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Sample records for allogenic adipose-derived stromal

  1. Acupoint Injection of Autologous Stromal Vascular Fraction and Allogeneic Adipose-Derived Stem Cells to Treat Hip Dysplasia in Dogs

    PubMed Central

    Marx, Camila; Silveira, Maiele Dornelles; Selbach, Isabel; da Silva, Ariel Silveira; Braga, Luisa Maria Gomes de Macedo; Camassola, Melissa; Nardi, Nance Beyer

    2014-01-01

    Stem cells isolated from adipose tissue show great therapeutic potential in veterinary medicine, but some points such as the use of fresh or cultured cells and route of administration need better knowledge. This study aimed to evaluate the effect of autologous stromal vascular fraction (SVF, n = 4) or allogeneic cultured adipose-derived stem cells (ASCs, n = 5) injected into acupuncture points in dogs with hip dysplasia and weak response to drug therapy. Canine ASCs have proliferation and differentiation potential similar to ASCs from other species. After the first week of treatment, clinical evaluation showed marked improvement compared with baseline results in all patients treated with autologous SVF and three of the dogs treated with allogeneic ASCs. On days 15 and 30, all dogs showed improvement in range of motion, lameness at trot, and pain on manipulation of the joints, except for one ASC-treated patient. Positive results were more clearly seen in the SVF-treated group. These results show that autologous SVF or allogeneic ASCs can be safely used in acupoint injection for treating hip dysplasia in dogs and represent an important therapeutic alternative for this type of pathology. Further studies are necessary to assess a possible advantage of SVF cells in treating joint diseases. PMID:25180040

  2. Insulin-secreting adipose-derived mesenchymal stromal cells with bone marrow-derived hematopoietic stem cells from autologous and allogenic sources for type 1 diabetes mellitus.

    PubMed

    Thakkar, Umang G; Trivedi, Hargovind L; Vanikar, Aruna V; Dave, Shruti D

    2015-07-01

    Stem cell therapy (SCT) is now the up-coming therapeutic modality for treatment of type 1 diabetes mellitus (T1DM). Our study was a prospective, open-labeled, two-armed trial for 10 T1DM patients in each arm of allogenic and autologous adipose-derived insulin-secreting mesenchymal stromal cells (IS-AD-MSC)+bone marrow-derived hematopoietic stem cell (BM-HSC) infusion. Group 1 received autologous SCT: nine male patients and one female patient; mean age, 20.2 years, disease duration 8.1 years; group 2 received allogenic SCT: six male patients and four female patients, mean age, 19.7 years and disease duration, 7.9 years. Glycosylated hemoglobin (HbA1c) was 10.99%; serum (S.) C-peptide, 0.22 ng/mL and insulin requirement, 63.9 IU/day in group 1; HbA1c was 11.93%, S.C-peptide, 0.028 ng/mL and insulin requirement, 57.55 IU/day in group 2. SCs were infused into the portal+thymic circulation and subcutaneous tissue under non-myelo-ablative conditioning. Patients were monitored for blood sugar, S.C-peptide, glutamic acid decarboxylase antibodies and HbA1c at 3-month intervals. Group 1 received mean SCs 103.14 mL with 2.65 ± 0.8 × 10(4) ISCs/kg body wt, CD34+ 0.81% and CD45-/90+/73+, 81.55%. Group 2 received mean SCs 95.33 mL with 2.07 ± 0.67 × 10(4) ISCs/kg body wt, CD34+ 0.32% and CD45-/90+/73+ 54.04%. No untoward effect was observed with sustained improvement in HbA1c and S.C-peptide in both groups with a decrease in glutamic acid decarboxylase antibodies and reduction in mean insulin requirement. SCT is a safe and viable treatment option for T1DM. Autologous IS-AD-MSC+ BM-HSC co-infusion offers better long-term control of hyperglycemia as compared with allogenic SCT. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  3. Allogeneic Adipose-Derived Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune Encephalomyelitis by Regulating Self-Reactive T Cell Responses and Dendritic Cell Function

    PubMed Central

    Gonzalez-Rey, Elena; Martin, Francisco; Oliver, F. Javier

    2017-01-01

    Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising therapy for autoimmune diseases, including multiple sclerosis (MS). Administration of MSCs to MS patients has proven safe with signs of immunomodulation but their therapeutic efficacy remains low. The aim of the current study has been to further characterize the immunomodulatory mechanisms of adipose tissue-derived MSCs (ASCs) in vitro and in vivo using the EAE model of chronic brain inflammation in mice. We found that murine ASCs (mASCs) suppress T cell proliferation in vitro via inducible nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 1/2 activities. mASCs also prevented the lipopolysaccharide- (LPS-) induced maturation of dendritic cells (DCs) in vitro. The addition of the COX-1/2 inhibitor indomethacin, but not the iNOS inhibitor L-NAME, reversed the block in DC maturation implicating prostaglandin (PG) E2 in this process. In vivo, early administration of murine and human ASCs (hASCs) ameliorated myelin oligodendrocyte protein- (MOG35-55-) induced EAE in C57Bl/6 mice. Mechanistic studies showed that mASCs suppressed the function of autoantigen-specific T cells and also decreased the frequency of activated (CD11c+CD40high and CD11c+TNF-α+) DCs in draining lymph nodes (DLNs). In summary, these data suggest that mASCs reduce EAE severity, in part, through the impairment of DC and T cell function. PMID:28250776

  4. Studies in fat grafting: Part IV. Adipose-derived stromal cell gene expression in cell-assisted lipotransfer.

    PubMed

    Garza, Rebecca M; Rennert, Robert C; Paik, Kevin J; Atashroo, David; Chung, Michael T; Duscher, Dominik; Januszyk, Michael; Gurtner, Geoffrey C; Longaker, Michael T; Wan, Derrick C

    2015-04-01

    Fat graft volume retention remains highly unpredictable, but addition of adipose-derived stromal cells to fat grafts has been shown to improve retention. The present study aimed to investigate the mechanisms involved in adipose-derived stromal cell enhancement of fat grafting. Adipose-derived stromal cells isolated from human lipoaspirate were labeled with green fluorescent protein and luciferase. Fat grafts enhanced with adipose-derived stromal cells were injected into the scalp and bioluminescent imaging was performed to follow retention of adipose-derived stromal cells within the fat graft. Fat grafts were also explanted at days 1, 5, and 10 after grafting for adipose-derived stromal cell extraction and single-cell gene analysis. Finally, CD31 immunohistochemical staining was performed on fat grafts enriched with adipose-derived stromal cells. Bioluminescent imaging demonstrated significant reduction in luciferase-positive adipose-derived stromal cells within fat grafts at 5 days after grafting. A similar reduction in viable green fluorescent protein-positive adipose-derived stromal cells retrieved from explanted grafts was also noted. Single-cell analysis revealed expression of multiple genes/markers related to cell survival and angiogenesis, including BMPR2, CD90, CD105, FGF2, CD248, TGFß1, and VEGFA. Genes involved in adipogenesis were not expressed by adipose-derived stromal cells. Finally, CD31 staining revealed significantly higher vascular density in fat grafts explanted at day 10 after grafting. Although adipose-derived stromal cell survival in the hypoxic graft environment decreases significantly over time, these cells provide multiple angiogenic growth factors. Therefore, improved fat graft volume retention with adipose-derived stromal cell enrichment may be attributable to improved graft vascularization.

  5. [Adipose-derived stromal cells: history, isolation, immunomodulatory properties and clinical perspectives].

    PubMed

    Bertheuil, N; Chaput, B; Ménard, C; Varin, A; Garrido, I; Grolleau, J L; Sensébé, L; Watier, E; Tarte, K

    2015-04-01

    Over the last decade, the clinical use of adipose-derived stromal/stem cells (ASC) in regenerative medicine is rapidly increasing. ASC belong to the mesenchymal stromal cells initially obtained from the bone marrow. Their limited differentiation capacity in vivo into functional mature cells has led to a reassessment of their mechanisms of action. One of the major clinical interests appears related to paracrine effects through a temporary production of trophic and immunomodulatory factors. Our purpose is to provide a review on the latest knowledge in the field of ASC, mechanisms of action, mainly immunomodulatory/immunosuppressive properties, methods of obtention, with a focus on clinical perspectives particularly in the field of cellular therapy and fat grafting technique in plastic surgery. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  6. Mechanical Stimulation Increases Knee Meniscus Gene RNA-level Expression in Adipose-derived Stromal Cells

    PubMed Central

    Meier, Elizabeth M.; Wu, Bin; Siddiqui, Aamir; Tepper, Donna G.; Longaker, Michael T.

    2016-01-01

    Background: Efforts have been made to engineer knee meniscus tissue for injury repair, yet most attempts have been unsuccessful. Creating a cell source that resembles the complex, heterogeneous phenotype of the meniscus cell remains difficult. Stem cell differentiation has been investigated, mainly using bone marrow mesenchymal cells and biochemical means for differentiation, resulting in no solution. Mechanical stimulation has been investigated to an extent with no conclusion. Here, we explore the potential for and effectiveness of mechanical stimulation to induce the meniscal phenotype in adipose-derived stromal cells. Methods: Human adipose-derived stromal cells were chosen for their fibrogenic nature and conduciveness for chondrogenesis. Biochemical and mechanical stimulation were investigated. Biochemical stimulation included fibrogenic and chondrogenic media. For mechanical stimulation, a custom-built device was used to apply constant, cyclical, uniaxial strain for up to 6 hours. Strain and frequency varied. Results: Under biochemical stimulation, both fibrogenic (collagen I, versican) and chondrogenic (collagen II, Sox9, aggrecan) genes were expressed by cells exposed to either fibrogenic or chondrogenic biochemical factors. Mechanical strain was found to preferentially promote fibrogenesis over chondrogenesis, confirming that tensile strain is an effective fibrogenic cue. Three hours at 10% strain and 1 Hz in chondrogenic media resulted in the highest expression of fibrochondrogenic genes. Although mechanical stimulation did not seem to affect protein level expression, biochemical means did affect protein level presence of collagen fibers. Conclusion: Mechanical stimulation can be a useful differentiation tool for mechanoresponsive cell types as long as biochemical factors are also integrated. PMID:27757329

  7. Characterization of a Murine Pressure Ulcer Model to Assess Efficacy of Adipose-derived Stromal Cells.

    PubMed

    Strong, Amy L; Bowles, Annie C; MacCrimmon, Connor P; Lee, Stephen J; Frazier, Trivia P; Katz, Adam J; Gawronska-Kozak, Barbara; Bunnell, Bruce A; Gimble, Jeffrey M

    2015-03-01

    As the world's population lives longer, the number of individuals at risk for pressure ulcers will increase considerably in the coming decades. In developed countries, up to 18% of nursing home residents suffer from pressure ulcers and the resulting hospital costs can account for up to 4% of a nation's health care budget. Although full-thickness surgical skin wounds have been used as a model, preclinical rodent studies have demonstrated that repeated cycles of ischemia and reperfusion created by exposure to magnets most closely mimic the human pressure ulcer condition. This study uses in vivo and in vitro quantitative parameters to characterize the temporal kinetics and histology of pressure ulcers in young, female C57BL/6 mice exposed to 2 or 3 ischemia-reperfusion cycles. This pressure ulcer model was validated further in studies examining the efficacy of adipose-derived stromal/stem cell administration. Optimal results were obtained with the 2-cycle model based on the wound size, histology, and gene expression profile of representative angiogenic and reparative messenger RNAs. When treated with adipose-derived stromal/stem cells, pressure ulcer wounds displayed a dose-dependent and significant acceleration in wound closure rates and improved tissue histology. These findings document the utility of this simplified preclinical model for the evaluation of novel tissue engineering and medical approaches to treat pressure ulcers in humans.

  8. Mechanical Stimulation Increases Knee Meniscus Gene RNA-level Expression in Adipose-derived Stromal Cells.

    PubMed

    Meier, Elizabeth M; Wu, Bin; Siddiqui, Aamir; Tepper, Donna G; Longaker, Michael T; Lam, Mai T

    2016-09-01

    Efforts have been made to engineer knee meniscus tissue for injury repair, yet most attempts have been unsuccessful. Creating a cell source that resembles the complex, heterogeneous phenotype of the meniscus cell remains difficult. Stem cell differentiation has been investigated, mainly using bone marrow mesenchymal cells and biochemical means for differentiation, resulting in no solution. Mechanical stimulation has been investigated to an extent with no conclusion. Here, we explore the potential for and effectiveness of mechanical stimulation to induce the meniscal phenotype in adipose-derived stromal cells. Human adipose-derived stromal cells were chosen for their fibrogenic nature and conduciveness for chondrogenesis. Biochemical and mechanical stimulation were investigated. Biochemical stimulation included fibrogenic and chondrogenic media. For mechanical stimulation, a custom-built device was used to apply constant, cyclical, uniaxial strain for up to 6 hours. Strain and frequency varied. Under biochemical stimulation, both fibrogenic (collagen I, versican) and chondrogenic (collagen II, Sox9, aggrecan) genes were expressed by cells exposed to either fibrogenic or chondrogenic biochemical factors. Mechanical strain was found to preferentially promote fibrogenesis over chondrogenesis, confirming that tensile strain is an effective fibrogenic cue. Three hours at 10% strain and 1 Hz in chondrogenic media resulted in the highest expression of fibrochondrogenic genes. Although mechanical stimulation did not seem to affect protein level expression, biochemical means did affect protein level presence of collagen fibers. Mechanical stimulation can be a useful differentiation tool for mechanoresponsive cell types as long as biochemical factors are also integrated.

  9. Ultrastructural features of human adipose-derived multipotent mesenchymal stromal cells.

    PubMed

    Manea, Claudiu Marius; Rusu, Mugurel Constantin; Constantin, Daniel; Mănoiu, Valentina Mariana; Moldovan, Lucia; Jianu, Adelina Maria

    2014-01-01

    Multipotent mesenchymal stromal cells (MMSCs) are plastic-adherent cells with a well-established phenotype. Equine, but not human, adipose MMSCs have been characterized ultrastructurally. The purpose of our study was to evaluate ultrastructurally the adipose-derived human MMSCs. Cell cultures were prepared from human lipoaspirate. The flow cytometry evaluation of surface markers of cultured cells confirmed the expected profile of MMSCs, that were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. We examined these human adipose-derived MMSCs in transmission electron microscopy (TEM) by Epon en-face embedding the fixed MMSCs. The main ultrastructural features of MMSCs were the extremely rich content of endosomal/vesicular elements, long mitochondria, dilated RER (rough endoplasmic reticulum) cisternae, and abundant intermediate filaments and microtubules. We found two types of MMSCS prolongations: (a) thick processes, with opposite, vesicular and filaments-rich, sides and (b) slender processes (pseudopodes and filopodes), with occasional proximal dilated segments housing mitochondria, vesicles and secretory granules. These TEM features of MMSCs characterized an in vitro cell population and could use to distinguish between different cell types in culture.

  10. Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

    SciTech Connect

    Li Huiwu; Dai Kerong . E-mail: krdai@163.com; Tang Tingting; Zhang Xiaoling; Yan Mengning; Lou Jueren

    2007-05-18

    In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a {beta}-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified by BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals.

  11. Adipose-derived stromal cells: Their identity and uses in clinical trials, an update

    PubMed Central

    Casteilla, Louis; Planat-Benard, Valérie; Laharrague, Patrick; Cousin, Béatrice

    2011-01-01

    In adults, adipose tissue is abundant and can be easily sampled using liposuction. Largely involved in obesity and associated metabolic disorders, it is now described as a reservoir of immature stromal cells. These cells, called adipose-derived stromal cells (ADSCs) must be distinguished from the crude stromal vascular fraction (SVF) obtained after digestion of adipose tissue. ADSCs share many features with mesenchymal stem cells derived from bone marrow, including paracrine activity, but they also display some specific features, including a greater angiogenic potential. Their angiogenic properties as well as their paracrine activity suggest a putative tumor-promoting role for ADSCs although contradictory data have been published on this issue. Both SVF cells and ADSCs are currently being investigated in clinical trials in several fields (chronic inflammation, ischemic diseases, etc.). Apart from a phase III trial on the treatment of fistula, most of these are in phase I and use autologous cells. In the near future, the end results of these trials should provide a great deal of data on the safety of ADSC use. PMID:21607134

  12. Adipose-derived stromal cells: Their identity and uses in clinical trials, an update.

    PubMed

    Casteilla, Louis; Planat-Benard, Valérie; Laharrague, Patrick; Cousin, Béatrice

    2011-04-26

    In adults, adipose tissue is abundant and can be easily sampled using liposuction. Largely involved in obesity and associated metabolic disorders, it is now described as a reservoir of immature stromal cells. These cells, called adipose-derived stromal cells (ADSCs) must be distinguished from the crude stromal vascular fraction (SVF) obtained after digestion of adipose tissue. ADSCs share many features with mesenchymal stem cells derived from bone marrow, including paracrine activity, but they also display some specific features, including a greater angiogenic potential. Their angiogenic properties as well as their paracrine activity suggest a putative tumor-promoting role for ADSCs although contradictory data have been published on this issue. Both SVF cells and ADSCs are currently being investigated in clinical trials in several fields (chronic inflammation, ischemic diseases, etc.). Apart from a phase III trial on the treatment of fistula, most of these are in phase I and use autologous cells. In the near future, the end results of these trials should provide a great deal of data on the safety of ADSC use.

  13. Therapeutic applications of human adipose-derived stromal cells for soft tissue reconstruction.

    PubMed

    Zielins, Elizabeth R; Luan, Anna; Brett, Elizabeth A; Longaker, Michael T; Wan, Derrick C

    2015-04-01

    Adipose derived stromal cells (ASCs) are a multipotent cell population derived from the stromal vascular fraction of lipoaspirate. Given their relatively broad differentiation potential and paracrine capabilities, ASCs represent a readily accessible, endogenous resource for novel reconstructive strategies. In particular, augmentation of autologous fat grafts with ASCs has already been employed clinically for restoration of soft tissue defects. While fat grafting alone remains highly unpredictable, enrichment of fat with supplemental ASCs, also known as cell-assisted lipotransfer (CAL), has been shown to significantly enhance volume retention. How addition of these cells to fat grafts results in improved outcomes, however, remains poorly understood. Furthermore, the safety of CAL in the setting of prior malignancy and post-radiation wound beds has yet to be fully determined, an important consideration for its use in cancer reconstruction. Thus, further studies to determine the how and why behind the efficacy of CAL are necessary before it can be widely adopted as a safe and reliable surgical technique.

  14. Functional Plasticity of Adipose-Derived Stromal Cells During Development of Obesity

    PubMed Central

    Zhu, Xiang-Yang; Ma, Shuangtao; Eirin, Alfonso; Woollard, John R.; Hickson, LaTonya J.; Sun, Dong; Lerman, Amir

    2016-01-01

    Obesity is a major risk factor for a number of chronic diseases, including diabetes, cardiovascular diseases, and cancer. Expansion of the adipose mass requires adipocyte precursor cells that originate from multipotent adipose-derived stromal cells (ASCs), which in turn also participate in repair activities. ASC function might decline in a disease milieu, but it remains unclear whether ASC function varies during the development of obesity. We tested the hypothesis that microenvironmental inflammatory changes during development of metabolic disorders in obesity affect ASC function. Domestic pigs were fed with an atherogenic (n = 7) or normal (n = 7) diet for 16 weeks. Abdominal adipose tissue biopsies were collected after 8, 12, and 16 weeks of diet for ASC isolation and immunohistochemistry of in situ ASCs and tumor necrosis factor-α (TNF-α). Longitudinal changes in proliferation, differentiation, and anti-inflammatory functions of ASCs were assessed. At 16 weeks, upregulated TNF-α expression in adipose tissue from obese pigs was accompanied by increased numbers of adipocyte progenitors (CD24+/CD34+) in adipose tissue and enlarged adipocyte size. In vitro, ASCs from obese pigs showed enhanced adipogenic and osteogenic propensity, which was abolished by anti-TNF-α treatment, whereas lean ASCs treated with TNF-α showed enhanced adipogenesis. Furthermore, obese ASCs showed increased senescence compared with lean ASCs, whereas their immunomodulatory capacity was preserved. Adipose tissue inflammation promotes an increase in resident adipocyte progenitors and upregulated TNF-α enhances ASC adipogenesis. Thus, adipose tissue anti-inflammatory strategies might be a novel target to attenuate obesity and its complications. Significance Adipose-derived stromal cell (ASC) function might decline in a disease milieu, but it remains unclear whether ASC function varies during the development of obesity. This study tested the hypothesis that microenvironmental inflammatory

  15. IL-6 is produced by adipose-derived stromal cells and promotes osteogenesis.

    PubMed

    Huh, Jeong-Eun; Lee, Soo Young

    2013-12-01

    Although Toll-like receptors (TLRs) have been implicated in the regulation of stem cell functions, their role in osteogenic differentiation of adipose-derived stromal cells (ASCs) has not been reported. We found that ASCs express a restricted subset of TLRs, including TLR1-TLR5, and that TLR agonists such as Pam3CSK4 (TLR1/2 agonist), polyinosinic:polycytidylic acid (TLR3 agonist), lipopolysaccharide (TLR4 agonist), and flagellin (TLR5 agonist), but not R848 (TLR7/8 agonist), consistently induced osteogenic differentiation in murine-derived ASCs, which coincided with the TLR expression pattern of ASCs. Cytokine expression profiles induced by TLR agonists and results from subsequent functional assays indicated that interleukin-6 (IL-6) together with soluble IL-6 receptor (sIL-6R) enhanced osteogenic differentiation of ASCs by activating STAT3. Small interfering RNA (siRNA)-mediated STAT3-silencing blunted osteogenesis and the expression of osteogenic markers, whereas STAT3 overexpression resulted in an increase in osteogenesis. Consistently, STAT3 inhibitor treatment reduced osteogenesis, STAT3 phosphorylation, and expression of osteogenic markers including osterix. Chromatin immunoprecipitation (ChIP) assays indicated that STAT3 binding to the STAT3-binding sites on the osterix promoter increased during IL-6-stimulated osteogenesis. Our results thus establish TLRs as novel regulators of ASCs which signal through IL-6/STAT3 pathway and induce osterix expression as a part of the osteogenesis.

  16. Therapeutic effect of human adipose-derived stromal cells cluster in rat hind-limb ischemia.

    PubMed

    Park, In-Su; Kang, Jo A; Kang, Jungmi; Rhie, Jong-Won; Kim, Sang-Heon

    2014-12-01

    We investigated whether transplantation of three-dimensional cell masses (3DCM) of human adipose-derived stromal cells (hASCs) cultured on a basic fibroblast growth factor-immobilized substrate improved hind limb functional recovery by stimulating angiogenesis in an immune-competent rat ischemic limb model. In vitro experiments confirmed that cells within 3DCMs differentiate toward the endothelial lineage one day after culture in normal medium. The therapeutic effect of 3DCMs was evaluated by transplanting hASCs, phosphate-buffered saline alone, and the 3DCM into rat ischemic hind limbs. Blood flow was enhanced in the ischemic hind limb in the 3DCM-injected group compared with the other groups. The ratio of human nuclear antigen (HNA) and hVEGF-positive cells was significantly higher in the 3DCM-injected group compared to hASC-injected group. Human VEGF was observed in most HNA-positive cells. Many hCD31 and hSMA-positive cells were observed in vessel-like structures in the 3DCM-injected group. The 3DCM transplantation improved cell retention and angiogenic effects compared with ASC transplantation. These findings suggest that transplantation of 3DCMs may be an effective stem cell therapy for hind limb ischemia.

  17. Adipose-derived stromal cells overexpressing vascular endothelial growth factor accelerate mouse excisional wound healing.

    PubMed

    Nauta, Allison; Seidel, Catharina; Deveza, Lorenzo; Montoro, Daniel; Grova, Monica; Ko, Sae Hee; Hyun, Jeong; Gurtner, Geoffrey C; Longaker, Michael T; Yang, Fan

    2013-02-01

    Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

  18. Pulsed Direct Current Electric Fields Enhance Osteogenesis in Adipose-Derived Stromal Cells

    PubMed Central

    Hammerick, Kyle E.; James, Aaron W.; Huang, Zubin; Prinz, Fritz B.

    2010-01-01

    Adipose-derived stromal cells (ASCs) constitute a promising source of cells for regenerative medicine applications. Previous studies of osteogenic potential in ASCs have focused on chemicals, growth factors, and mechanical stimuli. Citing the demonstrated role electric fields play in enhancing healing in bone fractures and defects, we investigated the ability of pulsed direct current electric fields to drive osteogenic differentiation in mouse ASCs. Employing 50 Hz direct current electric fields in concert with and without osteogenic factors, we demonstrated increased early osteoblast-specific markers. We were also able to establish that commonly reported artifacts of electric field stimulation are not the primary mediators of the observed effects. The electric fields caused marked changes in the cytoskeleton. We used atomic force microscopy–based force spectroscopy to record an increase in the cytoskeletal tension after treatment with electric fields. We abolished the increased cytoskeletal stresses with the rho-associated protein kinase inhibitor, Y27632, and did not see any decrease in osteogenic gene expression, suggesting that the pro-osteogenic effects of the electric fields are not transduced via cytoskeletal tension. Electric fields may show promise as candidate enhancers of osteogenesis of ASCs and may be incorporated into cell-based strategies for skeletal regeneration. PMID:19824802

  19. Transplantation of Adipose Derived Stromal Cells into the Developing Mouse Eye

    PubMed Central

    Yu, Song-Hee; Jang, Yu-Jin; Lee, Eun-Shil; Hwang, Dong-Youn; Jeon, Chang-Jin

    2010-01-01

    Adipose derived stromal cells (ADSCs) were transplanted into a developing mouse eye to investigate the influence of a developing host micro environment on integration and differentiation. Green fluorescent protein-expressing ADSCs were transplanted by intraocular injections. The age of the mouse was in the range of 1 to 10 days postnatal (PN). Survival dates ranged from 7 to 28 post transplantation (DPT), at which time immunohistochemistry was performed. The transplanted ADSCs displayed some morphological differentiations in the host eye. Some cells expressed microtubule associated protein 2 (marker for mature neuron), or glial fibrillary acid protein (marker for glial cell). In addition, some cells integrated into the ganglion cell layer. The integration and differentiation of the transplanted ADSCs in the 5 and 10 PN 7 DPT were better than in the host eye the other age ranges. This study was aimed at demonstrating how the age of host micro environment would influence the differentiation and integration of the transplanted ADSCs. However, it was found that the integration and differentiation into the developing retina were very limited when compared with other stem cells, such as murine brain progenitor cell. PMID:21245978

  20. Making the Switch: Alternatives to Fetal Bovine Serum for Adipose-Derived Stromal Cell Expansion

    PubMed Central

    Dessels, Carla; Potgieter, Marnie; Pepper, Michael S.

    2016-01-01

    Adipose-derived stromal cells (ASCs) are being used extensively in clinical trials. These trials require that ASCs are prepared using good manufacturing practices (GMPs) and are safe for use in humans. The majority of clinical trials in which ASCs are expanded make use of fetal bovine serum (FBS). While FBS is used traditionally in the research setting for in vitro expansion, it does carry the risk of xenoimmunization and zoonotic transmission when used for expanding cells destined for therapeutic purposes. In order to ensure a GMP quality product for cellular therapy, in vitro expansion of ASCs has been undertaken using xeno-free (XF), chemically-defined, and human blood-derived alternatives. These investigations usually include the criteria proposed by the International Society of Cellular Therapy (ISCT) and International Fat Applied Technology Society (IFATS). The majority of studies use these criteria to compare plastic-adherence, morphology, the immunophenotype and the trilineage differentiation of ASCs under the different medium supplemented conditions. Based on these studies, all of the alternatives to FBS seem to be suitable replacements; however, each has its own advantages and drawbacks. Very few studies have investigated the effects of the supplements on the immunomodulation of ASCs; the transcriptome, proteome and secretome; and the ultimate effects in appropriate animal models. The selection of medium supplementation will depend on the downstream application of the ASCs and their efficacy and safety in preclinical studies. PMID:27800478

  1. Adipose-derived Stromal Cells Overexpressing Vascular Endothelial Growth Factor Accelerate Mouse Excisional Wound Healing

    PubMed Central

    Nauta, Allison; Seidel, Catharina; Deveza, Lorenzo; Montoro, Daniel; Grova, Monica; Ko, Sae Hee; Hyun, Jeong; Gurtner, Geoffrey C; Longaker, Michael T; Yang, Fan

    2013-01-01

    Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12–15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10–12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired. PMID:23164936

  2. IFATS Collection: Adipose-Derived Stromal Cells Improve the Foreign Body Response

    PubMed Central

    Prichard, Heather L.; Reichert, William; Klitzman, Bruce

    2015-01-01

    Many implanted devices fail due to the formation of an avascular capsule surrounding the device. Additionally, fat has long been known to promote healing and vascularization. The goals of this study were to identify potential mechanisms of the provascular actions of adipose-derived stromal cells (ASCs) and to improve implant biocompatibility. First, adult ASCs and fibroblasts from rats were attached to polyurethane and polystyrene in vitro and their cytokine secretion profile was analyzed. Secretion of vascular endothelial growth factor (VEGF) from ASCs was 10 –70 times higher than fibroblasts after 3 and 6 days. Next, polyurethane, bare and with cellular coatings, was implanted subcutaneously in rats. The fibrous capsule surrounding bare polyurethane implants was 17%–32% thicker and the amount of collagen was 27% greater than the capsule surrounding ASC-coated implants. Finally, the microvessel density adjacent to ASC-coated polyurethane was approximately 50%–80% higher than bare polyurethane. In summary, ASCs attached to polyurethane have a dramatically increased VEGF production compared with fibroblasts in vitro, and these cells also produce an increased microvessel density in the surrounding tissue when implanted subcutaneously in rats. PMID:18436858

  3. Pulsed direct current electric fields enhance osteogenesis in adipose-derived stromal cells.

    PubMed

    Hammerick, Kyle E; James, Aaron W; Huang, Zubin; Prinz, Fritz B; Longaker, Michael T

    2010-03-01

    Adipose-derived stromal cells (ASCs) constitute a promising source of cells for regenerative medicine applications. Previous studies of osteogenic potential in ASCs have focused on chemicals, growth factors, and mechanical stimuli. Citing the demonstrated role electric fields play in enhancing healing in bone fractures and defects, we investigated the ability of pulsed direct current electric fields to drive osteogenic differentiation in mouse ASCs. Employing 50 Hz direct current electric fields in concert with and without osteogenic factors, we demonstrated increased early osteoblast-specific markers. We were also able to establish that commonly reported artifacts of electric field stimulation are not the primary mediators of the observed effects. The electric fields caused marked changes in the cytoskeleton. We used atomic force microscopy-based force spectroscopy to record an increase in the cytoskeletal tension after treatment with electric fields. We abolished the increased cytoskeletal stresses with the rho-associated protein kinase inhibitor, Y27632, and did not see any decrease in osteogenic gene expression, suggesting that the pro-osteogenic effects of the electric fields are not transduced via cytoskeletal tension. Electric fields may show promise as candidate enhancers of osteogenesis of ASCs and may be incorporated into cell-based strategies for skeletal regeneration.

  4. Effect of canine cortical bone demineralization on osteogenic differentiation of adipose-derived mesenchymal stromal cells.

    PubMed

    Jo, Kwangrae; Kim, Yongsun; Lee, Seung Hoon; Yoon, Yong Seok; Kim, Wan Hee; Kweon, Oh-Kyeong

    2017-08-01

    Demineralized bone allografts and mesenchymal stromal cells have been used to promote bone regeneration. However, the degree to which cortical bone should be demineralized for use in combination with adipose-derived mesenchymal stromal cells (Ad-MSCs) remains to be clarified. In this study, the in vitro osteogenic ability of Ad-MSCs on allografts was investigated in relation to the extent of demineralization. Three treatment groups were established by varying exposure time to 0.6 N HCL: partially demineralized (PDB; 12 h), fully demineralized (FDB; 48 h), and non-demineralized bone (NDB; 0 h, as a control). Allografts were prepared as discs 6 mm in diameter for in vitro evaluation, and their demineralization and structure were evaluated by micro-computed tomography and scanning electron microscopy. Ad-MSC adhesion and proliferation were measured by MTS assay, and osteogenesis-related gene expression was assessed by quantitative reverse transcription polymerase chain reaction. PDB and FDB demineralization rates were 57.13 and 92.30%, respectively. Moreover, Ad-MSC adhesion rates on NDB, PDB, and FDB were 53.41, 60.65, and 61.32%, respectively. Proliferation of these cells on FDB increased significantly after 2 days of culture compared to the other groups (P < 0.05). Furthermore, expression of the osteogenic genes ALP, BMP-7, and TGF-β in the FDB group on culture day 3 was significantly elevated in comparison to the other treatments. Given its biocompatibility and promotion of the osteogenic differentiation of Ad-MSCs, our results suggest that FDB may be a suitable scaffold for use in the repair of bone defects.

  5. Microcirculatory Response In Vivo on Local Intraarterial Infusion of Autogenic Adipose-derived Stem Cells or Stromal Vascular Fraction.

    PubMed

    Wang, Wei Z

    2016-09-01

    Both adipose-derived stem cells (ASCs) and stromal vascular fraction (SVF) have been demonstrated to have regenerative properties with therapeutic potential for numerous diseases through local or topical applications. However, it is unclear whether ASC or SVF can be delivered systemically through an intra-arterial infusion. The purpose of this study was to examine the microcirculatory response in vivo on local intraarterial infusion of autogenic ASCs or SVF in a vascular pedicle isolated rat cremaster microcirculation model.

  6. Patient factors influencing the concentration of stromal vascular fraction (SVF) for adipose-derived stromal cell (ASC) therapy in dogs.

    PubMed

    Astor, Donniel E; Hoelzler, Michael G; Harman, Robert; Bastian, Richard P

    2013-07-01

    The objective of this study was to determine whether patient factors influence the concentration of the stromal vascular fraction (SVF) in fat for adipose-derived stromal cell (ASC) therapy in dogs. A total of 1265 dogs underwent adipose collection surgeries by veterinarians for processing by the Vet-Stem laboratory and data on cell counts and patient factors were collected. Body condition score (BCS) and breed size did not significantly affect the viable cells per gram (VCPG) of adipose tissue that represents the viable SVF. Age significantly affected the VCPG, with dogs in age quartile 1 having a significantly higher VCPG than those in quartile 2 (P = 0.003) and quartile 4 (P = 0.002). Adipose tissue collected at the falciform location had significantly fewer VCPG than tissue collected at the thoracic wall and inguinal locations (P < 0.001). When the interaction of gender and location was evaluated, there were significantly fewer VCPG in tissue collected at the falciform location than at the thoracic wall and inguinal locations in female spayed dogs (P < 0.001) and male neutered dogs (P < 0.001), but not in female intact dogs (P = 0.743) or male intact dogs (P = 0.208). It was concluded that specific patient factors should be taken into consideration in order to obtain the maximal yield of VCPG from an adipose collection procedure.

  7. Evaluation of adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis.

    PubMed

    Frisbie, David D; Kisiday, John D; Kawcak, Chris E; Werpy, Natasha M; McIlwraith, C Wayne

    2009-12-01

    The purpose of this study was the assessment of clinical, biochemical, and histologic effects of intraarticular administered adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis. Osteoarthritis was induced arthroscopically in the middle carpal joint of all horses, the contralateral joint being sham-operated. All horses received treatment on Day 14. Eight horses received placebo treatment and eight horses received adipose-derived stromal vascular fraction in their osteoarthritis-affected joint. The final eight horses were treated the in osteoarthritis-affected joint with bone marrow-derived mesenchymal stem cells. Evaluations included clinical, radiographic, synovial fluid analysis, gross, histologic, histochemical, and biochemical evaluations. No adverse treatment-related events were observed. The model induced a significant change in all but two parameters, no significant treatment effects were demonstrated, with the exception of improvement in synovial fluid effusion PGE2 levels with bone marrow-derived mesenchymal stem cells when compared to placebo. A greater improvement was seen with bone marrow-derived mesenchymal stem cells when compared to adipose-derived stromal vascular fraction and placebo treatment. Overall, the findings of this study were not significant enough to recommend the use of stem cells for the treatment of osteoarthritis represented in this model.

  8. Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

    PubMed

    Wang, Wei Z; Fang, Xin-Hua; Williams, Shelley J; Stephenson, Linda L; Baynosa, Richard C; Wong, Nancy; Khiabani, Kayvan T; Zamboni, William A

    2013-01-01

    Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90/CD45, CD105/CD45, and CD34/CD31) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p < 0.05). Among the stromal vascular fraction cells, 51.1 ± 3.7 percent expressed CD90/CD45, 7.5 ± 1.0 percent expressed CD105/CD45, and 26.4 ± 3.8 percent expressed CD34/CD31. CD34/CD31 adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105/CD45 adipose-derived stem cells (p < 0.05). Adipose-derived stem cells had a higher rate of apoptosis and necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.

  9. Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

    SciTech Connect

    Takahara, Kiyoshi; Ii, Masaaki; Inamoto, Teruo; Komura, Kazumasa; Ibuki, Naokazu; Minami, Koichiro; Uehara, Hirofumi; Hirano, Hajime; Nomi, Hayahito; Kiyama, Satoshi; Asahi, Michio; Azuma, Haruhito

    2014-04-18

    Highlights: • AdSC transplantation exhibits inhibitory effect on tumor progressions of PCa cells. • AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway. • High expression of the TGF-β1 gene in AdSCs. - Abstract: Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.

  10. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    PubMed Central

    Cordeiro, Ingrid R.; Lopes, Daiana V.; Abreu, José G.; Carneiro, Katia; Rossi, Maria I. D.; Brito, José M.

    2015-01-01

    ABSTRACT Human adipose-derived stromal cells (hADSC) are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1) regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues. PMID:26319582

  11. Modulation of vocal fold scar fibroblasts by adipose-derived stem/stromal cells.

    PubMed

    Kumai, Yoshihiko; Kobler, James B; Park, Hyoungshin; Galindo, Marilyn; Herrera, Victoria L M; Zeitels, Steven M

    2010-02-01

    To explore whether adipose-derived stem/stromal cells (ASCs) have therapeutic potential for treating scarred superficial lamina propria through the effects of secreted hepatocyte growth factor (HGF) on scar fibroblasts. In vitro study using coculture system. Scar fibroblasts (SFs) were isolated from ferret vocal folds electrocauterized 2 weeks previously. ASCs were isolated from ferret lipoaspirated subcutaneous abdominal fat. For coculture experiments, the two cell types were combined in Transwell plates for 6 days, followed by 1 or 3 days of monoculture after removing the upper chamber. Assays were then performed on cells and media from the bottom chamber. We measured: 1) the production of hyaluronic acid (HA), collagen and HGF via enzyme-linked immunosorbent assays, 2) the expression of alpha-smooth muscle actin (alpha-SMA), 3) cell proliferation, and 4) apoptosis of SFs (2, 3, and 4 via flow cytometry). Other experiments examined the effects of HGF on SFs and the effects of HGF neutralization in the coculture system. Coculture led to significant decreases in SF collagen production (P < .05), cell proliferation (P < .05), and alpha-SMA expression (P < .05), whereas HA production increased (P < .05). Coculture also increased HGF secretion from ASCs (P < .05). Neutralization of HGF abolished the inhibitory effects of ASCs on SF collagen synthesis (P < .05). ASCs influence SFs to adopt a less fibrotic profile. It appears that HGF is at least one of the soluble factors responsible for this effect. Implanted ASCs could potentially ameliorate vocal fold scar by acting as long-term, intrinsic sources of HGF.

  12. Effect of adipose-derived mesenchymal stromal cells on tendon healing in aging and estrogen deficiency: an in vitro co-culture model.

    PubMed

    Veronesi, Francesca; Della Bella, Elena; Torricelli, Paola; Pagani, Stefania; Fini, Milena

    2015-11-01

    Aging and estrogen deficiency play a pivotal role in reducing tenocyte proliferation, collagen turnover and extracellular matrix remodeling. Mesenchymal stromal cells are being studied as an alternative for tendon regeneration, but little is known about the molecular events of adipose-derived mesenchymal stromal cells (ADSCs) on tenocytes in tendons compromised by aging and estrogen deficiency. The present in vitro study aims to compare the potential therapeutic effects of ADSCs, harvested from healthy young (sham) and aged estrogen-deficient (OVX) subjects, for tendon healing. An indirect co-culture system was set up with ADSCs, isolated from OVX or sham rats, and tenocytes from OVX rats. Cell proliferation, healing rate and gene expression were evaluated in both a standard culture condition and a microwound-healing model. It was observed that tenocyte proliferation, healing rate and collagen expression improved after the addition of sham ADSCs in both culture situations. OVX ADSCs also increased tenocyte proliferation and healing rate but less compared with sham ADSCs. Decorin and Tenascin C expression increased in the presence of OVX ADSCs. Findings suggest that ADSCs might be a promising treatment for tendon regeneration in advanced age and estrogen deficiency. However, some differences between allogenic and autologous cells were found and should be investigated in further in vivo studies. It appears that allogenic ADSCs improve tenocyte proliferation, collagen expression and the healing rate more than autologous cells. Autologous cells increase collagen expression only in the absence of an injury and increase Decorin and Tenascin C more than allogenic cells. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  13. Osteogenic Ability of Canine Adipose-Derived Mesenchymal Stromal Cell Sheets in Relation to Culture Time.

    PubMed

    Kuk, Minyong; Kim, Yongsun; Lee, Seung Hoon; Kim, Wan Hee; Kweon, Oh-Kyeong

    2016-01-01

    Cell sheets could be used for bone regeneration without requiring a scaffold and can be easily produced from autologous mesenchymal stromal cells (MSCs). We compared the osteogenic potential of MSC-derived cell sheets in relation to culture time. Undifferentiated cell sheets (U-CS) and osteogenic differentiated cell sheets (O-CS) were generated using canine adipose-derived MSCs. Undifferentiated cells (UCs) were used as the control. Osteogenic differentiation was assessed by assaying alkaline phosphatase (ALP) activity. Expression of osteogenesis-related genes was evaluated by reverse transcription-polymerase chain reaction at 4, 7, 14, and 21 days after initiation of culture. The calcium content in cells was measured, and the cells were stained with Alizarin red S (ARS). The mRNA expression of transforming growth factor-β in U-CS and O-CS at day 4 was higher than that in UCs (p < 0.05). The level of bone morphogenetic protein 7 mRNA in O-CS increased significantly at day 4 and was significantly higher than that of U-CS at day 7. The mRNA level of runt-related transcription factor-2 in both sheet types increased significantly at 7 days of culture. The mRNA level of ALP in O-CS and U-CS increased significantly at day 7, and ALP activity was highest at days 7 and 14, respectively (p < 0.05). The mRNA level of osteocalcin in U-CS and O-CS increased significantly at day 21. O-CS and U-CS showed negative ARS staining but their calcium contents increased marginally at day 21. The O-CS cells started to aggregate at days 10-12, and only a partial sheet remained at day 21. The upregulation of expression of genes related to osteogenic differentiation, peak in ALP activity, and morphological changes in cell sheets suggest that the optimal time for application of O-CS and U-CS is between 7 and 10 days and after 14 days of culture, respectively.

  14. Role of adipose-derived stromal cells in pedicle skin flap survival in experimental animal models.

    PubMed

    Foroglou, Pericles; Karathanasis, Vasileios; Demiri, Efterpi; Koliakos, George; Papadakis, Marios

    2016-03-26

    The use of skin flaps in reconstructive surgery is the first-line surgical treatment for the reconstruction of skin defects and is essentially considered the starting point of plastic surgery. Despite their excellent usability, their application includes general surgical risks or possible complications, the primary and most common is necrosis of the flap. To improve flap survival, researchers have used different methods, including the use of adipose-derived stem cells, with significant positive results. In our research we will report the use of adipose-derived stem cells in pedicle skin flap survival based on current literature on various experimental models in animals.

  15. Role of adipose-derived stromal cells in pedicle skin flap survival in experimental animal models

    PubMed Central

    Foroglou, Pericles; Karathanasis, Vasileios; Demiri, Efterpi; Koliakos, George; Papadakis, Marios

    2016-01-01

    The use of skin flaps in reconstructive surgery is the first-line surgical treatment for the reconstruction of skin defects and is essentially considered the starting point of plastic surgery. Despite their excellent usability, their application includes general surgical risks or possible complications, the primary and most common is necrosis of the flap. To improve flap survival, researchers have used different methods, including the use of adipose-derived stem cells, with significant positive results. In our research we will report the use of adipose-derived stem cells in pedicle skin flap survival based on current literature on various experimental models in animals. PMID:27022440

  16. Dynamic imaging of allogeneic adipose-derived regenerative cells transplanted in ischemic hind limb of apolipoprotein E mouse model

    PubMed Central

    Zheng, Yi; Qin, Jinbao; Wang, Xin; Peng, Zhiyou; Hou, Peiyong; Lu, Xinwu

    2017-01-01

    Background Transplantation of allogeneic adipose-derived regenerative cells (ADRCs) is a promising treatment modality for severe ischemic diseases. However, minimal information is available on the in vivo effects, fate, and migration of ADRCs, as well as the mechanisms of their therapeutic angiogenesis. Materials and methods In this study, green fluorescent protein-expressing ADRCs (GFP-ADRCs) were obtained, labeled with acetylated 3-aminopropyltrimethoxysilane (APTS)-coated iron oxide nanoparticles (APTS NPs), and injected into an old apolipoprotein E knockout (ApoE-KO) mouse model with hind limb ischemia. Then, 3.0 T magnetic resonance imaging (MRI) was performed to dynamically trace the role of ADRCs targeting hind limb ischemia in the ApoE-KO mice model. Results Labeled cells were visualized as large hypointense spots in ischemic muscles by serial 3.0 T MRI scans during a 4-week follow-up. The presence of labeled GFP-ADRCs was confirmed by Prussian blue staining and fluorescence microscopy on postmortem specimens. Conclusion This study showed that allogeneic ADRCs offer great potential application for therapeutic angiogenesis in severe ischemic disease based on the efficacy and feasibility of ADRC transplantation and on the available amounts of tissue. PMID:28053524

  17. Adipose-Derived Cells (Stromal Vascular Fraction) Transplanted for Orthopedical or Neurological Purposes: Are They Safe Enough?

