Niacinamide/pharmacology ; Recovery of Function ; SOXF Transcription Factors/metabolism ; Sodium Selenite/pharmacology ; Trans-Activators/metabolism ; Transferrin/pharmacology

To investigate the potential of gene therapy for the treatment of chronic diseases such as hypertension, chronic heart failure, and chronic renal failure, we established the neonatal rat fibroblast line engineered to secrete the mutant human atrial natriuretic peptide (mhANP), and then transplanted the cell line into young spontaneously hypertensive rats (SHR) subcutaneously. We found that a single transplantation of the cell line caused an obvious rise in the concentration of mhANP in serum 7 d after transplantation ((135 +/- 8) vs (106 +/- 7) pg/mL, P < 0.01). The animals' blood pressure in test group was always remarkably lower than that of empty vector group within 42 d after transplantation, even though the blood pressure in all groups was constantly increasing in the process of ontogeny ((175 +/- 10) mm Hg vs (189 +/- 12) mm Hg, P < 0.05). A maximal blood pressure reduction of 33 mm Hg ((157 +/- 9) mm Hg vs (124 +/- 112) mm Hg, P < 0.01) was observed 14 d post cell transplantation. There was a marked increase in urine volume in test group from second week after treatment beginning ((5.9 +/- 0.7) mL/6 h vs (4.3 +/- 0.8) mL/6 h, P < 0.01) and the effect lasted 14 d ((6.1 +/- 1.1) mL/6 h vs (4.0 +/- 0.8) mL/6 h, P < 0.01), however the statistical difference in concentration of K+ and Na+ in serum and urine was not observed. The results suggested that subcutaneous implantation of fibroblasts-expressing mhANP significantly reduced blood pressure in young SHR during the period of ontogeny and efficiently improved their renal function and the somatic gene transfer of mhANP may have potential value in the treatment of human chronic diseases such as hypertension, chronic heart failure, and chronic renal failure.
Animals ; Atrial Natriuretic Factor ; genetics ; physiology ; Cell Line ; Fibroblasts ; cytology ; metabolism ; transplantation ; Gene Expression ; Genetic Therapy ; methods ; Humans ; Hypertension ; genetics ; physiopathology ; therapy ; Male ; Mutation ; Rats ; Rats, Inbred SHR ; Transfection ; Urination

OBJECTIVETo study the effect of tissue-engineered skin loaded with keratinocyte growth factor (KGF) nanocapsules for skin defect on athymic mice.

METHODSThe acellular dermal matrix (ADM) loaded with KGF-ADM was constructed by means of phacoemulsification solvent evaporation and low temperature drying. The human epidermal stem cells and fibroblasts were captured and identified, then cultivated on the surface of the KGF-ADM. The cell growth was observed. The tissue-engineered skin without KGF was used as sham group. The autogenous skin graft was used as control group. 2 and 6 weeks after the skin was transplanted to the back of athymic mice, the contraction and histological healing of the transplanted skins were observed respectively. Then the immunofluorescence examination with anti-human K10-FITC and beta1-integrin-Cy3 were applied to detect the origin, growth and differentiation of epidermal and dermal cells in tissue-engineered skin.

RESULTSThe epidermal stem cells grew well and attached tightly on KGF-ADM. There were small round stem cells and polygonal terminally-differentiated cells, which appeared a partly cloning growth and a tendency of merging. The tissue-engineered skin with KGF nanocapsules gained better result in repairing the skin defects as compared with the blank group and the control group 2 and 6 weeks after transplantation. The regenerative skin cells could connect and mix closely with the athymic mouse skin cells on the border of skin defect. Meanwhile, the regenerative skin existed some contraction. The histological observation with HE staining showed that the regenerative skin possessed intact epidermis with several cell layers and normal keratose stratum, among which there were still some beta1-integrin (+) cells which represented epidermal stem cells or transient amplifying cells when they were tested by immunofluorescence after 6 weeks of transplantation.

CONCLUSIONSThe tissue-engineered skin loaded with KGF nanocapsules had a better result in repairing athymic mice skin defects than common tissue-engineered skin without KGF nanocapsules or skin auto-graft.

Animals ; Cell Culture Techniques ; Cells, Cultured ; Dermatologic Surgical Procedures ; Dermis ; cytology ; Epidermis ; cytology ; Fibroblast Growth Factor 7 ; Fibroblasts ; cytology ; Humans ; Mice ; Mice, Nude ; Nanocapsules ; Skin ; cytology ; injuries ; Skin Transplantation ; Tissue Engineering ; methods ; Tissue Scaffolds

BACKGROUND/AIMS: Bone marrow-derived cells (BMDC) contribute to tissue maintenance under many kinds of pathologic conditions. We carried out a study to see how BMDC play a role in the treatment of experimental murine colitis. METHODS: We divided the animals into 3 groups and treated them with 50% ethanol (control group), 2,4,6-trinitrobenzene sulfinic acid colitis (TNBS group), and TNBS+bone marrow transplant (BMT group). To induce colitis, TNBS (5.0 mg/mouse) dissolved in 50% ethanol was injected into anus weekly for two weeks. Bone marrow transplantations were performed using bone marrow of male transgenic mouse (donor) with green fluoresence protein (GFP) into female wild type mouse (recipient) three weeks before TNBS instillation. All animals were sacrificed, and colons were extracted one week after the last TNBS instillation. We measured microscopic scores of mucosal injury and investigated the GFP expression for bone marrow engraftment. The immunostaining of vimentin and alpha-smooth muscle actin (alpha-SMA) for myofibroblasts was performed. RESULTS: The score of mucosal injury in the TNBS group was much more severe than those in control, and reduced significantly by BMT (p<0.05). GFP-positive cells were almost deposited in pericryptal niche of BMT group but not at all in both control and TNBS group. Most of myofibroblasts stained with both vimentin and SMA also infiltrated into pericryptal niche. But, the number of myofibroblasts stained with vimentin and SMA in both control and TNBS group was smaller than that in BMT group. CONCLUSIONS: BMDC deposited on pericryptal niche might have a significant role in repairing acute experimental murine colitis.
Actins/metabolism ; Acute Disease ; Animals ; *Bone Marrow Transplantation ; Colitis/chemically induced/pathology/*surgery ; Female ; Fibroblasts/cytology ; Intestinal Mucosa/cytology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Transplantation, Homologous ; Trinitrobenzenesulfonic Acid/*toxicity ; Vimentin/metabolism

OBJECTIVETo construct of tissue engineering skin including active composite dermal matrix.

METHODSThe human fibroblasts and bovine collagen with type I were inoculated on the surface of porcine acellular dermal matrix (PADM) for construction of active dermal substitute, then epidermal cells were inoculated on the dermal matrix for gas-liquid interface culture. The tissue-engineering skin was observed by histological examinations.

RESULTSThe structure of fibroblasts in collagen was intact, which was used to construct composite dermal matrix with PADM. The epithelial structure of tissue-engineering skin was similar to that of normal skin with good cell differentiation. Some phenomena were showed in epidermis: basic layer, stratum spinosum, granular layer and stratum corneum, desmosomes.

CONCLUSIONFibroblasts-Collagen-PADM can be an optimal dermal matrix for construction of tissue-engineering skin.

Animals ; Cattle ; Cell Culture Techniques ; Collagen Type I ; Dermis ; transplantation ; Epidermis ; cytology ; Extracellular Matrix ; transplantation ; Fibroblasts ; cytology ; Humans ; Skin ; cytology ; Skin, Artificial ; Swine ; Tissue Engineering