    PubMed Central

    Zolocinska, Aleksandra; Stepien, Karolina; Lubina-Dabrowska, Natalia; Maciagowska, Marzena; Mazur, Slawomir; Zdanowicz, Urszula; Smigielski, Robert; Stepien, Adam

    2016-01-01

    Although mesenchymal stem cells are used in numerous clinical trials, the safety of their application is still a matter of concern. We have analysed the clinical results of the autologous adipose-derived stem cell treatment (stromal vascular fraction (SVF) containing adipose-derived stem cells, endothelial progenitors, and blood mononuclear cells) for orthopedic (cartilage, bone, tendon, or combined joint injuries) and neurologic (multiple sclerosis) diseases. Methods of adipose tissue collection, cell isolation and purification, and resulting cell numbers, viability, and morphology were considered, and patient's age, sex, disease type, and method of cell administration (cell numbers per single application, treatment numbers and frequency, and methods of cell implantation) were analysed and searched for the unwanted clinical effects. Results of cellular therapy were compared retrospectively to those obtained with conventional medication without SVF application. SVF transplantation was always the accessory treatment of patients receiving “standard routine” therapies of their diseases. Clinical experiments were approved by the Bioethical Medical Committees supervising the centers where patients were hospitalised. The conclusion of the study is that none of the treated patients developed any serious adverse event, and autologous mesenchymal stem (stromal) cell clinical application is a safe procedure resulting in some beneficial clinical effects (not analysed in this study). PMID:27698672

  18. Gender-dimorphic effects of adipose-derived stromal vascular fractions on HUVECs exposed to oxidative stress

    PubMed Central

    Lim, Soyeon; Kim, Il-Kwon; Choi, Jung-Won; Seo, Hyang-Hee; Lim, Kyu Hee; Lee, Seahyoung; Lee, Hoon-Bum; Kim, Sang Woo; Hwang, Ki-Chul

    2017-01-01

    Stromal vascular fractions (SVFs) are a heterogeneous collection of cells within adipose tissue that are being studied for various clinical indications. In this study, we aimed to determine whether SVF transplantation into impaired tissues has differential effects on inflammatory and angiogenetic properties with regard to gender. As reactive oxygen species have been implicated in cardiovascular disease development, we investigated differences in gene and protein expression related to inflammation and angiogenesis in HUVECs co-cultured with adipose-derived SVFs from male (M group) and female (F group) individuals under oxidative stress conditions. The expression of several inflammatory (interleukin (IL)-33) and angiogenetic (platelet-derived growth factor (PDGF)) factors differed dramatically between male and female donors. Anti-inflammatory and pro-angiogenetic responses were observed in HUVECs co-cultured with SVFs under oxidative stress conditions, and these characteristics may exhibit partially differential effects according to gender. Using network analysis, we showed that co-culturing HUVECs with SVFs ameliorated pyroptosis/apoptosis via an increase in oxidative stress. Activation of caspase-1 and IL-1B was significantly altered in HUVECs co-cultured with SVFs from female donors. These findings regarding gender-dimorphic regulation of adipose-derived SVFs provide valuable information that can be used for evidence-based gender-specific clinical treatment of SVF transplantation for understanding of cardiovascular disease, allowing for the development of additional treatment. PMID:28824330

  19. Cell-Assisted Lipotransfer Using Autologous Adipose-Derived Stromal Cells for Alleviation of Breast Cancer-Related Lymphedema.

    PubMed

    Toyserkani, Navid Mohamadpour; Jensen, Charlotte Harken; Sheikh, Søren Paludan; Sørensen, Jens Ahm

    2016-07-01

    : Lymphedema is one of the most frequent side effects following cancer treatment, and treatment opportunities for it are currently lacking. Stem cell therapy has been proposed as a possible novel treatment modality. This study was the first case in which freshly isolated adipose-derived stromal cells were used to treat lymphedema. Treatment was given as a cell-assisted lipotransfer in which 4.07 × 10(7) cells were injected with 10 ml of lipoaspirate in the axillary region. Four months after treatment, the patient reported a great improvement in daily symptoms, reduction in need for compression therapy, and volume reduction of her affected arm. There were no adverse events. The outcome for this patient provides support for the potential use of cellular therapy for lymphedema treatment. We have begun a larger study to further test the feasibility and safety of this procedure (ClinicalTrials.gov Identifier NCT02592213). Lymphedema is a very debilitating side effect of cancer treatment and has very few treatment options. Stem cell therapy has the potential to change the treatment paradigm from a conservative to a more curative approach. Freshly isolated, autologous, adipose-derived stromal cells were combined with a fat-graft procedure to treat lymphedema. The treated patient had great improvement in daily symptoms, a reduced need for compression therapy, and a reduction in arm volume after 4 months. There were no adverse events. The use of cellular therapy for lymphedema treatment is supported by this patient's outcome. A phase II study has begun to further test its feasibility and safety. ©AlphaMed Press.

  20. Platelet-Rich Plasma Influences Expansion and Paracrine Function of Adipose-Derived Stromal Cells in a Dose-Dependent Fashion.

    PubMed

    Willemsen, Joep C N; Spiekman, Maroesjka; Stevens, H P Jeroen; van der Lei, Berend; Harmsen, Martin C

    2016-03-01

    Lipofilling is a treatment modality to restore tissue volume. Both platelet-rich plasma and adipose-derived stromal cells have been reported to augment the efficacy of lipofilling, yet results are not conclusive. The authors hypothesized that the variation reported in literature is caused by a dose-dependent influence of platelet-rich plasma on adipose-derived stromal cells. Whole blood (n = 3) was used to generate platelet-rich plasma, which was diluted with Dulbecco's Modified Eagle Medium to 15%, 5%, and 1.7%, with 15% platelet-poor plasma and 10% fetal calf serum as controls. Pooled adipose-derived stromal cells (n = 3) were cultured in these media. Gene expression was assessed, along with angiogenic sprouting of endothelial cells by conditioned medium and platelet-rich plasma. platelet-rich plasma in culture medium affected the expression of genes in a dose-dependent manner. The 15% concentration stimulated proliferation almost eightfold. Mesenchymal markers were unaffected. Interestingly, expression of collagens type 1 and 3 increased at lower concentrations, whereas transforming growth factor-β showed reduced expression in lower concentrations. Proangiogenic gene expression was unaltered or strongly reduced in a dose-dependent manner. platelet-rich plasma promoted endothelial sprouting and survival in a dose-dependent manner; however, conditioned medium from adipose-derived stromal cells exposed to platelet-rich plasma blocked endothelial sprouting capabilities. The dose-dependent influence of platelet-rich plasma on the therapeutic capacity of adipose-derived stromal cells conditioned medium in vitro warrants caution in clinical trials.

  1. Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo and in vitro

    PubMed Central

    Baer, Patrick C

    2014-01-01

    Adipose tissue is a rich, ubiquitous and easily accessible source for multipotent stromal/stem cells and has, therefore, several advantages compared to other sources of mesenchymal stromal/stem cells. Several studies have tried to identify the origin of the stromal/stem cell population within adipose tissue in situ. This is a complicated attempt because no marker has currently been described which unambiguously identifies native adipose-derived stromal/stem cells (ASCs). Isolated and cultured ASCs are a non-uniform preparation consisting of several subsets of stem and precursor cells. Cultured ASCs are characterized by their expression of a panel of markers (and the absence of others), whereas their in vitro phenotype is dynamic. Some markers were expressed de novo during culture, the expression of some markers is lost. For a long time, CD34 expression was solely used to characterize haematopoietic stem and progenitor cells, but now it has become evident that it is also a potential marker to identify an ASC subpopulation in situ and after a short culture time. Nevertheless, long-term cultured ASCs do not express CD34, perhaps due to the artificial environment. This review gives an update of the recently published data on the origin and phenotype of ASCs both in vivo and in vitro. In addition, the composition of ASCs (or their subpopulations) seems to vary between different laboratories and preparations. This heterogeneity of ASC preparations may result from different reasons. One of the main problems in comparing results from different laboratories is the lack of a standardized isolation and culture protocol for ASCs. Since many aspects of ASCs, such as the differential potential or the current use in clinical trials, are fully described in other recent reviews, this review further updates the more basic research issues concerning ASCs’ subpopulations, heterogeneity and culture standardization. PMID:25126376

  2. Human and Autologous Adipose-derived Stromal Cells Increase Flap Survival in Rats Independently of Host Immune Response.

    PubMed

    Toyserkani, Navid Mohamadpour; Jensen, Charlotte Harken; Andersen, Ditte Caroline; Sheikh, Søren Paludan; Sørensen, Jens Ahm

    2017-07-22

    There is a rising interest in adipose-derived stromal cells for clinical use; however, it is unknown whether freshly isolated stromal cells (SVF) or culture-expanded cells (ASCs) are more efficacious. We therefore aimed to compare the 2 cellular therapies in an in vivo model of angiogenesis, the ischemic flap in rats, which induces acute ischemia. We also aimed to determine the importance of cell presence and the host immune response. A total of 96 rats (n = 12 in each group) were used, and in each rat, a caudally based random flap measuring 2 × 7 cm was made. The study was conducted in 3 phases. First, each rat was treated with human SVF cells, human ASCs, or vehicle. Second, each rat was treated with human SVF, human SVF lysate, or vehicle. Finally, each rat was treated with rat (autologous) SVF cells or vehicle. Flap survival, vessel density, and stromal cell retention were evaluated after 7 days. The mean survival rates for SVF treatment regardless of human or autologous origin were significantly increased as compared with the control group. Adipose stem/stromal cell and SVF lysate injection did not increase flap survival. Vessel density was increased for human and rat SVF and human ASC but not for SVF lysate. Human cells were not detected in the flaps after 7 days. Flap survival increased with SVF treatment regardless of human or autologous origin, suggesting that increased flap survival is independent of the host immune response. All cell injections lead to increased vessel density, but it did not necessarily lead to increased flap survival. Further research should elaborate which molecular events make SVF treatment more efficacious than ASC.

  3. Effect of TGF-β1 Stimulation on the Secretome of Human Adipose-Derived Mesenchymal Stromal Cells.

    PubMed

    Rodríguez, Tania M; Saldías, Alejandro; Irigo, Marcelo; Zamora, Jorge Velasco; Perone, Marcelo J; Dewey, Ricardo A

    2015-08-01

    Adipose tissue is an attractive source of mesenchymal stromal cells (MSCs) owing to the relative ease of obtaining large volumes with more MSC abundance compared with other sources. Increasing evidence supports the fact that trophic factors secreted by MSCs play a pivotal therapeutic role. Several strategies in regenerative medicine use MSCs, mainly exploiting their immunosuppressive effect and homing capacity to sites of damage. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that, depending on the cell niche, can display either anti-inflammatory or proinflammatory effects. TGF-β1 expression increases in various tissues with damage, especially when accompanied by inflammation. Thus, we analyzed the effect of TGF-β1 on the secretion by adipose-derived mesenchymal stromal cells (ASCs) of a panel of 80 cytokines/chemokines using an antibody array. To avoid a possible effect of fetal bovine serum (FBS) on ASCs secretion, we performed our analysis by culturing cells in FBS-free conditions, only supplemented with 0.1% of bovine serum albumin. We report the cytokine profile secreted by ASCs. We also found that TGF-β1 exposure modulates 8 chemokines and 18 cytokines, including TGF-β1 and -β2, and other important cytokines involved in immunosuppression, allergic responses, and bone resorption.

  4. Effect of TGF-β1 Stimulation on the Secretome of Human Adipose-Derived Mesenchymal Stromal Cells

    PubMed Central

    Rodríguez, Tania M.; Saldías, Alejandro; Irigo, Marcelo; Zamora, Jorge Velasco; Perone, Marcelo J.

    2015-01-01

    Adipose tissue is an attractive source of mesenchymal stromal cells (MSCs) owing to the relative ease of obtaining large volumes with more MSC abundance compared with other sources. Increasing evidence supports the fact that trophic factors secreted by MSCs play a pivotal therapeutic role. Several strategies in regenerative medicine use MSCs, mainly exploiting their immunosuppressive effect and homing capacity to sites of damage. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that, depending on the cell niche, can display either anti-inflammatory or proinflammatory effects. TGF-β1 expression increases in various tissues with damage, especially when accompanied by inflammation. Thus, we analyzed the effect of TGF-β1 on the secretion by adipose-derived mesenchymal stromal cells (ASCs) of a panel of 80 cytokines/chemokines using an antibody array. To avoid a possible effect of fetal bovine serum (FBS) on ASCs secretion, we performed our analysis by culturing cells in FBS-free conditions, only supplemented with 0.1% of bovine serum albumin. We report the cytokine profile secreted by ASCs. We also found that TGF-β1 exposure modulates 8 chemokines and 18 cytokines, including TGF-β1 and -β2, and other important cytokines involved in immunosuppression, allergic responses, and bone resorption. Significance Mesenchymal stromal cells (MSCs) secrete a broad spectrum of bioactive macromolecules that are both immunoregulatory and serve to structure regenerative microenvironments in fields of tissue injury. Increases or decreases in the production of TGF-β1 have been linked to numerous disease states, including autoimmune diseases and cancer. The secretome of MSCs stimulated with TGF-β1 is largely unknown. Thus, the present study makes an important contribution toward a better understanding of how MSCs could be affected by a cytokine normally upregulated in various diseases. PMID:26025982

  5. Ectopic osteogenic capacity of freshly isolated adipose-derived stromal vascular fraction cells supported with platelet-rich plasma: A simulation of intraoperative procedure.

    PubMed

    Najman, Stevo J; Cvetković, Vladimir J; Najdanović, Jelena G; Stojanović, Sanja; Vukelić-Nikolić, Marija Đ; Vučković, Ivica; Petrović, Dragan

    2016-10-01

    Bone defects represent a serious problem in cranio-maxillofacial surgery. Autologous adipose-derived stromal vascular fraction (SVF) cells in combination with biological factors and bone substitutes were previously proposed as alternative to bone grafting. By simulating an intraoperative procedure we examined osteogenic capacity of the combination of two autologous components, freshly isolated adipose-derived SVF cells, and platelet-rich plasma (PRP), delivered on bone mineral matrix (BMM) carrier (SPB group) in mice ectopic bone forming model. Implantation of BMM only (B group) was a control. The presence of adipose-derived stem cells (ADSCs) in SVF was detected by immunocytochemical analysis. Expression of bone- and endothelial-related genes was compared between freshly isolated SVF and ADSCs obtained from SVF after in vitro cultivation. The implants were analyzed using expression analysis of bone-related genes at one, two, four and eight weeks and histochemical, immunohistochemical and histomorphometrical analyses at two and eight weeks after implantation. Freshly isolated adipose-derived SVF contained ADSCs and exhibited promising osteogenic and vasculogenic capacity. At two and four weeks, significantly higher expression of bone-related genes was detected in SPB group compared to B group. The signs of osteogenic process were more pronounced in SPB than in B implants. By the end of experiment, percentage of infiltrated tissue and vascularization was significantly higher in SPB than in B implants. Adipose-derived SVF cells, PRP and BMM rapidly initiated osteogenesis what makes this combination promising candidate for treatment of bone defects.

  6. Shift toward Mechanical Isolation of Adipose-derived Stromal Vascular Fraction: Review of Upcoming Techniques

    PubMed Central

    Kotamarti, Vasanth S.; Sherman, Lauren S.; Keith, Jonathan D.; Lee, Edward S.; Granick, Mark S.; Rameshwar, Pranela

    2016-01-01

    Background: Standard isolation of adipose stromal vascular fraction (SVF) requires the use of collagenase and is considered more than “minimally manipulated” by current good manufacturing practice requirements. Alternatively, nonenzymatic isolation methods have surfaced using physical forces to separate cells from the adipose matrix. The purpose of this study was to review the literature on the use of mechanical isolation protocols and compare the results. The implication for use as a standard procedure in practice is discussed. Methods: A systematic review of the literature was performed on mechanical isolation of SVF with a search of six terms on PubMed and Medline databases. One thousand sixty-six articles were subject to evaluation by predetermined inclusion and exclusion criteria. Results: Two level 2 evidence articles and 7 in vitro studies were selected. SVF was isolated using automated closed systems or by subjecting the lipoaspirate to centrifugation only or by shaking or vortexing followed by centrifugation. Six articles reported isolation in laboratory settings and three inside the operating room. Stromal vascular cells expressed CD34, and CD44, CD73, CD90, and CD105, and differentiated along adipogenic and osteogenic lineages. When compared with enzymatic methods, mechanical isolation required less time but yielded fewer cells. Both case–control studies reported improved volume retention with cell-supplemented fat grafts for breast reconstruction. Conclusions: Mechanical isolation methods are alternatives to circumvent safety issues posed by enzymatic protocols. However, randomized comparative studies with long-term clinical outcomes using mechanically isolated stromal vascular cells are needed to identify their ideal clinical applications. PMID:27757339

  7. Periodontal tissue regeneration by transplantation of rat adipose-derived stromal cells in combination with PLGA-based solid scaffolds.

    PubMed

    Akita, Daisuke; Morokuma, Masakazu; Saito, Yoko; Yamanaka, Katsuyuki; Akiyama, Yuko; Sato, Momoko; Mashimo, Takayuki; Toriumi, Taku; Arai, Yoshinori; Kaneko, Tadashi; Tsukimura, Naoki; Isokawa, Keitaro; Ishigami, Tomohiko; Honda, Masaki J

    2014-01-01

    Regeneration of damaged periodontium is challenging due to its multi-tissue composition. Mesenchymalstem cell-based approaches using adipose-derived stromal cells (ASCs) may contribute to periodontal reconstruction, particularly when combined with the use of scaffolds to maintain a space for new tissue growth. The aim of this study was to assess the regenerative potential of ASCs derived from inbred or outbred rats in combination with novel solid scaffolds composed of PLGA (Poly D,L-lactic-co-glycolic acid) (PLGA-scaffolds). Cultured ASCs seeded onto PLGA scaffolds (ASCs/PLGA) or PLGA-scaffolds (PLGA) alone were transplanted into periodontal fenestration defects created in F344 or Sprague Dawley (SD) rats. Micro-CT analysis showed a significantly higher percentage of bone growth in the ASCs/PLGA groups compared with the PLGA-alone groups at five weeks after surgery. Similarly, histomorphometric analysis demonstrated thicker growth of periodontal ligament and cementum layers in the ASCs/PLGA-groups compared with the PLGA-alone groups. In addition, transplanted DiI-labeled ASCs were observed in the periodontal regenerative sites. The present investigation demonstrated the marked ability of ASCs in combination with PLGA scaffolds to repair periodontal defects.

  8. Obesity-associated dysregulation of calpastatin and MMP-15 in adipose-derived stromal cells results in their enhanced invasion.

    PubMed

    Strong, Amy L; Semon, Julie A; Strong, Thomas A; Santoke, Tatyana T; Zhang, Shijia; McFerrin, Harris E; Gimble, Jeffrey M; Bunnell, Bruce A

    2012-12-01

    Adipose tissue maintains a subpopulation of cells, referred to as adipose-derived stromal/stem cells (ASCs), which have been associated with increased breast cancer tumorigenesis and metastasis. For ASCs to affect breast cancer cells, it is necessary to delineate how they mobilize and home to cancer cells, which requires mobilization and invasion through extracellular matrix barriers. In this study, ASCs were separated into four different categories based on the donor's obesity status and depot site of origin. ASCs isolated from the subcutaneous abdominal adipose tissue of obese patients (Ob(+)Ab(+)) demonstrated increased invasion through Matrigel as well as a chick chorioallantoic membrane, a type I collagen-rich extracellular matrix barrier. Detailed mRNA and protein analyses revealed that calpain-4, calpastatin, and MMP-15 were associated with increased invasion, and the silencing of each protease or protease inhibitor confirmed their role in ASC invasion. Thus, the data indicate that both the donor's obesity status and depot site of origin distinguishes the properties of subcutaneous-derived ASCs with respect to enhanced invasion and this is associated with the dysregulation of calpain-4, calpastatin, and MMP-15.

  9. Enhanced angiogenic effect of adipose-derived stromal cell spheroid with low-level light therapy in hindlimb ischemia mice

    NASA Astrophysics Data System (ADS)

    Park, In-Su; Ahn, Jin-Chul; Chung, Phil-Sang

    2014-02-01

    Adipose-derived stromal cells (ASCs) are attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted ASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level laser therapy (LLLT) on transplanted human ASCs (hASCs) spheroid in a hindlimb ischemia animal model. LLLT, hASCs spheroid and hASCs spheroid transplantation with LLLT (spheroid + LLLT) were applied to the ischemic hindlimbs in athymic mice. The survival, differentiation and secretion of vascular endothelial growth (VEGF) of spheroid ASCs were evaluated by immunohistochemistry. The spheroid + LLLT group enhanced the tissue regeneration, including angiogenesis, compared with other groups. The spheroid contributed tissue regeneration via differentiation and secretion of growth factors. In the spheroid + LLLT group, the survival of spheroid hASCs was increased by the decreased apoptosis of spheroid hASCs in the ischemic hindlimb. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASCs group and spheroid group. These data suggest that LLLT is an effective biostimulator of spheroid hASCs in tissue regeneration that enhances the survival of ASCs and stimulates the secretion of growth factors in the ischemic hindlimb.

  10. Hyaluronan preserves the proliferation and differentiation potentials of long-term cultured murine adipose-derived stromal cells

    SciTech Connect

    Chen, P.-Y.; Huang, Lynn L.H. . E-mail: lynn@mail.ncku.edu.tw; Hsieh, H.-J. . E-mail: hjhsieh@ntu.edu.tw

    2007-08-17

    For long-term culture, murine adipose-derived stromal cells (mADSCs) at latter passages demonstrated a marked decline in proliferative activity, exhibited senescent morphology and reduced differentiation potentials, particularly osteogenesis. To extend the lifespan of mADSCs, two culture conditions containing hyaluronan (HA) was compared in our study, one as a culture medium supplement (SHA), and the other where HA was pre-coated on culture surface (CHA). mADSCs cultivated with SHA exhibited a prolonged lifespan, reduced cellular senescence, and enhanced osteogenic potential compared to regular culture condition (control). Upon CHA treatment, mADSCs tended to form cell aggregates with gradual growth profiles, while their differentiation activities remained similar to SHA groups. After transferring mADSCs from CHA to control surface, they were shown to have an extended lifespan and an increase of osteogenic potential. Our results suggested that HA can be useful for preserving the proliferation and differentiation potentials of long-term cultured mADSCs.

  11. Studies in Fat Grafting: Part IV. Adipose-derived stromal cell gene expression in cell-assisted lipotransfer

    PubMed Central

    Garza, Rebecca M.; Rennert, Robert C.; Paik, Kevin J.; Atashroo, David; Chung, Michael T.; Duscher, Dominik; Januszyk, Michael; Gurtner, Geoffrey C.; Longaker, Michael T.; Wan, Derrick C.

    2014-01-01

    Background Fat graft volume retention remains highly unpredictable, but addition of adipose-derived stromal cells (ASCs) to fat grafts has been shown to improve retention. The present study aimed to investigate the mechanisms involved in ASC enhancement of fat grafting. Methods ASCs isolated from human lipoaspirate were labeled with green fluorescent protein (GFP) and luciferase. Fat grafts enhanced with ASCs were injected into the scalp and bioluminescent imaging was performed to follow retention of ASCs within the fat graft. Fat grafts were also explanted at days 1, 5, and 10 post-grafting for ASC extraction and single-cell gene analysis. Finally, CD31 immunohistochemical staining was performed on fat grafts enriched with ASCs. Results Bioluminescent imaging demonstrated significant reduction in luciferase+ ASCs within fat grafts at five days post-grafting. A similar reduction in viable GFP+ ASCs retrieved from explanted grafts was also noted. Single cell analysis revealed expression of multiple genes/markers related to cell survival and angiogenesis including BMPR2, CD90, CD105, FGF2, CD248, TGFβ1, and VEGFA. Genes involved in adipogenesis were not expressed by ASCs. Finally, CD31 staining revealed significantly higher vascular density in fat grafts explanted at day 10 post-grafting. Conclusions Although ASC survival in the hypoxic graft environment decreases significantly over time, these cells provide multiple angiogenic growth factors. Therefore, improved fat graft volume retention with ASC enrichment may be due to improved graft vascularization. PMID:25502860

  12. Extracellular matrix components of adipose derived stromal cells promote alignment, organization, and maturation of cardiomyocytes in vitro.

    PubMed

    Przybyt, Ewa; van Luyn, Marja J A; Harmsen, Martin C

    2015-05-01

    Adipose derived stromal cells (ADSC) are relevant therapeutic agents to treat myocardial infarction (MI) in clinical trials. Soluble factors secreted by ADSC, such as growth factors and cytokines, suppress inflammation and apoptosis while promoting angiogenesis and the proliferation of cardiomyocytes (CM). Moreover, ADSC synthesize extracellular matrix (ECM) components into the intercellular space which might contribute to their therapeutic capacity. Thus, ADSC might directly modulate the post-MI microenvironment through a combination of paracrine and juxtacrine signaling. In this study, the juxtacrine role of ADSC and ADSC-derived ECM on the organization and maturation of CM was investiagated. Human ADSC synthesized and deposited a heterogenous and complex mixture of ECM components such as collagen I, III, IV, fibronectin, elastin as well as the matricellular protein periostin. Cocultures of rodent CM with human ADSC or with human ADSC-derived ECM components enhanced the cardiomyocyte alignment, their intercellular connections and the maturation of their sarcomeres, while the proliferation rate of the CM was increased and their level of hypertrophy reduced. The use of human ADSC-derived ECM could serve both to augment in vitro tissue-engineered myocardial constructs and to improve myocardial remodeling after infarction. © 2014 Wiley Periodicals, Inc.

  13. Autologous transplants of Adipose-Derived Adult Stromal (ADAS) cells afford dopaminergic neuroprotection in a model of Parkinson's disease.

    PubMed

    McCoy, Melissa K; Martinez, Terina N; Ruhn, Kelly A; Wrage, Philip C; Keefer, Edward W; Botterman, Barry R; Tansey, Keith E; Tansey, Malú G

    2008-03-01

    Adult adipose contains stromal progenitor cells with neurogenic potential. However, the stability of neuronal phenotypes adopted by Adipose-Derived Adult Stromal (ADAS) cells and whether terminal neuronal differentiation is required for their consideration as alternatives in cell replacement strategies to treat neurological disorders is largely unknown. We investigated whether in vitro neural induction of ADAS cells determined their ability to neuroprotect or restore function in a lesioned dopaminergic pathway. In vitro-expanded naïve or differentiated ADAS cells were autologously transplanted into substantia nigra 1 week after an intrastriatal 6-hydroxydopamine injection. Neurochemical and behavioral measures demonstrated neuroprotective effects of both ADAS grafts against 6-hydroxydopamine-induced dopaminergic neuron death, suggesting that pre-transplantation differentiation of the cells does not determine their ability to survive or neuroprotect in vivo. Therefore, we investigated whether equivalent protection by naïve and neurally-induced ADAS grafts resulted from robust in situ differentiation of both graft types into dopaminergic fates. Immunohistological analyses revealed that ADAS cells did not adopt dopaminergic cell fates in situ, consistent with the limited ability of these cells to undergo terminal differentiation into electrically active neurons in vitro. Moreover, re-exposure of neurally-differentiated ADAS cells to serum-containing medium in vitro confirmed ADAS cell phenotypic instability (plasticity). Lastly, given that gene expression analyses of in vitro-expanded ADAS cells revealed that both naïve and differentiated ADAS cells express potent dopaminergic survival factors, ADAS transplants may have exerted neuroprotective effects by production of trophic factors at the lesion site. ADAS cells may be ideal for ex vivo gene transfer therapies in Parkinson's disease treatment.

  14. Qualitative and quantitative differences of adipose-derived stromal cells from superficial and deep subcutaneous lipoaspirates: a matter of fat.

    PubMed

    Di Taranto, Giuseppe; Cicione, Claudia; Visconti, Giuseppe; Isgrò, Maria A; Barba, Marta; Di Stasio, Enrico; Stigliano, Egidio; Bernardini, Camilla; Michetti, Fabrizio; Salgarello, Marzia; Lattanzi, Wanda

    2015-08-01

    Subcutaneous fat represents a valuable reservoir of adipose-derived stem cells (ASCs) in the stromal vascular fraction (SVF), widely exploited in regenerative medicine applications, being easily harvested through lipoaspiration. The lack of standardized procedures for autologous fat grafting guided research efforts aimed at identifying possible differences related to the harvesting site, which may affect cell isolation yield, cell growth properties and clinical outcomes. Subcutaneous fat features a complex architecture: the superficial fascia separates superficial adipose tissue (SAT) from deep layer tissue (DAT). We aimed to unravel the differences between SAT and DAT, considering morphological structure, SVF composition, and ASC properties. SAT and DAT were collected from female donors and comparatively analyzed to evaluate cellular yield and viability, morphology, immunophenotype and molecular profile. ASCs were isolated in primary culture and used for in vitro differentiation assays. SAT and DAT from cadaver donors were also analyzed through histology and immunohistochemistry to assess morphology and cell localization within the hypoderm. Liposuctioned SAT contained a higher stromal tissue compound, along with a higher proportion of CD105-positive cells, compared with DAT from the same harvesting site. Also, cells isolated from SAT displayed increased multipotency and stemness features. All differences were mainly evidenced in specimens harvested from the abdominal region. According to our results, SAT features overall increased stem properties. Given that subcutaneous adipose tissue is currently exploited as the gold standard source for high-yield isolation of adult stem cells, these results may provide precious hints toward the definition of standardized protocols for microharvesting. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  15. [Effects of rat allogeneic adipose-derived stem cells on the early neovascularization of autologous fat transplantation].

    PubMed

    Tian, Tian; Jia, Chiyu; Liu, Yi; Liu, Zhen; Hu, Guodong; Wang, Ruichen; Chang, Chunjuan

    2014-12-01

    To investigate the effects of allogeneic adipose-derived stem cells (ADSCs) of rat on the early neovascularization of autologous fat transplantation. (1) Experiment 1. Adipose tissue was collected from both inguinal regions of two SD rats to isolate, culture, and purify ADSCs through collagen enzyme digestion, density gradient centrifugation, and adherence method. The fourth passage of cells were collected for morphologic observation, detection of expressions of surface markers CD34, CD49d, CD106, and CD45 of ADSCs with flow cytometer, identification of adipogenic and osteogenic differentiation, and determination of the cell proliferation ability with thiazolyl blue method. (2) Experiment 2. Another 30 SD rats were divided into allogeneic adipose granule (AG) group (A, n = 6), autologous AG group (B, n = 8), autologous ADSCs+autologous AG group (C, n = 8), and allogeneic ADSCs+autologous AG group (D, n = 8) according to the random number table. The fourth passage of ADSCs were obtained from adipose tissue from one side of inguinal region of SD rats in group C. Adipose tissue obtained from one side of inguinal region of SD rats of the other 3 groups was abandoned. The AG was prepared from another side of inguinal region of SD rats in the 4 groups. The mixture of 0.6 g AG from one rat and 1 mL DMEM/F12 nutrient solution was injected subcutaneously into the back of another rat in group A, and so on. Autologous AG was injected into its own body of the rats in group B. The mixture of 1 mL autologous ADSCs mixture which contains 3.0 × 10⁶ cells per mililitre autologous ADSCs combined with autologous AG was injected into the rats in group C. The mixture of 1 mL allogeneic ADSCs mixture which contains 3.0 × 10⁶ cells per mililitre ADSCs extractived from the former 2 rats in experiment 1 combined with autologous AG was injected into the rats in group D. At 7 days post transplantation, fat transplants were harvested for gross observation, measurement of wet weight

  16. Periodontal Tissue Regeneration Using Syngeneic Adipose-Derived Stromal Cells in a Mouse Model.

    PubMed

    Lemaitre, Mathieu; Monsarrat, Paul; Blasco-Baque, Vincent; Loubières, Pascale; Burcelin, Rémy; Casteilla, Louis; Planat-Bénard, Valérie; Kémoun, Philippe

    2017-02-01

    Current treatment of periodontitis is still associated with a high degree of variability in clinical outcomes. Recent advances in regenerative medicine by mesenchymal cells, including adipose stromal cells (ASC) have paved the way to improved periodontal regeneration (PD) but little is known about the biological processes involved. Here, we aimed to use syngeneic ASCs for periodontal regeneration in a new, relevant, bacteria-induced periodontitis model in mice. Periodontal defects were induced in female C57BL6/J mice by oral gavage with periodontal pathogens. We grafted 2 × 10(5) syngeneic mouse ASCs expressing green fluorescent protein (GFP) (GFP+/ASC) within a collagen vehicle in the lingual part of the first lower molar periodontium (experimental) while carrier alone was implanted in the contralateral side (control). Animals were sacrificed 0, 1, 6, and 12 weeks after treatment by GFP+/ASC or vehicle graft, and microscopic examination, immunofluorescence, and innovative bio-informatics histomorphometry methods were used to reveal deep periodontium changes. From 1 to 6 weeks after surgery, GFP+ cells were identified in the periodontal ligament (PDL), in experimental sites only. After 12 weeks, cementum regeneration, the organization of PDL fibers, the number of PD vessels, and bone morphogenetic protein-2 and osteopontin expression were greater in experimental sites than in controls. Specific stromal cell subsets were recruited in the newly formed tissue in ASC-implanted periodontium only. These data suggest that ASC grafting in diseased deep periodontium, relevant to human pathology, induces a significant improvement of the PDL microenvironment, leading to a recovery of tooth-supporting tissue homeostasis. Stem Cells Translational Medicine 2017;6:656-665.

  17. Periodontal Tissue Regeneration Using Syngeneic Adipose-Derived Stromal Cells in a Mouse Model.

    PubMed

    Lemaitre, Mathieu; Monsarrat, Paul; Blasco-Baque, Vincent; Loubières, Pascale; Burcelin, Rémy; Casteilla, Louis; Planat-Bénard, Valérie; Kémoun, Philippe

    2016-09-16

    : Current treatment of periodontitis is still associated with a high degree of variability in clinical outcomes. Recent advances in regenerative medicine by mesenchymal cells, including adipose stromal cells (ASC) have paved the way to improved periodontal regeneration (PD) but little is known about the biological processes involved. Here, we aimed to use syngeneic ASCs for periodontal regeneration in a new, relevant, bacteria-induced periodontitis model in mice. Periodontal defects were induced in female C57BL6/J mice by oral gavage with periodontal pathogens. We grafted 2 × 10(5) syngeneic mouse ASCs expressing green fluorescent protein (GFP) (GFP+/ASC) within a collagen vehicle in the lingual part of the first lower molar periodontium (experimental) while carrier alone was implanted in the contralateral side (control). Animals were sacrificed 0, 1, 6, and 12 weeks after treatment by GFP+/ASC or vehicle graft, and microscopic examination, immunofluorescence, and innovative bio-informatics histomorphometry methods were used to reveal deep periodontium changes. From 1 to 6 weeks after surgery, GFP+ cells were identified in the periodontal ligament (PDL), in experimental sites only. After 12 weeks, cementum regeneration, the organization of PDL fibers, the number of PD vessels, and bone morphogenetic protein-2 and osteopontin expression were greater in experimental sites than in controls. Specific stromal cell subsets were recruited in the newly formed tissue in ASC-implanted periodontium only. These data suggest that ASC grafting in diseased deep periodontium, relevant to human pathology, induces a significant improvement of the PDL microenvironment, leading to a recovery of tooth-supporting tissue homeostasis.

  18. Treatment of Crohn's-Related Rectovaginal Fistula With Allogeneic Expanded-Adipose Derived Stem Cells: A Phase I-IIa Clinical Trial.

    PubMed

    García-Arranz, Mariano; Herreros, Maria Dolores; González-Gómez, Carolina; de la Quintana, Paloma; Guadalajara, Héctor; Georgiev-Hristov, Tihomir; Trébol, Jacobo; Garcia-Olmo, Damián

    2016-11-01

    : The aim of this clinical trial was to determine the safety and feasibility of expanded allogeneic adipose-derived stem cells to treat Crohn's-related rectovaginal fistula (CRRVF). We designed a phase I-II clinical trial (https://ClinicalTrials.gov, NCT00999115) to treat 10 patients with CRRVF. Patients receiving biological therapy during follow-up were excluded. Curettage was performed, and a vaginal or rectal flap was added if the surgeon considered it necessary. The therapeutic protocol included intralesional injection of 20 million stem cells in the vaginal walls (submucosal area) and fistula tract. Healing was evaluated 12 weeks later. If the fistula had not healed, a second dose of 40 million stem cells was administered. Patient follow-up was 52 weeks from last cell injection. Healing was defined as re-epithelialization of both vaginal and rectal sides and absence of vaginal drainage. Cytokines and immunological blood tests were monitored. Serious adverse events or rejection issues were not observed. Five patients were excluded because biologic drugs were required to treat a Crohn's disease flare-up during follow-up. Cytokine profiles and immunotoxicity assays showed no statistically significant alterations. Sixty percent of the nonexcluded patients achieved a complete healing. Expanded allogeneic adipose-derived stem-cell injection is a safe and feasible therapy for treating CRRVF, and the healing success rate seems promising (60%). The results of this trial encourage further exploration into this therapy. This may be the first publication in which allogeneic stem cells to treat rectovaginal fistula in Crohn´s disease seem to be a feasible and safe treatment. Additional studies are necessary to confirm the efficacy profile of the allogeneic stem cells strategy in a controlled design. ©AlphaMed Press.

  19. Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells.

    PubMed

    Ye, Lanfeng; Chen, Lin; Feng, Fan; Cui, Junhui; Li, Kaide; Li, Zhiyong; Liu, Lei

    2015-10-01

    Tooth loss is presently a global epidemic and tooth regeneration is thought to be a feasible and ideal treatment approach. Choice of cell source is a primary concern in tooth regeneration. In this study, the odontogenic differentiation potential of two non-dental-derived stem cells, adipose-derived stromal cells (ADSCs) and bone marrow-derived stromal cells (BMSCs), were evaluated both in vitro and in vivo. ADSCs and BMSCs were induced in vitro in the presence of tooth germ cell-conditioned medium (TGC-CM) prior to implantation into the omentum majus of rats, in combination with inactivated dentin matrix (IDM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of odontogenic-related genes. Immunofluorescence and immunohistochemical assays were used to detect the protein levels of odontogenic-specific genes, such as DSP and DMP-1 both in vitro and in vivo. The results suggest that both ADSCs and BMSCs have odontogenic differentiation potential. However, the odontogenic potential of BMSCs was greater compared with ADSCs, showing that BMSCs are a more appropriate cell source for tooth regeneration.

  20. Hypoxia enhances angiogenesis in an adipose-derived stromal cell/endothelial cell co-culture 3D gel model.

    PubMed

    Xie, Qiang; Xie, Jing; Zhong, Juan; Cun, Xiangzhu; Lin, Shiyu; Lin, Yunfeng; Cai, Xiaoxiao

    2016-04-01

    This study aimed to investigate the influence of hypoxia on angiogenesis in a 3D gel, with co-culturing adipose-derived stromal cells (ASCs) and endothelial cells (ECs). ASCs from green fluorescent protein-labeled mice and ECs from red fluorescent protein-labeled mice were co-cultured in 3D collagen gels at 1:1 ratio, in normal and hypoxic oxygen conditions, and morphology of angiogenesis was observed using confocal laser scanning microscopy. To discover changes in growth factors between monoculture ASCs and ECs, transwell co-cultures of ASCs and ECs were applied. Semi-quantitative PCR was performed to explore mRNA expression of growth factors. Enhanced angiogenesis was observed in 3D gels implanted with 1:1 mixture of ASCs and ECs after 7 days hypoxia. Genes including VEGFA/B, EGF-1, HIF-1a, IGF-1, PDGF, TGF-β1 and BMP-2/4 in ECs, both monoculture and co-culture, were significantly enhanced after being cultured under hypoxia. In comparison, genes VEGFA/B, EGF-1, HIF-1a, TGF-β1 and BMP-2 in ASCs increased. In all, factors VEGFA/B, EGF-1, HIF-1a, TGF-β1 and BMP-2 increased in both ASCs and ECs after being cultured in hypoxia no matter whether as monoculture or co-culture. Co-culture of ASCs and ECs at 1:1 ratio in a 3D gel under hypoxia promoted angiogenesis. Those growth factors which were increased in both ASCs and ECs, indicate that VEGFA/B, EGF-1, HIF-1a, TGF-β1 and BMP-2 might be responsible for enhancement in angiogenesis triggered by hypoxia. © 2016 John Wiley & Sons Ltd.

  1. Dura Mater Stimulates Human Adipose-Derived Stromal Cells to Undergo Bone Formation in Mouse Calvarial Defects

    PubMed Central

    Levi, Benjamin; Nelson, Emily R.; Li, Shuli; James, Aaron W.; Hyun, Jeong S.; Montoro, Daniel T.; Lee, Min; Glotzbach, Jason P.; Commons, George W.; Longaker, Michael T.

    2015-01-01

    Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2–Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair. PMID:21656608

  2. DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival.

    PubMed

    Rogulska, Olena; Petrenko, Yuri; Petrenko, Alexander

    2017-04-01

    Off-the-shelf availability of human adipose-derived mesenchymal stromal cells (ASCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. Favorably, such cell processing protocols should not contain xenogeneic or toxic components, such as fetal bovine serum (FS) and dimethyl sulfoxide (DMSO). The objective of the study was to assess the sensitivity of ASCs to DMSO-free cryopreservation protocol depending on their expansion conditions: conventional, based on the application of FS or xeno-free, using PL as a medium supplement. ASCs expansion was carried out in α-MEM supplemented either with FS or PL. For DMSO- and xeno-free cryopreservation ASCs were pretreated with different concentrations of sucrose during 24 h of culture. Pretreated ASCs were cryopreserved in α-MEM containing 100-300 mM of sucrose with the cooling rate of 1 degree/min. ASCs were tested for survival (Trypan Blue test), viability (MTT test), recovery (Alamar Blue test), proliferation and ability to multilineage differentiation. The optimal concentrations of sucrose for ASCs pretreatment and as an additive in cryoprotective solution, which provided highest cell survival, comprised 100 and 200 mM, correspondingly. Survival and recovery rates of platelet lysate (PL)-expanded ASCs after DMSO-free cryopreservation comprised 59 and 51%, and were higher than in FS-cultured cells. After DMSO-free cryopreservation PL-processed ASCs had a shorter population doubling time and higher capacity for osteogenic differentiation than FS-processed cultures. The described DMSO- and xeno-free processing may form the basis for the development of safe and efficient protocols for manufacturing and banking of ASCs, providing their off-the-shelf availability for regenerative medicine applications.

  3. Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation.

    PubMed

    Herzmann, Nicole; Salamon, Achim; Fiedler, Tomas; Peters, Kirsten

    2017-01-01

    Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.

  4. Do IL-3/GM-CSF effect on the myofibroblastic differentiation of human adipose derived stromal cells?

    PubMed

    Lee, Jae-Sun; Tae, Son-Seung; Kim, Deok-Yeol; Han, Seung-Kyu; Kim, Woo-Kyung; Dhong, Eun-Sang

    2017-06-15

    Capsular contracture is an incurable complication after silicone-based implant surgery. Myofibroblast is the predominant cell in the contracted capsule. We hypothesized that human adipose derive stromal cells (hASCs) together with fibroblast may show a similar phenotypic characteristics of myofibroblast after the treatment of inflammatory cytokines in vitro. Interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) were treated in the culture of hASCs and HDFs. Lyn peptide inhibitor was applied as an inhibitor. The changes of cell surface markers (CD105, CD73, CD34, CD45, CD31, CD325 and CD146) were assessed. The expression of various cytokines related to wound contraction were tested such as TGF-β, α-SMA, HGF, FGF, ENT-1, and TSP-1. Myo-D, α-SMA, and glial fibrillary acidic protein (GFAP) were evaluated by blotting and immunocytochemical staining. The collagen-gel contraction assay was performed for the functional contraction of myofibroblastic phenotype. The expression of α-SMA, Myo-D and GFAP after the treatment of IL-3/GM-CSF showed similar results in hASCs and HDFs. Enhanced expression of TGF- β was observed in HDFs and the increase of ENT-1 and TSP-1 was significant in hASCs. Collagen-gel with HDFs contracted significantly within 24h after the treatment of IL-3/GM-CSF, and the contraction was inhibited by Lyn peptide inhibitor. But in hASCs, the gel-contraction was not significant. IL-3/ GM-CSF effected on the myofibroblastic differentiation of hASCs as well as it did on HDFs. But hASCs did not show the phenotypic gel-contraction within 24h. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Toward reconstruction of the subcutaneous fat layer with the use of adipose-derived stromal cell-seeded collagen matrices.

    PubMed

    Werner, Katharina; Jakubietz, Michael G; Jakubietz, Rafael G; Schmidt, Karsten; Muhr, Christian; Bauer-Kreisel, Petra; Blunk, Torsten

    2014-12-01

    Complex injuries of the upper and lower extremities often result in scarring and subsequent adhesion formation, which may cause severe pain and distinctly reduce range of motion. In revision surgery, replacement of the missing subcutaneous tissue is desirable to prevent new adhesions, to cushion scarred tendons and nerves and to regain tissue elasticity. Therefore, the objective of this study was the in vitro evaluation of cell-seeded collagen matrices to serve as the basis for the reconstruction of the subcutaneous adipose tissue layer. Five commercially available acellular dermal collagen matrices were seeded with human adipose-derived stromal cells (hASC). Size and shape stability of cell-matrix constructs were assessed and cell adhesion onto the matrix surface was evaluated histologically. Adipogenic differentiation of hASC on matrices was evaluated by means of histological staining, triglyceride quantification, and quantitative real-time polymerase chain reaction gene expression analysis. The collagen matrix Permacol was the only cell-seeded material that exhibited excellent size and shape stability. For Permacol and Strattice, successful seeding with continuous cell layers on top of the matrices was observed. For both matrices, histological staining, triglyceride quantification and messenger RNA expression of adipogenic transcription factors indicated substantial adipogenic differentiation of hASC after long-term induction as well as after short-term induction of only 4 days. Of all matrices investigated, only Permacol exhibited adequate handling stability and the development of a thin adipose tissue layer on top of the matrix. Thus, this matrix appears promising to be used in the development of a subcutaneous cushioning layer after complex injuries involving large scar formation. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  6. Tumor necrosis factor improves vascularization in osteogenic grafts engineered with human adipose-derived stem/stromal cells.

    PubMed

    Hutton, Daphne L; Kondragunta, Renu; Moore, Erika M; Hung, Ben P; Jia, Xiaofeng; Grayson, Warren L

    2014-01-01

    The innate immune response following bone injury plays an important role in promoting cellular recruitment, revascularization, and other repair mechanisms. Tumor necrosis factor-α (TNF) is a prominent pro-inflammatory cytokine in this cascade, and has been previously shown to improve bone formation and angiogenesis in a dose- and timing-dependent manner. This ability to positively impact both osteogenesis and vascular growth may benefit bone tissue engineering, as vasculature is essential to maintaining cell viability in large grafts after implantation. Here, we investigated the effects of exogenous TNF on the induction of adipose-derived stem/stromal cells (ASCs) to engineer pre-vascularized osteogenic tissue in vitro with respect to dose, timing, and co-stimulation with other inflammatory mediators. We found that acute (2-day), low-dose exposure to TNF promoted vascularization, whereas higher doses and continuous exposure inhibited vascular growth. Co-stimulation with platelet-derived growth factor (PDGF), another key factor released following bone injury, increased vascular network formation synergistically with TNF. ASC-seeded grafts were then cultured within polycaprolactone-fibrin composite scaffolds and implanted in nude rats for 2 weeks, resulting in further tissue maturation and increased angiogenic ingrowth in TNF-treated grafts. VEGF-A expression levels were significantly higher in TNF-treated grafts immediately prior to implantation, indicating a long-term pro-angiogenic effect. These findings demonstrate that TNF has the potential to promote vasculogenesis in engineered osteogenic grafts both in vitro and in vivo. Thus, modulation and/or recapitulation of the immune response following bone injury may be a beneficial strategy for bone tissue engineering.

  7. Effect of Adipose-Derived Stromal Cells and BMP12 on Intrasynovial Tendon Repair: A Biomechanical, Biochemical, and Proteomics Study

    PubMed Central

    Gelberman, Richard H.; Shen, Hua; Kormpakis, Ioannis; Rothrauff, Benjamin; Yang, Guang; Tuan, Rocky S.; Xia, Younan; Sakiyama-Elbert, Shelly; Silva, Matthew J.; Thomopoulos, Stavros

    2016-01-01

    The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor- and cell-based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin-based scaffold. Control groups consisted of repair-only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC+BMP12 treatment for range of motion or tensile properties outcomes versus repair-only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC+BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC+BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC+BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions. PMID:26445383

  8. Microcirculatory Response In Vivo on Local Intraarterial Infusion of Autogenic Adipose-derived Stem Cells or Stromal Vascular Fraction

    PubMed Central

    2016-01-01

    Background: Both adipose-derived stem cells (ASCs) and stromal vascular fraction (SVF) have been demonstrated to have regenerative properties with therapeutic potential for numerous diseases through local or topical applications. However, it is unclear whether ASC or SVF can be delivered systemically through an intra-arterial infusion. The purpose of this study was to examine the microcirculatory response in vivo on local intraarterial infusion of autogenic ASCs or SVF in a vascular pedicle isolated rat cremaster microcirculation model. Materials and Methods: Fat tissue was surgically harvested from the flanks of male Sprague–Dawley rats (n = 12) and processed for SVF isolation. Some SVF samples were cultured for 24 hours for ASC purification. The autogenic SVF (1 × 105) cells (n = 6) or purified ASC (1 × 105) cells (n = 6) cells were infused into the microcirculation of cremaster muscle at a speed of 0.05 mL/min through the cannulation of femoral artery. As this is a vascular pedicle isolated preparation, the infused SVF or ASC cells went nowhere but the cremaster muscle. The video image of the microcirculation was monitored in real time during infusion. Results: Arteriole diameter was measured as A1 (100–160 µm), A2 (40–80 µm), and A3/A4 (10–30 µm). Capillary perfusion was quantified in 18 capillary fields of each muscle. There was a significant increase in the diameter of terminal arterioles (P = 0.049) and the capillary density (P = 0.02) after ASC intraarterial infusion. However, a significant cell aggregation, embolisms, and arterial obstruction were observed in the microcirculation in every case during SVF infusion. Conclusions: Intraarterial infusion is an appropriate route for the delivery of autogenic ASCs, but not of SVF. SVF-induced microembolisms were the reason for narrowing or blocking the lumen of terminal arterioles, resulting in no flow in the corresponding capillaries. PMID:27757364

  9. Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia.

    PubMed

    Kishimoto, Satoko; Inoue, Ken-Ichi; Nakamura, Shingo; Hattori, Hidemi; Ishihara, Masayuki; Sakuma, Masashi; Toyoda, Shigeru; Iwaguro, Hideki; Taguchi, Isao; Inoue, Teruo; Yoshida, Ken-Ichiro

    2016-06-01

    Heparin/protamine micro/nanoparticles (LH/P-MPs) were recently developed as low-molecular weight, biodegradable carriers for adipose-derived stromal cells (ADSCs). These particles can be used for a locally delivered stem cell therapy that promotes angiogenesis. LH/P-MPs bind to the cell surface of ADSCs and promote cell-to-cell interaction and aggregation of ADSCs. Cultured ADSC/LH/P-MP aggregates remain viable. Here, we examined the ability of these aggregates to rescue limb loss in a mouse model of hindlimb ischemia. Unilateral hindlimb ischemia was induced in adult male BALB/c mice by ligation of the iliac artery and hindlimb vein. For allotransplantation of ADSCs from the same inbred strain, we injected ADSC alone or ADSC/LH/P-MP aggregates or control medium (sham-treated) directly into the ischemic muscles. Ischemic limb blood perfusion, vessel density, and vessel area were recorded. The extent of ischemic limb necrosis or limb loss was assessed on postoperative days 2, 7, and 14. Compared with the sham-treatment control, treatment with ADSCs alone showed modest effects on blood perfusion recovery and increased the number of α-SMA-positive vessels. Response to ADSC/LH/P-MP aggregates was significantly greater than ADSCs alone for every endpoint. ADSC/LH/P-MP aggregates more effectively prevented the loss of ischemic hindlimbs than ADSCs alone or the sham-treatment. The LH/P-MPs augmented the effects of ADSCs on angiogenesis and reversal of limb ischemia. Use of ADSC/LH/P-MP aggregates offers a novel and convenient treatment method and potentially represents a promising new therapeutic approach to inducing angiogenesis in ischemic diseases. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  10. Successful management of an equine carpal chip fracture by intra-articularly injected adipose-derived stromal vascular fraction after arthroscopic removal.

    PubMed

    Tyrnenopoulou, P; Karayannopoulou, M; Angelopoulou, S; Pyrros, A; Mparous, E; Koliakos, G; Diakakis, N

    2016-01-01

    Carpal chip fractures are common causes of lameness in racehorses. Due to disadvantages in surgical management, adjuvant treatment modalities are usually necessary. Adipose-derived stem cells (ADSCs) have the potential to differentiate into other cell types including bone and cartilage cells. Adipose-derived stromal vascular fraction (SVF) is produced during ADSCs isolation from adipose tissue. The purpose of this report was to present the successful management of a grade III chip fracture in the right carpus of a 5-year-old Thoroughbred gelding by intra-articularly injected autologous SVF one month after the arthroscopic removal of the fracture. This treatment resulted in lameness improvement and short rehabilitation period to previous racing activities. High performance levels and no recurrent injuries were recorded during a twenty month follow-up period.

  11. Successful management of an equine carpal chip fracture by intra-articularly injected adipose-derived stromal vascular fraction after arthroscopic removal

    PubMed Central

    Tyrnenopoulou, P.; Karayannopoulou, M.; Angelopoulou, S.; Pyrros, A.; Mparous, E.; Koliakos, G.; Diakakis, N.

    2016-01-01

    Carpal chip fractures are common causes of lameness in racehorses. Due to disadvantages in surgical management, adjuvant treatment modalities are usually necessary. Adipose-derived stem cells (ADSCs) have the potential to differentiate into other cell types including bone and cartilage cells. Adipose-derived stromal vascular fraction (SVF) is produced during ADSCs isolation from adipose tissue. The purpose of this report was to present the successful management of a grade III chip fracture in the right carpus of a 5-year-old Thoroughbred gelding by intra-articularly injected autologous SVF one month after the arthroscopic removal of the fracture. This treatment resulted in lameness improvement and short rehabilitation period to previous racing activities. High performance levels and no recurrent injuries were recorded during a twenty month follow-up period. PMID:27656232

  12. Treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study.

    PubMed

    Zheng, Guoping; Huang, Lanfang; Tong, Haijiang; Shu, Qiang; Hu, Yaoqin; Ge, Menghua; Deng, Keqin; Zhang, Liuya; Zou, Bin; Cheng, Baoli; Xu, Jianguo

    2014-04-04

    Recent studies have demonstrated that mesenchymal stem cells (MSCs) modulate the immune response and reduce lung injury in animal models. Currently, no clinical studies of the effects of MSCs in acute respiratory distress syndrome (ARDS) exist. The objectives of this study were first to examine the possible adverse events after systemic administration of allogeneic adipose-derived MSCs in ARDS patients and second to determine potential efficacy of MSCs on ARDS. Twelve adult patients meeting the Berlin definition of acute respiratory distress syndrome with a PaO2/FiO2 ratio of < 200 were randomized to receive allogeneic adipose-derived MSCs or placebo in a 1:1 fashion. Patients received one intravenous dose of 1 × 106 cells/kg of body weight or saline. Possible side effects were monitored after treatment. Acute lung injury biomarkers, including IL-6, IL-8 and surfactant protein D (SP-D), were examined to determine the effects of MSCs on lung injury and inflammation. There were no infusion toxicities or serious adverse events related to MSCs administration and there were no significant differences in the overall number of adverse events between the two groups. Length of hospital stay, ventilator-free days and ICU-free days at day 28 after treatment were similar. There were no changes in biomarkers examined in the placebo group. In the MSCs group, serum SP-D levels at day 5 were significantly lower than those at day 0 (p = 0.027) while the changes in IL-8 levels were not significant. The IL-6 levels at day 5 showed a trend towards lower levels as compared with day 0, but this trend was not statistically significant (p = 0.06). Administration of allogeneic adipose-derived MSCs appears to be safe and feasible in the treatment of ARDS. However, the clinical effect with the doses of MSCs used is weak, and further optimization of this strategy will probably be required to reach the goal of reduced alveolar epithelial injury in ARDS. Clinical trials.gov, NCT

  13. Clinical-scale expansion of Adipose derived Stromal Cells starting from Stromal Vascular Fraction in a single-use bioreactor: Proof of concept for autologous applications.

    PubMed

    Gadelorge, Mélanie; Bourdens, Marion; Espagnolle, Nicolas; Bardiaux, Clémence; Murrell, Julie; Savary, Lenaig; Ribaud, Sylvain; Chaput, Benoît; Sensebe, Luc

    2016-12-11

    Adipose derived stromal cells (ASC) are adult multipotent cells increasingly used for cell therapy due to their differentiation potential, their paracrine effect and their convenience. ASC are currently selected from stromal vascular fraction (SVF) of adipose tissue and expanded in 2D flasks following Good Manufacturing Practices. This process is limited in surface area, labor-intensive and expensive, especially for autologous applications requiring selection and expansion steps for every patient. Closed and automated bioreactors offer an alternative for scalable and cost-effective production of ASCs. This study investigated a single-use stirred-tank bioreactor that can expand ASC from SVF on microcarriers. A preliminary microcarrier screening in static and spinner flask conditions was performed to evaluate the best candidate for adhesion, amplification and harvest. The selected microcarrier was used for process development in the bioreactor. The first experiments showed poor selectivity and growth of the ASC from the SVF (n = 2). The process was then adjusted by two means: 1. decreasing the platelet lysate in the medium for enhancing cell adherence and 2. adding a shear protectant (Pluronic F68). Following these modifications, we demonstrated that the number of population doublings of ASCs from SVF was not significantly different between the bioreactor and the 2D controls (n = 3). In addition, the ASC characterization after culture showed that cells maintained their clonogenic potential, phenotype, differentiation potential and immunosuppressive capacities. This study provides the proof of concept that isolation and amplification of functional ASC from SVF can be performed in a stirred-tank bioreactor combined with microcarriers. This article is protected by copyright. All rights reserved.

  14. Adipose-Derived Stromal Cells Protect Intervertebral Disc Cells in Compression: Implications for Stem Cell Regenerative Disc Therapy

    PubMed Central

    Sun, Zhen; Luo, Beier; Liu, Zhi-Heng; Samartzis, Dino; Liu, Zhongyang; Gao, Bo; Huang, Liangliang; Luo, Zhuo-Jing

    2015-01-01

    Introduction: Abnormal biomechanics plays a role in intervertebral disc degeneration. Adipose-derived stromal cells (ADSCs) have been implicated in disc integrity; however, their role in the setting of mechanical stimuli upon the disc's nucleus pulposus (NP) remains unknown. As such, the present study aimed to evaluate the influence of ADSCs upon NP cells in compressive load culture. Methods: Human NP cells were cultured in compressive load at 3.0MPa for 48 hours with or without ADSCs co-culture (the ratio was 50:50). We used flow cytometry, live/dead staining and scanning electron microscopy (SEM) to evaluate cell death, and determined the expression of specific apoptotic pathways by characterizing the expression of activated caspases-3, -8 and -9. We further used real-time (RT-) PCR and immunostaining to determine the expression of the extracellular matrix (ECM), mediators of matrix degradation (e.g. MMPs, TIMPs and ADAMTSs), pro-inflammatory factors and NP cell phenotype markers. Results: ADSCs inhibited human NP cell apoptosis via suppression of activated caspase-9 and caspase-3. Furthermore, ADSCs protected NP cells from the degradative effects of compressive load by significantly up-regulating the expression of ECM genes (SOX9, COL2A1 and ACAN), tissue inhibitors of metalloproteinases (TIMPs) genes (TIMP-1 and TIMP-2) and cytokeratin 8 (CK8) protein expression. Alternatively, ADSCs showed protective effect by inhibiting compressive load mediated increase of matrix metalloproteinases (MMPs; MMP-3 and MMP-13), disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs; ADAMTS-1 and 5), and pro-inflammatory factors (IL-1beta, IL-6, TGF-beta1 and TNF-alpha). Conclusions: Our study is the first in vitro study assessing the impact of ADSCs on NP cells in an un-physiological mechanical stimulation culture environment. Our study noted that ADSCs protect compressive load induced NP cell death and degradation by inhibition of activated caspase-9 and -3

  15. In Vivo Evaluation of Adipose-Derived Stromal Cells Delivered with a Nanofiber Scaffold for Tendon-to-Bone Repair

    PubMed Central

    Lipner, Justin; Shen, Hua; Cavinatto, Leonardo; Liu, Wenying; Havlioglu, Necat; Xia, Younan; Galatz, Leesa M.

    2015-01-01

    Rotator cuff tears are common and cause a great deal of lost productivity, pain, and disability. Tears are typically repaired by suturing the tendon back to its bony attachment. Unfortunately, the structural (e.g., aligned collagen) and compositional (e.g., a gradient in mineral) elements that produce a robust attachment in the healthy tissue are not regenerated during healing, and the repair is prone to failure. Two features of the failed healing response are deposition of poorly aligned scar tissue and loss of bone at the repair site. Therefore, the objective of the current study was to improve tendon-to-bone healing by promoting aligned collagen deposition and increased bone formation using a biomimetic scaffold seeded with pluripotent cells. An aligned nanofibrous poly(lactic-co-glycolic acid) scaffold with a gradient in mineral content was seeded with adipose-derived stromal cells (ASCs) and implanted at the repair site of a rat rotator cuff model. In one group, cells were transduced with the osteogenic factor bone morphogenetic protein 2 (BMP2). The healing response was examined in four groups (suture only, acellular scaffold, cellular scaffold, and cellular BMP2 scaffold) using histologic, bone morphology, and biomechanical outcomes at 14, 28, and 56 days. Histologically, the healing interface was dominated by a fibrovascular scar response in all groups. The acellular scaffold group showed a delayed healing response compared to the other groups. When examining bone morphology parameters, bone loss was evident in the cellular BMP2 group compared to other groups at 28 days. When examining repair-site mechanical properties, strength and modulus were decreased in the cellular BMP2 groups compared to other groups at 28 and 56 days. These results indicated that tendon-to-bone healing in this animal model was dominated by scar formation, preventing any positive effects of the implanted biomimetic scaffold. Furthermore, cells transduced with the osteogenic factor

  16. Treatment of faecal incontinence using allogeneic-adipose-derived mesenchymal stem cells: a study protocol for a pilot randomised controlled trial

    PubMed Central

    Park, Eun Jung; Kang, Jeonghyun; Baik, Seung Hyuk

    2016-01-01

    Introduction Faecal incontinence is a distressing condition with recurrent uncontrolled passage of faecal material. Although faecal incontinence may cause psychological depression and social isolation, previous treatments have been limited. Recently, regenerative treatment has been developed using mesenchymal stem cells. Especially, there are possibilities that adipose-tissue-derived stem cells can be effective to treat a degenerated anal sphincter that is causing faecal incontinence. Therefore, this study aimed to investigate the safety and efficacy of using allogeneic-adipose-derived mesenchymal stem cells in the treatment of the anal sphincter of patients with faecal incontinence. Methods and analysis This study is a randomised, prospective, dose escalation, placebo-controlled, single-blinded, single-centre trial with two parallel groups. The safety test is performed by an injection of allogeneic-adipose-derived mesenchymal stem cells (ALLO-ASCs) into the anal sphincter with dose escalation (3×107, 6×107 and 9×107 cells, sequentially). After confirming the safety of the stem cells, an efficacy test is performed by this dose in the experimental group. The experimental group will receive ALLO-ASCs mixed with fibrin glue into the anal sphincter, and the placebo group will receive 0.9% normal saline injection mixed with fibrin glue. The primary end point is to assess the safety of ALLO-ASCs after the injection into the anal sphincter, and the secondary end point is to compare the efficacy of ALLO-ASC injection with fibrin glue in patients with faecal incontinence. Ethics and dissemination The study protocol was approved by the Ministry of Food and Drug Safety and the Ministry of Health & Welfare, in the Republic of Korea. The informed consent form was approved by the institutional review board of Gangnam Severance Hospital (IRB approval number 3-2014-0271). Dissemination of the results will be presented at a conference and in peer-reviewed publications. Trial

  17. Osteochondral tissue formation through adipose-derived stromal cell differentiation on biomimetic polycaprolactone nanofibrous scaffolds with graded insulin and Beta-glycerophosphate concentrations.

    PubMed

    Erisken, Cevat; Kalyon, Dilhan M; Wang, Hongjun; Ornek-Ballanco, Ceren; Xu, Jiahua

    2011-05-01

    The ability to fabricate tissue engineering scaffolds containing systematic gradients in the distributions of stimulators provides additional means for the mimicking of the important gradients observed in native tissues. Here the concentration distributions of two bioactive agents were varied concomitantly for the first time (one increasing, whereas the other decreasing monotonically) in between the two sides of a nanofibrous scaffold. This was achieved via the application of a new processing method, that is, the twin-screw extrusion and electrospinning method, to generate gradients of insulin, a stimulator of chondrogenic differentiation, and β-glycerophosphate (β-GP), for mineralization. The graded poly(ɛ-caprolactone) mesh was seeded with human adipose-derived stromal cells and cultured over 8 weeks. The resulting tissue constructs were analyzed for and revealed indications of selective differentiation of human adipose-derived stromal cells toward chondrogenic lineage and mineralization as functions of position as a result of the corresponding concentrations of insulin and β-GP. Chondrogenic differentiation of the stem cells increased at insulin-rich locations and mineralization increased at β-GP-rich locations.

  18. Combined use of decellularized allogeneic artery conduits with autologous transdifferentiated adipose-derived stem cells for facial nerve regeneration in rats.

    PubMed

    Sun, Fei; Zhou, Ke; Mi, Wen-juan; Qiu, Jian-hua

    2011-11-01

    Natural biological conduits containing seed cells have been widely used as an alternative strategy for nerve gap reconstruction to replace traditional nerve autograft techniques. The purpose of this study was to investigate the effects of a decellularized allogeneic artery conduit containing autologous transdifferentiated adipose-derived stem cells (dADSCs) on an 8-mm facial nerve branch lesion in a rat model. After 8 weeks, functional evaluation of vibrissae movements and electrophysiological assessment, retrograde labeling of facial motoneurons and morphological analysis of regenerated nerves were performed to assess nerve regeneration. The transected nerves reconstructed with dADSC-seeded artery conduits achieved satisfying regenerative outcomes associated with morphological and functional improvements which approached those achieved with Schwann cell (SC)-seeded artery conduits, and superior to those achieved with artery conduits alone or ADSC-seeded artery conduits, but inferior to those achieved with nerve autografts. Besides, numerous transplanted PKH26-labeled dADSCs maintained their acquired SC-phenotype and myelin sheath-forming capacity inside decellularized artery conduits and were involved in the process of axonal regeneration and remyelination. Collectively, our combined use of decellularized allogeneic artery conduits with autologous dADSCs certainly showed beneficial effects on nerve regeneration and functional restoration, and thus represents an alternative approach for the reconstruction of peripheral facial nerve defects. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Treatment of faecal incontinence using allogeneic-adipose-derived mesenchymal stem cells: a study protocol for a pilot randomised controlled trial.

    PubMed

    Park, Eun Jung; Kang, Jeonghyun; Baik, Seung Hyuk

    2016-02-17

    Faecal incontinence is a distressing condition with recurrent uncontrolled passage of faecal material. Although faecal incontinence may cause psychological depression and social isolation, previous treatments have been limited. Recently, regenerative treatment has been developed using mesenchymal stem cells. Especially, there are possibilities that adipose-tissue-derived stem cells can be effective to treat a degenerated anal sphincter that is causing faecal incontinence. Therefore, this study aimed to investigate the safety and efficacy of using allogeneic-adipose-derived mesenchymal stem cells in the treatment of the anal sphincter of patients with faecal incontinence. This study is a randomised, prospective, dose escalation, placebo-controlled, single-blinded, single-centre trial with two parallel groups. The safety test is performed by an injection of allogeneic-adipose-derived mesenchymal stem cells (ALLO-ASCs) into the anal sphincter with dose escalation (3 × 10(7), 6 × 10(7) and 9 × 10(7) cells, sequentially). After confirming the safety of the stem cells, an efficacy test is performed by this dose in the experimental group. The experimental group will receive ALLO-ASCs mixed with fibrin glue into the anal sphincter, and the placebo group will receive 0.9% normal saline injection mixed with fibrin glue. The primary end point is to assess the safety of ALLO-ASCs after the injection into the anal sphincter, and the secondary end point is to compare the efficacy of ALLO-ASC injection with fibrin glue in patients with faecal incontinence. The study protocol was approved by the Ministry of Food and Drug Safety and the Ministry of Health & Welfare, in the Republic of Korea. The informed consent form was approved by the institutional review board of Gangnam Severance Hospital (IRB approval number 3-2014-0271). Dissemination of the results will be presented at a conference and in peer-reviewed publications. NCT02384499; Pre-results. Published by the BMJ

  20. In Vitro and In Vivo Effects of Metformin on Osteopontin Expression in Mice Adipose-Derived Multipotent Stromal Cells and Adipose Tissue

    PubMed Central

    Basińska, Katarzyna; Chrząstek, Klaudia; Marycz, Krzysztof

    2015-01-01

    Metformin is applied not only as antidiabetic drug, but also in the treatment of obesity or as antiaging drug. The first part of the research discussed the effect of metformin at concentrations of 1 mM, 5 mM, and 10 mM on the morphology, ultrastructure, and proliferation potential of mice adipose-derived multipotent mesenchymal stromal cells (ASCs) in vitro. Additionally, we determined the influence of metformin on mice adipose tissue metabolism. This study has shown for the first time that metformin inhibits the proliferative potential of ASCs in vitro in a dose- and time-dependent manner. In addition, we have found a significant correlation between the activity of ASCs and osteopontin at the mRNA and protein level. Furthermore, we have demonstrated that 5 mM and 10 mM metformin have cytotoxic effect on ASCs, causing severe morphological, ultrastructural, and apoptotic changes. The reduced level of OPN in the adipose tissue of metformin-treated animals strongly correlated with the lower expression of Ki67 and CD105 and increased caspase-3. The metformin influenced also circulating levels of OPN, which is what was found with systemic and local action of metformin. The results are a valuable source of information regarding the in vitro effect of metformin on adipose-derived stem cells. PMID:26064989

  1. Analysis of the material properties of early chondrogenic differentiated adipose-derived stromal cells (ASC) using an in vitro three-dimensional micromass culture system

    SciTech Connect

    Xu, Yue; Balooch, Guive; Chiou, Michael; Bekerman, Elena; Ritchie, Robert O.; Longaker, Michael T. . E-mail: Longaker@stanford.edu

    2007-07-27

    Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitro model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation.

  2. Functional recovery and neural differentiation after transplantation of allogenic adipose-derived stem cells in a canine model of acute spinal cord injury

    PubMed Central

    Ryu, Hak-Hyun; Lim, Ji-Hey; Byeon, Ye-Eun; Park, Jeong-Ran; Seo, Min-Soo; Lee, Young-Won; Kim, Wan Hee

    2009-01-01

    In this study, we evaluated if the implantation of allogenic adipose-derived stem cells (ASCs) improved neurological function in a canine spinal cord injury model. Eleven adult dogs were assigned to three groups according to treatment after spinal cord injury by epidural balloon compression: C group (no ASCs treatment as control), V group (vehicle treatment with PBS), and ASC group (ASCs treatment). ASCs or vehicle were injected directly into the injured site 1 week after spinal cord injury. Pelvic limb function after transplantation was evaluated by Olby score. Magnetic resonance imaging, somatosensory evoked potential (SEP), histopathologic and immunohistichemical examinations were also performed. Olby scores in the ASC group increased from 2 weeks after transplantation and were significantly higher than C and V groups until 8 weeks (p < 0.05). However, there were no significant differences between the C and V groups. Nerve conduction velocity based on SEP was significantly improved in the ASC group compared to C and V groups (p < 0.05). Positive areas for Luxol fast blue staining were located at the injured site in the ASC group. Also, GFAP, Tuj-1 and NF160 were observed immunohistochemically in cells derived from implanted ASCs. These results suggested that improvement in neurological function by the transplantation of ASCs in dogs with spinal cord injury may be partially due to the neural differentiation of implanted stem cells. PMID:19934591

  3. Therapeutic Potential of Human Adipose-Derived Stem/Stromal Cell Microspheroids Prepared by Three-Dimensional Culture in Non-Cross-Linked Hyaluronic Acid Gel.

    PubMed

    Mineda, Kazuhide; Feng, Jingwei; Ishimine, Hisako; Takada, Hitomi; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Kanayama, Koji; Kato, Harunosuke; Mashiko, Takanobu; Hashimoto, Ichiro; Nakanishi, Hideki; Kurisaki, Akira; Yoshimura, Kotaro

    2015-12-01

    Three-dimensional culture of mesenchymal stem/stromal cells for spheroid formation is known to enhance their therapeutic potential for regenerative medicine. Spheroids were prepared by culturing human adipose-derived stem/stromal cells (hASCs) in a non-cross-linked hyaluronic acid (HA) gel and compared with dissociated hASCs and hASC spheroids prepared using a nonadherent dish. Preliminary experiments indicated that a 4% HA gel was the most appropriate for forming hASC spheroids with a relatively consistent size (20-50 µm) within 48 hours. Prepared spheroids were positive for pluripotency markers (NANOG, OCT3/4, and SOX-2), and 40% of the cells were SSEA-3-positive, a marker of the multilineage differentiating stress enduring or Muse cell. In contrast with dissociated ASCs, increased secretion of cytokines such as hepatocyte growth factor was detected in ASC spheroids cultured under hypoxia. On microarray ASC spheroids showed upregulation of some pluripotency markers and downregulation of genes related to the mitotic cell cycle. After ischemia-reperfusion injury to the fat pad in SCID mice, local injection of hASC spheroids promoted tissue repair and reduced the final atrophy (1.6%) compared with that of dissociated hASCs (14.3%) or phosphate-buffered saline (20.3%). Part of the administered hASCs differentiated into vascular endothelial cells. ASC spheroids prepared in a HA gel contain undifferentiated cells with therapeutic potential to promote angiogenesis and tissue regeneration after damage. This study shows the therapeutic value of human adipose-derived stem cell spheroids prepared in hyarulonic acid gel. The spheroids have various benefits as an injectable cellular product and show therapeutic potential to the stem cell-depleted conditions such as diabetic chronic skin ulcer. ©AlphaMed Press.

  4. Extracellular matrix and α5β1 integrin signaling control the maintenance of bone formation capacity by human adipose-derived stromal cells

    PubMed Central

    Di Maggio, Nunzia; Martella, Elisa; Frismantiene, Agne; Resink, Therese J.; Schreiner, Simone; Lucarelli, Enrico; Jaquiery, Claude; Schaefer, Dirk J.; Martin, Ivan; Scherberich, Arnaud

    2017-01-01

    Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5β1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5β1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion. PMID:28290502

  5. Adipose-derived stromal cell cluster with light therapy enhance angiogenesis and skin wound healing in mice.

    PubMed

    Park, In-Su; Chung, Phil-Sang; Ahn, Jin Chul

    2015-07-03

    Human adipose-derived mesenchymal stem cells (hASCs) are attractive cell source for skin tissue engineering. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted cluster hASC in a skin wound animal model. The hASCs were cultured in monolayer or clusters. The LLLT, hASCs, hASC clusters, and hASC clusters transplantation with LLLT (cluster + LLLT) were applied to the wound bed in athymic mice. Wound healing was assessed by gross evaluation and by hematoxylin and eosin staining, and elastin van gieson histochemistry. The survival, differentiation, and secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) of the cluster ASC were evaluated by immunohistochemistry and Western blotting. The cluster + LLLT group enhanced the wound healing, including neovascularization and regeneration of skin appendages, compared with the cluster group. The secretion of growth factors was stimulated in the cluster + LLLT group compared with the ASCs and cluster group. These data suggest that LLLT is an effective biostimulator of cluster hASCs in wound healing that enhances the survival of hASCs and stimulates the secretion of growth factors in the wound bed.

  6. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.

    PubMed

    Naaijkens, B A; Niessen, H W M; Prins, H-J; Krijnen, P A J; Kokhuis, T J A; de Jong, N; van Hinsbergh, V W M; Kamp, O; Helder, M N; Musters, R J P; van Dijk, A; Juffermans, L J M

    2012-04-01

    Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

  7. Improvement of Mouth Functional Disability in Systemic Sclerosis Patients over One Year in a Trial of Fat Transplantation versus Adipose-Derived Stromal Cells

    PubMed Central

    Onesti, Maria Giuseppina; Fioramonti, Paolo; Carella, Sara; Fino, Pasquale; Marchese, Cinzia; Scuderi, Nicolò

    2016-01-01

    Background. Systemic sclerosis (SSc) is a multisystem disease characterized by cutaneous and visceral fibrosis. Face and mouth changes include telangiectasia, sicca syndrome, and thinning and reduction of mouth width (microcheilia) and opening (microstomia). We applied autologous fat transplantation compared with autologous adipose-derived stromal cells (ADSCs) injection to evaluate the clinical improvement of mouth opening. Methods. From February to May 2013 ten consecutive SSc patients were enrolled from the outpatient clinic of Plastic Surgery Department of Sapienza University of Rome. Patients were divided into two groups as follows: 5 patients were treated with fat transplantation and 5 patients received infiltration of ADSCs produced by cell factory of our institution. To value mouth opening, we use the Italian version of Mouth Handicap in Systemic Sclerosis Scale (IvMHISS). Mouth opening was assessed in centimetres (Maximal Mouth Opening, MMO). In order to evaluate compliance and physician and patient satisfaction, we employed a Questionnaire of Satisfaction and the Visual Analogic Scale (VAS) performed before starting study and 1 year after the last treatment. Results and Conclusion. We noticed that both procedures obtained significant results but neither one emerged as a first-choice technique. The present clinical experimentation should be regarded as a starting point for further experimental research and clinical trials. PMID:26880939

  8. Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro

    PubMed Central

    Zhao, Lili; Yang, Gongshe; Zhao, Xin

    2014-01-01

    Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca2+ transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway. PMID:25522345

  9. Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels

    PubMed Central

    Yang, Gang; Xiao, Zhenghua; Ren, Xiaomei; Long, Haiyan; Ma, Kunlong; Qian, Hong; Guo, Yingqiang

    2017-01-01

    Heart failure often develops after acute myocardial infarction because the injured myocardial tissue fails to recover or regenerate. Stem cell transplantation using adult cell sources, such as adipose-derived stromal vascular fraction (SVF), draws extensive attention. In this study, SVF cells were isolated from rat adipose tissue and cultivated on enzyme-crosslinked gelatin hydrogels. Morphological features of cell development and spontaneous beating behavior from these cells were observed and recorded. Cardiac phenotypes were characterized via immunofluorescence staining, and the expression of cardiac-specific genes was measured via RT-PCR. The functional assessment of SVF-derived cardiomyocyte-like cells (SVF-CMs) was performed by detecting cellular calcium transient activities and pharmacological responses. Results showed that most SVF-CMs exhibited elongated myotubule shapes and expressed cardiac troponin I strongly. SVF-CMs expressed cardiac-specific RNA (including transcription factors GATA binding protein 4) and myocyte enhancer factor 2c, as well as the structural proteins, namely, sarcomere actinin alpha 2, cardiac troponin I type 3, cardiac troponin T type 2, and cardiac gap junction protein alpha 1. Their beating mode, calcium activities, and pharmacological responses were similar to those of native CMs. Spontaneously beating SVF-CMs can be derived from adipose tissue-derived SVFs, and enzyme-crosslinked gelatin hydrogel promoted the cardiac differentiation of SVF cells. PMID:28155919

  10. Human adipose derived stromal/stem cells (hASCs) protect against STZ-induced hyperglycemia; analysis of hASC-derived paracrine effectors

    PubMed Central

    Kono, Tatsuyoshi M.; Sims, Emily K.; Moss, Dan R.; Yamamoto, Wataru; Ahn, Geonyoung; Diamond, Julie; Tong, Xin; Day, Kathleen H.; Territo, Paul R.; Hanenberg, Helmut; Traktuev, Dmitry O.; March, Keith L.; Evans-Molina, Carmella

    2014-01-01

    Adipose-derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)-derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved β cell mass, and increased β cell proliferation in STZ-treated NOD-SCID mice. Co-culture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with pro-inflammatory cytokines. Analysis of hASC-derived factors revealed VEGF and TIMP-1 to be highly abundant factors secreted by hASCs. Notably, TIMP-1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP-1 blockade was able to abrogate in vitro pro-survival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP-1 was increased in the serum of injected mice, while recombinant TIMP-1 increased viability in INS-1 cells treated with IL-1β, IFN-γ and TNF-α. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP-1, are able to mitigate against β cell death in rodent and in vitro models of Type 1 diabetes through a combination of local paracrine as well as systemic effects. PMID:24519994

  11. Degenerative Suspensory Ligament Desmitis (DSLD) in Peruvian Paso Horses Is Characterized by Altered Expression of TGFβ Signaling Components in Adipose-Derived Stromal Fibroblasts

    PubMed Central

    Luo, Wei; Sandy, John; Li, Jun; Brounts, Sabrina; Galante, Jorge; Plaas, Anna

    2016-01-01

    Equine degenerative suspensory ligament desmitis (DSLD) in Peruvian Paso horses typically presents at 7–15 years and is characterized by lameness, focal disorganization of collagen fibrils, and chondroid deposition in the body of the ligament. With the aim of developing a test for disease risk (that can be used to screen horses before breeding) we have quantified the expression of 76 TGFβ-signaling target genes in adipose-derived stromal fibroblasts (ADSCs) from six DSLD-affected and five unaffected Paso horses. Remarkably, 35 of the genes showed lower expression (p<0.05) in cells from DSLD-affected animals and this differential was largely eliminated by addition of exogenous TGFβ1. Moreover, TGFβ1-mediated effects on expression were prevented by the TGFβR1/2 inhibitor LY2109761, showing that the signaling was via a TGFβR1/2 complex. The genes affected by the pathology indicate that it is associated with a generalized metabolic disturbance, since some of those most markedly altered in DSLD cells (ATF3, MAPK14, ACVRL1 (ALK1), SMAD6, FOS, CREBBP, NFKBIA, and TGFBR2) represent master-regulators in a wide range of cellular metabolic responses. PMID:27902739

  12. Short-term preconditioning enhances the therapeutic potential of adipose-derived stromal/stem cell-conditioned medium in cisplatin-induced acute kidney injury.

    PubMed

    Overath, Jürgen M; Gauer, Stefan; Obermüller, Nicholas; Schubert, Ralf; Schäfer, Richard; Geiger, Helmut; Baer, Patrick C

    2016-03-15

    The development of new strategies to preserve renal function after acute kidney injury (AKI) is necessary due to limited clinical intervention options. The organ-protective effects of mesenchymal stromal/stem cells (MSCs) and their conditioned medium (CM) have been investigated demonstrating that both separately promoted tubular recovery and ameliorated the outcome of AKI. Nevertheless, strategies to optimise the regenerative potential of both are highly needed. Here we investigated the effects of CM from adipose-derived MSCs (ASCs) preincubated in a hypoxic environment (Hyp). Protective factors were investigated by PCR analysis and a protein array in vitro. The expression of 64 of the 308 proteins assayed was found to be more than two-fold increased after Hyp. CM of Hyp-pretreated ASCs (pCM) was used to enhance regeneration in a mouse model of cisplatin-induced AKI (cisAKI). Renal function was assessed by measurements of markers for AKI and serum cytokine levels. The pCM significantly ameliorated serum creatinine and neutrophil gelatinase-associated lipocalin values, and also the levels of inflammatory cytokines IL-1β and IL-6 in the serum of mice with AKI. Our work clearly showed that a Hyp preconditioning significantly increases the release of protective factors in ASCs and enhances the therapeutic effects of CM in cisAKI in mice.

  13. Connective Tissue Growth Factor in Regulation of RhoA Mediated Cytoskeletal Tension Associated Osteogenesis of Mouse Adipose-Derived Stromal Cells

    PubMed Central

    Xu, Yue; Wagner, Diane R.; Bekerman, Elena; Chiou, Michael; James, Aaron W.; Carter, Dennis; Longaker, Michael T.

    2010-01-01

    Background Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways. Methods/Principal Findings Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm2), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm2) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis. Conclusions/Significance We conclude that CTGF is important in the regulation of cytoskeletal tension mediated ASC osteogenic differentiation. PMID:20585662

  14. Comparison of human adipose stromal vascular fraction and adipose-derived mesenchymal stem cells for the attenuation of acute renal ischemia/reperfusion injury

    PubMed Central

    Zhou, Liuhua; Song, Qun; Shen, Jiangwei; Xu, Luwei; Xu, Zheng; Wu, Ran; Ge, Yuzheng; Zhu, Jiageng; Wu, Jianping; Dou, Quanliang; Jia, Ruipeng

    2017-01-01

    Stem cells therapy has been suggested as a promising option for the treatment of acute kidney injury (AKI). This study was performed to compare the abilities of xenogenic transplantation of human adipose stromal vascular fraction (SVF) and adipose-derived mesenchymal stem cells (AdMSCs) to facilitate the recovery of renal function and structure in a rat model of ischemia/reperfusion (IR) induced AKI. SVF or AdMSCs were transplanted to the injured kidney through intra-parenchymal injection. Significantly improved renal function and reduced tubular injury were observed in SVF and AdMSCs groups. Administration of SVF or AdMSCs contributed to significantly improved cell proliferation and markedly reduced cell apoptosis in parallel with reduced microvascular rarefaction in injured kidney. IR injury resulted in higher levels of inflammatory cytokines, whereas xenogenic transplantation of SVF or AdMSCs reduced but not induced inflammatory cytokines expression. Additionally, in vitro study showed that administration of SVF or AdMSCs could also significantly promote the proliferation and survival of renal tubular epithelial cells underwent hypoxia/reoxygenation injury through secreting various growth factors. However, cell proliferation was significantly promoted in SVF group than in AdMSCs group. In conclusion, our study demonstrated that administration of SVF or AdMSCs was equally effective in attenuating acute renal IR injury. PMID:28276451

  15. Degenerative Suspensory Ligament Desmitis (DSLD) in Peruvian Paso Horses Is Characterized by Altered Expression of TGFβ Signaling Components in Adipose-Derived Stromal Fibroblasts.

    PubMed

    Luo, Wei; Sandy, John; Trella, Katie; Gorski, Daniel; Gao, Shuguang; Li, Jun; Brounts, Sabrina; Galante, Jorge; Plaas, Anna

    2016-01-01

    Equine degenerative suspensory ligament desmitis (DSLD) in Peruvian Paso horses typically presents at 7-15 years and is characterized by lameness, focal disorganization of collagen fibrils, and chondroid deposition in the body of the ligament. With the aim of developing a test for disease risk (that can be used to screen horses before breeding) we have quantified the expression of 76 TGFβ-signaling target genes in adipose-derived stromal fibroblasts (ADSCs) from six DSLD-affected and five unaffected Paso horses. Remarkably, 35 of the genes showed lower expression (p<0.05) in cells from DSLD-affected animals and this differential was largely eliminated by addition of exogenous TGFβ1. Moreover, TGFβ1-mediated effects on expression were prevented by the TGFβR1/2 inhibitor LY2109761, showing that the signaling was via a TGFβR1/2 complex. The genes affected by the pathology indicate that it is associated with a generalized metabolic disturbance, since some of those most markedly altered in DSLD cells (ATF3, MAPK14, ACVRL1 (ALK1), SMAD6, FOS, CREBBP, NFKBIA, and TGFBR2) represent master-regulators in a wide range of cellular metabolic responses.

  16. Osteogenesis for postoperative temporal bone defects using human ear adipose-derived stromal cells and tissue engineering: an animal model study.

    PubMed

    Kim, Yeon Ju; Park, Seung Gu; Shin, Beomyong; Kim, Jangho; Kim, Seung Won; Choo, Oak-Sung; Yin, Xiang Yun; Min, Byoung Hyun; Choung, Yun-Hoon

    2017-09-05

    Mastoidectomy, the removal of infected mastoid bones, is a common surgical procedure for the treatment of chronic otitis media. Persistent and recurrent otorrhea and accumulation of keratin debris following open cavity mastoidectomy are still bothersome issues for both patients and otologists. In this study, we used human ear adipose-derived stromal cells (hEASCs) in combination with polycaprolactone (PCL) scaffolds and osteogenic differentiation medium (ODM) to regenerate temporal bone defects. The hEASCs showed stem cell phenotypes, and these characteristics were maintained up to passage 5. Mastoid bulla and cranial bone defects were induced in Sprague-Dawley rats using AgNO3 and burr hole drilling, respectively, and the rats were then divided into five groups: (1) control, (2) hEASCs, (3) hEASCs + ODM, (4) hEASCs + PCL scaffolds, and (5) hEASCs + PCL scaffolds + ODM. Osteogenesis was evaluated by micro-computed tomography and histology. Compared with the control group, the groups transplanted with hEASCs and PCL scaffolds had significantly higher bone formation along the periphery of the mastoid bulla area. Moreover, ODM synergistically enhanced bone formation in mastoid bulla defects. Our results suggest that combining hEASCs with PCL scaffolds represents a promising method for anatomical and functional reconstruction of postoperative temporal bone defects following mastoidectomy. This article is protected by copyright. All rights reserved. © 2017 Wiley Periodicals, Inc.

  17. Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels.

    PubMed

    Yang, Gang; Xiao, Zhenghua; Ren, Xiaomei; Long, Haiyan; Ma, Kunlong; Qian, Hong; Guo, Yingqiang

    2017-02-03

    Heart failure often develops after acute myocardial infarction because the injured myocardial tissue fails to recover or regenerate. Stem cell transplantation using adult cell sources, such as adipose-derived stromal vascular fraction (SVF), draws extensive attention. In this study, SVF cells were isolated from rat adipose tissue and cultivated on enzyme-crosslinked gelatin hydrogels. Morphological features of cell development and spontaneous beating behavior from these cells were observed and recorded. Cardiac phenotypes were characterized via immunofluorescence staining, and the expression of cardiac-specific genes was measured via RT-PCR. The functional assessment of SVF-derived cardiomyocyte-like cells (SVF-CMs) was performed by detecting cellular calcium transient activities and pharmacological responses. Results showed that most SVF-CMs exhibited elongated myotubule shapes and expressed cardiac troponin I strongly. SVF-CMs expressed cardiac-specific RNA (including transcription factors GATA binding protein 4) and myocyte enhancer factor 2c, as well as the structural proteins, namely, sarcomere actinin alpha 2, cardiac troponin I type 3, cardiac troponin T type 2, and cardiac gap junction protein alpha 1. Their beating mode, calcium activities, and pharmacological responses were similar to those of native CMs. Spontaneously beating SVF-CMs can be derived from adipose tissue-derived SVFs, and enzyme-crosslinked gelatin hydrogel promoted the cardiac differentiation of SVF cells.

  18. Effect of TiO2 nanoparticles on adipose derived stromal cell differentiation, morphology, ECM deposition and its susceptibility to bacterial infections

    NASA Astrophysics Data System (ADS)

    Mironava, Tatsiana; Xu, Yan; Rafailovich, Miriam

    The growing annual production of Titanium dioxide (TiO2) nanoparticles is proportional to an increase in the chances of occupational and consumer exposure. Considering, that these nanoparticles are currently being used in multiple personal care products many concerns have arisen about their health impact. Human skin is in constant contact with the external environment and is one of the most important routes of exposure to TiO2. In this study we have investigated the effect of two forms of TiO2, rutile and anatase, on human adipose derived stromal cells (ADSCs). Here, we focus on the effects of TiO2 exposure on intracellular lipid accumulation and expression of adipogenic markers; on whether different forms of TiO2 have similar effects on cell function; and whether nanoparticle localization inside cells correlates with loss of cell function. In addition presence of bacteria on the skin is taken into account in its complex interaction with ADSCs and TiO2 nanoparticles. Altogether, the present study indicates that nanosized TiO2 particles adversely effects the differentiation of ADSCs, have profound effects on cell function and increase the rate of bacterial infection.

  19. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system.

    PubMed

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-05-15

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage.

  20. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    SciTech Connect

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-05-15

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.

  1. Platelet-Derived Growth Factor BB Enhances Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal Stromal/Stem Cells.

    PubMed

    Hung, Ben P; Hutton, Daphne L; Kozielski, Kristen L; Bishop, Corey J; Naved, Bilal; Green, Jordan J; Caplan, Arnold I; Gimble, Jeffrey M; Dorafshar, Amir H; Grayson, Warren L

    2015-09-01

    Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFRβ within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.

  2. Tissue harvest by means of suction-assisted or third-generation ultrasound-assisted lipoaspiration has no effect on osteogenic potential of human adipose-derived stromal cells.

    PubMed

    Panetta, Nicholas J; Gupta, Deepak M; Kwan, Matthew D; Wan, Derrick C; Commons, George W; Longaker, Michael T

    2009-07-01

    Human adipose-derived stromal cells readily undergo osteogenic differentiation in vitro and in vivo. Thus, interest in their potential role in skeletal tissue engineering continues to escalate. Very little is known regarding the effects that energy delivered by means of third-generation ultrasound-assisted lipoaspiration may have on the osteogenic potential of these cells. The authors investigated whether differences in adipose-derived stromal cell yield, and the in vitro proliferation and osteogenic potential of these cells obtained by suction-assisted lipoaspiration or third-generation ultrasound-assisted lipoaspiration, exist. Adipose-derived stromal cells were harvested from lipoaspiration specimens of patients undergoing elective suction-assisted lipoaspiration and third-generation ultrasound-assisted lipoaspiration. Harvested cells were seeded to evaluate proliferative capacity and in vitro osteogenic potential. Alkaline phosphatase and alizarin red staining were performed to evaluate early and terminal osteogenic differentiation, respectively. Quantitative real-time polymerase chain reaction analysis was used to examine osteogenic gene expression patterns of RUNX2/CFBA1 (early differentiation) and osteocalcin (late differentiation). No significant differences in the proliferative capacity (n = 3), alkaline phosphatase staining (n = 3), or extracellular matrix mineralization (n = 3) of suction-assisted lipoaspiration- or third-generation ultrasound-assisted lipoaspiration-derived cells were appreciated. Transcript levels of markers of early and terminal osteogenic differentiation were not significantly different (n = 3). These findings suggest that exposure of adipose-derived stromal cells to ultrasound energy during tissue harvest by means of third-generation ultrasound-assisted lipoaspiration does not impart a negative consequence toward their proliferative capacity or osteogenic potential. Thus, the cells harvested using third-generation ultrasound

  3. Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial.

    PubMed

    Panés, Julián; García-Olmo, Damián; Van Assche, Gert; Colombel, Jean Frederic; Reinisch, Walter; Baumgart, Daniel C; Dignass, Axel; Nachury, Maria; Ferrante, Marc; Kazemi-Shirazi, Lili; Grimaud, Jean C; de la Portilla, Fernando; Goldin, Eran; Richard, Marie Paule; Leselbaum, Anne; Danese, Silvio

    2016-09-24

    Complex perianal fistulas in Crohn's disease are challenging to treat. Allogeneic, expanded, adipose-derived stem cells (Cx601) are a promising new therapeutic approach. We aimed to assess the safety and efficacy of Cx601 for treatment-refractory complex perianal fistulas in patients with Crohn's disease. We did this randomised, double-blind, parallel-group, placebo-controlled study at 49 hospitals in seven European countries and Israel from July 6, 2012, to July 27, 2015. Adult patients (≥18 years) with Crohn's disease and treatment-refractory, draining complex perianal fistulas were randomly assigned (1:1) using a pre-established randomisation list to a single intralesional injection of 120 million Cx601 cells or 24 mL saline solution (placebo), with stratification according to concomitant baseline treatment. Treatment was administered by an unmasked surgeon, with a masked gastroenterologist and radiologist assessing the therapeutic effect. The primary endpoint was combined remission at week 24 (ie, clinical assessment of closure of all treated external openings that were draining at baseline, and absence of collections >2 cm of the treated perianal fistulas confirmed by masked central MRI). Efficacy was assessed in the intention-to-treat (ITT) and modified ITT populations; safety was assessed in the safety population. This study is registered with ClinicalTrials.gov, number NCT01541579. 212 patients were randomly assigned: 107 to Cx601 and 105 to placebo. A significantly greater proportion of patients treated with Cx601 versus placebo achieved combined remission in the ITT (53 of 107 [50%] vs 36 of 105 [34%]; difference 15·2%, 97·5% CI 0·2-30·3; p=0·024) and modified ITT populations (53 of 103 [51%] vs 36 of 101 [36%]; 15·8%, 0·5-31·2; p=0·021). 18 (17%) of 103 patients in the Cx601 group versus 30 (29%) of 103 in the placebo group experienced treatment-related adverse events, the most common of which were anal abscess (six in the Cx601 group vs nine

  4. Evaluation of adipose-derived stromal vascular fraction from the lateral tailhead, inguinal region, and mesentery of horses

    PubMed Central

    Metcalf, Garrett L.; McClure, Scott R.; Hostetter, Jesse M.; Martinez, Rudy F.; Wang, Chong

    2016-01-01

    Use of mesenchymal stem cells (MSCs) found in the stromal vascular fraction (SVF) of equine adipose tissue has promising applications for regenerative therapies. The most commonly used source of equine adipose tissue is the subcutaneous tailhead. The objective of this study was to compare 3 adipose depot sites in horses and determine the viability and cellular yield, capillary density, gene expression for selected markers, and colony-forming unit fibroblasts (CFU-Fs) in adipose tissue taken from these sites. Adipose tissue was excised from the area lateral to the tailhead, the inguinal region, and the small colon mesentery of 6 horses. Lipoaspirate was also collected from the area lateral to the tailhead. Stromal vascular fraction (SVF) was prepared in duplicate from the 3 different adipose tissue depots. The total nucleated and dead cell counts was determined manually using a hemocytometer and percent viability was calculated. Mass and volume of adipose were determined in order to calculate density and factor-VIII immunohistochemical staining was used to determine vascular density in the excisional adipose tissue samples from each horse. Quantitative polymerase chain reaction (qPCR) was used to quantify gene expression for selected cellular markers from each site. There were significant differences in viability, yield of nucleated cells/gram of adipose tissue, vascular density, gene expression, and CFU-Fs among adipose depots. Adipose from the mesentery yielded the highest number of nucleated cells/gram of tissue and the highest vascular density and percentage of CFU-Fs. In the horse, both the anatomical site of collection and the method of tissue collection significantly impact the yield and composition of cells in the SVF. Further study is needed to assess whether one adipose source is superior for harvesting mesenchymal stem cells (MSCs) and whether the differences among sources are clinically relevant for in-vivo treatment of musculoskeletal injuries in horses

  5. Evaluation of adipose-derived stromal vascular fraction from the lateral tailhead, inguinal region, and mesentery of horses.

    PubMed

    Metcalf, Garrett L; McClure, Scott R; Hostetter, Jesse M; Martinez, Rudy F; Wang, Chong

    2016-10-01

    Use of mesenchymal stem cells (MSCs) found in the stromal vascular fraction (SVF) of equine adipose tissue has promising applications for regenerative therapies. The most commonly used source of equine adipose tissue is the subcutaneous tailhead. The objective of this study was to compare 3 adipose depot sites in horses and determine the viability and cellular yield, capillary density, gene expression for selected markers, and colony-forming unit fibroblasts (CFU-Fs) in adipose tissue taken from these sites. Adipose tissue was excised from the area lateral to the tailhead, the inguinal region, and the small colon mesentery of 6 horses. Lipoaspirate was also collected from the area lateral to the tailhead. Stromal vascular fraction (SVF) was prepared in duplicate from the 3 different adipose tissue depots. The total nucleated and dead cell counts was determined manually using a hemocytometer and percent viability was calculated. Mass and volume of adipose were determined in order to calculate density and factor-VIII immunohistochemical staining was used to determine vascular density in the excisional adipose tissue samples from each horse. Quantitative polymerase chain reaction (qPCR) was used to quantify gene expression for selected cellular markers from each site. There were significant differences in viability, yield of nucleated cells/gram of adipose tissue, vascular density, gene expression, and CFU-Fs among adipose depots. Adipose from the mesentery yielded the highest number of nucleated cells/gram of tissue and the highest vascular density and percentage of CFU-Fs. In the horse, both the anatomical site of collection and the method of tissue collection significantly impact the yield and composition of cells in the SVF. Further study is needed to assess whether one adipose source is superior for harvesting mesenchymal stem cells (MSCs) and whether the differences among sources are clinically relevant for in-vivo treatment of musculoskeletal injuries in horses.

  6. Phenotypical and Functional Characteristics of In Vitro-Expanded Adipose-Derived Mesenchymal Stromal Cells From Patients With Systematic Sclerosis.

    PubMed

    Capelli, Chiara; Zaccara, Eleonora; Cipriani, Paola; Di Benedetto, Paola; Maglione, Wanda; Andracco, Romina; Di Luca, Gabriele; Pignataro, Francesca; Giacomelli, Roberto; Introna, Martino; Vitali, Claudio; Del Papa, Nicoletta

    2017-05-09

    Mesenchymal stromal cells (MSCs) have received attention as an ideal source of regenerative cells because of their multipotent differentiation potential. Adipose tissue is an attractive source of MSCs. Recent studies have shown that autologous fat grafting may be effective in the treatment of systemic sclerosis (SSc), but no specific study exists that aimed at investigating whether adipose tissue-derived stromal cells (ADSCs) from SSc patients maintain normal phenotypic and functional characteristics. The purpose of the current study was to investigate whether ADSCs from patients with SSc (SSc-ADSCs) are phenotypically and functionally identical to those from healthy controls (HC-ADSCs). Adipose tissue samples were obtained from 10 patients with SSc and from 8 HCs. Both MSC populations were evaluated for their capacity to (a) express specific MSC surface antigens by flow cytometry analysis, (b) proliferate, (c) differentiate along the adipogenic and osteogenic lineages, (d) suppress in vitro lymphocyte proliferation induced by a mitogenic stimulus, and (e) support endothelial cell (EC) tube formation. ADSCs from SSc patients and HCs showed similar surface phenotype and multilineage differentiation capabilities. In PBMC proliferation inhibition assays, no significant differences were observed between SSc- and HC-ADSCs. Using ADSC/EC cocultures, both SSc- and HC-ADSCs improved tube formation by both HC- and SSc-ECs. This effect was enhanced under hypoxic conditions in all of the cocultures. SSc-ADSCs exhibited the same phenotypic pattern, proliferation and differentiation potentials, and immunosuppressive properties as those from HCs. The proangiogenic activity shown by SSc-ADSCs, namely, under hypoxic conditions, suggests that autologous ADSC grafting may represent a possible therapeutic option for SSc.

  7. Fluorescent immortalized human adipose derived stromal cells (hASCs-TS/GFP+) for studying cell drug delivery mediated by microvesicles.

    PubMed

    Coccè, Valentina; Balducci, Luigi; Falchetti, Maria Laura; Pascucci, Luisa; Ciusani, Emilio; Brini, Anna Teresa; Sisto, Francesca; Piovani, Giovanna; Alessandri, Giulio; Parati, Eugenio; Cabeza, Laura; Pessina, Augusto

    2017-03-27

    A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASC) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigate for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would be less resource and time consuming and it could possibly contribute to simplification of GMP procedures. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. TGFβ signalling pathway regulates angiogenesis by endothelial cells, in an adipose-derived stromal cell/endothelial cell co-culture 3D gel model.

    PubMed

    Lin, Shiyu; Xie, Jing; Gong, Tao; Shi, Sirong; Zhang, Tao; Fu, Na; Ye, Ling; Wang, Min; Lin, Yunfeng

    2015-12-01

    This study aimed to investigate the role of the TGFβ signalling pathway in angiogenesis in a three-dimensional (3D) collagen gel model, with co-culture between adipose-derived stromal cells (ASCs) and endothelial cells (ECs). A 3D collagen gel, implanted with green fluorescent protein-labelled mouse ASCs and red fluorescent protein-labelled mouse ECs, was established in vitro. Phenomena of angiogenesis with or without type I TGFβ receptor inhibitor (LY2157299) treatment, were observed 7 days post-implantation, using confocal laser scanning microscopy. To detect expression of angiogenesis-related genes, semi-quantitative PCR and quantitative real-time PCR were conducted. Zymography was performed to explore secretion of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) after treatment with LY2157299 of 5, 10, 20 to 50 μm concentrations, for 24 h. Angiogenesis was found to be attenuated in co-culture gels after ASC and EC treatment with LY2157299. Genes VEGF-A, VEGF-B, VE-ca, FGF-1, FGF-2, PDGF, HGF, BMP-4 were significantly reduced in the presence of LY2157299 in both mono-cultured and co-cultured ECs. Furthermore, reduction in co-cultured ECs was prominent relative to mono-cultured ECs, while the same results did not occur to ASCs. We further confirmed that gelatinases secreted by ECs were reduced in a dose-dependent manner, after treatment with LY2157299. These results indicate that in ASC/EC co-culture, the TGFβ signalling pathway regulated angiogenesis via EC activity. Co-cultured ECs were regulated more significantly than mono-cultured ECs suggesting that inhibition of TGFβRI may regulate paracrine secretion of ASCs to further modulate EC angiogenesis. © 2015 John Wiley & Sons Ltd.

  9. The JAK/STAT3 signalling pathway regulated angiogenesis in an endothelial cell/adipose-derived stromal cell co-culture, 3D gel model.

    PubMed

    Xue, Changyue; Xie, Jiamin; Zhao, Dan; Lin, Shiyu; Zhou, Tengfei; Shi, Sirong; Shao, Xiaoru; Lin, Yunfeng; Zhu, Bofeng; Cai, Xiaoxiao

    2017-02-01

    The aim of the study was to investigate the role of the JAK/STAT3 signalling pathway in angiogenesis. The model established in vitro, involved a 3D collagen gel being implanted with endothelial cells (ECs) from red fluorescent protein-labelled mice, and adipose-derived stromal cells (ASCs) from green fluorescent protein-labelled mice. Phenomena of angiogenesis, after treatment by the inhibitor and the activator of JAK/STAT3 pathway respectively, were observed using confocal laser scanning microscopy. Transwell co-culture of ECs and ASCs was used to elucidate mechanisms. Stattic, inhibitor of JAK/STAT3 pathway, attenuated angiogenesis in the model. In contrast, angiogenesis was promoted after treatment of Olanzapine, an activator. We found that protein levels of VEGFA and cyclin D1 were regulated by the JAK/STAT3 pathway, and flow cytometry further confirmed variations in cell cycle parameters of ECs and ASCs. Genes VEGFA/B, VEGFR2, MMP-2, MMP-9, IGF-1 and b-FGF were down-regulated by Stattic in ECs, while Olanzapine significantly up-regulated mRNA levels of these genes. As for ASCs, genes VEGFA, MMP-2, MMP-9, IGF-1 and b-FGF were modulated by the JAK/STAT3 pathway. Angiogenesis in the 3D collagen gel was regulated by the JAK/STAT3 pathway which involved changes in vessel length, vessel diameter and sprout number. The underlying mechanism was that the JAK/STAT3 signalling pathway regulated angiogenesis by modulation of numbers of angiogenesis-related growth factors and by direct regulation of cell cycle. © 2016 John Wiley & Sons Ltd.

  10. Human adipose-derived mesenchymal stromal cell pigment epithelium–derived factor cytotherapy modifies genetic and epigenetic profiles of prostate cancer cells

    PubMed Central

    Zolochevska, Olga; Shearer, Joseph; Ellis, Jayne; Fokina, Valentina; Shah, Forum; Gimble, Jeffrey M.; Figueiredo, Marxa L.

    2014-01-01

    Background aims Adipose-derived mesenchymal stromal cells (ASCs) are promising tools for delivery of cytotherapy against cancer. However, ASCs can exert profound effects on biological behavior of tumor cells. Our study aimed to examine the influence of ASCs on gene expression and epigenetic methylation profiles of prostate cancer cells as well as the impact of expressing a therapeutic gene on modifying the interaction between ASCs and prostate cancer cells. Methods ASCs were modified by lentiviral transduction to express either green fluorescent protein as a control or pigment epithelium–derived factor (PEDF) as a therapeutic molecule. PC3 prostate cancer cells were cultured in the presence of ASC culture–conditioned media (CCM), and effects on PC3 or DU145. Ras cells were examined by means of real-time quantitative polymerase chain reaction, EpiTect methyl prostate cancer–focused real-time quantitative polymerase chain reaction arrays, and luciferase reporter assays. Results ASCs transduced with lentiviral vectors were able to mediate expression of several tumor-inhibitory genes, some of which correlated with epigenetic methylation changes on cocultured PC3 prostate cancer cells. When PC3 cells were cultured with ASC-PEDF CCM, we observed a shift in the balance of gene expression toward tumor inhibition, which suggests that PEDF reduces the potential tumor-promoting activity of unmodified ASCs. Conclusions These results suggest that ASC-PEDF CCM can promote reprogramming of tumor cells in a paracrine manner. An improved understanding of genetic and epigenetic events in prostate cancer growth in response to PEDF paracrine therapy would enable a more effective use of ASC-PEDF, with the goal of achieving safer yet more potent anti-tumor effects. PMID:24424267

  11. MiR-124 Promote Neurogenic Transdifferentiation of Adipose Derived Mesenchymal Stromal Cells Partly through RhoA/ROCK1, but Not ROCK2 Signaling Pathway.

    PubMed

    Wang, Ye; Wang, Desheng; Guo, Dawen

    2016-01-01

    Some recent studies suggest that multiple miRNAs might regulate neurogenic transdifferentiation of mesenchymal stromal cells (MSCs). In the present study, we hypothesized that the miR-124 can repress the expression of RhoA upon the neurogenesis of adipose derived MSCs (ADMSCs). MiRNA expression dynamics during neurogenic transdifferentiation of ADMSCs were measured. The expression of neuron-specific enolase (NSE), Tuj-1 (Neuron-specific class III beta-tubulin) and glial fibrillary acidic protein (GFAP), as well as electrophysiological properties, were detected after neurogenic transdifferentiation. The targeting of miR-124 over RhoA was verified by dual luciferase assay, qRT-PCR and western blot. The functions of miR-124 and the RhoA/ROCK signaling pathway were studied using gain and loss of function experiments in vitro. MiR-124 is significantly upregulated during neurogenic transdifferentiation of ADMSCs. Knockdown of endogenous miR-124 hampered neurogenic transdifferentiation and the acquired electrophysiological properties. MiR-124 could directly target RHOA mRNA and repress its expression, through which it increased the proportion of transdifferentiated (transdiff.) cells with positive NSE, Tuj-1 and GFAP. RhoA/ROCK1, but not ROCK2 is a downstream signaling pathway of miR-124 in the process of transdifferentiation. MiR-124 is an important miRNA modulating neurogenic transdifferentiation of ADMSCs at least partly via the miR-124/RhoA/ROCK1 signaling pathway. These findings provided some fundamental information for future use of ADMSCs as an agent for regenerative medicine and cell therapy for neurological diseases.

  12. Effective wound healing in streptozotocin-induced diabetic rats by adipose-derived stromal cell transplantation in plasma-gel containing fragmin/protamine microparticles.

    PubMed

    Sumi, Yuki; Ishihara, Masayuki; Kishimoto, Satoko; Takikawa, Makoto; Hattori, Hidemi; Takikawa, Megumi; Azuma, Ryuichi; Nakamura, Shingo; Fujita, Masanori; Kiyosawa, Tomoharu

    2014-01-01

    We investigated the effectiveness of the application of inbred adipose-derived stromal cells (IR-ASCs) in high inbred rat plasma (IRP) (6%)-Dulbecco modified Eagle medium (DMEM) gel with fragmin/protamine microparticles (F/P MPs) (IR-ASCs + IRP-DMEM gel + F/P MPs) on wound healing in streptozotocin-induced diabetic rats. F/P MPs have previously been used as a cell carrier for IR-ASCs in inbred Fisher 344 rats and for preservation and controlled release of various cytokines in IRP-DMEM gel. We applied IR-ASCs + IRP-DMEM gel + F/P MPs to full-thickness skin excisions on the backs of the diabetic rats. The statistical significance of wound closure was evaluated on postwounding days 3, 7, 10, and 14, and the skin area surrounding the wound was removed for histological examination on days 7 and 14. The wound closure rate and histological examination of wounds treated with IR-ASCs + IRP-DMEM gel + F/P MPs demonstrated significantly advanced epithelialization, capillary formation, and granulation tissue formation. When DiI-labeled IR-ASCs + IRP-DMEM gel + F/P MPs were applied to full-thickness skin wounds on the backs of the diabetic rats, histological observation at 2 weeks showed appearances of both DiI-labeled granulation tissue and CD31-immunostained microvessels in the transplant areas. A portion of the transplanted IR-ASCs + IRP-DMEM gel + F/P MPs had been taken up into the granulation tissues to promote wound healing. Thus, IR-ASCs + IRP-DMEM gel + F/P MPs were effective for repairing healing-impaired wounds such as those arising in the diabetic rats.

  13. The neuro-glial properties of adipose-derived adult stromal (ADAS) cells are not regulated by Notch 1 and are not derived from neural crest lineage.

    PubMed

    Wrage, Philip C; Tran, Thi; To, Khai; Keefer, Edward W; Ruhn, Kelly A; Hong, John; Hattangadi, Supriya; Treviño, Isaac; Tansey, Malú G

    2008-01-16

    We investigated whether adipose-derived adult stromal (ADAS) are of neural crest origin and the extent to which Notch 1 regulates their growth and differentiation. Mouse ADAS cells cultured in media formulated for neural stem cells (NSC) displayed limited capacity for self-renewal, clonogenicity, and neurosphere formation compared to NSC from the subventricular zone in the hippocampus. Although ADAS cells expressed Nestin, GFAP, NSE and Tuj1 in vitro, exposure to NSC differentiation supplements did not induce mature neuronal marker expression. In contrast, in mesenchymal stem cell (MSC) media, ADAS cells retained their ability to proliferate and differentiate beyond 20 passages and expressed high levels of Nestin. In neuritizing cocktails, ADAS cells extended processes, downregulated Nestin expression, and displayed depolarization-induced Ca(2+) transients but no spontaneous or evoked neural network activity on Multi-Electrode Arrays. Deletion of Notch 1 in ADAS cell cultures grown in NSC proliferation medium did not significantly alter their proliferative potential in vitro or the differentiation-induced downregulation of Nestin. Co-culture of ADAS cells with fibroblasts that stably expressed the Notch ligand Jagged 1 or overexpression of the Notch intracellular domain (NICD) did not alter ADAS cell growth, morphology, or cellular marker expression. ADAS cells did not display robust expression of neural crest transcription factors or genes (Sox, CRABP2, and TH); and lineage tracing analyses using Wnt1-Cre;Rosa26R-lacZ or -EYFP reporter mice confirmed that fewer than 2% of the ADAS cell population derived from a Wnt1-positive population during development. In summary, although media formulations optimized for MSCs or NSCs enable expansion of mouse ADAS cells in vitro, we find no evidence that these cells are of neural crest origin, that they can undergo robust terminal differentiation into functionally mature neurons, and that Notch 1 is likely to be a key

  14. Del-1 Overexpression in Endothelial Cells Increases Vascular Density in Tissue-Engineered Implants Containing Endothelial Cells and Adipose-Derived Mesenchymal Stromal Cells

    PubMed Central

    Ciucurel, Ema C.

    2014-01-01

    We used a combination of strategies to stimulate the vascularization of tissue-engineered constructs in vivo including a modular approach to build larger tissues from individual building blocks (“modules”) mixed together. Each building block included vascular cells by design; modules were submillimeter-sized collagen gels with an outer layer of endothelial cells (ECs), and with embedded adipose-derived mesenchymal stromal cells (adMSCs) to support EC survival and blood vessel maturation in vivo. We transduced the ECs that coat the modules with a lentiviral construct to overexpress the angiogenic extracellular matrix (ECM) protein Developmental endothelial locus-1 (Del-1). Upon injection of modules in a subcutaneous SCID/Bg mouse model, there was an increase in the number of blood vessels for implants with ECs transduced to overexpress Del-1 compared with control implants (with enhanced green fluorescent protein [eGFP]–transduced ECs) over the 21-day duration of the study. The greatest difference between Del-1 and eGFP implants and the highest number of blood vessels were observed 7 days after transplantation. The day-7 Del-1 implants also had increased SMA+ staining compared with control, suggesting increased blood vessel maturation through recruitment of SMA+ smooth muscle cells or pericytes to stabilize the newly formed blood vessels. Perfusion studies (microcomputed tomography, ultrasound imaging, and systemic injection of fluorescent UEA-1 or dextran) showed that some of the newly formed blood vessels (both donor derived and host derived, in both Del-1 and eGFP implants) were perfused and connected to the host vasculature as early as 7 days after transplantation, and at later time points as well. Nevertheless, perfusion of the implants was limited in some cases, suggesting that further improvements are necessary to normalize the vasculature at the implant site. PMID:24151812

  15. MiR-124 Promote Neurogenic Transdifferentiation of Adipose Derived Mesenchymal Stromal Cells Partly through RhoA/ROCK1, but Not ROCK2 Signaling Pathway

    PubMed Central

    Wang, Ye; Wang, Desheng; Guo, Dawen

    2016-01-01

    Objective Some recent studies suggest that multiple miRNAs might regulate neurogenic transdifferentiation of mesenchymal stromal cells (MSCs). In the present study, we hypothesized that the miR-124 can repress the expression of RhoA upon the neurogenesis of adipose derived MSCs (ADMSCs). Methods MiRNA expression dynamics during neurogenic transdifferentiation of ADMSCs were measured. The expression of neuron-specific enolase (NSE), Tuj-1 (Neuron-specific class III beta-tubulin) and glial fibrillary acidic protein (GFAP), as well as electrophysiological properties, were detected after neurogenic transdifferentiation. The targeting of miR-124 over RhoA was verified by dual luciferase assay, qRT-PCR and western blot. The functions of miR-124 and the RhoA/ROCK signaling pathway were studied using gain and loss of function experiments in vitro. Results MiR-124 is significantly upregulated during neurogenic transdifferentiation of ADMSCs. Knockdown of endogenous miR-124 hampered neurogenic transdifferentiation and the acquired electrophysiological properties. MiR-124 could directly target RHOA mRNA and repress its expression, through which it increased the proportion of transdifferentiated (transdiff.) cells with positive NSE, Tuj-1 and GFAP. RhoA/ROCK1, but not ROCK2 is a downstream signaling pathway of miR-124 in the process of transdifferentiation. Conclusion MiR-124 is an important miRNA modulating neurogenic transdifferentiation of ADMSCs at least partly via the miR-124/RhoA/ROCK1 signaling pathway. These findings provided some fundamental information for future use of ADMSCs as an agent for regenerative medicine and cell therapy for neurological diseases. PMID:26745800

  16. Cotransplantation of adipose-derived mesenchymal stromal cells and endothelial cells in a modular construct drives vascularization in SCID/bg mice.

    PubMed

    Butler, Mark J; Sefton, Michael V

    2012-08-01

    A modular approach to adipose tissue engineering was explored by embedding adipose-derived mesenchymal stromal cells (adMSC) in sub-mm-sized collagen rods or "modules" and coating with human microvascular endothelial cells (HMEC). After subcutaneous injection into a SCID/Bg mouse, HMEC on modules containing embedded adMSC appeared to detach from the modules to form vessels as early as day 3, as confirmed by the human EC-specific UEA-1 lectin stain, and these vessels persisted for up to 90 days. Vessel numbers decreased over 14 days, but vessel size increased suggesting a maturing of the vasculature. Vessel perfusion with the host was confirmed at 21 days by microCT. HMEC on modules without embedded adMSC remained attached to the module surface at day 3 and UEA-1 staining disappeared over 14 days suggesting cell death. It appeared that cotransplantation with adMSC had an anti-apoptotic and proangiogenic effect on HMEC. The early revascularization strategy may be successful in supporting adMSC viability and differentiation, as a preliminary study suggests progressive fat accumulation in the HMEC+adMSC implants: ∼60% of the implant area stained positive for Oil Red O by day 90. adMSC-embedded modules without HMEC surface coating did not show similar levels of Oil Red O staining. All implant volumes decreased over the time course of the experiment, yet HMEC+adMSC module implants were larger than adMSC-only implants at day 90. Collagen gel is mechanically weak and contracts in vivo making it unsuitable as a biomaterial for adipose tissue engineering where volume maintenance is critical. When combined with an appropriate biomaterial, the modular approach to adipose tissue engineering may represent a successful strategy to engineer soft tissue substitutes of clinical relevance.

  17. Cotransplantation of Adipose-Derived Mesenchymal Stromal Cells and Endothelial Cells in a Modular Construct Drives Vascularization in SCID/bg Mice

    PubMed Central

    Butler, Mark J.

    2012-01-01

    A modular approach to adipose tissue engineering was explored by embedding adipose-derived mesenchymal stromal cells (adMSC) in sub-mm-sized collagen rods or “modules” and coating with human microvascular endothelial cells (HMEC). After subcutaneous injection into a SCID/Bg mouse, HMEC on modules containing embedded adMSC appeared to detach from the modules to form vessels as early as day 3, as confirmed by the human EC-specific UEA-1 lectin stain, and these vessels persisted for up to 90 days. Vessel numbers decreased over 14 days, but vessel size increased suggesting a maturing of the vasculature. Vessel perfusion with the host was confirmed at 21 days by microCT. HMEC on modules without embedded adMSC remained attached to the module surface at day 3 and UEA-1 staining disappeared over 14 days suggesting cell death. It appeared that cotransplantation with adMSC had an anti-apoptotic and proangiogenic effect on HMEC. The early revascularization strategy may be successful in supporting adMSC viability and differentiation, as a preliminary study suggests progressive fat accumulation in the HMEC+adMSC implants: ∼60% of the implant area stained positive for Oil Red O by day 90. adMSC-embedded modules without HMEC surface coating did not show similar levels of Oil Red O staining. All implant volumes decreased over the time course of the experiment, yet HMEC+adMSC module implants were larger than adMSC-only implants at day 90. Collagen gel is mechanically weak and contracts in vivo making it unsuitable as a biomaterial for adipose tissue engineering where volume maintenance is critical. When combined with an appropriate biomaterial, the modular approach to adipose tissue engineering may represent a successful strategy to engineer soft tissue substitutes of clinical relevance. PMID:22655687

  18. The Relationship between the Bcl-2/Bax Proteins and the Mitochondria-Mediated Apoptosis Pathway in the Differentiation of Adipose-Derived Stromal Cells into Neurons

    PubMed Central

    Wang, Quanquan; Zhang, Lili; Yuan, Xiaodong; Ou, Ya; Zhu, Xuhong; Cheng, Zanzan; Zhang, Pingshu; Wu, Xiaoying; Meng, Yan; Zhang, Liping

    2016-01-01

    Our objective is to study the relationship between the regulatory proteins Bcl-2/Bax and mitochondria-mediated apoptosis during the differentiation of adipose-derived stromal cells (ADSCs) into neurons. Immunocytochemistry and western blotting showed that the cells weakly expressed neuron-specific enolase (NSE) in the non-induced group and expressed NSE more strongly in the groups induced for 1 h, 3 h, 5 h and 8 h. NSE expression peaked at 5 h (P < 0.05), although there was no significant difference between 5 and 8 h (P > 0.05). Bcl-2 expression gradually decreased over time in the non-induced group (P < 0.05). However, Bax, caspase-9, Cyt-c and caspase-3 expression gradually increased and peaked at 8 h (P < 0.05). Transmission electron microscopy revealed karyopyknosis, chromatin edge setting, mitochondria swelling and cavitation in cells at 5 h, and the mitochondrial membrane potential decreased over time, as demonstrated by laser scanning confocal microscopy. After a 5 h induction, cells differentiated into typical neurons and expressed Bcl-2, which inhibited apoptosis. Bax showed a strong apoptosis-promoting capacity, leading to changes in the mitochondrial membrane potential and structure, and then triggered the caspase-independent apoptotic response through the mitochondrial pathway. At the same time, Cyt-c was directly or indirectly released from the mitochondria to the cytoplasm to trigger the caspase-dependent apoptotic response through the mitochondrial pathway. Therefore, Bcl-2/Bax play an important role in regulating caspase-dependent and caspase-independent apoptosis mediated by the mitochondrial pathway during the differentiation of ADSCs into neurons. PMID:27706181

  19. Effects of administration of adipose-derived stromal vascular fraction and platelet-rich plasma to dogs with osteoarthritis of the hip joints.

    PubMed

    Upchurch, David A; Renberg, Walter C; Roush, James K; Milliken, George A; Weiss, Mark L

    2016-09-01

    OBJECTIVE To evaluate effects of simultaneous intra-articular and IV injection of autologous adipose-derived stromal vascular fraction (SVF) and platelet-rich plasma (PRP) to dogs with osteoarthritis of the hip joints. ANIMALS 22 client-owned dogs (12 placebo-treated [control] dogs and 10 treated dogs). PROCEDURES Dogs with osteoarthritis of the hip joints that caused signs of lameness or discomfort were characterized on the basis of results of orthopedic examination, goniometry, lameness score, the Canine Brief Pain Inventory (CBPI), a visual analogue scale, and results obtained by use of a pressure-sensing walkway at week 0 (baseline). Dogs received a simultaneous intraarticular and IV injection of SVF and PRP or a placebo. Dogs were examined again 4, 8, 12, and 24 weeks after injection. RESULTS CBPI scores were significantly lower for the treatment group at week 24, compared with scores for the control group. Mean visual analogue scale score for the treatment group was significantly higher at week 0 than at weeks 4, 8, or 24. Dogs with baseline peak vertical force (PVF) in the lowest 25th percentile were compared, and the treatment group had a significantly higher PVF than did the control group. After the SVF-PRP injection, fewer dogs in the treated group than in the control group had lameness confirmed during examination. CONCLUSIONS AND CLINICAL RELEVANCE For dogs with osteoarthritis of the hip joints treated with SVF and PRP, improvements in CBPI and PVF were evident at some time points, compared with results for the control group.

  20. LL-37 stimulates the functions of adipose-derived stromal/stem cells via early growth response 1 and the MAPK pathway.

    PubMed

    Yang, Yoolhee; Choi, Hyunju; Seon, Mira; Cho, Daeho; Bang, Sa Ik

    2016-04-19

    LL-37 is a naturally occurring antimicrobial peptide found in the wound bed and assists wound repair. No published study has characterized the role of LL-37 in the function(s) of human mesenchymal stem cells (MSCs). This study investigated the functions of adipose-derived stromal/stem cells (ASCs) activated by LL-37 by performing both in vitro assays with cultured cells and in vivo assays with C57BL/6 mice with hair loss. Human ASCs were isolated from healthy donors with written informed consent. To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay. Early growth response 1 (EGR1) mRNA expression was determined by microarray and real-time PCR analyses. The protein levels of EGR1 and regenerative factors were analyzed by specific enzyme-linked immunosorbent assays and western blotting. LL-37 treatment enhanced the proliferation and migration of human ASCs expressing formyl peptide receptor like-1. Microarray and real-time PCR data showed that EGR1 expression was rapidly and significantly increased by LL-37 treatment. LL-37 treatment also enhanced the production of EGR1. Moreover, small interfering RNA-mediated knockdown of EGR1 inhibited LL-37-enhanced ASC proliferation and migration. Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37. EGR1 has a strong paracrine capability and can influence angiogenic factors in ASCs; therefore, we evaluated the secretion levels of vascular endothelial growth factor, thymosin beta-4, monocyte chemoattractant protein-1, and stromal cell-derived factor-1. LL-37 treatment increased the secretion of these regenerative factors. Moreover, treatment with the conditioned medium of ASCs

  1. Human breast adipose-derived stem cells transfected with the stromal cell-derived factor-1 receptor CXCR4 exhibit enhanced viability in human autologous free fat grafts.

    PubMed

    Xu, Fang-tian; Li, Hong-mian; Yin, Qing-Shui; Liu, Da-lie; Nan, Hua; Zhao, Pei-ran; Liang, Shuang-wu

    2014-01-01

    The main complication of autologous free fat tissue transplantation is fat resorption and calcification due to the ischemic necrosis of fat. The promotion of transplant neovascularization soon after autologous free fat grafts may reduce these outcomes. In adulthood, stromal cell-derived factor-1 (SDF-1) and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4) are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. We hypothesized that CXCR4 may improve the long-term survival of free fat tissue transplants by recruiting endothelial progenitor cells (EPCs) and may therefore improve graft revascularization. In this study, we aimed to determine the effect of human breast adipose-derived stem cells (HBASCs) transfected with the CXCR4 gene on the survival rate of human autologous free fat transplants in nude mice. Human breast adipose-derived stem cells (HBASCs) were expanded ex vivo for 3 passages, labeled with green fluorescent protein (GFP) and transfected with CXCR4 or left untransfected. Autologous fat tissues were mixed with the GFP-labeled, CXCR4-transfected HBASCs (group A), GFP-labeled HBASCs (group B), the known vascularization-promoting agent VEGF (group C), or medium (group D) and then injected subcutaneously into 32 nude mice at 4 spots in a random fashion. Six months later, the transplanted tissue volume and histology were evaluated, and neo-vascularization was quantified by counting the capillaries. CXCR4 and SDF-1α mRNA expression in the transplants was determined using real-time quantitative PCR analysis (qPCR). The data revealed that the control (group D) transplant volume survival was 28.3 ± 4.5%. Mixing CXCR4-transfected (group A) and untransfected (group B) HBASCs significantly increased transplant volume survival (79.5 ± 8.3% and 67.2 ± 5.9%, respectively), whereas VEGF-transfected HBASCs (group C) were less effective (41.2 ± 5.1%). Histological analysis revealed that both types

  2. Hypoxia Inhibits De Novo Vascular Assembly of Adipose-Derived Stromal/Stem Cell Populations, but Promotes Growth of Preformed Vessels

    PubMed Central

    Hutton, Daphne L.

    2016-01-01

    Vascularization is critical for cell survival within tissue-engineered grafts. Adipose-derived stromal/stem cells (ASCs) are widely used in tissue engineering applications as they are a clinically relevant source of stem cells and endothelial progenitor cells. ASCs have previously been shown to self-assemble into pericyte-stabilized vascular networks in normoxic (20% O2) cultures. This capacity for de novo vascular assembly may accelerate graft vascularization in vivo rather than relying solely on angiogenic ingrowth. However, oxygen depletion within large cell-seeded grafts will be rapid, and it is unclear how this worsening hypoxic environment will impact the vascular assembly of the transplanted cells. The objectives of this study were to determine whether ASC-derived vessels could grow in hypoxia and to assess whether the vessel maturity (i.e., individual cells vs. preformed vessels) influenced this hypoxic response. Utilizing an in vitro vascularization model, ASCs were encapsulated within fibrin gels and cultured in vitro for up to 6 days in either normoxia (20% O2) or hypoxia (0.2% or 2% O2). In a subsequent experiment, vessels were allowed to preform in normoxia for 6 days before an additional 6 days of either normoxia or hypoxia. Viability, vessel growth, pericyte coverage, proliferation, metabolism, and angiogenic factor expression were assessed for each experimental approach. Vessel growth was dramatically inhibited in both moderate and severe hypoxia (47% and 11% total vessel length vs. normoxia, respectively), despite maintaining high cell viability and upregulating endogenous expression of vascular endothelial growth factor in hypoxia. Bromodeoxyuridine labeling indicated significantly reduced proliferation of endothelial cells in hypoxia. In contrast, when vascular networks were allowed to preform for 6 days in normoxia, vessels not only survived but also continued to grow more in hypoxia than those maintained in normoxia. These findings demonstrate

  3. Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture.

    PubMed

    Schubert, Susanna; Brehm, Walter; Hillmann, Aline; Burk, Janina

    2017-09-19

    For clinical applications of multipotent mesenchymal stromal cells (MSCs), serum-free culture is preferable to standardize cell products and prevent contamination with pathogens. In contrast to human MSCs, knowledge on serum-free culture of large animal MSCs is limited, despite its relevance for preclinical studies and development of veterinary cellular therapeutics. This study aimed to evaluate the suitability of a commercially available serum-free human MSC medium for culturing equine adipose-derived MSCs in comparison with human adipose MSCs. Enzyme-free isolation by explant technique and expansion of equine and human cells in the serum-free medium were feasible. However, serum-free culture altered the morphology and complicated handling of equine MSCs, with cell aggregation and spontaneous detachment of multilayers, compared to culture in standard medium supplemented with fetal bovine serum. Furthermore, proliferation and the surface immunophenotype of equine cells were more variable compared to the controls and appeared to depend on the lot of the serum-free medium. Particularly the expression of CD90 was different between experimental groups (P < 0.05), with lower percentages of CD90(+) cells found in equine MSC samples cultured in serum-free medium (5.21-83.40%) compared to standard medium (86.20-99.50%). Additionally, small subpopulations expressing MSC exclusion markers such as CD14 (0.28-11.60%), CD34 (0.00-9.87%), CD45 (0.35-10.50%), or MHCII (0.00-3.67%) were found in equine samples after serum-free culture. In contrast, human samples displayed a more consistent morphology and a consistent CD29(+) (98.60-99.90%), CD73(+) (94.60-98.40%), CD90(+) (99.60-99.90%), and CD105(+) (97.40-99.80%) immunophenotype after culture in serum-free medium. The obtained data demonstrate that the serum-free medium was suitable for human MSC culture but did not lead to entirely satisfactory results in equine MSCs. This underlines that requirements regarding serum

  4. Intraoperative use of enriched collagen and elastin matrices with freshly isolated adipose-derived stem/stromal cells: a potential clinical approach for soft tissue reconstruction.

    PubMed

    Alharbi, Ziyad; Almakadi, Sultan; Opländer, Christian; Vogt, Michael; Rennekampff, Hans-Oliver; Pallua, Norbert

    2014-02-20

    Adipose tissue contains a large number of multipotent cells, which are essential for stem cell-based therapies. The combination of this therapy with suitable commercial clinically used matrices, such as collagen and elastin matrices (i.e. dermal matrices), is a promising approach for soft tissue reconstruction. We previously demonstrated that the liposuction method affects the adherence behaviour of freshly isolated adipose-derived stem/stromal cells (ASCs) on collagen and elastin matrices. However, it remains unclear whether freshly isolated and uncultured ASCs could be directly transferred to matrices during a single transplantation operation without additional cell culture steps. After each fat harvesting procedure, ASCs were isolated and directly seeded onto collagen and elastin matrices. Different time intervals (i.e. 1, 3 and 24 h) were investigated to determine the time interval needed for cellular attachment to the collagen and elastin matrices. Resazurin-based vitality assays were performed after seeding the cells onto the collagen and elastin matrices. In addition, the adhesion and migration of ASCs on the collagen and elastin matrices were visualised using histology and two-photon microscopy. A time-dependent increase in the number of viable ASCs attached to the collagen and elastin matrices was observed. This finding was supported by mitochondrial activity and histology results. Importantly, the ASCs attached and adhered to the collagen and elastin matrices after only 1 h of ex vivo enrichment. This finding was also supported by two-photon microscopy, which revealed the presence and attachment of viable cells on the upper layer of the construct. Freshly isolated uncultured ASCs can be safely seeded onto collagen and elastin matrices for ex vivo cellular enrichment of these constructs after liposuction. Although we observed a significant number of seeded cells on the matrices after a 3-h enrichment time, we also observed an adequate number of isolated

  5. Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone.

    PubMed

    Wittenburg, Gretel; Flade, Viktoria; Garbe, Annette I; Lauer, Günter; Labudde, Dirk

    2014-05-01

    We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR). Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression. Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for

  6. A prospective, non-randomized, no placebo-controlled, phase Ib clinical trial to study the safety of the adipose derived stromal cells-stromal vascular fraction in idiopathic pulmonary fibrosis

    PubMed Central

    2013-01-01

    Introduction Regenerative medicine and particular adult stem cells represent an alternative option with several fruitful therapeutic applications in patients suffering from chronic lung diseases including idiopathic pulmonary fibrosis (IPF). Nevertheless, lack of knowledge regarding the origin and the potential of mesenchymal stem cells (MSCs) to differentiate into fibroblasts has limited their use for the treatment of this dismal disease. Patients and methods To this end, we conducted a phase Ib, non-randomized, clinical trial to study the safety of three endobronchial infusions of autologous adipose derived stromal cells (ADSCs)-stromal vascular fraction (SVF) (0.5 million cells per kgr of body weight per infusion) in patients with IPF (n=14) of mild to moderate disease severity (forced vital capacity –FVC>50% predicted value and diffusion lung capacity for carbon monoxide-DLCO>35% of predicted value). Our primary end-point was incidence of treatment emergent adverse events within 12 months. Alterations of functional, exercise capacity and quality of life parameters at serial time points (baseline, 6 and 12 months after first infusion) were exploratory secondary end-points. Results No cases of serious or clinically meaningful adverse events including short-term infusional toxicities as well as long-term ectopic tissue formation were recorded in all patients. Detailed safety monitoring through several time-points indicated that cell-treated patients did not deteriorate in both functional parameters and indicators of quality of life. Conclusions The clinical trial met its primary objective demonstrating an acceptable safety profile of endobronchially administered autologous ADSCs-SVF. Our findings accelerate the rapidly expanded scientific knowledge and indicate a way towards future efficacy trials. PMID:23855653

  7. [Comparison between kinds of myofascial flap encapsulating adipose-derived stromal cells carrier complex in terms of adipogenic efficacy in vivo].

    PubMed

    Li, Hongmian; Gao, Jianhua; Lu, Feng; Li, Hua; Chen, Xiaowei; Fu, Bingchuan

    2009-02-01

    To compare two kinds of myofascial flap encapsulating adipose-derived stromal cells (ADSCs) in adipogenic efficacy in vivo, and to provide experimental basis for the efficient transplantation of free adipose tissue. ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits (aged 3- 4-months-old, male and female, weighing 2.0-2.5 kg), and primary culture and subculture of ADSCs were conducted. When the cells at passage 3 covered 70%-80% of the bottom of the culture flask, BrdU (10 microg/mL) was applied to label the cells for 48 hours before performing immunofluorescence staining. Oil red O staining observation was conducted to those cells 2 weeks after being induced towards adipocyte, alizarin red staining observation was performed 3 weeks after being induced towards osteoblast, and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte. Besides, after being induced towards adipocyte for 2 weeks, 1 x 10(7) ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I (10 mm x 10 mm x 5 mm/piece) to prepare cell carrier complex. The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial flap was adopted to encapsulate the complex; group B in which dextral gluteus maximus fascial flap with no specific vessel pedicle was applied to encapsulate the complex. Rabbits in each group went through autogenous ADSCs transplant and self control. The implants were dislodged 8 weeks after operation, HE staining and immunohistochemistry staining were performed to testify cambium, the wet weight and micro vessel count of the cambium in each group were tested, immunofluorescence staining was performed to determine the origin of cambium and microvascular endothelium. The nucleus of ADSCs positive for BrdU labeling showed green fluorescence under fluorescence microscope, with the positive labeling ratio of ADSCs above 90%. For ADSCs at passage 3, the formation of red

  8. Allogeneic Platelet Releasate Preparations Derived via a Novel Rapid Thrombin Activation Process Promote Rapid Growth and Increased BMP-2 and BMP-4 Expression in Human Adipose-Derived Stem Cells.

    PubMed

    McLaughlin, Michael; Gagnet, Paul; Cunningham, Elizabeth; Yeager, Randi; D'Amico, Michael; Guski, Katie; Scarpone, Michael; Kuebler, Daniel

    2016-01-01

    The administration of human adipose-derived stem cells (ASCs) represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR). ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase) and BMP-2 (4.7 ± 1.3-fold increase) and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease) and FGF-2 (33 ± 9.0-fold decrease). No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications.

  9. Low-frequency, low-magnitude vibrations (LFLM) enhances chondrogenic differentiation potential of human adipose derived mesenchymal stromal stem cells (hASCs).

    PubMed

    Marycz, Krzysztof; Lewandowski, Daniel; Tomaszewski, Krzysztof A; Henry, Brandon M; Golec, Edward B; Marędziak, Monika

    2016-01-01

    The aim of this study was to evaluate if low-frequency, low-magnitude vibrations (LFLM) could enhance chondrogenic differentiation potential of human adipose derived mesenchymal stem cells (hASCs) with simultaneous inhibition of their adipogenic properties for biomedical purposes. We developed a prototype device that induces low-magnitude (0.3 g) low-frequency vibrations with the following frequencies: 25, 35 and 45 Hz. Afterwards, we used human adipose derived mesenchymal stem cell (hASCS), to investigate their cellular response to the mechanical signals. We have also evaluated hASCs morphological and proliferative activity changes in response to each frequency. Induction of chondrogenesis in hASCs, under the influence of a 35 Hz signal leads to most effective and stable cartilaginous tissue formation through highest secretion of Bone Morphogenetic Protein 2 (BMP-2), and Collagen type II, with low concentration of Collagen type I. These results correlated well with appropriate gene expression level. Simultaneously, we observed significant up-regulation of α3, α4, β1 and β3 integrins in chondroblast progenitor cells treated with 35 Hz vibrations, as well as Sox-9. Interestingly, we noticed that application of 35 Hz frequencies significantly inhibited adipogenesis of hASCs. The obtained results suggest that application of LFLM vibrations together with stem cell therapy might be a promising tool in cartilage regeneration.

  10. Low-frequency, low-magnitude vibrations (LFLM) enhances chondrogenic differentiation potential of human adipose derived mesenchymal stromal stem cells (hASCs)

    PubMed Central

    Lewandowski, Daniel; Tomaszewski, Krzysztof A.; Henry, Brandon M.; Golec, Edward B.; Marędziak, Monika

    2016-01-01

    The aim of this study was to evaluate if low-frequency, low-magnitude vibrations (LFLM) could enhance chondrogenic differentiation potential of human adipose derived mesenchymal stem cells (hASCs) with simultaneous inhibition of their adipogenic properties for biomedical purposes. We developed a prototype device that induces low-magnitude (0.3 g) low-frequency vibrations with the following frequencies: 25, 35 and 45 Hz. Afterwards, we used human adipose derived mesenchymal stem cell (hASCS), to investigate their cellular response to the mechanical signals. We have also evaluated hASCs morphological and proliferative activity changes in response to each frequency. Induction of chondrogenesis in hASCs, under the influence of a 35 Hz signal leads to most effective and stable cartilaginous tissue formation through highest secretion of Bone Morphogenetic Protein 2 (BMP-2), and Collagen type II, with low concentration of Collagen type I. These results correlated well with appropriate gene expression level. Simultaneously, we observed significant up-regulation of α3, α4, β1 and β3 integrins in chondroblast progenitor cells treated with 35 Hz vibrations, as well as Sox-9. Interestingly, we noticed that application of 35 Hz frequencies significantly inhibited adipogenesis of hASCs. The obtained results suggest that application of LFLM vibrations together with stem cell therapy might be a promising tool in cartilage regeneration. PMID:26966645

  11. Elimination of allogeneic multipotent stromal cells by host macrophages in different models of regeneration

    PubMed Central

    Arutyunyan, Irina; Elchaninov, Andrey; Fatkhudinov, Timur; Makarov, Andrey; Kananykhina, Evgeniya; Usman, Natalia; Bolshakova, Galina; Glinkina, Valeria; Goldshtein, Dmitry; Sukhikh, Gennady

    2015-01-01

    Allogeneic multipotent stromal cells were previously thought to be poorly recognized by host immune system; the prolonged survival in host environments was explained by their immune privileged status. As long as the concept is currently reconsidered, the routes of elimination of allogeneic multipotent stromal cells by host immunity must be taken into account. This is necessary for correct comprehension of their therapeutic action. The study was focused upon survival of umbilical cord-derived allogeneic multipotent stromal cells in different rat models of tissue regeneration induced by partial hepatectomy or by critical limb ischemia. The observations were carried out by means of vital labeling of the cells with PKH26 prior to injection, in combination with differential immunostaining of host macrophages with anti-CD68 antibody. According to the results, allogeneic multipotent stromal cells are specifically eliminated by host immune system; the efficacy can reach 100%. Massive clearance of transplanted cells by host macrophages is accompanied by appropriation of the label by the latter, and this is a pronounced case of misleading presentation of exogenous label by host cells. The study emphasizes the role of macrophages in host response and also the need of additional criteria for correct data interpretation. PMID:26191137

  12. Assessment of intravenous adipose-derived allogeneic mesenchymal stem cells for the treatment of feline chronic kidney disease: a randomized, placebo-controlled clinical trial in eight cats.

    PubMed

    Quimby, Jessica M; Webb, Tracy L; Randall, Elissa; Marolf, Angela; Valdes-Martinez, Alex; Dow, Steve W

    2016-02-01

    Feline chronic kidney disease (CKD) is characterized by chronic tubulointerstitial nephritis, and inflammation contributes to the progression of renal fibrosis. Mesenchymal stem cells (MSCs) have demonstrated anti-inflammatory and antifibrotic effects in rodent CKD models. However, few randomized trials evaluating the effectiveness of MSC therapy for diseases in companion animals have been reported. The purpose of this study was to evaluate the effectiveness of allogeneic MSCs for the treatment of feline CKD using a randomized, placebo-controlled trial. MSCs were isolated from the cryopreserved adipose tissues of specific pathogen-free research cats and culture expanded. CKD cats were enrolled in a randomized, placebo-controlled, blinded one-way crossover clinical study. Four CKD cats were randomized to receive 2 × 10(6) MSCs/kg intravenously at 2, 4 and 6 weeks. Four CKD cats were randomized to receive placebo, with two cats crossing over to the MSC treatment group and one cat failing to complete the trial. Complete blood counts, chemistry and urinalysis were performed at weeks 0, 2, 4, 6 and 8. Glomerular filtration rate (GFR) via nuclear scintigraphy and urine protein:creatinine ratio (UPC) were determined at weeks 0 and 8. Six cats received three doses of allogeneic MSC culture expanded from cryopreserved adipose without adverse effects. No significant change in serum creatinine, blood urea nitrogen, potassium, phosphorus, GFR by nuclear scintigraphy, UPC or packed cell volume was seen in cats treated with MSCs. Individual changes in GFR were 12%, 8%, 8%, 2%, -13% and -67% in treated cats compared with 16%, 36% and 0% in placebo-treated cats. While administration of MSC culture expanded from cryopreserved adipose was not associated with adverse effects, significant improvement in renal function was not observed immediately after administration. Long-term follow-up is necessary to determine whether MSC administration affects disease progression in cats with CKD

  13. Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses

    PubMed Central

    Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka

    2017-01-01

    Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)—the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs’ and IECs’ morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome. PMID:28771165

  14. Effective expansion of human adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles-coated substratum with human platelet-rich plasma.

    PubMed

    Kishimoto, Satoko; Ishihara, Masayuki; Mori, Yasutaka; Takikawa, Megumi; Hattori, Hidemi; Nakamura, Shingo; Sato, Toshinori

    2013-12-01

    Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet-rich plasma (PRP). In this study, we investigated the capability of F/P NP-coated plates to act as a substratum for the proliferation of human adipose-derived stromal cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP-coated plates and grew optimally, with a doubling time of 30 and 32 h in low-concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor-2 (FGF-2) on the F/P NP-coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF-2 on F/P NP-coated plates pretreated with PRP and FGF-2 in a concentration-dependent manner. Thus, F/P NP-coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low-concentration PRP medium supplemented with FGF-2. No xenogeneic serum is required. Copyright © 2012 John Wiley & Sons, Ltd.

  15. Adipose-derived mesenchymal stromal (stem) cells differentiate to osteoblast and chondroblast lineages upon incubation with conditioned media from dental pulp stem cell-derived osteoblasts and auricle cartilage chondrocytes.

    PubMed

    Carbone, A; Valente, M; Annacontini, L; Castellani, S; Di Gioia, S; Parisi, D; Rucci, M; Belgiovine, G; Colombo, C; Di Benedetto, A; Mori, G; Lo Muzio, L; Maiorella, A; Portincasa, A; Conese, M

    2016-01-01

    The potential of adipose-derived mesenchymal stromal (stem) cells (ADSCs) to differentiate into either osteoblasts or chondrocytes is controversial. In this study we investigated the multicapacity potential of ADSCs to differentiate towards adipocyte, osteoblast, and chondrocyte lineages when cells are seeded onto plastic in comparison with incubation with conditioned media (CM) obtained from differentiated cell types.ADSCs, obtained from liposuctions, were characterized for mesenchymal and hematopoietic markers by cytofluorimetry. Their differentiation capacity towards adipocytes, osteoblasts, and chondrocytes was investigated by histochemistry methods (Oil-Red-O staining, Safranin O and Alizarin Red staining, respectively). Dental pulp stem cells (DPSCs) and dedifferentiated auricle derived-chondrocytes were differentiated towards osteoblastic and chondrocytic lineages respectively, and the CM obtained from these cultures was used to induce differentiation of ADSCs. ADSCs were positive for mesenchymal markers (CD29, CD105, CD73, CD44), but not for hematopoietic lineage markers (CD14, CD34, CD45) and this behavior was conserved from the isolation up to the fifth passage. While ADSCs were readily differentiated in adipocytes, they were not towards chondrocytes and osteoblastic lineages, a behavior different from that of bone marrow-derived MSCs that differentiated into the three lineages at two weeks post-induction. Only ADSCs treated with CM from cultured chondrocytes and DPSCs, produced glycosaminoglycans and mineralized matrix. These results indicate that ADSCs need growth/morphogenic factor supplementation from the tissue environment to be appropriately differentiated to mesodermic lineages.

  16. Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses.

    PubMed

    Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka; Marycz, Krzysztof

    2017-08-03

    Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)-the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs' and IECs' morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome.

  17. First-in-man intraglandular implantation of stromal vascular fraction and adipose-derived stem cells plus platelet-rich plasma in irradiation-induced gland damage: a case study.

    PubMed

    Comella, Kristin; Bell, Walter

    2017-01-01

    Stromal vascular fraction (SVF) is a mixture of cells which can be isolated from a mini-lipoaspirate of fat tissue. Platelet-rich plasma (PRP) is a mixture of growth factors and other nutrients which can be obtained from peripheral blood. Adipose-derived stem/stromal cells (ADSCs) can be isolated from fat tissue and expanded in culture. The SVF includes a variety of different cells such as ADSCs, pericytes, endothelial/progenitor cells, and a mix of different growth factors. The adipocytes (fat cells) can be removed via centrifugation. Here, we describe the rationale and, to our knowledge, the first clinical implementation of SVF and PRP followed by repeat dosing of culture-expanded ADSCs into a patient with severe xerostomia postirradiation. Approximately 120 mLs of adipose tissue was removed via mini-lipoaspirate procedure under local anesthetic. The SVF was prepared from half of the fat and resuspended in PRP. The mixture was delivered via ultrasound directly into the submandibular and parotid glands on both the right and left sides. The remaining 60 mLs of fat was processed to culture-expand ADSCs. The patient received seven follow-up injections of the ADSCs plus PRP at 5, 8, 16, 18, 23, 28, and 31 months postliposuction. The subject was monitored over a period of 31 months for safety (adverse events), glandular size via ultrasound and saliva production. Throughout the 31-month monitoring period, no safety events such as infection or severe adverse events were reported. The patient demonstrated an increase in gland size as measured by ultrasound which corresponded to increased saliva production. Overall, the patient reported improved quality of life and willingness to continue treatments. The strong safety profile and preliminary efficacy results warrant larger studies to determine if this is a feasible treatment plan for patients postradiation.

  18. Basic Fibroblast Growth Factor Inhibits Apoptosis and Promotes Proliferation of Adipose-Derived Mesenchymal Stromal Cells Isolated from Patients with Type 2 Diabetes by Reducing Cellular Oxidative Stress

    PubMed Central

    2017-01-01

    Type 2 diabetes (T2D) is a chronic metabolic disorder affecting increasing number of people in developed countries. Therefore new strategies for treatment of T2D and its complications are of special interest. Nowadays, cellular therapies involving mesenchymal stromal cells that reside in adipose tissue (ASCs) constitute a promising approach; however, there are still many obstacles concerning safety and effectiveness that need to be overcome before ASCs could be engaged for the treatment of diabetes mellitus. One of the challenges is preventing ASCs from deterioration caused by elevated oxidative stress present in diabetes milieu. In the current study we investigated the effect of basic fibroblast growth factor (bFGF) treatment on ASCs isolated from patients with diagnosed T2D. We demonstrate here that cell exposition to bFGF in 5 and 10 ng/mL dosages results in improved morphology, increased proliferative activity, reduced cellular senescence and apoptosis, and decreased oxidative stress, indicating recovery of ASCs' function impaired by T2D. Therefore our results provide a support for bFGF as a potential therapeutic agent for improving stem cell-based approaches for the treatment of diabetes mellitus and its complications. PMID:28168007

  19. Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering.

    PubMed

    Parvizi, Mojtaba; Bolhuis-Versteeg, Lydia A M; Poot, André A; Harmsen, Martin C

    2016-07-01

    Occluding artery disease causes a high demand for bioartificial replacement vessels. We investigated the combined use of biodegradable and creep-free poly (1,3-trimethylene carbonate) (PTMC) with smooth muscle cells (SMC) derived by biochemical or mechanical stimulation of adipose tissue-derived stromal cells (ASC) to engineer bioartificial arteries. Biochemical induction of cultured ASC to SMC was done with TGF-β1 for 7d. Phenotype and function were assessed by qRT-PCR, immunodetection and collagen contraction assays. The influence of mechanical stimulation on non-differentiated and pre-differentiated ASC, loaded in porous tubular PTMC scaffolds, was assessed after culturing under pulsatile flow for 14d. Assays included qRT-PCR, production of extracellular matrix and scanning electron microscopy. ASC adhesion and TGF-β1-driven differentiation to contractile SMC on PTMC did not differ from tissue culture polystyrene controls. Mesenchymal and SMC markers were increased compared to controls. Interestingly, pre-differentiated ASC had only marginal higher contractility than controls. Moreover, in 3D PTMC scaffolds, mechanical stimulation yielded well-aligned ASC-derived SMC which deposited ECM. Under the same conditions, pre-differentiated ASC-derived SMC maintained their SMC phenotype. Our results show that mechanical stimulation can replace TGF-β1 pre-stimulation to generate SMC from ASC and that pre-differentiated ASC keep their SMC phenotype with increased expression of SMC markers.

  20. The effect and safety of polylactic acid and adipose-derived stromal vascular fraction cell as an injectable bulking agent in urologic field: a 24-week follow-up study.

    PubMed

    Lee, Seong Ho; Ko, Kyungtae; Choo, Min Soo; Lee, Won Ki; Jeong, Hyun Cheol; Cho, Sung Tae; Kim, Sung Yong; Kim, Hayoung; Kang, Won Hwa; Kim, Gun Poong; Yang, Dae Yul

    2015-02-01

    The aim of this study is to evaluate whether polylactic acid (PLA) microspheres and adipose-derived stromal vascular fraction (SVF) cells have appropriate properties as an injectable bulking agent in urologic field. Forty male Sprague-Dawley rats (2-week-old) were randomized into two groups. A total of 0.05 mL of PLA microsphere suspension and 0.05 mL of PLA microsphere suspension mixed with PKH26-labeled SVF cells were injected into bladder wall in group I and group II, respectively. At 2, 8, 16, and 24 weeks of PLA microspheres injection, the volumes of implants were measured and bladder tissues including implants were analyzed and compared grossly and histologically between groups. The distant organs were examined histologically to determine migration of PLA microspheres. At 24 weeks of implantation, 65-70% of injected volume was maintained and there was no significant difference between groups. In histological analyses, injected PLA microspheres were localized in muscular layer of bladder without infiltration into adjacent layer. From 8 to 16 weeks of injection, hybrid tissues contained collagen and actin were observed between PLA microspheres and these findings were more clear in group II. PHK26-labeled SVF cells were identified by fluorescence microscopy at all time points. There was no migration of PLA microspheres to other organs and no abnormality in weight gain and hematologic values. These results suggest the possibility of PLA microspheres as a potentially useful bulking agent in urologic field. And further investigation is needed to know synergic effect of SVF cells.

  1. Pigment epithelial-derived factor and melanoma differentiation associated gene-7 cytokine gene therapies delivered by adipose-derived stromal/mesenchymal stem cells are effective in reducing prostate cancer cell growth.

    PubMed

    Zolochevska, Olga; Yu, Gang; Gimble, Jeffrey M; Figueiredo, Marxa L

    2012-05-01

    Adipose-derived stromal/mesenchymal stem cells (ASC) have gained interest as promising tools for delivering cancer therapy. Adipose tissue can be obtained readily in amounts sufficient for ASC isolation, which can be expanded rapidly, allowing its use at low passage numbers, and can be transduced by viral and nonviral means. Our goal was to examine the potential of ASC to deliver cytokine gene therapies melanoma differentiation associated gene-7 (MDA-7) or pigment epithelial-derived factor (PEDF) to cancer cells. These novel cytokines are a potent proapoptotic and an antiangiogenesis mediator, respectively, with potential as antitumor agents. Expression of cytokine therapies did not adversely affect ASC biology, and these cells were still able to differentiate and retain normal viability. The ASC cytokine therapies were efficient in reducing tumor cell growth in coculture and also in suppressing in vitro angiogenesis phenotypes. We also observed that ASC retained their innate ability to migrate toward tumor cells in coculture, and this ability could be blocked by inhibition of CXCR4 signaling. The ASC were found to be nontumorigenic in vitro using a soft agar assay, as well as in vivo, utilizing 2 prostate cancer xenograft models. The ASC-MDA7 only reduced tumor growth in the TRAMP-C2-Ras (TC2Ras) prostate cancer model. The ASC-PEDF, however, reduced growth in both the TC2Ras and the PC3 highly aggressive prostate cancer models, and it was able to completely prevent prostate tumor establishment in vivo. In conclusion, ASC expressing PEDF and MDA7 could effectively reduce prostate tumor growth in vivo, suggesting ASC-cytokine therapies might have translational applications, especially the PEDF modality.

  2. Immunomodulatory Effects of Adipose-Derived Stem Cells: Fact or Fiction?

    PubMed Central

    Leto Barone, Angelo A.; Khalifian, Saami; Lee, W. P. Andrew; Brandacher, Gerald

    2013-01-01

    Adipose-derived stromal cells (ASCs) are often referred to as adipose-derived stem cells due to their potential to undergo multilineage differentiation. Their promising role in tissue engineering and ability to modulate the immune system are the focus of extensive research. A number of clinical trials using ASCs are currently underway to better understand the role of such cell niche in enhancing or suppressing the immune response. If governable, such immunoregulatory role would find application in several conditions in which an immune response is present (i.e., autoimmune conditions) or feared (i.e., solid organ or reconstructive transplantation). Although allogeneic ASCs have been shown to prevent acute GvHD in both preclinical and clinical studies, their potential warrants further investigation. Well-designed and standardized clinical trials are necessary to prove the role of ASCs in the treatment of immune disorders or prevention of tissue rejection. In this paper we analyze the current literature on the role of ASCs in immunomodulation in vitro and in vivo and discuss their potential in regulating the immune system in the context of transplantation. PMID:24106704

  3. Decellularized Iliotibial Band Recolonized with Allogenic Homotopic Fibroblasts or Bone Marrow-Derived Mesenchymal Stromal Cells.

    PubMed

    Gögele, Clemens; Schwarz, Silke; Ondruschka, Benjamin; Hammer, Niels; Schulze-Tanzil, Gundula

    2017-05-10

    Decellularized scaffolds present promising biomimetic approaches in various fields of tissue engineering. Different tissues have been selected for decellularization, among them extracellular matrix (ECM)-rich tissues such as tendons, ligaments and cartilage. The dense ECM of ligaments is particularly challenging to achieve a completely non-immunogenic ECM void of any cells. Here, the methods for decellularization adapted to ligamentous tissue of the iliotibial band (ITB) are presented along with cell isolation and several recolonization techniques using allogenic ITB-derived fibroblasts or mesenchymal stromal cells (MSCs).

  4. Frozen adipose-derived mesenchymal stem cells maintain high capability to grow and differentiate.

    PubMed

    Minonzio, Greta; Corazza, Mattia; Mariotta, Luca; Gola, Mauro; Zanzi, Michele; Gandolfi, Eugenio; De Fazio, Domenico; Soldati, Gianni

    2014-10-01

    In recent years, there has been a shift toward tissue-engineering strategies using stem cells for plastic and reconstructive surgical procedures. Therefore, it is important to develop safe and reproducible protocols for the extraction of adipose-derived stromal cells (ASCs) to allow cells to be stored in liquid nitrogen for future needs. The aspirated liposuction obtained from healthy donors were immediately processed after the suction using a protocol developed in our laboratory. The resulting stromal vascular fraction (SVF) was then characterized by the presence of adipose-derived stromal cells, at later stage frozen in liquid nitrogen. After that, cells were thawed and again characterized by adipose-derived stromal cells, cellular survival, differentiation ability and Colony Forming Unit-Fibroblast like colonies (CFU-F). Extraction and freezing of cells contained in the stromal vascular fraction demonstrate that thawed cells maintain the full capability to grow and differentiate in culture. The advent of adipose-derived stromal cells use in tissue engineering will assume a wide role in esthetic restoration in plastic surgery. It is thus important to develop clinically translatable protocols for the preparation and storage of adipose-derived stromal cells. Our results show that adipose-derived stromal cells in serum free can easily be frozen and stored in liquid nitrogen with retention of 85% of cell viability and 180,890 cell/g yield plus normal proliferative capacity and differentiation potential compared with fresh controls. These observations set the basis for adipose-derived stromal cells banking. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Synergistic antibacterial effect of co-administering adipose-derived mesenchymal stromal cells and Ophiophagus hannah L-amino acid oxidase in a mouse model of methicillin-resistant Staphylococcus aureus-infected wounds.

    PubMed

    Mot, Yee Yik; Othman, Iekhsan; Sharifah, Syed Hassan

    2017-01-23

    Mesenchymal stromal cells (MSCs) and Ophiophagus hannah L-amino acid oxidase (Oh-LAAO) have been reported to exhibit antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). Published data have indicated that synergistic antibacterial effects could be achieved by co-administration of two or more antimicrobial agents. However, this hypothesis has not been proven in a cell- and protein-based combination. In this study, we investigate if co-administration of adipose-derived MSCs and Oh-LAAO into a mouse model of MRSA-infected wounds would be able to result in a synergistic antibacterial effect. MSCs and Oh-LAAO were isolated and characterized by standard methodologies. The effects of the experimental therapies were evaluated in C57/BL6 mice. The animal study groups consisted of full-thickness uninfected and MRSA-infected wound models which received Oh-LAAO, MSCs, or both. Oh-LAAO was administered directly on the wound while MSCs were delivered via intradermal injections. The animals were housed individually with wound measurements taken on days 0, 3, and 7. Histological analyses and bacterial enumeration were performed on wound biopsies to determine the efficacy of each treatment. Immunophenotyping and differentiation assays conducted on isolated MSCs indicated expression of standard cell surface markers and plasticity which corresponds to published data. Characterization of Oh-LAAO by proteomics, enzymatic, and antibacterial assays confirmed the identity, purity, and functionality of the enzyme prior to use in our subsequent studies. Individual treatments with MSCs and Oh-LAAO in the infected model resulted in reduction of MRSA load by one order of magnitude to the approximate range of 6 log10 colony-forming units (CFU) compared to untreated controls (7.3 log10 CFU). Similar wound healing and improvements in histological parameters were observed between the two groups. Co-administration of MSCs and Oh-LAAO reduced bacterial burden by

  6. Adipose-derived stem cells for periodontal tissue regeneration.

    PubMed

    Tobita, Morikuni; Mizuno, Hiroshi

    2011-01-01

    Mesenchymal stem cells can effectively regenerate destroyed periodontal tissue. Because periodontal tissues are complex, mesenchymal stem cells that can differentiate into many tissue types would aid periodontal tissue regeneration. Indeed, periodontal tissue regeneration using mesenchymal stem cells derived from adipose tissue or bone marrow has been performed in experimental animal models, such as rat, canine, swine, and monkey. We have shown that rat periodontal tissue can be regenerated with adipose-derived stem cells. Adipose tissue contains a large number of stromal cells and is relatively easy to obtain in large quantities, and thus constitutes a very convenient stromal cell source. In this chapter, we introduce a rat periodontal tissue regeneration model using adipose-derived stem cells.

  7. Polymeric film of 6-arm-poly(ethylene glycol) amine graphene oxide with poly (ε-caprolactone): Adherence and growth of adipose derived mesenchymal stromal cells culture on rat bladder

    NASA Astrophysics Data System (ADS)

    Durán, Marcela; Durán, Nelson; Luzo, Angela C. M.; Duarte, Adriana S. S.; Volpe, Bruno B.; Ceragioli, Helder J.; Andrade, Patricia F.; De Souza, Joel G.; Fávaro, Wagner J.

    2017-06-01

    Nanotechnology has been more present in different fields related to health. The need to find a durable material, of easy use, and which does not interfere significantly in the growth and differentiation of stem cells for the construction of a scaffold for use in urologic surgery, with the purpose of reducing infections, regeneration times and even graft rejection during reconstitution in patients with urethral stricture was conducted a broad survey of information about this and came to the consensus of this project: using graphene oxide, a widely studied nanomaterials which has been presenting numerous beneficial results when in contact with the adipose-derived stem cells. Advanced techniques for the growth, differentiation and proliferation of adipose-derived stem cells were used, as well as the characterization of graphene oxide sheets. For this study, it was prepared the graphene oxide/6 ARM-Poly (ethylene glycol) amine films with poly (ε-caprolactone). The graphene suspension in organic solvent was prepared by using an ultrasonicator bath and subsequently, the film was formed by solvent evaporation. Total characterization of graphene oxide/6 ARM-PEG-amine/ poly (ε-caprolactone) film was carried out. It was tested growth and adhesion of adipose-derived stem cells on the film, as well as, were verified the histopathological effects of this scaffold when implanted in the urinary bladder to repair the lesion. Our results demonstrated that this scaffold with adipose-derived stem cells enhanced the repair in rat urinary bladder defect model, resulting in a regular bladder. Improved organized muscle bundles and urothelial layer were observed in animals treated with this scaffold with adipose-derived stem cells compared with those treated only suture thread or scaffold. Thus, our biomaterial could be suitable for tissue engineered urinary tract reconstruction.

  8. Cranial reconstruction using allogeneic mesenchymal stromal cells: A phase 1 first-in-human trial.

    PubMed

    Morrison, David Anthony; Kop, Alan Matthew; Nilasaroya, Anastasia; Sturm, Marian; Shaw, Kathryn; Honeybul, Stephen

    2017-05-09

    Cranioplasty is necessary for patients that have undergone craniectomy following trauma, stroke or other causes of elevated intracranial pressure. This study assessed the effectiveness of treating cranial defects with allogeneic mesenchymal stromal cells (MSC) on a ceramic carrier and polymer scaffold, to produce viable bone and healing of a cranial void. Patients underwent a baseline computed tomography (CT) scan for construct design. Two sets of interlocking moulds were three-dimensional printed to enable shaping of two polymer meshes, which formed the boundaries of the construct corresponding to restoration of the skull interna and externa. In vitro expanded donor MSC were seeded onto ceramic granules in a good manufacturing practices facility. The inner mesh was placed in theatre, followed by the cell-loaded granules, and the outer mesh. Patients were followed-up at 3, 6 and 12 months and cosmesis assessed visually, while bone formation was assessed by CT scans at 1 day, 3 months and 12 months. Manufacture of the construct and surgery was uneventful for all three patients. Initial cosmesis was excellent with no complications. New bone formation was demonstrated by analysis of CT data; however, bone resorption was noted in all 3 cases on the 12-month CT scan. The lack of rigidity of the construct in an environment with continuous pulsatile movement may be preventing the formation of solid bone. It is possible to produce a customized allogeneic MSC construct for cranial reconstruction to replace cranial bone with good cosmesis, using a combination of medical computer modelling, rapid-prototyping and tissue engineering. Copyright © 2017 John Wiley & Sons, Ltd.

  9. Adipose-derived stem cells and periodontal tissue engineering.

    PubMed

    Tobita, Morikuni; Mizuno, Hiroshi

    2013-01-01

    Innovative developments in the multidisciplinary field of tissue engineering have yielded various implementation strategies and the possibility of functional tissue regeneration. Technologic advances in the combination of stem cells, biomaterials, and growth factors have created unique opportunities to fabricate tissues in vivo and in vitro. The therapeutic potential of human multipotent mesenchymal stem cells (MSCs), which are harvested from bone marrow and adipose tissue, has generated increasing interest in a wide variety of biomedical disciplines. These cells can differentiate into a variety of tissue types, including bone, cartilage, fat, and nerve tissue. Adipose-derived stem cells have some advantages compared with other sources of stem cells, most notably that a large number of cells can be easily and quickly isolated from adipose tissue. In current clinical therapy for periodontal tissue regeneration, several methods have been developed and applied either alone or in combination, such as enamel matrix proteins, guided tissue regeneration, autologous/allogeneic/xenogeneic bone grafts, and growth factors. However, there are various limitations and shortcomings for periodontal tissue regeneration using current methods. Recently, periodontal tissue regeneration using MSCs has been examined in some animal models. This method has potential in the regeneration of functional periodontal tissues because the various secreted growth factors from MSCs might not only promote the regeneration of periodontal tissue but also encourage neovascularization of the damaged tissues. Adipose-derived stem cells are especially effective for neovascularization compared with other MSC sources. In this review, the possibility and potential of adipose-derived stem cells for regenerative medicine are introduced. Of particular interest, periodontal tissue regeneration with adipose-derived stem cells is discussed.

  10. Adipose-Derived Stem Cells in Crohn's Rectovaginal Fistula

    PubMed Central

    García-Olmo, D.; Herreros, D.; De-La-Quintana, P.; Guadalajara, H.; Trébol, J.; Georgiev-Hristov, T.; García-Arranz, M.

    2010-01-01

    Therapeutic options for recto-vaginal fistula in the setting of Crohn's disease are limited and many data are available in the literature. The manuscript describes the history of a patient who has been the pioneer of our Clinical Trials in treating this disease in fistulizing Crohn's disease environment. We believe it is the first time that a patient with this disease has been treated by adipose-derived stem cells in allogeneic form. The conclusion of our study with Mary is that the use of mesenchymal stem cells derived from adipose tissue is secure, either in autologous or allogeneic form. Furthermore, we have proved that if we use multi-dose and multiple applications on a patient, it does not produce any adverse effect, which confirms us the safety of using these cells in patients at least in the fistulizing Crohn's disease environment. PMID:20224798

  11. Safety of allogeneic bone marrow derived mesenchymal stromal cell therapy in renal transplant recipients: the neptune study.

    PubMed

    Reinders, Marlies E J; Dreyer, Geertje J; Bank, Jonna R; Roelofs, Helene; Heidt, Sebastiaan; Roelen, Dave L; Zandvliet, Maarten L; Huurman, Volkert A L; Fibbe, Wim E; van Kooten, Cees; Claas, Frans H J; Rabelink, Ton J; de Fijter, Johan W

    2015-11-04

    Mesenchymal stromal cells (MSC) may serve as an attractive therapy in renal transplantation due to their immunosuppressive and reparative properties. While most studies have used autologous MSCs, allogeneic MSCs offer the advantage of immediate availability for clinical use. This is of major importance for indications where instant treatment is needed, for example allograft rejection or calcineurin inhibitor toxicity. Clinical studies using allogeneic MSCs are limited in number. Although these studies showed no adverse reactions, allogeneic MSCs could possibly elicit an anti-donor immune response, which may increase the incidence of rejection and impact the allograft survival in the long term. These safety issues should be addressed before further studies are planned with allogeneic MSCs in the solid organ transplant setting. 10 renal allograft recipients, 18-75 years old, will be included in this clinical phase Ib, open label, single center study. Patients will receive two doses of 1.5 × 10(6) per/kg body weight allogeneic bone marrow derived MSCs intravenously, at 25 and 26 weeks after transplantation, when immune suppression levels are reduced. The primary end point of this study is safety by assessing biopsy proven acute rejection (BPAR)/graft loss after MSC treatment. Secondary end points, all measured before and after MSC infusions, include: comparison of fibrosis in renal biopsy by quantitative Sirius Red scoring; de novo HLA antibody development and extensive immune monitoring; renal function measured by cGFR and iohexol clearance; CMV and BK infection and other opportunistic infections. This study will provide information on the safety of allogeneic MSC infusion and its effect on the incidence of BPAR/graft loss. NCT02387151.

  12. Intramuscular Transplantation of Allogeneic Mesenchymal Stromal Cells Derived from Equine Umbilical Cord

    PubMed Central

    Dias, Marianne Camargos; da Cruz Landim-Alvarenga, Fernanda; de Moraes, Carolina Nogueira; da Costa, Leonardo Dourado; Geraldini, Caroline Medeiros; de Vasconcelos Machado, Vânia Maria; Maia, Leandro

    2016-01-01

    Background and Objectives Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. Methods and Results For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs diluted in Hank’s Balanced Solution (HBSS) was performed on right and left sides of the rump muscle. As a control, HBSS injections were performed caudally in the same muscle. Muscle biopsies were obtained as a control 30 days before transplantation (D-30). The biopsies were collected again on day 2 (left side) and day 7 (right side) post transplantation and examined histologically. All procedures were preceded by ultrasound examination and blood sampling. Hematologic evaluation remained within normal limits and no differences were observed between time points (p>0.05). Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups (control and treated). At histological evaluation it was found only discrete inflammation signals between D-30×D2 (p<0.05) in the treated group, without differences (p> 0.05) between the groups at different time points. Conclusions Equine UC-MSCs under the experimental conditions did not promote severe inflammation that causes tissue damage or lead to its rejection by the host organism and therefore has a good potential for clinical use. PMID:27572709

  13. Intramuscular Transplantation of Allogeneic Mesenchymal Stromal Cells Derived from Equine Umbilical Cord.

    PubMed

    Dias, Marianne Camargos; Landim-Alvarenga, Fernanda da Cruz; de Moraes, Carolina Nogueira; da Costa, Leonardo Dourado; Geraldini, Caroline Medeiros; de Vasconcelos Machado, Vânia Maria; Maia, Leandro

    2016-11-30

    Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs diluted in Hank's Balanced Solution (HBSS) was performed on right and left sides of the rump muscle. As a control, HBSS injections were performed caudally in the same muscle. Muscle biopsies were obtained as a control 30 days before transplantation (D-30). The biopsies were collected again on day 2 (left side) and day 7 (right side) post transplantation and examined histologically. All procedures were preceded by ultrasound examination and blood sampling. Hematologic evaluation remained within normal limits and no differences were observed between time points (p>0.05). Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups (control and treated). At histological evaluation it was found only discrete inflammation signals between D-30×D2 (p<0.05) in the treated group, without differences (p> 0.05) between the groups at different time points. Equine UC-MSCs under the experimental conditions did not promote severe inflammation that causes tissue damage or lead to its rejection by the host organism and therefore has a good potential for clinical use.

  14. Immunogenicity of Decidual Stromal Cells in an Epidermolysis Bullosa Patient and in Allogeneic Hematopoietic Stem Cell Transplantation Patients

    PubMed Central

    Carlson, Lena-Maria; Erkers, Tom; Nava, Silvia; Molldén, Pia; Gustafsson, Britt; Qian, Hua; Li, Xiaoguang; Hashimoto, Takashi; Sadeghi, Behnam; Alheim, Mats; Ringdén, Olle

    2015-01-01

    Allogeneic mesenchymal stromal cells (MSCs) are widely used in regenerative medicine, but little is known about their immunogenicity. In this study, we monitored the therapeutic and immunogenic effects of decidual stromal cells (DSCs) from term placentas when used as a therapy for generalized severe junctional epidermolysis bullosa (JEB) (previously termed Herlitz JEB), a lethal condition caused by the lack of functional laminin-332. An 11-month-old JEB patient was treated with five infusions of allogeneic DSCs within a 3-month period. Amniotic membranes (AMs) were applied to severe wounds. After the treatment, wounds started to heal in the middle of the blisters, but the improvements were transient. After two infusions of DSCs, the JEB patient had developed multispecific anti-HLA class-I antibodies. No antibodies to laminin-332 were detected, but the patient had high levels of anti-bovine serum albumin antibodies, which could bind to DSCs. Peripheral blood mononuclear cells (PBMCs) from the patient had a higher proliferative response to DSCs than to third-party PBMCs, which contrasts with the pattern observed in healthy donors. Human DSCs and MSCs induced similar xenoreactivity in mice. Two of 16 allogeneic stem cell-transplanted patients, treated with DSCs for graft-versus-host disease or hemorrhagic cystitis, showed a positive flow cytometric crossmatch test. One patient had anti-HLA antibodies before DSC infusion, whereas the other had no anti-HLA antibodies at any time. AM and DSC infusions may have improved the healing process in the JEB patient, but DSCs appeared to induce anti-HLA antibodies. The risk of alloimmunization by DSCs seems to be low in immunocompromised patients. PMID:25658253

  15. Natural Killer Cells Differentiate Human Adipose-Derived Stem Cells and Modulate Their Adipogenic Potential.

    PubMed

    Rezzadeh, Kameron S; Hokugo, Akishige; Jewett, Anahid; Kozlowska, Anna; Segovia, Luis Andres; Zuk, Patricia; Jarrahy, Reza

    2015-09-01

    Natural killer cells are thought to represent more than 30 percent of all lymphocytes within the stromal vascular fraction of lipoaspirates. However, their physiologic interaction with adipocytes and their precursors has never been specifically examined. The authors hypothesized that natural killer cells, by means of cytokine secretion, are capable of promoting the differentiation of adipose-derived stem cells. Human natural killer cells purified from healthy donors' peripheral blood mononuclear cells were activated with a combination of interleukin-2 and anti-CD16 monoclonal antibody; natural killer cell supernatant was collected. Adipose-derived stem cells isolated from raw human lipoaspirates from healthy patients were treated with growth media, growth media with natural killer cell supernatant, adipogenic media, and adipogenic media with natural killer cells supernatant. Flow cytometric analysis was performed on cells using antibodies against B7H1, CD36, CD44, CD34, CD29, and MHC-1. Adipogenic-related gene expression (PPAR-γ, LPL, GPD-1, and aP2) was assessed. Oil Red O staining was performed as a functional assay of adipocyte differentiation and adipogenesis. Adipose-derived stem cells maintained in growth media with natural killer cell supernatant lost markers of "stemness," including CD44, CD34, and CD29; and expressed markers of differentiation, including B7H1 and MHC-1. Adipose-derived stem cells treated with natural killer cell supernatant accumulated small amounts of lipid after 10 days of natural killer cell supernatant treatment. Adipose-derived stem cells treated with natural killer cell supernatant showed altered expression of adipogenesis-associated genes compared with cells maintained in growth media. Adipose-derived stem cells maintained in adipogenic media with natural killer cell supernatant accumulated less lipid than those cells in adipogenic media alone. The authors demonstrate that, through secreted factors, natural killer cells are capable

  16. Effects of allogeneic bone marrow derived mesenchymal stromal cell therapy on voiding function in a rat model of Parkinson disease.

    PubMed

    Campeau, Lysanne; Soler, Roberto; Sittadjody, Sivanandane; Pareta, Rajesh; Nomiya, Masanori; Zarifpour, Mona; Opara, Emmanuel C; Yoo, James J; Andersson, Karl-Erik

    2014-03-01

    Cellular therapy induced transient urodynamic improvement in a rat model of Parkinson disease in which bladder dysfunction was noted after unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. We sought to prolong the effect by injecting allogeneic rat bone marrow mesenchymal stromal cells before and after microencapsulation into the substantia nigra pars compacta. Female rats underwent unilateral stereotactic injection of 6-hydroxydopamine in the medial forebrain bundle. Injection was performed in the ipsilateral substantia nigra pars compacta using vehicle alone or vehicle with nonmicroencapsulated or microencapsulated rat bone marrow derived mesenchymal stromal cells. Rats were evaluated by cystometry 7, 14, 28 and 42 days after treatment. Brains were extracted for immunostaining. At 42 days the nonmicroencapsulated group had lower threshold and intermicturition pressure, spontaneous activity and AUC than vehicle treated animals. Rats that received microencapsulated cells had lower threshold pressure at 28 days and lower spontaneous activity at 42 days than vehicle treated rats. Microencapsulated and nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells were noted in the substantia nigra pars compacta up to 42 days after transplantation. At 42 days tyrosine hydroxylase positive neurons were more numerous in the substantia nigra pars compacta of the nonmicroencapsulated group, followed by the microencapsulated and vehicle treated groups. Urodynamic effects of the 6-hydroxydopamine lesion persisted up to 42 days after vehicle injection. Transplantation of nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells improved urodynamic pressure by 42 days after treatment more markedly than microencapsulated cells. This was associated with more tyrosine hydroxylase positive neurons in the treated substantia nigra pars compacta of the nonmicroencapsulated group, suggesting that functional improvement requires a

  17. Skin Tissue Engineering: Application of Adipose-Derived Stem Cells

    PubMed Central

    Zimoch, Jakub; Biedermann, Thomas

    2017-01-01

    Perception of the adipose tissue has changed dramatically over the last few decades. Identification of adipose-derived stem cells (ASCs) ultimately transformed paradigm of this tissue from a passive energy depot into a promising stem cell source with properties of self-renewal and multipotential differentiation. As compared to bone marrow-derived stem cells (BMSCs), ASCs are more easily accessible and their isolation yields higher amount of stem cells. Therefore, the ASCs are of high interest for stem cell-based therapies and skin tissue engineering. Currently, freshly isolated stromal vascular fraction (SVF), which may be used directly without any expansion, was also assessed to be highly effective in treating skin radiation injuries, burns, or nonhealing wounds such as diabetic ulcers. In this paper, we review the characteristics of SVF and ASCs and the efficacy of their treatment for skin injuries and disorders. PMID:28337463

  18. Intra-Bone Marrow Transplantation of Endosteal Bone Marrow Cells Facilitates Allogeneic Hematopoietic and Stromal Cells Engraftment Dependent on Early Expression of CXCL-12.

    PubMed

    Chen, Chen; Su, Yingjun; Chen, Jianwu; Zhang, Dongliang; Song, Yajuan; Guo, Shuzhong

    2015-09-16

    Hematopoietic stem cell transplantation (HSCT) has been considered as an effective approach at inducing allogeneic hematopoietic reconstitution and immune tolerance. However, it remains critical to find the optimal HSCT delivery method and robust sources of hematopoietic stem cells (HSCs). We introduced a new method by infusing allogeneic endosteal bone marrow cells (BMCs) harvested from long bones endosteum through intra-bone marrow transplantation (IBBMT) into irradiated mice. Recipient mice that were transplanted with central BMCs or through intravenous bone marrow transplantation (IVBMT) were used as controls (n=6 per group). We compared the new method with each control group for allogeneic HSCs homing pattern, peripheral blood chimerism level, skin allograft survival time, and donor stromal cell percentage in recipient BM. AMD3100 was injected to determine whether chemokine stromal cell-derived factor-1 (CXCL-12) was critical for the new method. More allogeneic HSCs homed into spleen and bone marrow for the new method as compared to each control group. IBBMT of endosteal BMCs led to a higher peripheral blood chimerism and skin allograft survival. At 18 weeks, donor stromal cell percentage in recipient BMCs was higher for the new method than in each control group. By AMD3100 blockade at day 1, peripheral blood chimerism level and donor stromal cell percentage were significantly reduced as compared to the control group without AMD3100 blockade. Our study suggests that IBBMT of endosteal BMCs is an effective approach for HSCT in inducing allogeneic hematopoietic reconstitution. The advantage is dependent upon the early expression of CXCL-12 after bone marrow transplantation.

  19. Safety, tolerability and potential efficacy of injection of autologous adipose-derived stromal vascular fraction in the fingers of patients with systemic sclerosis: an open-label phase I trial.

    PubMed

    Granel, Brigitte; Daumas, Aurélie; Jouve, Elisabeth; Harlé, Jean-Robert; Nguyen, Pierre-Sébastien; Chabannon, Christian; Colavolpe, Nathalie; Reynier, Jean-Charles; Truillet, Romain; Mallet, Stéphanie; Baiada, Antoine; Casanova, Dominique; Giraudo, Laurent; Arnaud, Laurent; Veran, Julie; Sabatier, Florence; Magalon, Guy

    2015-12-01

    In patients with systemic sclerosis (scleroderma, SSc), impaired hand function greatly contributes to disability and reduced quality of life, and is insufficiently relieved by currently available therapies. Adipose tissue-derived stromal vascular fraction (SVF) is increasingly recognised as an easily accessible source of regenerative cells with therapeutic potential in ischaemic or autoimmune diseases. We aimed to measure for the first time the safety, tolerability and potential efficacy of autologous SVF cells local injections in patients with SSc with hand disability. We did an open-label, single arm, at one study site with 6-month follow-up among 12 female SSc patients with Cochin Hand Function Scale score >20/90. Autologous SVF was obtained from lipoaspirates, using an automated processing system, and subsequently injected into the subcutaneous tissue of each finger in contact with neurovascular pedicles. Primary outcome was the number and the severity of adverse events related to SVF-based therapy. Secondary endpoints were changes in hand disability and fibrosis, vascular manifestations, pain and quality of life from baseline to 2 and 6 months after cell therapy. All enrolled patients had surgery, and there were no dropouts or patients lost to follow-up. No severe adverse events occurred during the procedure and follow-up. Four minor adverse events were reported and resolved spontaneously. A significant improvement in hand disability and pain, Raynaud's phenomenon, finger oedema and quality of life was observed. This study outlines the safety of the autologous SVF cells injection in the hands of patients with SSc. Preliminary assessments at 6 months suggest potential efficacy needing confirmation in a randomised placebo-controlled trial on a larger population. GFRS (Groupe Francophone de Recherche sur la Sclérodermie). NCT01813279. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to

  20. Safety, tolerability and potential efficacy of injection of autologous adipose-derived stromal vascular fraction in the fingers of patients with systemic sclerosis: an open-label phase I trial

    PubMed Central

    Granel, Brigitte; Daumas, Aurélie; Jouve, Elisabeth; Harlé, Jean-Robert; Nguyen, Pierre-Sébastien; Chabannon, Christian; Colavolpe, Nathalie; Reynier, Jean-Charles; Truillet, Romain; Mallet, Stéphanie; Baiada, Antoine; Casanova, Dominique; Giraudo, Laurent; Arnaud, Laurent; Veran, Julie; Sabatier, Florence; Magalon, Guy

    2015-01-01

    Background In patients with systemic sclerosis (scleroderma, SSc), impaired hand function greatly contributes to disability and reduced quality of life, and is insufficiently relieved by currently available therapies. Adipose tissue-derived stromal vascular fraction (SVF) is increasingly recognised as an easily accessible source of regenerative cells with therapeutic potential in ischaemic or autoimmune diseases. We aimed to measure for the first time the safety, tolerability and potential efficacy of autologous SVF cells local injections in patients with SSc with hand disability. Methods We did an open-label, single arm, at one study site with 6-month follow-up among 12 female SSc patients with Cochin Hand Function Scale score >20/90. Autologous SVF was obtained from lipoaspirates, using an automated processing system, and subsequently injected into the subcutaneous tissue of each finger in contact with neurovascular pedicles. Primary outcome was the number and the severity of adverse events related to SVF-based therapy. Secondary endpoints were changes in hand disability and fibrosis, vascular manifestations, pain and quality of life from baseline to 2 and 6 months after cell therapy. Findings All enrolled patients had surgery, and there were no dropouts or patients lost to follow-up. No severe adverse events occurred during the procedure and follow-up. Four minor adverse events were reported and resolved spontaneously. A significant improvement in hand disability and pain, Raynaud's phenomenon, finger oedema and quality of life was observed. Interpretation This study outlines the safety of the autologous SVF cells injection in the hands of patients with SSc. Preliminary assessments at 6 months suggest potential efficacy needing confirmation in a randomised placebo-controlled trial on a larger population. Funding GFRS (Groupe Francophone de Recherche sur la Sclérodermie). Clinical Trials number NCT01813279. PMID:25114060

  1. Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

    PubMed

    Forte, Dorian; Ciciarello, Marilena; Valerii, Maria Chiara; De Fazio, Luigia; Cavazza, Elena; Giordano, Rosaria; Parazzi, Valentina; Lazzari, Lorenza; Laureti, Silvio; Rizzello, Fernando; Cavo, Michele; Curti, Antonio; Lemoli, Roberto M; Spisni, Enzo; Catani, Lucia

    2015-09-09

    Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of

  2. Allogeneic Transplantation of Periodontal Ligament-Derived Multipotent Mesenchymal Stromal Cell Sheets in Canine Critical-Size Supra-Alveolar Periodontal Defect Model

    PubMed Central

    Tsumanuma, Yuka; Iwata, Takanori; Kinoshita, Atsuhiro; Washio, Kaoru; Yoshida, Toshiyuki; Yamada, Azusa; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Izumi, Yuichi

    2016-01-01

    Abstract Periodontitis is a chronic inflammatory disease that induces the destruction of tooth-supporting tissues, followed by tooth loss. Although several approaches have been applied to periodontal regeneration, complete periodontal regeneration has not been accomplished. Tissue engineering using a combination of cells and scaffolds is considered to be a viable alternative strategy. We have shown that autologous transplantation of periodontal ligament-derived multipotent mesenchymal stromal cell (PDL-MSC) sheets regenerates periodontal tissue in canine models. However, the indications for autologous cell transplantation in clinical situations are limited. Therefore, this study evaluated the safety and efficacy of allogeneic transplantation of PDL-MSC sheets using a canine horizontal periodontal defect model. Canine PDL-MSCs were labeled with enhanced green fluorescent protein (EGFP) and were cultured on temperature-responsive dishes. Three-layered cell sheets were transplanted around denuded root surfaces either autologously or allogeneically. A mixture of β-tricalcium phosphate and collagen gel was placed on the bone defects. Eight weeks after transplantation, dogs were euthanized and subjected to microcomputed tomography and histological analyses. RNA and DNA were extracted from the paraffin sections to verify the presence of EGFP at the transplantation site. Inflammatory markers from peripheral blood sera were quantified using an enzyme-linked immunosorbent assay. Periodontal regeneration was observed in both the autologous and the allogeneic transplantation groups. The allogeneic transplantation group showed particularly significant regeneration of newly formed cementum, which is critical for the periodontal regeneration. Serum levels of inflammatory markers from peripheral blood sera showed little difference between the autologous and allogeneic groups. EGFP amplicons were detectable in the paraffin sections of the allogeneic group. These results suggest

  3. Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on Graft-Versus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation

    PubMed Central

    Nevruz, Oral; Avcu, Ferit; Ural, A. Uğur; Pekel, Aysel; Dirican, Bahar; Safalı, Mükerrem; Akdağ, Elvin; Beyzadeoğlu, Murat; İde, Tayfun; Sengül, Ali

    2013-01-01

    Objective: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic bone marrow transplantation (allo-BMT). While multipotent mesenchymal stromal cells (MSCs) demonstrate alloresponse in vitro and in vivo, they also have clinical applications toward prevention or treatment of GVHD. The aim of this study was to investigate the ability of MSCs to prevent or treat GVHD in a rat BMT model. Materials and Methods: The GVHD model was established by transplantation of Sprague Dawley rats’ bone marrow and spleen cells into lethally irradiated (950 cGy) SDxWistar rat recipients. A total of 49 rats were randomly assigned to 4 study and 3 control groups administered different GVHD prophylactic regimens including MSCs. After transplantation, clinical GVHD scores and survival status were monitored. Results: All irradiated and untreated control mice with GVHD died. MSCs inhibited lethal GVHD as efficiently as the standard GVHD prophylactic regimen. The gross and histopathological findings of GVHD and the ratio of CD4/CD8 expression decreased. The subgroup given MSCs displayed higher in vivo proportions of CD25+ T cells and plasma interleukin-2 levels as compared to conventional GVHD treatment after allo-BMT. Conclusion: Our results suggest that clinical use of MSCs in both prophylaxis against and treatment of established GVHD is effective. This study supports the use of MSCs in the prophylaxis and treatment of GVHD after allo-BMT; however, large scale studies are needed. Conflict of interest:None declared. PMID:24385804

  4. Mechanically Isolated Stromal Vascular Fraction Provides a Valid and Useful Collagenase-Free Alternative Technique: A Comparative Study.

    PubMed

    Chaput, Benoit; Bertheuil, Nicolas; Escubes, Marina; Grolleau, Jean-Louis; Garrido, Ignacio; Laloze, Jérôme; Espagnolle, Nicolas; Casteilla, Louis; Sensebé, Luc; Varin, Audrey

    2016-10-01

    The use of stromal vascular fraction and adipose-derived stromal cells in tissue regeneration is now being increasingly investigated, and studies have demonstrated that adipose-derived stromal cells present differentiation and immunomodulatory capacities. The development of a rapid, inexpensive, and enzyme-free technique to isolate adipose-derived stromal cell-enriched stromal vascular fraction is a major goal for stem cell therapy. Therefore, the authors compared innovative mechanical procedures to the gold standard technique, collagenase digestion. Stromal vascular fraction was prepared from 21 liposuctions using either enzymatic digestion or two different mechanical methods: high vortexing/centrifugation and dissociation by intersyringe processing. The effects of tissue processing on cell count, viability, proliferation, clonogenic enrichment, and the phenotypes of the different native cell were determined. Adipose-derived stromal cell phenotypes from the different protocols, and their differentiation and immunosuppressive potential, were compared. Enzymatic digestion isolated more viable cells than dissociation by intersyringe processing and vortexing/centrifugation. The expansion rate and clonogenic enrichment were higher for stromal vascular fraction isolated with collagenase. The proportion of adipose-derived stromal cells was higher in stromal vascular fraction extracted with dissociation than with enzymatic digestion and vortexing/centrifugation (p < 0.01). Interestingly, all cultured adipose-derived stromal cells displayed similar differentiation and immunosuppressive capacities. Enzymatic digestion extracts more adipose-derived stromal cells, but intersyringe dissociation enables the rapid extraction of adipose-derived stromal cell-enriched stromal vascular fraction. Moreover, mechanical methods enable adipose-derived stromal cell isolation with stemness and immunosuppressive properties, similar to enzymatic digestion. Such mechanical procedures could

  5. Delayed minimally invasive injection of allogenic bone marrow stromal cell sheets regenerates large bone defects in an ovine preclinical animal model.

    PubMed

    Berner, Arne; Henkel, Jan; Woodruff, Maria A; Steck, Roland; Nerlich, Michael; Schuetz, Michael A; Hutmacher, Dietmar W

    2015-05-01

    Cell-based tissue engineering approaches are promising strategies in the field of regenerative medicine. However, the mode of cell delivery is still a concern and needs to be significantly improved. Scaffolds and/or matrices loaded with cells are often transplanted into a bone defect immediately after the defect has been created. At this point, the nutrient and oxygen supply is low and the inflammatory cascade is incited, thus creating a highly unfavorable microenvironment for transplanted cells to survive and participate in the regeneration process. We therefore developed a unique treatment concept using the delayed injection of allogenic bone marrow stromal cell (BMSC) sheets to regenerate a critical-sized tibial defect in sheep to study the effect of the cells' regeneration potential when introduced at a postinflammatory stage. Minimally invasive percutaneous injection of allogenic BMSCs into biodegradable composite scaffolds 4 weeks after the defect surgery led to significantly improved bone regeneration compared with preseeded scaffold/cell constructs and scaffold-only groups. Biomechanical testing and microcomputed tomography showed comparable results to the clinical reference standard (i.e., an autologous bone graft). To our knowledge, we are the first to show in a validated preclinical large animal model that delayed allogenic cell transplantation can provide applicable clinical treatment alternatives for challenging bone defects in the future.

  6. Osteogenesis of Adipose-Derived Stem Cells

    PubMed Central

    Grottkau, Brian E.; Lin, Yunfeng

    2013-01-01

    Current treatment options for skeletal repair, including immobilization, rigid fixation, alloplastic materials and bone grafts, have significant limitations. Bone tissue engineering offers a promising method for the repair of bone deficieny caused by fractures, bone loss and tumors. The use of adipose derived stem cells (ASCs) has received attention because of the self-renewal ability, high proliferative capacity and potential of osteogenic differentiation in vitro and in vivo studies of bone regeneration. Although cell therapies using ASCs are widely promising in various clinical fields, no large human clinical trials exist for bone tissue engineering. The aim of this review is to introduce how they are harvested, examine the characterization of ASCs, to review the mechanisms of osteogenic differentiation, to analyze the effect of mechanical and chemical stimuli on ASC osteodifferentiation, to summarize the current knowledge about usage of ASC in vivo studies and clinical trials, and finally to conclude with a general summary of the field and comments on its future direction. PMID:26273498

  7. Co-transplantation of multipotent mesenchymal stromal cells in allogeneic hematopoietic stem cell transplantation: A systematic review and meta-analysis.

    PubMed

    Kallekleiv, Merete; Larun, Lillebeth; Bruserud, Øystein; Hatfield, Kimberley Joanne

    2016-02-01

    Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment option for patients with hematological malignancies. Co-transplantation of multipotent mesenchymal stromal cells (MSCs) during allogeneic HSCT has been explored to enhance engraftment and decrease the risk of graft-versus-host disease (GVHD). We aimed to identify, evaluate and summarize the findings of all relevant controlled clinical studies to determine the potential benefits of MSC infusion during allogeneic HSCT, with regard to the outcomes engraftment, GVHD, post-transplant relapse and survival. We conducted a systematic search of electronic databases for relevant controlled clinical studies. Studies included patients of all ages with hematological malignancies receiving allogeneic HSCT with or without infusion of MSCs within a 24-h time frame of transplantation. Nine studies met our inclusion criteria, including three randomized, one non-randomized and five historically controlled trials, representing a total of 309 patients. Our meta-analyses did not reveal any statistically significant differences in donor engraftment or GVHD. A review of data regarding relapse and overall survival may result in a positive attitude toward intervention with MSCs, but due to heterogeneous reporting, it is difficult to draw any strict conclusions. None of the studies had overall serious risks of bias, but the quality of the evidence is low. Meta-analysis did not reveal any statistically significant effects of MSC co-transplantation, but the results must be interpreted with caution because of the weak study design and small study populations. We discuss further needs to explore the potential effects of MSCs in a HSCT setting. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Harvesting Technique Affects Adipose-Derived Stem Cell Yield

    PubMed Central

    Iyyanki, Tejaswi; Hubenak, Justin; Liu, Jun; Chang, Edward I.; Beahm, Elisabeth K.; Zhang, Qixu

    2015-01-01

    Background The success of an autologous fat graft depends in part on its total stromal vascular fraction (SVF) and adipose-derived stem cells (ASCs). However, variations in the yields of ASCs and SVF cells as a result of different harvesting techniques and donor sites are poorly understood. Objective To investigate the effects of adipose tissue harvesting technique and donor site on the yield of ASCs and SVF cells. Methods Subcutaneous fat tissues from the abdomen, flank, or axilla were harvested from patients of various ages by mechanical liposuction, direct surgical excision, or Coleman's technique with or without centrifugation. Cells were isolated and then analyzed with flow cytometry to determine the yields of total SVF cells and ASCs (CD11b−, CD45−, CD34+, CD90+, D7-FIB+). Differences in ASC and total SVF yields were assessed with one-way analysis of variance. Differentiation experiments were performed to confirm the multilineage potential of cultured SVF cells. Results Compared with Coleman's technique without centrifugation, direct excision yielded significantly more ASCs (P < .001) and total SVF cells (P = .007); liposuction yielded significantly fewer ASCs (P < .001) and total SVF cells (P < .05); and Coleman's technique with centrifugation yielded significantly more total SVF cells (P < .005), but not ASCs. The total number of SVF cells in fat harvested from the abdomen was significantly larger than the number in fat harvested from the flank or axilla (P < .05). Cultured SVF cells differentiated to adipocytes, osteocytes, and chondrocytes. Conclusions Adipose tissue harvested from the abdomen through direct excision or Coleman's technique with centrifugation was found to yield the most SVF cells and ASCs. PMID:25791999

  9. Response to Intravenous Allogeneic Equine Cord Blood-Derived Mesenchymal Stromal Cells Administered from Chilled or Frozen State in Serum and Protein-Free Media

    PubMed Central

    Williams, Lynn B.; Co, Carmon; Koenig, Judith B.; Tse, Crystal; Lindsay, Emily; Koch, Thomas G.

    2016-01-01

    Equine mesenchymal stromal cells (MSC) are commonly transported, chilled or frozen, to veterinary clinics. These MSC must remain viable and minimally affected by culture, transport, or injection processes. The safety of two carrier solutions developed for optimal viability and excipient use were evaluated in ponies, with and without allogeneic cord blood-derived (CB) MSC. We hypothesized that neither the carrier solutions nor CB-MSC would elicit measurable changes in clinical, hematological, or biochemical parameters. In nine ponies (study 1), a bolus of HypoThermosol® FRS (HTS-FRS), CryoStor® CS10 (CS10), or saline was injected IV (n = 3/treatment). Study 2, following a 1-week washout period, 5 × 107 pooled allogeneic CB-MSCs were administered IV in HTS-FRS following 24 h simulated chilled transport. Study 3, following another 1-week washout period 5 × 107 pooled allogeneic CB-MSCs were administered IV in CS10 immediately after thawing. Nine ponies received CB-MSCs in study 2 and 3, and three ponies received the cell carrier media without cells. CB-MSCs were pooled in equal numbers from five unrelated donors. In all studies, ponies were monitored with physical examination, and blood collection for 7 days following injection. CD4 and CD8 lymphocyte populations were also evaluated in each blood sample. In all three studies, physical exam, complete blood cell count, serum biochemistry, and coagulation panel did not deviate from established normal ranges. Proportions of CD4+ and CD8+ lymphocytes increased at 168 h postinjection in CB-MSC treatment groups regardless of the carrier solution. Decreases in CD4+/CD8+ double positive populations were observed at 24 and 72 h in CB-MSC-treated animals. There was no difference in viability between CB-MSCs suspended in HTS-FRS and CS10. HTS-FRS and CS10 used for low volume excipient injection of MSC suspensions were not associated with short-term adverse reactions. HTS-FRS and CS10 both adequately

  10. Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

    PubMed Central

    Tan, Chengbo; Wang, Zifeng; Hamauchi, Shuji; Abumiya, Takeo; Kazumata, Ken; Ito, Tsuneo; Kudo, Kohsuke; Takamoto, Shigeru; Houkin, Kiyohiro

    2016-01-01

    Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs) as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs). Platelet concentrates (PC) were harvested from healthy volunteers and made into single donor-derived PL (sPL). The PL mixtures (mPL) were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA). The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO) agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS). No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke. PMID:27840648

  11. The Effect of Antiseptics on Adipose-Derived Stem Cells.

    PubMed

    Kim, Bong-Sung; Ott, Veronica; Boecker, Arne Hendrick; Stromps, Jan-Philipp; Paul, Nora Emilie; Alharbi, Ziyad; Cakmak, Ercan; Bernhagen, Jürgen; Bucala, Richard; Pallua, Norbert

    2017-03-01

    Although chemical antiseptics are the most basic measure to control wound infection and frequently come into contact with subcutaneous adipose tissue, no studies have evaluated their toxicity on adipose tissue and its cell fractions. In the present study, the effects of five different antiseptics on adipose-derived stem cells were evaluated. Human adipose-derived stem cells were harvested from healthy donors. Adipose-derived stem cell viability was measured after treatment with different concentrations of antiseptics over 5 days. Furthermore, the effect on the proliferation, adipogenic differentiation, and apoptosis/necrosis of adipose-derived stem cells was analyzed. Finally, the mRNA expression of the stem cell markers CD29, CD34, CD73, CD90, and CD105 was detected. Octenisept and Betaisodona significantly reduced cell proliferation and differentiation and led to considerable adipose-derived stem cell necrosis. Octenisept decreased stem cell viability at the lowest concentrations tested, and all stem cell markers were down-regulated by Octeniseptr and Betaisodona. Lavasept and Prontosan both led to reduced stem cell viability, proliferation, and differentiation, and increased apoptosis/necrosis, although the effects were less pronounced compared with Octenisept and Betaisodona. Adipose-derived stem cells survived treatment with mafenide acetate even at high concentrations, and mafenide acetate showed minimal negative effects on their proliferation, adipogenic differentiation, cell death, and stem cell marker expression. Mafenide acetate may be regarded as a feasible antiseptic for the treatment of wounds with exposed adipose tissue because of its low adipose-derived stem cell toxicity. Lavasept and Prontosan are possible alternatives to mafenide acetate. Octenisept and Betaisodona, by contrast, may be used only in highly diluted solutions. Therapeutic, V.

  12. Tracking Intracavernously Injected Adipose-Derived Stem Cells to Bone Marrow

    PubMed Central

    Lin, Guiting; Qiu, Xuefeng; Fandel, Thomas; Banie, Lia; Wang, Guifang; Lue, Tom F.; Lin, Ching-Shwun

    2012-01-01

    Intracavernous (IC) injection of stem cells (SCs) has been shown to improve erectile function in various erectile dysfunction (ED) animal models. However, the tissue distribution of the injected cells remains unknown. In this study we tracked IC injected adipose-derived stem cells (ADSCs) in various tissues. Rat paratesticular fat was processed for ADSC isolation and culture. The animals were then subject to cavernous nerve (CN) crush injury or sham operation, followed by IC injection of one million autologous or allogeneic ADSCs that were labeled with 5-ethynyl-2-deoxyuridine (EdU). Another group of rats received IC injection of EdU-labeled allogeneic penile smooth muscle cells (PSMCs). At 2 and 7 days post-injection, penises and femoral bone marrow were processed for histological analyses. Whole femoral bone marrows were also analyzed for EdU-positive cells by flow cytometry. The results show that ADSCs exited the penis within days of IC injection and migrated preferentially to bone marrow. Allogenicity did not affect ADSC's bone marrow appearance either at 2 or 7 days, while CN injury reduced the number of ADSCs in bone marrow significantly at 7 but not 2 days. The significance of these results in relation to SC therapy for ED is discussed. PMID:21796145

  13. Autologous and allogeneic marrow stromal cells are safe and effective for the treatment of acute kidney injury.

    PubMed

    Tögel, Florian; Cohen, Arthur; Zhang, Ping; Yang, Ying; Hu, Zhuma; Westenfelder, Christof

    2009-04-01

    Acute kidney injury (AKI) is a major clinical problem associated with high morbidity and mortality. Likely due to its complex pathophysiology, therapies with a single pharmacological agent have generally failed to improve outcomes. In contrast, stem cell-based interventions utilize these cells' ability to simultaneously target multiple pathophysiological components of AKI and thus represent a promising new tool for the treatment of AKI. The aims of the this study were to investigate the long-term outcome and safety of treatment with autologous and allogeneic mesenchymal stem cells (MSCs) after AKI and the role of vascular endothelial growth factor (VEGF) as one of the principal paracrine mediators of renoprotection of MSCs. MSC administration after AKI was not associated with adverse events and proved to be renoprotective in animals with severe renal failure. Identical doses of autologous MSC were more effective than allogeneic. At 3 months, MSCs were not engrafted in any tissues except in the bone marrow in 50% of animals given the highest allogeneic cell dose. There was no long-term fibrotic response in the kidneys attributable to MSC therapy, and animals with severe AKI were protected from development of fibrotic lesions after AKI. Furthermore, this study establishes VEGF as a critical factor mediating renal recovery. VEGF knockdown by small-interfering RNA reduced effectiveness of MSCs significantly and decreased survival. In summary, our results show that both autologous and allogeneic MSC are safe and effective in AKI, and importantly, reduce late renal fibrosis and loss of renal function in surviving animals and that VEGF is a critical factor in renoprotection by MSCs. Together, we posit that these data provide further justification for the conduct of clinical trails in which AKI is treated with MSC.

  14. Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues.

    PubMed

    Chen, Yu-Jen; Liu, Hui-Yu; Chang, Yun-Tsui; Cheng, Ying-Hung; Mersmann, Harry J; Kuo, Wen-Hung; Ding, Shih-Torng

    2016-03-31

    Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

  15. MicroRNA regulation of adipose derived stem cells in aging rats.

    PubMed

    Fei, Jia; Tamski, Holly; Cook, Carla; Santanam, Nalini

    2013-01-01

    Perturbations in abdominal fat secreted adipokines play a key role in metabolic syndrome. This process is further altered during the aging process, probably due to alterations in the preadipocytes (aka. stromal vascular fraction cells-SVF cells or adipose derived stem cells-ASCs) composition and/or function. Since microRNAs regulate genes involved both in development and aging processes, we hypothesized that the impaired adipose function with aging is due to altered microRNA regulation of adipogenic pathways in SVF cells. Alterations in mRNA and proteins associated with adipogenic differentiation (ERK5 and PPARg) but not osteogenic (RUNX2) pathways were observed in SVF cells isolated from visceral adipose tissue with aging (6 to 30 mo) in female Fischer 344 x Brown Norway Hybrid (FBN) rats. The impaired differentiation capacity with aging correlated with altered levels of miRNAs involved in adipocyte differentiation (miRNA-143) and osteogenic pathways (miRNA-204). Gain and loss of function studies using premir or antagomir-143 validated the age associated adipocyte dysfunction. Our studies for the first time indicate a role for miRNA mediated regulation of SVF cells with aging. This discovery is important in the light of the findings that dysfunctional adipose derived stem cells contribute to age related chronic diseases.

  16. Vasopressin-induced Ca(2+) signals in human adipose-derived stem cells.

    PubMed

    Tran, Tran Doan Ngoc; Gimble, Jeffrey M; Cheng, Henrique

    2016-03-01

    Intracellular Ca(2+) signals are essential for stem cell differentiation due to their ability to control signaling pathways involved in this process. Arginine vasopression (AVP) is a neurohypophyseal hormone that increases intracellular Ca(2+) concentration during adipogenesis via V1a receptors, Gq-proteins and the PLC-IP3 pathway in human adipose-derived stromal/stem cells (hASCs). These Ca(2+) signals originate through calcium release from pools within the endoplasmic reticulum and the extracellular space. AVP supplementation to the adipogenic media inhibits adipogenesis and key adipocyte marker genes. This review focuses on the intersection between AVP, Ca(2+) signals and ASC differentiation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

    SciTech Connect

    Fujimura, Juri; E-mail: juri-f@nms.ac.jp; Ogawa, Rei; Mizuno, Hiroshi; Fukunaga, Yoshitaka; Suzuki, Hidenori

    2005-07-22

    Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate into neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders.

  18. Local delivery of allogeneic bone marrow and adipose tissue-derived mesenchymal stromal cells for cutaneous wound healing in a porcine model.

    PubMed

    Hanson, Summer E; Kleinbeck, Kyle R; Cantu, David; Kim, Jaeyhup; Bentz, Michael L; Faucher, Lee D; Kao, W John; Hematti, Peiman

    2016-02-01

    Wound healing remains a major challenge in modern medicine. Bone marrow- (BM) and adipose tissue- (AT) derived mesenchymal stromal/stem cells (MSCs) are of great interest for tissue reconstruction due to their unique immunological properties and regenerative potential. The purpose of this study was to characterize BM and AT-MSCs and evaluate their effect when administered in a porcine wound model. MSCs were derived from male Göttingen Minipigs and characterized according to established criteria. Allogeneic BM- or AT-MSCs were administered intradermally (1 x 10(6) cells) into partial-thickness wounds created on female animals, and covered with Vaseline® gauze or fibrin in a randomized pattern. Animals were euthanized at 7, 10, 14 and 21 days. Tissues were analyzed visually for healing and by microscopic examination for epidermal development and remodelling. Polymerase chain reaction (PCR) was used to detect the presence of male DNA in the specimens. All wounds were healed by 14 days. MSC-injected wounds were associated with improved appearance and faster re-epithelialization compared to saline controls. Evaluation of rete ridge depth and architecture showed that MSC treatment promoted a faster rate of epidermal maturation. Male DNA was detected in all samples at days 7 and 10, suggesting the presence of MSCs. We showed the safety, feasibility and potential efficacy of local injection of allogeneic BM- and AT-MSCs for treatment of wounds in a preclinical model. Our data in this large animal model support the potential use of BM- and AT-MSC for treatment of cutaneous wounds through modulation of healing and epithelialization. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Experimental bladder regeneration using a poly-l-lactide/silk fibroin scaffold seeded with nanoparticle-labeled allogenic bone marrow stromal cells

    PubMed Central

    Yudintceva, Natalia M; Nashchekina, Yulia A; Blinova, Miralda I; Orlova, Nadezhda V; Muraviov, Alexandr N; Vinogradova, Tatiana I; Sheykhov, Magomed G; Shapkova, Elena Y; Emeljannikov, Dmitriy V; Yablonskii, Petr K; Samusenko, Igor A; Mikhrina, Anastasiya L; Pakhomov, Artem V; Shevtsov, Maxim A

    2016-01-01

    In the present study, a poly-l-lactide/silk fibroin (PL-SF) bilayer scaffold seeded with allogenic bone marrow stromal cells (BMSCs) was investigated as a potential approach for bladder tissue engineering in a model of partial bladder wall cystectomy in rabbits. The inner porous layer of the scaffold produced from silk fibroin was designed to promote cell proliferation and the outer layer produced from poly-l-lactic acid to serve as a waterproof barrier. To compare the feasibility and efficacy of BMSC application in the reconstruction of bladder defects, 12 adult male rabbits were divided into experimental and control groups (six animals each) that received a scaffold seeded with BMSCs or an acellular one, respectively. For BMSC tracking in the graft in in vivo studies using magnetic resonance imaging, cells were labeled with superparamagnetic iron oxide nanoparticles. In vitro studies demonstrated high intracellular incorporation of nanoparticles and the absence of a toxic influence on BMSC viability and proliferation. Following implantation of the graft with BMSCs into the bladder, we observed integration of the scaffold with surrounding bladder tissues (as detected by magnetic resonance imaging). During the follow-up period of 12 weeks, labeled BMSCs resided in the implanted scaffold. The functional activity of the reconstructed bladder was confirmed by electromyography. Subsequent histological assay demonstrated enhanced biointegrative properties of the PL-SF scaffold with cells in comparison to the control graft, as related to complete regeneration of the smooth muscle and urothelium tissues in the implant. Confocal microscopy studies confirmed the presence of the superparamagnetic iron oxide nanoparticle-labeled BMSCs in newly formed bladder layers, thus indicating the role of stem cells in bladder regeneration. The results of this study demonstrate that application of a PL-SF scaffold seeded with allogenic BMSCs can enhance biointegration of the graft in

  20. Macrophage Response to Allogeneic Adipose Tissue-Derived Stromal Cells in Hyaluronan-Based Hydrogel in a Porcine Vocal Fold Injury Model.

    PubMed

    King, Suzanne N; Woo, Joo Hyun; Tang, Sharon; Thibeault, Susan L

    2017-06-01

    Adipose tissue-derived stromal cells (ASC) embedded in hyaluronan scaffold is a beneficial prophylactic treatment for vocal fold (VF) surgical scar. Here, we investigated the macrophage inflammatory response to allogeneic ASC-constructs and identified changes in lamina propria extracellular matrix. Pig ASC were characterized and transfected with GFP+ lentivirus. Thirty-three pigs underwent VF biopsies, and after 3 days, gel alone, gel+pASC, placebo, or pASC alone was injected into wound bed. Animals were sacrificed 3, 7, or 26 days post-injection. Flow cytometry; qPCR for NF-α, TGFβ, IL-10, IL-4, IFNγ, IL-12, FGF2, Col1A1, and HGF; and immunohistochemistry for collagen, elastin, HA, and fibronectin were performed to characterize macrophage phenotype, quantify cytokine transcription, analyze extracellular matrix remodeling, and track GFP+ cells. No significant differences were found in SWC3+/SWC9+ phenotype or mRNA expression between cells+gel, gel, or placebo. The ASC alone exhibited significantly greater collagen, gel alone resulted in significantly less hyaluronan, and gel+pASC significantly more fibronectin (all P < .05). The pASC-GFP+ were detected 26 days post-injection. The ASC-constructs were biocompatible; they did not influence the macrophage inflammatory response or provoke increases in collagen expression. Long-term engraftment was confirmed.

  1. The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells.

    PubMed

    Pelagiadis, Iordanis; Stiakaki, Eftichia; Choulaki, Christianna; Kalmanti, Maria; Dimitriou, Helen

    2015-10-01

    The recognition of the role of Mesenchymal Stromal Cells (MSC) in hematopoiesis, as part of the bone marrow microenvironment, renewed the interest for cord blood (CB) ex vivo expansion as a source of HSC for transplantation. MSC from children are recognized to have different biological properties compared to the ones from adults. The current study focuses on the evaluation of the effects of children's bone marrow MSC on the ex vivo expansion capacity of both allogeneic cord blood and autologous bone marrow (BM) CD34(+) hematopoietic stem cells (HSCs) when used as a cell feeder layer with or without recombinant cytokines. Our results showed that children's bone marrow-derived MSC expand more primitive populations in co culture with CD34 and that the expansion is further enhanced when the culture is supplemented with growth factors. No additive effect was seen either with the early- or late-acting growth factors' cocktails used. Biological features of CB hematopoietic progenitors seem to make them more suitable than their BM counterparts for ex vivo expansion. Clinical implementation will be facilitated by methodological standardization and guidelines' establishment.

  2. A Nerve Conduit Containing a Vascular Bundle and Implanted With Bone Marrow Stromal Cells and Decellularized Allogenic Nerve Matrix.

    PubMed

    Kaizawa, Yukitoshi; Kakinoki, Ryosuke; Ikeguchi, Ryosuke; Ohta, Soichi; Noguchi, Takashi; Takeuchi, Hisataka; Oda, Hiroki; Yurie, Hirofumi; Matsuda, Shuichi

    2017-02-16

    Cells, scaffolds, growth factors, and vascularity are essential for nerve regeneration. Previously, we reported that the insertion of a vascular bundle and the implantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a nerve conduit promoted peripheral nerve regeneration. In this study, the efficacy of nerve conduits containing a vascular bundle, BM-MSCs, and thermally decellularized allogenic nerve matrix (DANM) was investigated using a rat sciatic nerve model with a 20-mm defect. Lewis rats were used as the sciatic nerve model and for the preparation of BM-MSCs, and Dark Agouti rats were used for the preparation of the DANM. The revascularization and the immunogenicity of the DANM were investigated histologically. The regeneration of nerves through nerve conduits containing vessels, BM-MSCs, and DANM (VBD group) was evaluated based on electrophysiological, morphometric, and reinnervated muscle weight measurements and compared with that of vessel-containing conduits that were implanted with BM-MSCs (VB group). The DANM that was implanted into vessel-containing tubes (VCTs) was revascularized by neovascular vessels that originated from the inserted vascular bundle 5-7 days after surgery. The number of CD8+ cells found in the DANM in the VCT was significantly smaller than that detected in the untreated allogenic nerve segment. The regenerated nerve in the VBD group was significantly superior to that in the VB group with regard to the amplitude of the compound muscle action potential detected in the pedal adductor muscle; the number, diameter, and myelin thickness of the myelinated axons; and the tibialis anterior muscle weight at 12 and 24 weeks. The additional implantation of the DANM into the BM-MSC-implanted VCT optimized the axonal regeneration through the conduit. Nerve conduits constructed with vascularity, cells, and scaffolds could be an effective strategy for the treatment of peripheral nerve injuries with significant segmental defects.

  3. Short-, middle- and long-term safety of superparamagnetic iron oxide-labeled allogeneic bone marrow stromal cell transplantation in rat model of lacunar infarction.

    PubMed

    Tan, Chengbo; Shichinohe, Hideo; Abumiya, Takeo; Nakayama, Naoki; Kazumata, Ken; Hokari, Masaaki; Hamauchi, Shuji; Houkin, Kiyohiro

    2015-06-01

    Recently, both basic and clinical studies demonstrated that bone marrow stromal cell (BMSC) transplantation therapy can promote functional recovery of patients with CNS disorders. A non-invasive method for cell tracking using MRI and superparamagnetic iron oxide (SPIO)-based labeling agents has been applied to elucidate the behavior of transplanted cells. However, the long-term safety of SPIO-labeled BMSCs still remains unclear. The aim of this study was to investigate the short-, middle- and long-term safety of the SPIO-labeled allogeneic BMSC transplantation. For this purpose, BMSCs were isolated from transgenic rats expressing green fluorescent protein (GFP) and were labeled with SPIO. The Na/K ATPase pump inhibitor ouabain or vehicle was stereotactically injected into the right striatum of wild-type rats to induce a lacunar lesion (n = 22). Seven days after the insult, either BMSCs or SPIO solution were stereotactically injected into the left striatum. A 7.0-Tesla MRI was performed to serially monitor the behavior of BMSCs in the host brain. The animals were sacrificed after 7 days (n = 7), 6 weeks (n = 6) or 10 months (n = 9) after the transplantation. MRI demonstrated that BMSCs migrated to the damage area through the corpus callosum. Histological analysis showed that activated microglia were present around the bolus of donor cells 7 days after the allogeneic cell transplantation, although an immunosuppressive drug was administered. The SPIO-labeled BMSCs resided and started to proliferate around the route of the cell transplantation. Within 6 weeks, large numbers of SPIO-labeled BMSCs reached the lacunar infarction area from the transplantation region through the corpus callosum. Some SPIO nanoparticles were phagocytized by microglia. After 10 months, the number of SPIO-positive cells was lower compared with the 7-day and 6-week groups. There was no tumorigenesis or severe injury observed in any of the animals. These findings suggest that

  4. Immunosuppressive effects of multipotent mesenchymal stromal cells on graft-versus-host disease in rats following allogeneic bone marrow transplantation.

    PubMed

    Nevruz, Oral; Avcu, Ferit; Ural, A Uğur; Pekel, Aysel; Dirican, Bahar; Safalı, Mükerrem; Akdağ, Elvin; Beyzadeoğlu, Murat; Ide, Tayfun; Sengül, Ali

    2013-09-01

    Amaç: Graft versus host hastalığı (GVHH) , başarılı bir kemik iliği nakli için önemli bir engel oluşturmaktadır. Multipotent mezenşimal stromal hücrelerin (MSH) immünsupresif etkileri, in vivo ve in vitro olarak gösterilmiş olmakla birlikte, GVHH’ nı önleme yönünde klinik uygulamalarda bulunmaktadır . Gereç ve Yöntemler: Bu çalışmanın amacı ratlarda kemik iliği nakli sonrası oluşturulan GVHH’nı önleme ve tedavi etmede MSH nin etkinliğinin incelenmesidir. Bu amaçla 49 Sprague Dawley cinsi rat rastegele 4 çalışma, 3 kontrol grubuna ayrılmış ve gruplara MSH de içeren farklı GVHH önleyici tedaviler uygulanmıştır. Kemik iliği nakli sonrası GVHH skorlaması ve yaşama süreleri incelenmiştir. Bulgular: Tüm ışınlanmış ve önleyici tedavi verilmemiş ratlar ölmüştür. MSH nin önleyici uygulamaları, standart GVHD önleyici tedavileri kadar etkin bulunmuştur. MSH uygulamaları, GVHH nın gözlemsel ve histolojik bulgularını ve CD4+/CD8+ oranını azaltmaktadır.Ayrıca MSH uygulanan gruplarda CD25+ T hücrelerinin in vivo oranıda daha yüksek olup, Allojeneik kemik iliği nakli sonrası standart GVHH tedavisi uygulananlara göre plazma İnterlökin-2 seviyesinin daha yüksek olarak saptanmıştır. Sonuç: Bulgularımız MSH uygulamasının, GVHH nın hem önlenme hem de tedavi edilmesinde etkin olduğunu göstermiştir. Ancak bu bulguların geniş ölçekli çalışmalarla desteklenmesi gerekmektedir.

  5. Systemic treatment of acute liver failure with adipose derived stem cells.

    PubMed

    Pascual-Miguelañez, Isabel; Salinas-Gomez, Javier; Fernandez-Luengas, David; Villar-Zarra, Karen; Clemente, Luz Vega; Garcia-Arranz, Mariano; Olmo, Damian Garcia

    2015-04-01

    The definitive treatment for liver failure is, currently, liver transplantation. Research into other possible treatments, focused on achieving regeneration of the liver parenchyma, have led to the development of methods to generate hepatocytes from stem cells. In our study, we transplant allogenic adipose-derived stem cells (ASCs), not previously differentiated to hepatocytes, to treat acute liver failure induced by intraperitoneal administration of carbon tetrachloride (CCl4) in a Sprague-Dawley rat model. The ASCs were delivered via the tail vein, having previously been labeled with PKH26, a fluorescent membrane marker. Two control groups were established, Group 1(n = 15) consisting of olive oil (5 mL/kg) and Group 2(n = 15): 1 × 10(6) PKH26-labeled ASCs. Further, two study groups, Group 3(n = 30): CCl4 dissolved in olive oil and Group 4(n = 30): CCl4 dissolved in olive oil and 1 × 10(6) PKH26-labeled ASCs completed the experimental design. Blood samples were analyzed, finding AST and ALT levels significantly higher in treatment over control groups at 24 and 48 hours. The mortality rates were statistically different between control groups and Group 3 (Group 1-3 p = .04, Group 2-3 p = .04) and between Groups 3 and 4 (p = .02). Examining the liver parenchyma, a significantly higher number of ASCs were observed in Group 4 than in Group 2 at all time points (p = .00). The intravenous injection of allogenic ASCs in this model of CCl4-induced liver failure reduced the mortality in treated animals. ASCs injected in the rat tail vein were found in the liver in animals with induced acute liver failure.

  6. Unrelated donor allogeneic hematopoietic stem cell transplantation for patients with hemoglobinopathies using a reduced-intensity conditioning regimen and third-party mesenchymal stromal cells.

    PubMed

    Kharbanda, Sandhya; Smith, Angela R; Hutchinson, Stephanie K; McKenna, David H; Ball, James B; Lamb, Lawrence S; Agarwal, Rajni; Weinberg, Kenneth I; Wagner, John E

    2014-04-01

    Allogeneic hematopoietic stem cell transplantation for patients with a hemoglobinopathy can be curative but is limited by donor availability. Although positive results are frequently observed in those with an HLA-matched sibling donor, use of unrelated donors has been complicated by poor engraftment, excessive regimen-related toxicity, and graft-versus-host disease (GVHD). As a potential strategy to address these obstacles, a pilot study was designed that incorporated both a reduced-intensity conditioning and mesenchymal stromal cells (MSCs). Six patients were enrolled, including 4 with high-risk sickle cell disease (SCD) and 2 with transfusion-dependent thalassemia major. Conditioning consisted of fludarabine (150 mg/m(2)), melphalan (140 mg/m(2)), and alemtuzumab (60 mg for patients weighing > 30 kg and .9 mg/kg for patients weighing <30 kg). Two patients received HLA 7/8 allele matched bone marrow and 4 received 4-5/6 HLA matched umbilical cord blood as the source of HSCs. MSCs were of bone marrow origin and derived from a parent in 1 patient and from an unrelated third-party donor in the remaining 5 patients. GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. One patient had neutropenic graft failure, 2 had autologous hematopoietic recovery, and 3 had hematopoietic recovery with complete chimerism. The 2 SCD patients with autologous hematopoietic recovery are alive. The remaining 4 died either from opportunistic infection, GVHD, or intracranial hemorrhage. Although no infusion-related toxicity was seen, the cotransplantation of MSCs was not sufficient for reliable engraftment in patients with advanced hemoglobinopathy. Although poor engraftment has been observed in nearly all such trials to date in this patient population, there was no evidence to suggest that MSCs had any positive impact on engraftment. Because of the lack of improved engraftment and unacceptably high transplant-related mortality, the study was prematurely terminated

  7. Study on viability and chondrogenic differentiation of cryopreserved adipose tissue-derived mesenchymal stromal cells for future use in regenerative medicine.

    PubMed

    González-Fernández, M L; Pérez-Castrillo, S; Ordás-Fernández, P; López-González, M E; Colaço, B; Villar-Suárez, V

    2015-10-01

    Adipose-derived mesenchymal stromal cells are promising as a regenerative therapy tool for defective tissues in mesenchymal lineage, including fat, bone, cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation is an essential fundamental technology. The aim of this study is to define an adequate protocol for the cryopreservation of adipose-derived mesenchymal stromal cells, by comparing various protocols so as to determine the effects of cryopreservation on viability and chondrogenic differentiation potential of adipose-derived stem cells upon freeze-thawing of AT-MSCs colonies cryopreserved with standard and modified protocols, using flow cytometry and confocal microscopy. The study concludes that adipose-derived mesenchymal stromal cells could be long-term cryopreserved without any loss of their proliferative or differentiation potential. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Potential of adipose-derived stem cells in muscular regenerative therapies

    PubMed Central

    Forcales, Sonia-V

    2015-01-01

    Regenerative capacity of skeletal muscles resides in satellite cells, a self-renewing population of muscle cells. Several studies are investigating epigenetic mechanisms that control myogenic proliferation and differentiation to find new approaches that could boost regeneration of endogenous myogenic progenitor populations. In recent years, a lot of effort has been applied to purify, expand and manipulate adult stem cells from muscle tissue. However, this population of endogenous myogenic progenitors in adults is limited and their access is difficult and invasive. Therefore, other sources of stem cells with potential to regenerate muscles need to be examined. An excellent candidate could be a population of adult stromal cells within fat characterized by mesenchymal properties, which have been termed adipose-derived stem cells (ASCs). These progenitor adult stem cells have been successfully differentiated in vitro to osteogenic, chondrogenic, neurogenic and myogenic lineages. Autologous ASCs are multipotent and can be harvested with low morbidity; thus, they hold promise for a range of therapeutic applications. This review will summarize the use of ASCs in muscle regenerative approaches. PMID:26217219

  9. Hair regeneration using adipose-derived stem cells.

    PubMed

    Jin, Su-Eon; Sung, Jong-Hyuk

    2016-03-01

    Adipose-derived stem cells (ASCs) have been used in tissue repair and regeneration. Recently, it was reported that ASC transplantation promotes hair growth in animal experiments, and a conditioned medium of ASCs (ASC-CM) induced the proliferation of hair-compositing cells in vitro. However, ASCs and their conditioned medium have shown limited effectiveness in clinical settings. ASC preconditioning is one strategy that can be used to enhance the efficacy of ASCs and ASC-CM. Therefore, we highlighted the functional role of ASCs in hair cycle progression and also the advantages and disadvantages of their application in hair regeneration. In addition, we introduced novel ASC preconditioning methods to enhance hair regeneration using ASC stimulators, such as vitamin C, platelet-derived growth factor, hypoxia, and ultraviolet B.

  10. Replenishment of type VII collagen and re-epithelialization of chronically ulcerated skin after intradermal administration of allogeneic mesenchymal stromal cells in two patients with recessive dystrophic epidermolysis bullosa.

    PubMed

    Conget, Paulette; Rodriguez, Fernando; Kramer, Susanne; Allers, Carolina; Simon, Valeska; Palisson, Francis; Gonzalez, Sergio; Yubero, Maria J

    2010-05-01

    In animal models it has been shown that mesenchymal stromal cells (MSC) contribute to skin regeneration and accelerate wound healing. We evaluated whether allogeneic MSC administration resulted in an improvement in the skin of two patients with recessive dystrophic epidermolysis bullosa (RDEB; OMIM 226600). Patients had absent type VII collagen immunohistofluorescence and since birth had suffered severe blistering and wounds that heal with scarring. Vehicle or 0.5 x 10(6) MSC were infused intradermally in intact and chronic ulcerated sites. One week after intervention, in MSC-treated skin type VII collagen was detected along the basement membrane zone and the dermal-epidermal junction was continuous. Re-epithelialization of chronic ulcerated skin was observed only near MSC administration sites. In both patients the observed clinical benefit lasted for 4 months. Thus intradermal administration of allogeneic MSC associates with type VII collagen replenishment at the dermal-epidermal junction, prevents blistering and improves wound healing in unconditioned patients with RDEB.

  11. Role of adipose-derived stem cells in wound healing.

    PubMed

    Hassan, Waqar Ul; Greiser, Udo; Wang, Wenxin

    2014-01-01

    Impaired wound healing remains a challenge to date and causes debilitating effects with tremendous suffering. Recent advances in tissue engineering approaches in the area of cell therapy have provided promising treatment options to meet the challenges of impaired skin wound healing such as diabetic foot ulcers. Over the last few years, stem cell therapy has emerged as a novel therapeutic approach for various diseases including wound repair and tissue regeneration. Several different types of stem cells have been studied in both preclinical and clinical settings such as bone marrow-derived stem cells, adipose-derived stem cells (ASCs), circulating angiogenic cells (e.g., endothelial progenitor cells), human dermal fibroblasts, and keratinocytes for wound healing. Adipose tissue is an abundant source of mesenchymal stem cells, which have shown an improved outcome in wound healing studies. ASCs are pluripotent stem cells with the ability to differentiate into different lineages and to secrete paracrine factors initiating tissue regeneration process. The abundant supply of fat tissue, ease of isolation, extensive proliferative capacities ex vivo, and their ability to secrete pro-angiogenic growth factors make them an ideal cell type to use in therapies for the treatment of nonhealing wounds. In this review, we look at the pathogenesis of chronic wounds, role of stem cells in wound healing, and more specifically look at the role of ASCs, their mechanism of action and their safety profile in wound repair and tissue regeneration.

  12. Human adipose-derived stem cells stimulate neuroregeneration.

    PubMed

    Masgutov, Ruslan F; Masgutova, Galina A; Zhuravleva, Margarita N; Salafutdinov, Ilnur I; Mukhametshina, Regina T; Mukhamedshina, Yana O; Lima, Luciana M; Reis, Helton J; Kiyasov, Andrey P; Palotás, András; Rizvanov, Albert A

    2016-08-01

    Traumatic brain injuries and degenerative neurological disorders such as Alzheimer's dementia, Parkinson's disease, amyotrophic lateral sclerosis and many others are characterized by loss of brain cells and supporting structures. Restoring microanatomy and function using stem cells is a promising therapeutic approach. Among the many various sources, adipose-derived stem cells (ADSCs) are one of the most easily harvested alternatives, they multiply rapidly, and they demonstrate low immunogenicity with an ability to differentiate into several cell types. The objective of this study was to evaluate the effect of xenotransplanted human ADSCs on post-traumatic regeneration of rat sciatic nerve. Peripheral reconstruction following complete sciatic transection and autonerve grafting was complemented by intra-operative injection of hADSCs into the proximal and distal stumps. The injury caused gliosis and apoptosis of sensory neurons in the lumbar 5 (L5) ganglia in the control rodents; however, animals treated with hADSCs demonstrated a smaller amount of cellular loss. Formation of amputation neuroma, which hinders axonal repair, was less prominent in the experimental group, and immunohistochemical analysis of myelin basic protein showed good myelination 65 days after surgery. At this point, control groups still exhibited high levels of microglia/macrophage-specific marker Iba-1 and proliferating cell nuclear antigen, the mark of an ongoing inflammation and incomplete axonal growth 2 months after the injury. This report demonstrates that hADSCs promote neuronal survival in the spinal ganglion, fuel axonal repair and stimulate the regeneration of peripheral nerves.

  13. Identification and characterization of pig adipose-derived progenitor cells.

    PubMed

    Zhang, Shuang; Bai, Chunyu; Zheng, Dong; Gao, Yuhua; Fan, Yanan; Li, Lu; Guan, Weijun; Ma, Yuehui

    2016-10-01

    Adipose-derived stem cells (ADSCs) are multipotent, and can be differentiated into many cell types in vitro. In this study, tissues from pigs were chosen to identify and characterize ADSCs. Primary ADSCs were sub-cultured to passage 28. The surface markers of ADSCs: CD29, CD71, CD73, CD90, and CD166 were detected by reverse-transcription polymerase chain reaction assays and the markers CD29, CD44, CD105, and vimentin were detected by immunofluorescence. Growth curves and the capacity of clone-forming were performed to test the proliferation of ADSCs. Karyotype analysis showed that ADSCs cultured in vitro were genetically stable. To assess the differentiation capacity of the ADSCs, cells were induced to differentiate into osteoblasts, adipocytes, epithelial cells, neural cells, and hepatocyte-like cells. The results suggest that ADSCs from pigs showed similar biological characteristics with those separated from other species, and their multi-lineage differentiation shows potential as an application for cellular therapy in an animal model.

  14. Adult Adipose-Derived Stem Cell Attachment to Biomaterials

    PubMed Central

    Prichard, Heather L; Reichert, William M; Klitzman, Bruce

    2007-01-01

    Attachment of adipose-derived stem cells (ASC) to biomaterials prior to implantation is a possible strategy for mediating inflammation and wound healing. In this study, the ASC percent coverage was measured on common medical grade biosensor materials subjected to different surface treatments. Cell coverage on silicone elastomer (poly dimethylsiloxane) was below 20% for all surface treatments. Polyimide (Kapton), polyurethane (Pellethane) and tissue culture polystyrene all exhibited >50% coverage for surfaces treated with fibronectin (Fn), fibronectin plus avidin/biotin (dual ligand), and oxygen plasma plus fibronectin treatments (Fn O2). The fibronectin treatment performed as well or better on polyimide, polyurethane, and tissue culture polystyrene compared to the dual ligand and fibronectin oxygen plasma treated surfaces. Cell detachment with increasing shear stresses was <25% for each attachment method on both polyimide and polyurethane. The effects of attachment methods on the basic cell functions of proliferation, metabolism, ATP concentration, and caspase-3 activity were analyzed yielding proliferation profiles that were very similar among all of the materials. No significant differences in metabolism, intracellular ATP, or intracellular caspase-3 activity were observed for any of the attachment methods on either polyimide or polyurethane. PMID:17074385

  15. Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation. Complete prevention of recurrence of autoimmune diseases in MRL/MP-Ipr/Ipr mice by transplantation of bone marrow plus bones (stromal cells) from the same donor.

    PubMed

    Ishida, T; Inaba, M; Hisha, H; Sugiura, K; Adachi, Y; Nagata, N; Ogawa, R; Good, R A; Ikehara, S

    1994-03-15

    MRL/MP-Ipr/Ipr (MRL/Ipr) mice possess radioresistant (9.5 Gy) abnormal stem cells and show a recurrence of autoimmune diseases within 5 mo of conventional allogeneic bone marrow transplantation. We recently have found that the MHC preference exists between hemopoietic stem cells and stromal cells; when bones are engrafted, donor-derived stromal cells present in the engrafted bones can migrate into the recipient bone marrows, which are replaced with both donor-derived stromal cells and hematopoietic cells. Based on these findings, we attempted to prevent the recurrence of autoimmune diseases in MRL/Ipr mice by the transplantation of both bone marrow cells and bone (as a source of stromal cells). MRL/Ipr mice were irradiated (8.5 Gy) and then reconstituted with C57BL/6 bone marrow cells plus bone grafts. The mice survived more than 48 wk after this treatment. Immunohistologic studies revealed that the mice were completely free from both lymphadenopathy and autoimmune diseases such as lupus nephritis and rheumatoid arthritis. Sera from these mice showed normal levels of circulating immune complexes and rheumatoid factors. Normal functions of both T cells and B cells were noted. Abnormal T cells such as Thy-1+B220+ cells present in nontreated MRL/Ipr mice could not be seen in the thus-treated mice. In addition, to our surprise, spleen cells from treated mice showed completely normal in vitro primary anti-SRBC responses. These results indicate that stromal cells in allogeneic bone marrow transplantation play a crucial role not only in the prevention of graft failure but also in the successful cooperation among APCs, T cells, and B cells. Although MRL/Ipr mice are radiosensitive and usually die of interstitial pneumonia or fatty liver due to the side effects of radiation, it should be noted that this strategy allows a reduction in the radiation dose (9.5 Gy-->8.5 Gy), and that these mice can survive more than 48 wk without showing any symptoms of autoimmune diseases.

  16. Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells.

    PubMed

    Kono, Shota; Kazama, Tomohiko; Kano, Koichiro; Harada, Kayoko; Uechi, Masami; Matsumoto, Taro

    2014-01-01

    It has been reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multilineage differentiation potential similar to that observed in mesenchymal stem cells. Since DFAT cells can be prepared from a small quantity of adipose tissue, they could facilitate cell-based therapies in small companion animals such as cats. The present study examined whether multipotent DFAT cells can be generated from feline adipose tissue, and the properties of DFAT cells were compared with those of adipose-derived stem cells (ASCs). DFAT cells and ASCs were prepared from the floating mature adipocyte fraction and the stromal vascular fraction, respectively, of collagenase-digested feline omental adipose tissue. Both cell types were evaluated for growth kinetics, colony-forming unit fibroblast (CFU-F) frequency, immunophenotypic properties, and multilineage differentiation potential. DFAT cells and ASCs could be generated from approximately 1g of adipose tissue and were grown and subcultured on laminin-coated dishes. The frequency of CFU-Fs in DFAT cells (35.8%) was significantly higher than that in ASCs (20.8%) at passage 1 (P1). DFAT cells and ASCs displayed similar immunophenotypes (CD44(+), CD90(+), CD105(+), CD14(-), CD34(-) and CD45(-)). Alpha-smooth muscle actin-positive cells were readily detected in ASCs (15.2±7.2%) but were rare in DFAT cells (2.2±3.2%) at P1. Both cell types exhibited adipogenic, osteogenic, chondrogenic, and smooth muscle cell differentiation potential in vitro. In conclusion, feline DFAT cells exhibited similar properties to ASCs but displayed higher CFU-F frequency and greater homogeneity. DFAT cells, like ASCs, may be an attractive source for cell-based therapies in cats.

  17. Adipose-Derived Regenerative Cell Therapy for Burn Wound Healing: A Comparison of Two Delivery Methods

    PubMed Central

    Foubert, Philippe; Gonzalez, Andreina D.; Teodosescu, Stephan; Berard, Felipe; Doyle-Eisele, Melanie; Yekkala, Krishna; Tenenhaus, Mayer; Fraser, John K.

    2016-01-01

    Objective: The use of noncultured autologous stromal vascular fraction or clinical grade adipose-derived regenerative cells (ADRCs) is a promising strategy to promote wound healing and tissue repair. Nevertheless, issues regarding the optimal mode of administration remain unclear. The purpose of this study was to compare the effects of local injection and topical spray delivery of ADRCs in a porcine model of thermal burns. Approach: Full-thickness thermal burns were created on the dorsum of 10 Gottingen minipigs. Two days following injury, wounds underwent fascial excision and were randomized to receive control vehicle or freshly isolated autologous ADRCs delivered by either multiple injections into or surrounding the wound bed, or by spray onto the wound surface (0.25 × 106 viable cells/cm2). Healing was evaluated by planimetry, histopathology, and immunohistochemistry at day 7, 12, 16, 21, and 28 posttreatment. Results: In vitro analysis demonstrated that there was no substantial loss of cell number or viability attributable to the spray procedure. Planimetric assessment revealed that delivery of ADRCs by either local injection or topical spray increased wound reepithelialization relative to control at day 14. No significant difference in wound reepithelialization was observed between both delivery approaches. In addition, on day 7 posttreatment, blood vessel density was greater in wounds receiving local or topical spray ADRCs than in the wounds treated with vehicle control. Histopathologic analysis suggests that ADRC treatment may modulate the inflammatory response by reducing neutrophil infiltration at day 7 and 12 posttreatment, irrespective of the route of administration. Conclusions: These data demonstrate that local injection and spray delivery of ADRCs modulate inflammation and improve wound angiogenesis and epithelialization. Importantly, both delivery routes exhibited similar effects on wound healing. Given the greater ease-of-use associated with

  18. The role of SDF-1 in homing of human adipose-derived stem cells.

    PubMed

    Stuermer, Ewa K; Lipenksy, Alexandra; Thamm, Oliver; Neugebauer, Edmund; Schaefer, Nadine; Fuchs, Paul; Bouillon, Bertil; Koenen, Paola

    2015-01-01

    One of the putative pathophysiological mechanisms of chronic wounds is a disturbed homing of stem cells. In this project, the stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor (CXCR) 4 and SDF-1/CXCR7 pathway were focused in human adipose-derived stem cells (ASCs). ASCs were incubated with acute (AWF) or chronic wound fluid (CWF) to analyze their effects by quantitative real-time polymerase chain reaction (SDF-1, CXCR4, CXCR7, TIMP3), enzyme-linked immunosorbent assay (SDF-1 in WFs and supernatant), and transwell migration assay with/without antagonization. Whereas SDF-1 amounted 73.5 pg/mL in AWF, it could not be detected in CWF. Incubation with AWF led to a significant enhancement (129.7 pg/mL vs. 95.5 pg/mL), whereas CWF resulted in a significant reduction (30 pg/mL vs. 95.5 pg/mL) of SDF-1 in ASC supernatant. The SDF-1 receptor CXCR7 was detected on ASCs. AWF but not CWF significantly induced ASC migration, which was inhibited by CXCR4 and CXCR7 antagonists. Expressions of SDF-1, CXCR4, and CXCR7 were significantly stimulated by AWF while TIMP3 expression was reduced. In conclusion, an uncontrolled inflammation in the chronic wound environment, indicated by a reduced SDF-1 expression, resulted in a decreased ASC migration. A disturbed SDF-1/CXCR4 as well as SDF-1/CXCR7 pathway seems to play an important role in the impaired healing of chronic wounds.

  19. Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells

    PubMed Central

    López, Melany; Bollag, Roni J.; Yu, Jack C.; Isales, Carlos M.; Eroglu, Ali

    2016-01-01

    The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS)-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate), two polymers (PVA and ficoll), two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide), a disaccharide (trehalose), and a calcium chelator (EGTA) to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum) were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the plating

  20. Adipose-derived stem cells retain their regenerative potential after methotrexate treatment

    SciTech Connect

    Beane, Olivia S.; Fonseca, Vera C.; Darling, Eric M.

    2014-10-01

    In musculoskeletal tissues like bone, chemotherapy can impair progenitor cell differentiation and proliferation, resulting in decreased bone growth and mineralization throughout a patient's lifetime. In the current study, we investigated the effects of chemotherapeutics on adipose-derived stem cell (ASC) function to determine whether this cell source could be a candidate for repairing, or even preventing, chemotherapy-induced tissue damage. Dose-dependent proliferation rates of ASCs and normal human fibroblasts (NHFs) were quantified after treatment with cytarabine (CY), etoposide (ETO), methotrexate (MTX), and vincristine (VIN) using a fluorescence-based assay. The influence of MTX on the multipotency of ASCs and freshly isolated stromal vascular fraction (SVF) cells was also evaluated using lineage-specific stains and spectrophotometry. ASC and NHF proliferation were equally inhibited by exposure to CY and ETO; however, when treated with MTX and VIN, ASCs exhibited greater resistance. This was especially apparent for MTX-treated samples, with ASC proliferation showing no inhibition for clinically relevant MTX doses ranging from 0.1 to 50 μM. Additional experiments revealed that the differentiation potential of ASCs was not affected by MTX treatment and that upregulation of dihydrofolate reductase possibly contributed to this response. Moreover, SVF cells, which include ASCs, exhibited similar resistance to MTX impairment, with respect to cellular proliferation, clonogenicity, and differentiation capability. Therefore, we have shown that the regenerative properties of ASCs resist the cytotoxicity of MTX, identifying these cells as a potential key for repairing musculoskeletal damage in patients undergoing chemotherapy. - Highlights: • Long-term effects of chemotherapeutics can include musculoskeletal dysfunction. • A screen of common drugs showed disparate effects on ASCs and fibroblasts. • One drug, methotrexate, did not impair ASC growth characteristics

  1. Cellular Therapy With Human Autologous Adipose-Derived Adult Stem Cells for Advanced Keratoconus.

    PubMed

    Alió Del Barrio, Jorge L; El Zarif, Mona; de Miguel, María P; Azaar, Albert; Makdissy, Norman; Harb, Walid; El Achkar, Ibrahim; Arnalich-Montiel, Francisco; Alió, Jorge L

    2017-08-01

    The aim of this phase 1 study was to preliminarily evaluate the safety and efficacy of autologous adipose-derived adult stem cell (ADASC) implantation within the corneal stroma of patients with advanced keratoconus. Five consecutive patients were selected. Autologous ADASCs were obtained by elective liposuction. ADASCs (3 × 10) contained in 1 mL saline were injected into the corneal stroma through a femtosecond-assisted 9.5-mm diameter lamellar pocket under topical anesthesia. Patients were reviewed at 1 day, 1 week, 1, 3, and 6 months postoperatively. Visual function, manifest refraction, slit-lamp biomicroscopy, intraocular pressure, endothelial cell density, corneal topography, corneal optical coherence tomography, and corneal confocal biomicroscopy were recorded. No intraoperative or postoperative complications were recorded, with full corneal transparency recovery within 24 hours. Four patients completed the full follow-up. All patients improved their visual function (mean: 1 line of unaided and spectacle-corrected distance vision and 2 lines of rigid contact lens distance vision). Manifest refraction and topographic keratometry remained stable. Corneal optical coherence tomography showed a mean improvement of 16.5 μm in the central corneal thickness, and new collagen production was observed as patchy hyperreflective areas at the level of the stromal pocket. Confocal biomicroscopy confirmed the survival of the implanted stem cells at the surgical plane. Intraocular pressure and endothelial cell density remained stable. Cellular therapy of the human corneal stroma in vivo with autologous ADASCs appears to be safe. Stem cells survive in vivo with intrastromal new collagen production. Future studies with larger samples are required to confirm these preliminary results.

  2. Adipose Stromal Cells Repair Pressure Ulcers in Both Young and Elderly Mice: Potential Role of Adipogenesis in Skin Repair

    PubMed Central

    Strong, Amy L.; Bowles, Annie C.; MacCrimmon, Connor P.; Frazier, Trivia P.; Lee, Stephen J.; Wu, Xiying; Katz, Adam J.; Gawronska-Kozak, Barbara; Bunnell, Bruce A.

    2015-01-01

    More than 2.5 million patients in the U.S. require treatment for pressure ulcers annually, and the elderly are at particularly high risk for pressure ulcer development. Current therapy for pressure ulcers consists of conservative medical management for shallow lesions and aggressive debridement and surgery for deeper lesions. The current study uses a murine model to address the hypothesis that adipose-derived stromal/stem cell (ASC) treatment would accelerate and enhance pressure ulcer repair. The dorsal skin of both young (2 months old [mo]) and old (20 mo) C57BL/6J female mice was sandwiched between external magnets for 12 hours over 2 consecutive days to initiate a pressure ulcer. One day following the induction, mice were injected with ASCs isolated from congenic mice transgenic for the green fluorescent protein under a ubiquitous promoter. Relative to phosphate-buffered saline-treated controls, ASC-treated mice displayed a cell concentration-dependent acceleration of wound closure, improved epidermal/dermal architecture, increased adipogenesis, and reduced inflammatory cell infiltration. The ASC-induced improvements occurred in both young and elderly recipients, although the expression profile of angiogenic, immunomodulatory, and reparative mRNAs differed as a function of age. The results are consistent with clinical reports that fat grafting improved skin architecture in thermal injuries; the authors of this published study have invoked ASC-based mechanisms to account for their clinical outcomes. Thus, the current proof-of-principle study sets the stage for clinical translation of autologous and/or allogeneic ASC treatment of pressure ulcers. Significance Adipose-derived stromal/stem cells (ASCs) promote the healing of pressure ulcer wounds in both young and old mice. ASCs enhance wound healing rates through adipogenic differentiation and regeneration of the underlying architecture of the skin. PMID:25900728

  3. Adipose stromal cells repair pressure ulcers in both young and elderly mice: potential role of adipogenesis in skin repair.

    PubMed

    Strong, Amy L; Bowles, Annie C; MacCrimmon, Connor P; Frazier, Trivia P; Lee, Stephen J; Wu, Xiying; Katz, Adam J; Gawronska-Kozak, Barbara; Bunnell, Bruce A; Gimble, Jeffrey M

    2015-06-01

    More than 2.5 million patients in the U.S. require treatment for pressure ulcers annually, and the elderly are at particularly high risk for pressure ulcer development. Current therapy for pressure ulcers consists of conservative medical management for shallow lesions and aggressive debridement and surgery for deeper lesions. The current study uses a murine model to address the hypothesis that adipose-derived stromal/stem cell (ASC) treatment would accelerate and enhance pressure ulcer repair. The dorsal skin of both young (2 months old [mo]) and old (20 mo) C57BL/6J female mice was sandwiched between external magnets for 12 hours over 2 consecutive days to initiate a pressure ulcer. One day following the induction, mice were injected with ASCs isolated from congenic mice transgenic for the green fluorescent protein under a ubiquitous promoter. Relative to phosphate-buffered saline-treated controls, ASC-treated mice displayed a cell concentration-dependent acceleration of wound closure, improved epidermal/dermal architecture, increased adipogenesis, and reduced inflammatory cell infiltration. The ASC-induced improvements occurred in both young and elderly recipients, although the expression profile of angiogenic, immunomodulatory, and reparative mRNAs differed as a function of age. The results are consistent with clinical reports that fat grafting improved skin architecture in thermal injuries; the authors of this published study have invoked ASC-based mechanisms to account for their clinical outcomes. Thus, the current proof-of-principle study sets the stage for clinical translation of autologous and/or allogeneic ASC treatment of pressure ulcers. Adipose-derived stromal/stem cells (ASCs) promote the healing of pressure ulcer wounds in both young and old mice. ASCs enhance wound healing rates through adipogenic differentiation and regeneration of the underlying architecture of the skin. ©AlphaMed Press.

  4. Hair Regeneration Treatment Using Adipose-Derived Stem Cell Conditioned Medium: Follow-up With Trichograms

    PubMed Central

    Suga, Hirotaka

    2015-01-01

    Objective: Adipose-derived stem cells secrete various growth factors that promote hair growth. This study examined the effects of adipose-derived stem cell-conditioned medium on alopecia. Methods: Adipose-derived stem cell-conditioned medium was intradermally injected in 22 patients (11 men and 11 women) with alopecia. Patients received treatment every 3 to 5 weeks for a total of 6 sessions. Hair numbers were counted using trichograms before and after treatment. A half-side comparison study was also performed in 10 patients (8 men and 2 women). Results: Hair numbers were significantly increased after treatment in both male (including those without finasteride administration) and female patients. In the half-side comparison study, the increase in hair numbers was significantly higher on the treatment side than on the placebo side. Conclusion: Treatment using adipose-derived stem cell-conditioned medium appears highly effective for alopecia and may represent a new therapy for hair regeneration. PMID:25834689

  5. Adipose-Derived Mesenchymal Stem Cells in Autoimmune Disorders: State of the Art and Perspectives for Systemic Sclerosis.

    PubMed

    Maria, Alexandre T J; Maumus, Marie; Le Quellec, Alain; Jorgensen, Christian; Noël, Danièle; Guilpain, Philippe

    2017-04-01

    Mesenchymal stromal/stem cells (MSC) are non-hematopoietic multipotent progenitor cells, first described in bone marrow in the middle of last century. Since then, MSC have been the objects of a myriad of publications, progressively increasing our knowledge on their potentialities and bringing high expectancies for their regenerative properties. During the same period, numerous tissues, such as adipose tissue, placenta, or umbilical cord, have been used as alternative sources of MSC in comparison with bone marrow. In particular, considering the accessibility and ease to harvest fat tissue, adipose-derived MSC have gained interest above bone marrow-derived MSC. More recently, the discovery of MSC immunomodulatory properties made MSC-based therapy progressively slip from the field of regenerative medicine to the one of autoimmunity. Indeed, in this group of disorders caused by aberrant activation of the immune system resulting in loss of self-tolerance and auto-reactivity, conventional immunosuppressant may be harmful. One advantage of MSC-based therapy would lie in their immune plasticity, resulting in space and time-limited immunosuppression. More specifically, among autoimmune disorders, systemic sclerosis appears as a peculiar multifaceted disease, in which autoimmune phenomena coexist with vascular abnormalities and multi-visceral fibrosis. Considering the pleiotropic effects of MSC, displaying immunomodulatory, angiogenic and antifibrotic capabilities, MSC-based therapy could counteract the three main pathogenic axes of systemic sclerosis and might thus represent a complete breakthrough in this intractable disease with unmet medical need. In this article, while reviewing most recent literature on MSC biology, we itemize their current applications in the field of autoimmunity and shed light onto the potential use of adipose-derived MSC as an innovative strategy to cure systemic sclerosis.

  6. Angiogenesis after transplantation of auto- and allogenic cells.

    PubMed

    Fatkhudinov, T Kh; Bol'shakova, G B; Komissarova, S V; Arutyunyan, I V; Rzhaninova, A A; Goldstein, D V

    2010-10-01

    Neoangiogenesis after transplantation of auto- and allogenic mononuclears and multipotent stromal cells from the bone marrow was studied on the model of inflammatory angiogenesis. Transplanted auto- and allogenic cells stimulate the formation of new blood vessels in the granulation tissue, this manifesting in an increase in the quantity and volume density of blood vessels. The most pronounced angiogenesis was observed after transplantation of allogenic mononuclears and multipotent stromal cells. It was associated with intense inflammatory infiltration, with less numerous and mature collagen fibers in the granulation tissue. Injection of allogenic cells led to stimulation and chronization of inflammation, infiltration with inflammatory and poorly differentiated cells, and more pronounced and lasting angiogenesis. However, neither auto-, nor allogenic transplanted labeled cells were detected in the walls of new blood vessels. Hence, it seems that bone marrow mononuclears and multipotent stromal cells stimulated angiogenesis mainly at the expense of production of angiogenic factors, and after transplantation of allogenic cells also by stimulating the inflammation.

  7. Analysis of type II diabetes mellitus adipose-derived stem cells for tissue engineering applications

    PubMed Central

    Minteer, Danielle Marie; Young, Matthew T; Lin, Yen-Chih; Over, Patrick J; Rubin, J Peter; Gerlach, Jorg C

    2015-01-01

    To address the functionality of diabetic adipose-derived stem cells in tissue engineering applications, adipose-derived stem cells isolated from patients with and without type II diabetes mellitus were cultured in bioreactor culture systems. The adipose-derived stem cells were differentiated into adipocytes and maintained as functional adipocytes. The bioreactor system utilizes a hollow fiber–based technology for three-dimensional perfusion of tissues in vitro, creating a model in which long-term culture of adipocytes is feasible, and providing a potential tool useful for drug discovery. Daily metabolic activity of the adipose-derived stem cells was analyzed within the medium recirculating throughout the bioreactor system. At experiment termination, tissues were extracted from bioreactors for immunohistological analyses in addition to gene and protein expression. Type II diabetic adipose-derived stem cells did not exhibit significantly different glucose consumption compared to adipose-derived stem cells from patients without type II diabetes (p > 0.05, N = 3). Expression of mature adipocyte genes was not significantly different between diabetic/non-diabetic groups (p > 0.05, N = 3). Protein expression of adipose tissue grown within all bioreactors was verified by Western blotting.The results from this small-scale study reveal adipose-derived stem cells from patients with type II diabetes when removed from diabetic environments behave metabolically similar to the same cells of non-diabetic patients when cultured in a three-dimensional perfusion bioreactor, suggesting that glucose transport across the adipocyte cell membrane, the hindrance of which being characteristic of type II diabetes, is dependent on environment. The presented observation describes a tissue-engineered tool for long-term cell culture and, following future adjustments to the culture environment and increased sample sizes, potentially for anti-diabetic drug testing. PMID:26090087

  8. Adipose-derived stem cells inhibit epidermal melanocytes through an interleukin-6-mediated mechanism.

    PubMed

    Kim, Deok-Woo; Jeon, Byung-Joon; Hwang, Na-Hyun; Kim, Min-Sook; Park, Seung-Ha; Dhong, Eun-Sang; Yoon, Eul-Sik; Lee, Byung-Il

    2014-09-01

    Several investigators have postulated that human adipose-derived stem cells can be used for skin rejuvenation, but there have been few reports about their direct effects on human epidermal melanocytes. The authors studied the effects on melanocytes, and the causative agent of those effects was further investigated in this study. Human epidermal melanocytes were divided into three groups and cultured in adipose-derived stem cell-conditioned medium, human dermal fibroblast-conditioned medium, or control medium. Concentrations of melanogenic cytokines in these media were measured using enzyme-linked immunosorbent assay kits. After 3 and 7 days of incubation, cell proliferation, melanin content, tyrosinase activity, and melanogenic gene expression were measured. Interleukin-6-neutralizing antibodies were mixed with adipose-derived stem cell-conditioned medium in which human epidermal melanocytes were cultured, and melanocyte growth and melanogenesis were measured again. Interleukin-6 concentrations in adipose-derived stem cell- and human epidermal melanocyte-conditioned media were 1373 and 495 pg/ml, respectively. Both types of medium suppressed melanocyte proliferation and melanin synthesis (p < 0.05), but adipose-derived stem cell-conditioned medium was more effective than human dermal fibroblast-conditioned medium in inhibition of human epidermal melanocyte proliferation, melanin synthesis, and tyrosinase activity (p < 0.05). Interleukin-6-neutralizing antibody sufficiently reversed the antimelanogenic effects of adipose-derived stem cell-conditioned medium such that human epidermal melanocyte proliferation, melanin content, tyrosinase activity, and tyrosinase mRNA levels were restored (p < 0.05). Adipose-derived stem cell-conditioned medium inhibited melanocyte proliferation and melanin synthesis by down-regulating melanogenic enzymes. Interleukin-6 plays a pivotal role in inhibition of melanocytes.

  9. Analysis of type II diabetes mellitus adipose-derived stem cells for tissue engineering applications.

    PubMed

    Minteer, Danielle Marie; Young, Matthew T; Lin, Yen-Chih; Over, Patrick J; Rubin, J Peter; Gerlach, Jorg C; Marra, Kacey G

    2015-01-01

    To address the functionality of diabetic adipose-derived stem cells in tissue engineering applications, adipose-derived stem cells isolated from patients with and without type II diabetes mellitus were cultured in bioreactor culture systems. The adipose-derived stem cells were differentiated into adipocytes and maintained as functional adipocytes. The bioreactor system utilizes a hollow fiber-based technology for three-dimensional perfusion of tissues in vitro, creating a model in which long-term culture of adipocytes is feasible, and providing a potential tool useful for drug discovery. Daily metabolic activity of the adipose-derived stem cells was analyzed within the medium recirculating throughout the bioreactor system. At experiment termination, tissues were extracted from bioreactors for immunohistological analyses in addition to gene and protein expression. Type II diabetic adipose-derived stem cells did not exhibit significantly different glucose consumption compared to adipose-derived stem cells from patients without type II diabetes (p > 0.05, N = 3). Expression of mature adipocyte genes was not significantly different between diabetic/non-diabetic groups (p > 0.05, N = 3). Protein expression of adipose tissue grown within all bioreactors was verified by Western blotting.The results from this small-scale study reveal adipose-derived stem cells from patients with type II diabetes when removed from diabetic environments behave metabolically similar to the same cells of non-diabetic patients when cultured in a three-dimensional perfusion bioreactor, suggesting that glucose transport across the adipocyte cell membrane, the hindrance of which being characteristic of type II diabetes, is dependent on environment. The presented observation describes a tissue-engineered tool for long-term cell culture and, following future adjustments to the culture environment and increased sample sizes, potentially for anti-diabetic drug testing.

  10. Mesenchymal Stromal Cells from Osteoarthritic Synovium Are a Distinct Population Compared to Their Bone-Marrow Counterparts regarding Surface Marker Distribution and Immunomodulation of Allogeneic CD4+ T-Cell Cultures.

    PubMed

    Hagmann, Sebastien; Rimmele, Claudia; Bucur, Florin; Dreher, Thomas; Zeifang, Felix; Moradi, Babak; Gotterbarm, Tobias

    2016-01-01

    Introduction. The participation of an inflammatory joint milieu has been described in osteoarthritis (OA) pathogenesis. Mesenchymal stromal cells (MSCs) play an important role in modulating inflammatory processes. Based on previous studies in an allogeneic T-cell coculture model, we aimed at further determining the role of synovial MSCs in OA pathogenesis. Methods. Bone-marrow (BM) and synovial membrane (SM) MSCs from hip joints of late stage OA patients and CD4+ T-cells from healthy donors were analysed regarding surface marker expression before and after coculture. Proliferation upon CD3/CD28 stimulation and cytokine analyses were compared between MSCs. Results. SM-MSCs differed from BM-MSCs in several surface markers and their osteogenic differentiation potential. Cocultures of both MSCs with CD4+ T-cells resulted in recruitment of CD45RA+ FoxP3+ regulatory T-cells. Upon stimulation, only SM-MSCs suppressed CD4+ T-cell proliferation, while both SM-MSCs and BM-MSCs modified cytokine profiles through suppressing IL-2 and TNF-α as well as increasing IL-6 secretion. Conclusions. Synovial MSCs from OA joints are a unique fraction that can be distinguished from their bone-marrow derived counterparts. Their unique ability to suppress CD3/CD28 induced CD4+ T-cell proliferation makes them a potential target for future therapeutic approaches.

  11. Mesenchymal Stromal Cells from Osteoarthritic Synovium Are a Distinct Population Compared to Their Bone-Marrow Counterparts regarding Surface Marker Distribution and Immunomodulation of Allogeneic CD4+ T-Cell Cultures

    PubMed Central

    Bucur, Florin; Moradi, Babak

    2016-01-01

    Introduction. The participation of an inflammatory joint milieu has been described in osteoarthritis (OA) pathogenesis. Mesenchymal stromal cells (MSCs) play an important role in modulating inflammatory processes. Based on previous studies in an allogeneic T-cell coculture model, we aimed at further determining the role of synovial MSCs in OA pathogenesis. Methods. Bone-marrow (BM) and synovial membrane (SM) MSCs from hip joints of late stage OA patients and CD4+ T-cells from healthy donors were analysed regarding surface marker expression before and after coculture. Proliferation upon CD3/CD28 stimulation and cytokine analyses were compared between MSCs. Results. SM-MSCs differed from BM-MSCs in several surface markers and their osteogenic differentiation potential. Cocultures of both MSCs with CD4+ T-cells resulted in recruitment of CD45RA+ FoxP3+ regulatory T-cells. Upon stimulation, only SM-MSCs suppressed CD4+ T-cell proliferation, while both SM-MSCs and BM-MSCs modified cytokine profiles through suppressing IL-2 and TNF-α as well as increasing IL-6 secretion. Conclusions. Synovial MSCs from OA joints are a unique fraction that can be distinguished from their bone-marrow derived counterparts. Their unique ability to suppress CD3/CD28 induced CD4+ T-cell proliferation makes them a potential target for future therapeutic approaches. PMID:27516777

  12. Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells

    PubMed Central

    Sugii, Shigeki; Kida, Yasuyuki; Kawamura, Teruhisa; Suzuki, Jotaro; Vassena, Rita; Yin, Yun-Qiang; Lutz, Margaret K.; Berggren, W. Travis; Izpisúa Belmonte, Juan Carlos; Evans, Ronald M.

    2010-01-01

    Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFβ, fibronectin, and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance. PMID:20133714

  13. Equine allogeneic umbilical cord blood derived mesenchymal stromal cells reduce synovial fluid nucleated cell count and induce mild self-limiting inflammation when evaluated in an lipopolysaccharide induced synovitis model.

    PubMed

    Williams, L B; Koenig, J B; Black, B; Gibson, T W G; Sharif, S; Koch, T G

    2016-09-01

    Improvement has been reported following intra-articular (i.a.) injection of mesenchymal stromal cells (MSCs) in several species. These observations have led to the use of i.a. MSCs in equine practice with little understanding of the mechanisms by which perceived improvement occurs. To evaluate the effect of i.a. allogeneic umbilical cord blood (CB-) derived MSCs using a lipopolysaccharide (LPS) induced synovitis model. We hypothesised that i.a. CB-MSCs would reduce the inflammatory response associated with LPS injection. Randomised, blinded experimental study. Feasibility studies evaluated i.a. LPS or CB-MSCs alone into the tarsocrural joint. In the principal study, middle carpal joint synovitis was induced bilaterally with LPS and then CB-MSCs were injected into one middle carpal joint. Lameness, routine synovial fluid analysis, and synovial fluid biomarkers were evaluated at 0, 8, 24, 48 and 72 h. LPS injection alone resulted in transient lameness and signs of inflammation. In joints untreated with LPS, injection of 30 million CB-MSCs resulted in mild synovitis that resolved without treatment. Mild (grade 1-2) lameness in the CB-MSC-treated limb was observed in 2 horses and severe lameness (grade 4) in the 3rd, 24 h post injection. Lameness did not correlate with synovitis induced by CB-MSC injection. Simultaneous injection of LPS and CB-MSCs resulted in significant reduction in synovial fluid total nucleated, neutrophil and mononuclear cell numbers compared with contralateral LPS-only joints. No difference was detected in other parameters associated with synovial fluid analysis or in synovial fluid biomarkers. The incidence of lameness was only different from baseline at 8 h, where horses were lame in CB-MSC limbs. Allogeneic CB-MSCs reduced synovial fluid cell populations and stimulated mild self-limiting inflammation in the synovitis model. Continued evaluation of the effects of i.a. CB-MSC therapy on synovitis in horses is needed to evaluate anti- and

  14. Increased Adipogenesis of Human Adipose-Derived Stem Cells on Polycaprolactone Fiber Matrices

    PubMed Central

    Brännmark, Cecilia; Paul, Alexandra; Ribeiro, Diana; Magnusson, Björn; Brolén, Gabriella; Enejder, Annika; Forslöw, Anna

    2014-01-01

    With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells - differentiated to adipocytes in vitro - are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics – studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells. PMID:25419971

  15. Human Adipose-Derived Stem Cells Labeled with Plasmonic Gold Nanostars for Cellular Tracking and Photothermal Cancer Cell Ablation.

    PubMed

    Shammas, Ronnie L; Fales, Andrew M; Crawford, Bridget M; Wisdom, Amy J; Devi, Gayathri R; Brown, David A; Vo-Dinh, Tuan; Hollenbeck, Scott T

    2017-04-01

    Gold nanostars are unique nanoplatforms that can be imaged in real time and transform light energy into heat to ablate cells. Adipose-derived stem cells migrate toward tumor niches in response to chemokines. The ability of adipose-derived stem cells to migrate and integrate into tumors makes them ideal vehicles for the targeted delivery of cancer nanotherapeutics. To test the labeling efficiency of gold nanostars, undifferentiated adipose-derived stem cells were incubated with gold nanostars and a commercially available nanoparticle (Qtracker), then imaged using two-photon photoluminescence microscopy. The effects of gold nanostars on cell phenotype, proliferation, and viability were assessed with flow cytometry, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide metabolic assay, and trypan blue, respectively. Trilineage differentiation of gold nanostar-labeled adipose-derived stem cells was induced with the appropriate media. Photothermolysis was performed on adipose-derived stem cells cultured alone or in co-culture with SKBR3 cancer cells. Efficient uptake of gold nanostars occurred in adipose-derived stem cells, with persistence of the luminescent signal over 4 days. Labeling efficiency and signal quality were greater than with Qtracker. Gold nanostars did not affect cell phenotype, viability, or proliferation, and exhibited stronger luminescence than Qtracker throughout differentiation. Zones of complete ablation surrounding the gold nanostar-labeled adipose-derived stem cells were observed following photothermolysis in both monoculture and co-culture models. Gold nanostars effectively label adipose-derived stem cells without altering cell phenotype. Once labeled, photoactivation of gold nanostar-labeled adipose-derived stem cells ablates neighboring cancer cells, demonstrating the potential of adipose-derived stem cells as a vehicle for the delivery of site-specific cancer therapy.

  16. Administration of Adult Human Bone Marrow-Derived, Cultured, Pooled, Allogeneic Mesenchymal Stromal Cells in Critical Limb Ischemia Due to Buerger's Disease: Phase II Study Report Suggests Clinical Efficacy.

    PubMed

    Gupta, Pawan K; Krishna, Murali; Chullikana, Anoop; Desai, Sanjay; Murugesan, Rajkumar; Dutta, Santanu; Sarkar, Uday; Raju, Radhakrishnan; Dhar, Anita; Parakh, Rajiv; Jeyaseelan, Lakshmanan; Viswanathan, Pachaiyappan; Vellotare, Prasanth Kulapurathu; Seetharam, Raviraja N; Thej, Charan; Rengasamy, Mathiyazhagan; Balasubramanian, Sudha; Majumdar, Anish S

    2017-03-01

    Critical limb ischemia (CLI) due to Buerger's disease is a major unmet medical need with a high incidence of morbidity. This phase II, prospective, nonrandomized, open-label, multicentric, dose-ranging study was conducted to assess the efficacy and safety of i.m. injection of adult human bone marrow-derived, cultured, pooled, allogeneic mesenchymal stromal cells (BMMSC) in CLI due to Buerger's disease. Patients were allocated to three groups: 1 and 2 million cells/kg body weight (36 patients each) and standard of care (SOC) (18 patients). BMMSCs were administered as 40-60 injections in the calf muscle and locally, around the ulcer. Most patients were young (age range, 38-42 years) and ex-smokers, and all patients had at least one ulcer. Both the primary endpoints-reduction in rest pain (0.3 units per month [SE, 0.13]) and healing of ulcers (11% decrease in size per month [SE, 0.05])-were significantly better in the group receiving 2 million cells/kg body weight than in the SOC arm. Improvement in secondary endpoints, such as ankle brachial pressure index (0.03 [SE, 0.01] unit increase per month) and total walking distance (1.03 [SE, 0.02] times higher per month), were also significant in the group receiving 2 million cells/kg as compared with the SOC arm. Adverse events reported were remotely related or unrelated to BMMSCs. In conclusion, i.m. administration of BMMSC at a dose of 2 million cells/kg showed clinical benefit and may be the best regimen in patients with CLI due to Buerger's disease. However, further randomized controlled trials are required to confirm the most appropriate dose. Stem Cells Translational Medicine 2017;6:689-699.

  17. Human Adipose-Derived Stem Cells Impair Natural Killer Cell Function and Exhibit Low Susceptibility to Natural Killer-Mediated Lysis

    PubMed Central

    DelaRosa, Olga; Sánchez-Correa, Beatriz; Morgado, Sara; Ramírez, Cristina; del Río, Borja; Menta, Ramón; Lombardo, Eleuterio

    2012-01-01

    Human adipose-derived stem cells (hASCs) have been successfully used in treating numerous diseases. However, several aspects need to be considered, particularly in the context of allogeneic cell therapy. To better understand hASCs-host interactions, we studied the phenotype of hASCs and their modulatory effect on natural killer (NK) cells by using bone marrow-mesenchymal stem cells (hBM-MSCs) as a reference. The hASCs displayed a lower susceptibility to NK cell-mediated lysis and a lower expression of ligands for DNAM-1 when compared with hBM-MSCs. Moreover, here we demonstrated that hASCs and hBM-MSCs can modulate NK cells through the action of soluble factors such as indoleamine 2,3-dioxygenase. Altogether, these results suggest that for an adoptive cell therapy based on the transfer of allogeneic hASCs, the NK-hASCs crosstalk will not result in an immediate recognition of the transferred cells. Thus, hASCs may remain in the tissue long enough to balance the immune response before being cleared. PMID:21867426

  18. Arthroscopic, histological and MRI analyses of cartilage repair after a minimally invasive method of transplantation of allogeneic synovial mesenchymal stromal cells into cartilage defects in pigs

    PubMed Central

    Nakamura, Tomomasa; Sekiya, Ichiro; Muneta, Takeshi; Hatsushika, Daisuke; Horie, Masafumi; Tsuji, Kunikazu; Kawarasaki, Tatsuo; Watanabe, Atsuya; Hishikawa, Shuji; Fujimoto, Yasuhiro; Tanaka, Hozumi; Kobayashi, Eiji

    2012-01-01

    Background aims Transplantation of synovial mesenchymal stromal cells (MSCs) may induce repair of cartilage defects. We transplanted synovial MSCs into cartilage defects using a simple method and investigated its usefulness and repair process in a pig model. Methods The chondrogenic potential of the porcine MSCs was compared in vitro. Cartilage defects were created in both knees of seven pigs, and divided into MSCs treated and non-treated control knees. Synovial MSCs were injected into the defect, and the knee was kept immobilized for 10 min before wound closure. To visualize the actual delivery and adhesion of the cells, fluorescence-labeled synovial MSCs from transgenic green fluorescent protein (GFP) pig were injected into the defect in a subgroup of two pigs. In these two animals, the wounds were closed before MSCs were injected and observed for 10 min under arthroscopic control. The defects were analyzed sequentially arthroscopically, histologically and by magnetic resonance imaging (MRI) for 3 months. Results Synovial MSCs had a higher chondrogenic potential in vitro than the other MSCs examined. Arthroscopic observations showed adhesion of synovial MSCs and membrane formation on the cartilage defects before cartilage repair. Quantification analyses for arthroscopy, histology and MRI revealed a better outcome in the MSC-treated knees than in the non-treated control knees. Conclusions Leaving a synovial MSC suspension in cartilage defects for 10 min made it possible for cells to adhere in the defect in a porcine cartilage defect model. The cartilage defect was first covered with membrane, then the cartilage matrix emerged after transplantation of synovial MSCs. PMID:22309371

  19. Impact of different pancreatic microenvironments on improvement in hyperglycemia and insulin deficiency in diabetic rats after transplantation of allogeneic mesenchymal stromal cells.

    PubMed

    Katuchova, Jana; Tothova, Timea; Farkasova Iannaccone, Silvia; Toporcer, Tomas; Harvanova, Denisa; Hildebrand, Tomas; Kilik, Robert; Bacenkova, Darina; Frohlichova, Lucia; Rosocha, Jan; Bobrov, Nikita; Radonak, Jozef

    2012-11-01

    Mesenchymal stromal cells (MSCs) in the pancreatic microenvironment can improve diabetes mellitus (DM). The aim of the present study was to determine whether different pancreatic microenvironments influence the improvement of hyperglycemia and insulin deficiency. MSCs isolated from rat bone marrow were transplanted directly into different pancreatic microenvironments in male DM rats. DM was induced in the rats by streptozotocin injection. The rats were divided into 5 groups: normal control rats, DM control rats, and 3 experimental groups (DM rats plus MSCs injected into the head of the pancreas, the tail of the pancreas, or the whole pancreas). The body weight and blood glucose of the rats were monitored during the experiment after transplantation of the MSCs. Histopathologic and immunohistochemical analyses were used to detect the presence and number of islets and insulin production in the pancreatic tissue of the rats after MSC transplantation. At 28 days after MSC transplantation, we observed a statistically significant decrease in the blood glucose level and an increase in weight in DM rats compared with DM control rats (P < 0.0001 and P < 0.03, respectively). A comparison of each of the DM rat groups treated with MSCs showed no significant differences in the blood glucose levels or body weight. Our results suggest that transplantation of MSCs could improve DM in the pancreatic microenvironment in an animal model with streptozotocin-induced DM. The different pancreatic areas into which the MSCs were implanted had no significant influence on the improvement in hyperglycemia and insulin deficiency. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Arthroscopic Harvest of Adipose-Derived Mesenchymal Stem Cells From the Infrapatellar Fat Pad.

    PubMed

    Dragoo, Jason L; Chang, Wenteh

    2017-08-01

    The successful isolation of adipose-derived mesenchymal stem cells (ADSCs) from the arthroscopically harvested infrapatellar fat pad (IFP) would provide orthopaedic surgeons with an autologous solution for regenerative procedures. To demonstrate the quantity and viability of the mesenchymal stem cell population arthroscopically harvested from the IFP as well as the surrounding synovium. Descriptive laboratory study. The posterior border of the IFP, including the surrounding synovial tissue, was harvested arthroscopically from patients undergoing anterior cruciate ligament reconstruction. Tissue was then collected in an AquaVage adipose canister, followed by fat fractionization using syringe emulsification and concentration with an AdiPrep device. In the laboratory, the layers of tissue were separated and then digested with 0.3% type I collagenase. The pelleted stromal vascular fraction (SVF) cells were then immediately analyzed for viability, mesenchymal cell surface markers by fluorescence-activated cell sorting, and clonogenic capacity. After culture expansion, the metabolic activity of the ADSCs was assessed by an AlamarBlue assay, and the multilineage differentiation capability was tested. The transition of surface antigens from the SVF toward expanded ADSCs at passage 2 was further evaluated. SVF cells were successfully harvested with a mean yield of 4.86 ± 2.64 × 10(5) cells/g of tissue and a mean viability of 69.03% ± 10.75%, with ages ranging from 17 to 52 years (mean, 35.14 ± 13.70 years; n = 7). The cultured ADSCs composed a mean 5.85% ± 5.89% of SVF cells with a mean yield of 0.33 ± 0.42 × 10(5) cells/g of tissue. The nonhematopoietic cells (CD45(-)) displayed the following surface antigens as a percentage of the viable population: CD44(+) (52.21% ± 4.50%), CD73(+)CD90(+)CD105(+) (19.20% ± 17.04%), and CD44(+)CD73(+)CD90(+)CD105(+) (15.32% ± 15.23%). There was also a significant increase in the expression of ADSC markers CD73 (96.97% ± 1

  1. Purification of human adipose-derived stem cells from fat tissues using PLGA/silk screen hybrid membranes.

    PubMed

    Chen, Da-Chung; Chen, Li-Yu; Ling, Qing-Dong; Wu, Meng-Hsueh; Wang, Ching-Tang; Suresh Kumar, S; Chang, Yung; Munusamy, Murugan A; Alarfajj, Abdullah A; Wang, Han-Chow; Hsu, Shih-Tien; Higuchi, Akon

    2014-05-01

    The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 10⁴ cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. An Evaluation of the Stemness, Paracrine, and Tumorigenic Characteristics of Highly Expanded, Minimally Passaged Adipose-Derived Stem Cells

    PubMed Central

    El Atat, Oula; Antonios, Diane; Hilal, George; Hokayem, Nabil; Abou-Ghoch, Joelle; Hashim, Hussein; Serhal, Rim; Hebbo, Clara; Moussa, Mayssam; Alaaeddine, Nada

    2016-01-01

    The use of adipose-derived stem cells (ADSC) in regenerative medicine is rising due to their plasticity, capacity of differentiation and paracrine and trophic effects. Despite the large number of cells obtained from adipose tissue, it is usually not enough for therapeutic purposes for many diseases or cosmetic procedures. Thus, there is the need for culturing and expanding cells in-vitro for several weeks remain. Our aim is to investigate if long- term proliferation with minimal passaging will affect the stemness, paracrine secretions and carcinogenesis markers of ADSC. The immunophenotypic properties and aldehyde dehydrogenase (ALDH) activity of the initial stromal vascular fraction (SVF) and serially passaged ADSC were observed by flow cytometry. In parallel, the telomerase activity and the relative expression of oncogenes and tumor suppressor genes were assessed by q-PCR. We also assessed the cytokine secretion profile of passaged ADSC by an ELISA. The expanded ADSC retain their morphological and phenotypical characteristics. These cells maintained in culture for up to 12 weeks until P4, possessed stable telomerase and ALDH activity, without having a TP53 mutation. Furthermore, the relative expression levels of TP53, RB, and MDM2 were not affected while the relative expression of c-Myc decreased significantly. Finally, the levels of the secretions of PGE2, STC1, and TIMP2 were not affected but the levels of IL-6, VEGF, and TIMP 1 significantly decreased at P2. Our results suggest that the expansion of passaged ADSC does not affect the differentiation capacity of stem cells and does not confer a cancerous state or capacity in vitro to the cells. PMID:27632538

  3. Fat depot-specific gene signature and ECM remodeling of Sca1(high) adipose-derived stem cells.

    PubMed

    Tokunaga, Masakuni; Inoue, Mayumi; Jiang, Yibin; Barnes, Richard H; Buchner, David A; Chun, Tae-Hwa

    2014-06-01

    Stem cell antigen-1 (Sca1 or Ly6A/E) is a cell surface marker that is widely expressed in mesenchymal stem cells, including adipose-derived stem cells (ASCs). We hypothesized that the fat depot-specific gene signature of Sca1(high) ASCs may play the major role in defining adipose tissue function and extracellular matrix (ECM) remodeling in a depot-specific manner. Herein we aimed to characterize the unique gene signature and ECM remodeling of Sca1(high) ASCs isolated from subcutaneous (inguinal) and visceral (epididymal) adipose tissues. Sca1(high) ASCs are found in the adventitia and perivascular areas of adipose tissues. Sca1(high) ASCs purified with magnetic-activated cell sorting (MACS) demonstrate dendrite or round shape with the higher expression of cytokines and chemokines (e.g., Il6, Cxcl1) and the lower expression of a glucose transporter (Glut1). Subcutaneous and visceral fat-derived Sca1(high) ASCs particularly differ in the gene expressions of adhesion and ECM molecules. While the expression of the major membrane-type collagenase (MMP14) is comparable between the groups, the expressions of secreted collagenases (MMP8 and MMP13) are higher in visceral Sca1(high) ASCs than in subcutaneous ASCs. Consistently, slow but focal MMP-dependent collagenolysis was observed with subcutaneous adipose tissue-derived vascular stromal cells, whereas rapid and bulk collagenolysis was observed with visceral adipose tissue-derived cells in MMP-dependent and -independent manners. These results suggest that the fat depot-specific gene signatures of ASCs may contribute to the distinct patterns of ECM remodeling and adipose function in different fat depots.

  4. Mesenchymal Stem Cells from Bichat's Fat Pad: In Vitro Comparison with Adipose-Derived Stem Cells from Subcutaneous Tissue

    PubMed Central

    Broccaioli, Eugenio; Niada, Stefania; Rasperini, Giulio; Ferreira, Lorena Maria; Arrigoni, Elena; Yenagi, Vijay

    2013-01-01

    Abstract Adipose-derived stem/stromal cells (ASCs) are progenitor cells used in bone tissue engineering and regenerative medicine. Since Bichat's fat pad is easily accessible for dentists and maxillo-facial surgeons, we compared the features of ASCs from Bichat's fat pad (BFP-ASCs) with human ASCs from subcutaneous adipose tissue (SC-ASCs). BFP-ASCs isolated from a small amount of tissue were characterized for their stemness and multidifferentiative ability. They showed an important clonogenic ability and the typical mesenchymal stem cell immunophenotype. Moreover, when properly induced, osteogenic and adipogenic differentiation markers, such as alkaline phosphatase activity, collagen deposition and lipid vacuoles formation, were promptly observed. Growth of both BFP-ASCs and SC-ASCs in the presence of human serum and their adhesion to natural and synthetic scaffolds were also assessed. Both types of ASCs adapted rapidly to human autologous or heterologous sera, increasing their proliferation rate compared to standard culture condition, and all the cells adhered finely to bone, periodontal ligament, collagen membrane, and polyglycol acid filaments that are present in the oral cavity or are commonly used in oral surgery. At last, we showed that amelogenin seems to be an early osteoinductive factor for BFP-ASCs, but not SC-ASCs, in vitro. We conclude that Bichat's fat pad contains BFP-ASCs with stemness features that are able to differentiate and adhere to biological supports and synthetic materials. They are also able to proliferate in the presence of human serum. For all these reasons we propose BFP-ASCs for future therapies of periodontal defects and bone regeneration. PMID:23593563

  5. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

    SciTech Connect

    Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing Wang, Zehua

    2015-09-10

    Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.

  6. Reovirus safety study for proliferation and differentiation of human adipose-derived mesenchymal stem cells.

    PubMed

    Park, Jeong-Soo; Kim, Manbok

    2017-01-01

    Naturally occurring reoviruses are live replication-proficient viruses specifically infecting human cancer cells while sparing the normal counterparts. Stem cells can be highly susceptible to viral infection due to their innate high proliferation potential and other active signaling pathways of cells that might be involved in viral tropism. In the previous study, we showed that reoviruses could adversely affect murine embryonic stem cells' integrity in vitro and in vivo. Oncolytic viruses, delivered systemically face many hurdles that also impede their localization and infection of, metastatic tumors, due to a variety of immune and physical barriers. To overcome such hurdles to systemic delivery, several studies supported the idea that certain types of cells, including mesenchymal stem cells, might play a role as cell carriers for oncolytic viruses. Thus, it would be interesting to examine whether human adult stem cells such as human adipose-derived mesenchymal stem cells could be saved by the reoviral challenge. In this study, we report that biological activities such as proliferation and multipotency of human adipose-derived stem cells are not affected by wild-type reovirus challenge as evidenced by survival, osteogenic and adipogenic differentiation potential assays following treatment with reoviruses. Therefore, unlike murine embryonic stem cells, our study strongly suggests that human adipose-derived adult stem cells could be spared in vivo during wild-type reoviral anti-cancer therapeutics in a clinical setting. Furthermore, the results support the possible clinical use of human adipose-derived stem cells as an effective cell carrier of oncolytic reovirus to maximize their tumor tropism and anti-tumor activity.

  7. Isolation, Characterization, Differentiation, and Application of Adipose-Derived Stem Cells

    NASA Astrophysics Data System (ADS)

    Kuhbier, Jörn W.; Weyand, Birgit; Radtke, Christine; Vogt, Peter M.; Kasper, Cornelia; Reimers, Kerstin

    While bone marrow-derived mesenchymal stem cells are known and have been investigated for a long time, mesenchymal stem cells derived from the adipose tissue were identified as such by Zuk et al. in 2001. However, as subcutaneous fat tissue is a rich source which is much more easily accessible than bone marrow and thus can be reached by less invasive procedures, adipose-derived stem cells have moved into the research spotlight over the last 8 years.

  8. Adipose-derived Mesenchymal Stem Cells and Their Reparative Potential in Ischemic Heart Disease.

    PubMed

    Badimon, Lina; Oñate, Blanca; Vilahur, Gemma

    2015-07-01

    Adipose tissue has long been considered an energy storage and endocrine organ; however, in recent decades, this tissue has also been considered an abundant source of mesenchymal cells. Adipose-derived stem cells are easily obtained, show a strong capacity for ex vivo expansion and differentiation to other cell types, release a large variety of angiogenic factors, and have immunomodulatory properties. Thus, adipose tissue is currently the focus of considerable interest in the field of regenerative medicine. In the context of coronary heart disease, numerous experimental studies have supported the safety and efficacy of adipose-derived stem cells in the setting of myocardial infarction. These results have encouraged the clinical use of these stem cells, possibly prematurely. Indeed, the presence of cardiovascular risk factors, such as hypertension, coronary disease, diabetes mellitus, and obesity, alter and reduce the functionality of adipose-derived stem cells, putting in doubt the efficacy of their autologous implantation. In the present article, white adipose tissue is described, the stem cells found in this tissue are characterized, and the use of these cells is discussed according to the preclinical and clinical trials performed so far.

  9. Effects of nanoporous anodic titanium oxide on human adipose derived stem cells.

    PubMed

    Malec, Katarzyna; Góralska, Joanna; Hubalewska-Mazgaj, Magdalena; Głowacz, Paulina; Jarosz, Magdalena; Brzewski, Pawel; Sulka, Grzegorz D; Jaskuła, Marian; Wybrańska, Iwona

    The aim of current bone biomaterials research is to design implants that induce controlled, guided, successful, and rapid healing. Titanium implants are widely used in dental, orthopedic, and reconstructive surgery. A series of studies has indicated that cells can respond not only to the chemical properties of the biomaterial, but also, in particular, to the changes in surface topography. Nanoporous materials remain in focus of scientific queries due to their exclusive properties and broad applications. One such material is nanostructured titanium oxide with highly ordered, mutually perpendicular nanopores. Nanoporous anodic titanium dioxide (TiO2) films were fabricated by a three-step anodization process in propan-1,2,3-triol-based electrolyte containing fluoride ions. Adipose-derived stem cells offer many interesting opportunities for regenerative medicine. The important goal of tissue engineering is to direct stem cell differentiation into a desired cell lineage. The influence of nanoporous TiO2 with pore diameters of 80 and 108 nm on cell response, growth, viability, and ability to differentiate into osteoblastic lineage of human adipose-derived progenitors was explored. Cells were harvested from the subcutaneous abdominal fat tissue by a simple, minimally invasive, and inexpensive method. Our results indicate that anodic nanostructured TiO2 is a safe and nontoxic biomaterial. In vitro studies demonstrated that the nanotopography induced and enhanced osteodifferentiation of human adipose-derived stem cells from the abdominal subcutaneous fat tissue.

  10. Effects of nanoporous anodic titanium oxide on human adipose derived stem cells

    PubMed Central

    Malec, Katarzyna; Góralska, Joanna; Hubalewska-Mazgaj, Magdalena; Głowacz, Paulina; Jarosz, Magdalena; Brzewski, Pawel; Sulka, Grzegorz D; Jaskuła, Marian; Wybrańska, Iwona

    2016-01-01

    The aim of current bone biomaterials research is to design implants that induce controlled, guided, successful, and rapid healing. Titanium implants are widely used in dental, orthopedic, and reconstructive surgery. A series of studies has indicated that cells can respond not only to the chemical properties of the biomaterial, but also, in particular, to the changes in surface topography. Nanoporous materials remain in focus of scientific queries due to their exclusive properties and broad applications. One such material is nanostructured titanium oxide with highly ordered, mutually perpendicular nanopores. Nanoporous anodic titanium dioxide (TiO2) films were fabricated by a three-step anodization process in propan-1,2,3-triol-based electrolyte containing fluoride ions. Adipose-derived stem cells offer many interesting opportunities for regenerative medicine. The important goal of tissue engineering is to direct stem cell differentiation into a desired cell lineage. The influence of nanoporous TiO2 with pore diameters of 80 and 108 nm on cell response, growth, viability, and ability to differentiate into osteoblastic lineage of human adipose-derived progenitors was explored. Cells were harvested from the subcutaneous abdominal fat tissue by a simple, minimally invasive, and inexpensive method. Our results indicate that anodic nanostructured TiO2 is a safe and nontoxic biomaterial. In vitro studies demonstrated that the nanotopography induced and enhanced osteodifferentiation of human adipose-derived stem cells from the abdominal subcutaneous fat tissue. PMID:27789947

  11. Retinoic Acid Enhances the Differentiation of Adipose-Derived Stem Cells to Keratocytes In Vitro

    PubMed Central

    Lynch, Amy P.; Ahearne, Mark

    2017-01-01

    Purpose All-trans retinoic acid (RA) supplementation was investigated as a method of enhancing the differentiation of human adipose-derived stem cells (ASCs) to corneal keratocytes in vitro, in combination with a chemically defined serum-free medium. Methods Adipose-derived stem cells were cultured in monolayer and supplemented with 0.1, 1, or 10 μM RA for 14 days. The effects of RA on cell proliferation, migration, and extracellular matrix (ECM) accumulation were evaluated. In addition, the expression of phenotypic keratocyte markers was examined by reverse transcription polymerase chain reaction (PCR), immunocytochemistry, and Western blotting. Results Adipose-derived stem cells cultured with RA showed improved cell proliferation and ECM production. In addition, RA enhanced the expression of keratocyte-specific markers, keratocan, aldehyde dehydrogenase 3A1, lumican, and decorin, when compared to serum-free media alone. Furthermore, the presence of RA increased the amount of collagen type I while reducing the expression of fibrotic marker, α-smooth muscle actin. Conclusions These findings indicate that RA is a useful supplement for promoting a keratocyte phenotype in ASC. Translational Relevance This study is particularly important for the generation of biological corneal substitutes and next generation cell based therapies for corneal conditions. PMID:28138416

  12. Differential Mechanisms of Myocardial Conduction Slowing by Adipose Tissue-Derived Stromal Cells Derived from Different Species.

    PubMed

    Ten Sande, Judith N; Smit, Nicoline W; Parvizi, Mojtaba; van Amersfoorth, Shirley C M; Plantinga, Josée A; van Dessel, Pascal F H M; de Bakker, Jacques M T; Harmsen, Marco C; Coronel, Ruben

    2017-01-01

    Stem cell therapy is a promising therapeutic option to treat patients after myocardial infarction. However, the intramyocardial administration of large amounts of stem cells might generate a proarrhythmic substrate. Proarrhythmic effects can be explained by electrotonic and/or paracrine mechanisms. The narrow therapeutic time window for cell therapy and the presence of comorbidities limit the application of autologous cell therapy. The use of allogeneic or xenogeneic stem cells is a potential alternative to autologous cells, but differences in the proarrhythmic effects of adipose-derived stromal cells (ADSCs) across species are unknown. Using microelectrode arrays and microelectrode recordings, we obtained local unipolar electrograms and action potentials from monolayers of neonatal rat ventricular myocytes (NRVMs) that were cocultured with rat, human, or pig ADSCs (rADSCs, hADSCs, pADSCs, respectively). Monolayers of NRVMs were cultured in the respective conditioned medium to investigate paracrine effects. We observed significant conduction slowing in all cardiomyocyte cultures containing ADSCs, independent of species used (p < .01). All cocultures were depolarized compared with controls (p < .01). Only conditioned medium taken from cocultures with pADSCs and applied to NRVM monolayers demonstrated similar electrophysiological changes as the corresponding cocultures. We have shown that independent of species used, ADSCs cause conduction slowing in monolayers of NRVMs. In addition, pADSCs exert conduction slowing mainly by a paracrine effect, whereas the influence on conduction by hADSCs and rADSCs is preferentially by electrotonic interaction. Stem Cells Translational Medicine 2017;6:22-30.

  13. Differential Mechanisms of Myocardial Conduction Slowing by Adipose Tissue-Derived Stromal Cells Derived From Different Species.

    PubMed

    Ten Sande, Judith N; Smit, Nicoline W; Parvizi, Mojtaba; van Amersfoorth, Shirley C M; Plantinga, Josée A; van Dessel, Pascal F H M; de Bakker, Jacques M T; Harmsen, Marco C; Coronel, Ruben

    2016-08-02

    : Stem cell therapy is a promising therapeutic option to treat patients after myocardial infarction. However, the intramyocardial administration of large amounts of stem cells might generate a proarrhythmic substrate. Proarrhythmic effects can be explained by electrotonic and/or paracrine mechanisms. The narrow therapeutic time window for cell therapy and the presence of comorbidities limit the application of autologous cell therapy. The use of allogeneic or xenogeneic stem cells is a potential alternative to autologous cells, but differences in the proarrhythmic effects of adipose-derived stromal cells (ADSCs) across species are unknown. Using microelectrode arrays and microelectrode recordings, we obtained local unipolar electrograms and action potentials from monolayers of neonatal rat ventricular myocytes (NRVMs) that were cocultured with rat, human, or pig ADSCs (rADSCs, hADSCs, pADSCs, respectively). Monolayers of NRVMs were cultured in the respective conditioned medium to investigate paracrine effects. We observed significant conduction slowing in all cardiomyocyte cultures containing ADSCs, independent of species used (p < .01). All cocultures were depolarized compared with controls (p < .01). Only conditioned medium taken from cocultures with pADSCs and applied to NRVM monolayers demonstrated similar electrophysiological changes as the corresponding cocultures. We have shown that independent of species used, ADSCs cause conduction slowing in monolayers of NRVMs. In addition, pADSCs exert conduction slowing mainly by a paracrine effect, whereas the influence on conduction by hADSCs and rADSCs is preferentially by electrotonic interaction.

  14. Extracellular Vesicles Released from Human Umbilical Cord-Derived Mesenchymal Stromal Cells Prevent Life-Threatening Acute Graft-Versus-Host Disease in a Mouse Model of Allogeneic Hematopoietic Stem Cell Transplantation.

    PubMed

    Wang, Li; Gu, Zhenyang; Zhao, Xiaoli; Yang, Nan; Wang, Feiyan; Deng, Ailing; Zhao, Shasha; Luo, Lan; Wei, Huaping; Guan, Lixun; Gao, Zhe; Li, Yonghui; Wang, Lili; Liu, Daihong; Gao, Chunji

    2016-12-15

    Mesenchymal stromal cells (MSCs) are attractive agents for the prophylaxis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, safety concerns remain about their clinical application. In this study, we explored whether extracellular vesicles released from human umbilical cord-derived MSCs (hUC-MSC-EVs) could prevent aGVHD in a mouse model of allo-HSCT. hUC-MSC-EVs were intravenously administered to recipient mice on days 0 and 7 after allo-HSCT, and the prophylactic effects of hUC-MSC-EVs were assessed by observing the in vivo manifestations of aGVHD, histologic changes in target organs, and recipient mouse survival. We evaluated the effects of hUC-MSC-EVs on immune cells and inflammatory cytokines by flow cytometry and ProcartaPlex™ Multiplex Immunoassays, respectively. The in vitro effects of hUC-MSC-EVs were determined by mitogen-induced proliferation assays. hUC-MSC-EVs alleviated the in vivo manifestations of aGVHD and the associated histologic changes and significantly reduced the mortality of the recipient mice. Recipients treated with hUC-MSC-EVs had significantly lower frequencies and absolute numbers of CD3(+)CD8(+) T cells; reduced serum levels of IL-2, TNF-α, and IFN-γ; a higher ratio of CD3(+)CD4(+) and CD3(+)CD8(+) T cells; and higher serum levels of IL-10. An in vitro experiment demonstrated that hUC-MSC-EVs inhibited the mitogen-induced proliferation of splenocytes in a dose-dependent manner, and the cytokine changes were similar to those observed in vivo. This study indicated that hUC-MSC-EVs can prevent life-threatening aGVHD by modulating immune responses. These data provide the first evidence that hUC-MSC-EVs represent an ideal alternative in the prophylaxis of aGVHD after allo-HSCT.

  15. Origin of hemopoietic stromal progenitor cells in chimeras

    SciTech Connect

    Chertkov, J.L.; Drize, N.J.; Gurevitch, O.A.; Samoylova, R.S.

    1985-12-01

    Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice.

  16. Skeletal Muscle Regeneration in a Rat (Rattus norvegicus) Model with CorMatrix and Adipose Derived Stem Cells

    DTIC Science & Technology

    2015-07-16

    Model with CorMatrix and Adipose Derived Stem Cells ." PRINCIPAL INVESTIGATOR (Pl) I TRAINING COORDINATOR (TC): Major Lucas Neff DEPARTMENT...ability of the matrix to work in concert with adipose derived stem cells for further augmentation of healing. 3 FDGXXX Attachments: Attachment 1...additional pages if necessary.) PROTOCOL #: FDG20130044A DATE: 16 July 2015 PROTOCOL TITLE: "Skeletal Muscle Regeneration in a Rat (Rattus no1Vegicus

  17. Use of Adipose Derived Stem Cells to Treat Large Bone Defects

    DTIC Science & Technology

    2009-03-01

    innervation and the best way to achieve this is by using autologous stem cells. Our objective is to develop technology to use adipose-derived MSCs to treat...trauma. Most individuals have an adequate supply of fat tissue that can be used as a source of cells, but MSCs are in low abundance. Culture expansion...the inguinal fat pads of Sprague-Dawley rats, plated at 5,000 cells/cm2, and cultured in MSC growth media (GM) or osteogenic media (OM) (Lonza

  18. Therapeutic potential of human adipose-derived stem cells in neurological disorders.

    PubMed

    Chang, Keun-A; Lee, Jun-Ho; Suh, Yoo-Hun

    2014-01-01

    Stem cell therapy has been noted as a novel strategy to various diseases including neurological disorders such as Alzheimer's disease, Parkinson's disease, stroke, amyotrophic lateral sclerosis, and Huntington's disease that have no effective treatment available to date. The adipose-derived stem cells (ASCs), mesenchymal stem cells (MSCs) isolated from adipose tissue, are well known for their pluripotency with the ability to differentiate into various types of cells and immuno-modulatory property. These biological features make ASCs a promising source for regenerative cell therapy in neurological disorders. Here we discuss the recent progress of regenerative therapies in various neurological disorders utilizing ASCs.

  19. [Advance of adipose-derived stem cells in tendon tissue engineering].

    PubMed

    Yan, Mingming; Ni, Jiangdong

    2014-02-01

    Tendon tissue engineering is a novel therapeutic strategy for severe tendon injury and loss. Adipose derived stem cells (ASCs) have been studied extensively, due to their potency to differentiate into musculoskeletal tissue precursors such as osteoblasts, chondrocytes, adipocytes, and tendocytes under specific cues and high ability of proliferation. Resources of ASCs are ubiquitous and isolation of ASCs is secure, simple and minimally invasive. Mounting evidences demonstrate that ASCs may be involved in tendon tissue engineering and repair the severe injury of tendon under stimulation of various growth factors and other appropriate fittings.

  20. Tendon Regeneration with Tendon Hydrogel-Based Cell Delivery: A Comparison of Fibroblasts and Adipose-Derived Stem Cells.

    PubMed

    Chattopadhyay, Arhana; Galvez, Michael G; Bachmann, Michael; Legrand, Anais; McGoldrick, Rory; Lovell, Alberto; Jacobs, Mollie; Crowe, Chris; Umansky, Elise; Chang, James

    2016-09-01

    Tendon hydrogel is a promising biomaterial for improving repair strength after tendon injury. This study compares the capacity of fibroblasts and adipose-derived stem cells to proliferate, survive, and acquire tenogenic properties when seeded into tendon hydrogel in vitro and in vivo. The effect of cell density on hydrogel contraction was measured macroscopically. To assess tenogenic properties, RNA was isolated from cells seeded in vitro in hydrogel, and tenocyte markers were quantified. To assess in vitro proliferation and survival, MTS and live-dead assays were performed. Finally, to assess the in vivo survival of cells in hydrogel, subcutaneous injections were performed on rats and in vivo imaging was performed. At 0.5 million cells/ml, both the fibroblasts and adipose-derived stem cells induced minimal hydrogel contraction compared with higher cellular concentrations. Fibroblasts and adipose-derived stem cells seeded at 0.5 million cells/ml in tendon hydrogel up-regulated several tenocyte markers after 1 week. On MTS assay, fibroblasts and adipose-derived stem cells proliferated in hydrogel at similar rates. On live-dead assay, fibroblasts survived longer than adipose-derived stem cells. With use of the in vivo imaging system and histologic evaluation, fibroblasts survived longer than adipose-derived stem cells in hydrogel in vivo. Tendon healing is mediated by the proliferation, survival, and tenogenic differentiation of cells at the site of injury. Tendon hydrogel delivering dermal fibroblasts may improve and stimulate this process compared with adipose-derived stem cells. Future studies will be needed to evaluate the effects of this hydrogel-based cell delivery on chronic tendon injuries.

  1. Adipose-Derived Stem Cells Cocultured with Chondrocytes Promote the Proliferation of Chondrocytes

    PubMed Central

    2017-01-01

    Articular cartilage injury and defect caused by trauma and chronic osteoarthritis vascularity are very common, while the repair of injured cartilage remains a great challenge due to its limited healing capacity. Stem cell-based tissue engineering provides a promising treatment option for injured articular cartilage because of the cells potential for multiple differentiations. However, its application has been largely limited by stem cell type, number, source, proliferation, and differentiation. We hypothesized that (1) adipose-derived stem cells are ideal seed cells for articular cartilage repair because of their accessibility and abundance and (2) the microenvironment of articular cartilage could induce adipose-derived stem cells (ADSCs) to differentiate into chondrocytes. In order to test our hypotheses, we isolated stem cells from rabbit adipose tissues and cocultured these ADSCs with rabbit articular cartilage chondrocytes. We found that when ADSCs were cocultured with chondrocytes, the proliferation of articular cartilage chondrocytes was promoted, the apoptosis of chondrocytes was inhibited, and the osteogenic and chondrogenic differentiation of ADSCs was enhanced. The study on the mechanism of this coculture system indicated that the role of this coculture system is similar to the function of TGF-β1 in the promotion of chondrocytes. PMID:28133485

  2. Adipose-derived stem cells (ADSC) in the viability of random skin flap in rats.

    PubMed

    Suartz, Caio Vinicius; Gaiba, Silvana; França, Jerônimo Pereira de; Aloise, Antonio Carlos; Ferreira, Lydia Masako

    2014-01-01

    To evaluate the effects of the adipose-derived stem cells (ADSC) in the viability of random skin flap in rats. Thirty five adult male Wistar rats (weight 250-300 g) were used. ADSC were isolated from adult male rats (n=5). ADSC were separated, cultured and then analyzed. A dorsal skin flap measuring 10 x 4 cm was raised and a plastic barrier was placed between the flap and its bed. After the surgical procedure, the animals were randomized into two groups (n=15 each group), group control and group ADSC. In all groups the procedures were performed immediately after the surgery. The percentage of flap necrosis was measured on the seventh postoperative day. The ADSC were able to replicate in our culture conditions. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. The results were statistically significant showing that the ADSC decreased the area of necrosis (p<0.05). The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro. The administration of adipose-derived stem cells was effective to increase the viability of the random skin flaps in rats.

  3. Surface chemical functionalities affect the behavior of human adipose-derived stem cells in vitro

    NASA Astrophysics Data System (ADS)

    Liu, Xujie; Feng, Qingling; Bachhuka, Akash; Vasilev, Krasimir

    2013-04-01

    This study examines the effect of surface chemical functionalities on the behavior of human adipose-derived stem cells (hASCs) in vitro. Plasma polymerized films rich in amine (sbnd NH2), carboxyl (sbnd COOH) and methyl (sbnd CH3), were generated on hydroxyapatite (HAp) substrates. The surface chemical functionalities were characterized by X-ray photoelectron spectroscopy (XPS). The ability of different substrates to absorb proteins was evaluated. The results showed that substrates modified with hydrophilic functional group (sbnd COOH and sbnd NH2) can absorb more proteins than these modified with more hydrophobic functional group (sbnd CH3). The behavior of human adipose-derived stem cells (hASCs) cultured on different substrates was investigated in vitro: cell counting kit-8 (CCK-8) analysis was used to characterize cell proliferation, scanning electronic microscopy (SEM) analysis was used to characterize cell morphology and alkaline phosphatase (ALP) activity analysis was used to account for differentiation. The results of this study demonstrated that the sbnd NH2 modified surfaces encourage osteogenic differentiation; the sbnd COOH modified surfaces promote cell adhesion and spreading and the sbnd CH3 modified surfaces have the lowest ability to induce osteogenic differentiation. These findings confirmed that the surface chemical states of biomaterials can affect the behavior of hASCs in vitro.

  4. Adipose-derived adult stem cells: available technologies for potential clinical regenerative applications in dentistry.

    PubMed

    Catalano, Enrico; Cochis, Andrea; Varoni, Elena; Rimondini, Lia; Carrassi, Antonio; Azzimonti, Barbara

    2013-01-01

    Tissue homeostasis depends closely on the activity and welfare of adult stem cells. These cells represent a promising tool for biomedical research since they can aid in treatment and promote the regeneration of damaged organs in many human disorders. Adult stem cells indefinitely preserve their ability to self-renew and differentiate into various phenotypes; this capacity could be promoted in vitro by particular culture conditions (differentiation media) or spontaneously induced in vivo by exploiting the biochemical and mechanical properties of the tissue in which the stem cells are implanted. Among the different sources of adult stem cells, adipose tissue is an attractive possibility thanks to its ready availability and the standard extraction techniques at our disposal today. This review discusses the isolation, characterization, and differentiation of human adipose-derived adult stem cells, as well as regeneration strategies, therapeutic uses, and adverse effects of their delivery. In particular, since oral disorders (e.g., trauma, erosion, and chronic periodontitis) often cause the loss of dental tissue along with functional, phonetic, and aesthetic impairment, this review focuses on the application of human adipose-derived adult stem cells, alone or in combination with biomaterials, in treating oral diseases.

  5. Adipose-Derived Mesenchymal Stem Cells Ameliorate Lipid Metabolic Disturbance in Mice.

    PubMed

    Liu, Guang-Yang; Liu, Jin; Wang, You-Liang; Liu, Yang; Shao, Yong; Han, Yan; Qin, Ya-Ru; Xiao, Feng-Jun; Li, Peng-Fei; Zhao, Lan-Jun; Gu, En-Yan; Chen, Si-Yu; Gao, Li-Hua; Wu, Chu-Tse; Hu, Xian-Wen; Duan, Hai-Feng

    2016-09-01

    : Adipose-derived mesenchymal stem cells (AD-MSCs) have been shown to ameliorate hyperglycemia in diabetic animals and individuals. However, little is known about whether AD-MSCs affect lipid metabolism. Here we have demonstrated for the first time that AD-MSC infusion can significantly suppress the increase in body weight and remarkably improve dyslipidemia in db/db obese mice and diet-induced obesity mice. Induction of white fat tissue "browning" and activation of adenosine monophosphate-activated protein kinase and its downstream hormone-sensitive lipase in adipose tissue contribute to the antiobesity and lipid-lowering effects. Thus, AD-MSC infusion holds great therapeutic potential for dyslipidemia and associated cardiovascular diseases. Mesenchymal stem cells (MSCs) are considered one of the most promising types of stem cells for translational application because of their rich tissue sources, multilineage differentiation capacity, and easy amplification in vitro and unique immunobiological properties. This study demonstrated that adipose-derived MSCs (AD-MSCs) infusion can significantly suppress the increase in body weight and remarkably improve dyslipidemia in obese mice. Induction of white fat tissue "browning" and activation of adenosine monophosphate-activated protein kinase and its downstream hormone-sensitive lipase in adipose tissue were demonstrated to contribute to the antiobesity and lipid-lowering effects. Thus, AD-MSC infusion holds great therapeutic potential for dyslipidemia. ©AlphaMed Press.

  6. Biocellular Regenerative Medicine: Use of Adipose-Derived Stem/Stromal Cells and It's Native Bioactive Matrix.

    PubMed

    Alexander, Robert W

    2016-11-01

    "Using your own tissues to heal" represents a major health care paradigm change and is one of the most exciting minimally invasive options currently available. Biocellular regenerative therapies are rapidly improving in documentation and cellular analyses and are gaining good safety and efficacy profiles. Once considered purely experimental, they have entered into an accepted, translational period to clinical providers, backed by improving science supporting the basic hypotheses. It is a well-recognized and reported alternative to many traditional medical/surgical interventions.

  7. The effect of conjugating RGD into 3D alginate hydrogels on adipogenic differentiation of human adipose-derived stromal cells.

    PubMed

    Kang, Sun-Woong; Cha, Byung-Hyun; Park, Honghyun; Park, Kwang-Sook; Lee, Kuen Yong; Lee, Soo-Hong

    2011-05-12

    The effects of RGD peptide conjugation to alginate hydrogel on the adipogenic differentiation of ASCs was investigated. After 3 d of culture, RGD-modified alginate hydrogels significantly stimulated FAK and integrin α1 gene expressions and vinculin expression in ASCs. In addition, RGD-modified alginate hydrogels significantly enhanced the adipogenic differentiation of human ASCs to exhibit higher expression levels of oil red O staining and adipogenic genes compared to those of the control group (unmodified gels). These results suggest potential applications of RGD-modified alginate gels for adipose tissue regeneration.

  8. Original Research: Adipose-derived stem cells from younger donors, but not aging donors, inspire the host self-healing capability through its secreta.

    PubMed

    Ma, Ning; Qiao, Chenhui; Zhang, Weihua; Luo, Hong; Zhang, Xin; Liu, Donghai; Zang, Suhua; Zhang, Liang; Bai, Jingyun

    2017-01-01

    Adipose-derived stem cells demonstrate promising effects in promoting cutaneous wound healing, but the mechanisms are still not well defined and contradictory views are still debatable. In the present research, we established a mouse cutaneous wound model and investigated the effects of adipose-derived stem cells in wound healing. Adipocyte, adipose-derived stem cells, and epidermal keratinocyte stem cells were isolated from younger and aged donors according to the standard protocol. The conditioned medium either from adipose-derived stem cells or from adipocytes was used to treat epidermal keratinocyte cells. The results showed that adipocytes or adipose-derived stem cells isolated from younger donors demonstrated mild advantage over those cells isolated from aging donors. Adipose-derived stem cells showed stronger stimuli than adipocytes, and the adipose-derived stem cells or adipocytes from younger donors enabled to support higher growth rate of keratinocyte stem cells. The invasion of vasculature was observed at day 10 after posttransplantation in the mice bearing the keratinocyte stem cells or combination of keratinocyte stem cells with adipose-derived stem cells; however, simply inoculating keratinocyte stem cells from aging donors did not result in vasculature formation. Adipose-derived stem cells isolated from younger donors were able to inspire the host's self-healing capabilities, and age-associated factors should be taken into consideration when designing a feasible therapeutic treatment for skin regeneration. © 2016 by the Society for Experimental Biology and Medicine.

  9. Characterization of adipose-derived stem cells from subcutaneous and visceral adipose tissues and their function in breast cancer cells

    PubMed Central

    Ritter, Andreas; Friemel, Alexandra; Fornoff, Friderike; Adjan, Mouhib; Solbach, Christine

    2015-01-01

    Adipose-derived stem cells are capable of differentiating into multiple cell types and thus considered useful for regenerative medicine. However, this differentiation feature seems to be associated with tumor initiation and metastasis raising safety concerns, which requires further investigation. In this study, we isolated adipose-derived stem cells from subcutaneous as well as from visceral adipose tissues of the same donor and systematically compared their features. Although being characteristic of mesenchymal stem cells, subcutaneous adipose-derived stem cells tend to be spindle form-like and are more able to home to cancer cells, whereas visceral adipose-derived stem cells incline to be “epithelial”-like and more competent to differentiate. Moreover, compared to subcutaneous adipose-derived stem cells, visceral adipose-derived stem cells are more capable of promoting proliferation, inducing the epithelial-to-mesenchymal transition, enhancing migration and invasion of breast cancer cells by cell-cell contact and by secreting interleukins such as IL-6 and IL-8. Importantly, ASCs affect the low malignant breast cancer cells MCF-7 more than the highly metastatic MDA-MB-231 cells. Induction of the epithelial-to-mesenchymal transition is mediated by the activation of multiple pathways especially the PI3K/AKT signaling in breast cancer cells. BCL6, an important player in B-cell lymphoma and breast cancer progression, is crucial for this transition. Finally, this transition fuels malignant properties of breast cancer cells and render them resistant to ATP competitive Polo-like kinase 1 inhibitors BI 2535 and BI 6727. PMID:26439686

  10. Characterization of adipose-derived stem cells from subcutaneous and visceral adipose tissues and their function in breast cancer cells.

    PubMed

    Ritter, Andreas; Friemel, Alexandra; Fornoff, Friderike; Adjan, Mouhib; Solbach, Christine; Yuan, Juping; Louwen, Frank

    2015-10-27

    Adipose-derived stem cells are capable of differentiating into multiple cell types and thus considered useful for regenerative medicine. However, this differentiation feature seems to be associated with tumor initiation and metastasis raising safety concerns, which requires further investigation. In this study, we isolated adipose-derived stem cells from subcutaneous as well as from visceral adipose tissues of the same donor and systematically compared their features. Although being characteristic of mesenchymal stem cells, subcutaneous adipose-derived stem cells tend to be spindle form-like and are more able to home to cancer cells, whereas visceral adipose-derived stem cells incline to be "epithelial"-like and more competent to differentiate. Moreover, compared to subcutaneous adipose-derived stem cells, visceral adipose-derived stem cells are more capable of promoting proliferation, inducing the epithelial-to-mesenchymal transition, enhancing migration and invasion of breast cancer cells by cell-cell contact and by secreting interleukins such as IL-6 and IL-8. Importantly, ASCs affect the low malignant breast cancer cells MCF-7 more than the highly metastatic MDA-MB-231 cells. Induction of the epithelial-to-mesenchymal transition is mediated by the activation of multiple pathways especially the PI3K/AKT signaling in breast cancer cells. BCL6, an important player in B-cell lymphoma and breast cancer progression, is crucial for this transition. Finally, this transition fuels malignant properties of breast cancer cells and render them resistant to ATP competitive Polo-like kinase 1 inhibitors BI 2535 and BI 6727.

  11. Activin B Regulates Adipose-derived Mesenchymal Stem Cells to Promote Skin Wound Healing via Activation of the MAPK Signaling Pathway.

    PubMed

    Zhang, Lei; Xu, Pengcheng; Wang, Xueer; Zhang, Min; Yan, Yuan; Chen, Yinghua; Zhang, Lu; Zhang, Lin

    2017-04-07

    Adipose-derived stem cells (ADSCs) are multipotent stromal cells that can differentiate into a variety of cell types, including skin cells, and they can provide an abundant source of cells for skin tissue engineering and skin wound healing. The purpose of this study is to explore the therapeutic effects of activin B in combination with ADSCs and the possible signaling mechanism. In this study, we found that activin B was able to promote ADSC migration by inducing actin stress fiber formation in vitro. In vivo, activin B in combination with ADSCs was capable of enhancing α-SMA expression and wound closure. This combined treatment also promoted fibroblast and keratinocyte proliferation and accelerated re-epithelialization and collagen deposition. Moreover, activin B in combination with ADSCs boosted angiogenesis in the wound area. Further study of the mechanism revealed that activation of JNK and ERK signaling, but not p38 signaling, were required for activin B-induced ADSC actin stress fiber formation and cell migration. These results showed that activin B was able to activate JNK and ERK signaling pathways to induce actin stress fiber formation and ADSC migration to promote wound healing. These results suggest that combined treatment with activin B and ADSCs is a promising therapeutic strategy for the management of serious skin wounds.

  12. The differentiation of human adipose-derived stem cells (hASCs) into osteoblasts is promoted by low amplitude, high frequency vibration treatment.

    PubMed

    Prè, D; Ceccarelli, G; Gastaldi, G; Asti, A; Saino, E; Visai, L; Benazzo, F; Cusella De Angelis, M G; Magenes, G

    2011-08-01

    Several studies have demonstrated that tissue culture conditions influence the differentiation of human adipose-derived stem cells (hASCs). Recently, studies performed on SAOS-2 and bone marrow stromal cells (BMSCs) have shown the effectiveness of high frequency vibration treatment on cell differentiation to osteoblasts. The aim of this study was to evaluate the effects of low amplitude, high frequency vibrations on the differentiation of hASCs toward bone tissue. In view of this goal, hASCs were cultured in proliferative or osteogenic media and stimulated daily at 30Hz for 45min for 28days. The state of calcification of the extracellular matrix was determined using the alizarin assay, while the expression of extracellular matrix and associated mRNA was determined by ELISA assays and quantitative RT-PCR (qRT-PCR). The results showed the osteogenic effect of high frequency vibration treatment in the early stages of hASC differentiation (after 14 and 21days). On the contrary, no additional significant differences were observed after 28days cell culture. Transmission Electron Microscopy (TEM) images performed on 21day samples showed evidence of structured collagen fibers in the treated samples. All together, these results demonstrate the effectiveness of high frequency vibration treatment on hASC differentiation toward osteoblasts.

  13. Xeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Escobar, Carlos Hugo

    2016-01-01

    Molecules of animal or bacterial origin, which pose a risk for zoonoses or immune rejection, are commonly used for extraction, culture, and cryopreservation of mesenchymal stem cells. There is no sequential and orderly protocol for producing human adipose-derived stem cells (hASCs) under xeno-free conditions. After standardizing a human platelet lysate (hPL) production protocol, four human adipose tissue samples were processed through explants with fetal bovine serum (FBS)-supplemented or hPL-supplemented media for extracting the adipose-derived stem cells. The cells were cultivated in cell culture medium + hPL (5%) or FBS (10%). The cellular replication rate, immunophenotype, and differentiation potential were evaluated at fourth passage. Cellular viability was evaluated before and after cryopreservation of the cells, with an hPL-based solution compared with an FBS-based solution. The explants cultured in hPL-supplemented media showed earlier and faster hASC proliferation than did those supplemented with FBS. Likewise, cells grown in hPL-supplemented media showed a greater proliferation rate, without losing the immunophenotype. Osteogenic differentiation of xeno-free hASC was higher than the hASC produced in standard conditions. However, adipogenic differentiation was reduced in xeno-free hASC. Finally, the cells cryopreserved in an hPL-based solution showed a higher cellular viability than the cells cryopreserved in an FBS-based. In conclusion, we have developed a complete xeno-free protocol for extracting, culturing, and cryopreserving hASCs that can be safely implemented in clinical studies. Significance This study was performed to standardize a complete ordered protocol to produce xeno-free human adipose-derived mesenchymal stem cells (hASCs) as a safe therapeutic alternative. Cells were extracted by adipose tissue explants and then cultured and cryopreserved using human platelet lysate (hPL). Different scientific journals have published data regarding the use

  14. miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells.

    PubMed

    Zhang, Zi-ji; Zhang, Hao; Kang, Yan; Sheng, Pu-yi; Ma, Yuan-chen; Yang, Zi-bo; Zhang, Zhi-qi; Fu, Ming; He, Ai-shan; Liao, Wei-ming

    2012-03-01

    Human adipose-derived stem cells (hADSC) are capable of differentiating into an osteogenic lineage. It is believed that microRNAs (miRNAs) play important roles in regulating this osteogenic differentiation of human adipose-derived cells, although its molecular mechanism remains unclear. We investigated the miRNA expression profile during osteogenic differentiation of hADSCs, and assessed the roles of involved miRNAs during the osteogenic differentiation. We obtained and cultured human adipose-derived stems cells from donors who underwent elective liposuction or other abdominal surgery at our institution. miRNA expression profiles pre- and post-osteogenic induction were obtained using microarray essay, and differently expressed miRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of osteogenic proteins was detected using an enzyme-linked immunosorbent assay. Putative targets of the miRNAs were predicted using online software MiRanda, TargetScan, and miRBase. Eight miRNAs were found differently expressed pre- and post-osteogenic induction, among which four miRNAs (miR-17, miR-20a, miR-20b, and miR-106a) were up-regulated and four miRNAs (miR-31, miR-125a-5p, miR-125b, and miR-193a) were down-regulated. qRT-PCR analysis further confirmed the results. Predicted target genes of the differentially expressed miRNAs based on the overlap from three public prediction algorithms: MiRanda, TargetScan, and miRBase Target have the known functions of regulating stem cell osteogenic differentiation, self-renewal, signal transduction, and cell cycle control. We identified a group of miRNAs that may play important roles in regulating hADSC cell differentiation toward an osteoblast lineage. Further study of these miRNAs may elucidate the mechanism of hADSC differentiation into adipose tissue, and thus provide basis for tissue engineering. © 2011 Wiley Periodicals, Inc.

  15. The therapeutic effects of human adipose-derived stem cells in Alzheimer's disease mouse models.

    PubMed

    Chang, Keun-A; Kim, Hee Jin; Joo, Yuyoung; Ha, Sungji; Suh, Yoo-Hun

    2014-01-01

    Alzheimer's disease (AD) is an irreversible neurodegenerative disease, still lacking proper clinical treatment. Therefore, many researchers have focused on the possibility of therapeutic use of stem cells for AD. Adipose-derived stem cells (ASCs), mesenchymal stem cells (MSCs) isolated from adipose tissue, are well known for their pluripotency and their ability to differentiate into multiple tissue types and have immune modulatory properties similar to those of MSCs from other origins. Because of their biological properties, ASCs can be considered for cell therapy and neuroregeneration. Our recent results clearly showed the therapeutic potential of these cells after transplantation into Tg2576 mice (an AD mouse model). Intravenously or intracerebrally transplanted human ASCs (hASCs) greatly improved the memory impairment and the neuropathology, suggesting that hASCs have a high therapeutic potential for AD.

  16. Adipose-Derived Stem Cells for Tissue Engineering and Regenerative Medicine Applications

    PubMed Central

    Dai, Ru; Wang, Zongjie; Samanipour, Roya; Koo, Kyo-in; Kim, Keekyoung

    2016-01-01

    Adipose-derived stem cells (ASCs) are a mesenchymal stem cell source with properties of self-renewal and multipotential differentiation. Compared to bone marrow-derived stem cells (BMSCs), ASCs can be derived from more sources and are harvested more easily. Three-dimensional (3D) tissue engineering scaffolds are better able to mimic the in vivo cellular microenvironment, which benefits the localization, attachment, proliferation, and differentiation of ASCs. Therefore, tissue-engineered ASCs are recognized as an attractive substitute for tissue and organ transplantation. In this paper, we review the characteristics of ASCs, as well as the biomaterials and tissue engineering methods used to proliferate and differentiate ASCs in a 3D environment. Clinical applications of tissue-engineered ASCs are also discussed to reveal the potential and feasibility of using tissue-engineered ASCs in regenerative medicine. PMID:27057174

  17. [Nuclear heterogeneity and proliferative capacity of human adipose derived MSC-like cells].

    PubMed

    Lavrov, A V; Smirnichina, S A

    2010-01-01

    Adipose derived stem cells (ADSCs) are MSC-like cells which could be easily used for regenerative medicine. Here, the morphology and proliferative capacity of human ADSCs is discribed. ADSCs were analyzed after one month of cultivation at a density of 10 cells/cm2. 21 colonies were counted. Few atypical cells (huge nuclei and cytoplasm) were found in 9 out of 17 colonies analyzed. ANOVA demonstrated that colonies also differed (P = 0.0025) in nuclei dimensions and scatter in the dimensions in each colony. Nuclei dimensions and cell density logarithms correlated in reverse proportion (-0.7; P = 0.002). Thus, ADSCs were heterogeneous and represented two types of cells: small highly proliferative and large low proliferative cells. Cell heterogeneity observed in some colonies might be due to cells registered at different cell cycle phases. Stable and typical morphology, colony-formation capability and high proliferative capacity of cells indicate visceral adipose tissue as a rich source of ADSCs.

  18. Adipose-Derived Stem Cells in Functional Bone Tissue Engineering: Lessons from Bone Mechanobiology

    PubMed Central

    Bodle, Josephine C.; Hanson, Ariel D.

    2011-01-01

    This review aims to highlight the current and significant work in the use of adipose-derived stem cells (ASC) in functional bone tissue engineering framed through the bone mechanobiology perspective. Over a century of work on the principles of bone mechanosensitivity is now being applied to our understanding of bone development. We are just beginning to harness that potential using stem cells in bone tissue engineering. ASC are the primary focus of this review due to their abundance and relative ease of accessibility for autologous procedures. This article outlines the current knowledge base in bone mechanobiology to investigate how the knowledge from this area has been applied to the various stem cell-based approaches to engineering bone tissue constructs. Specific emphasis is placed on the use of human ASC for this application. PMID:21338267

  19. Implications for human adipose-derived stem cells in plastic surgery

    PubMed Central

    Banyard, Derek A; Salibian, Ara A; Widgerow, Alan D; Evans, Gregory R D

    2015-01-01

    Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs) that possess many of the same regenerative properties as other MSCs. However, the ubiquitous presence of ADSCs and their ease of access in human tissue have led to a burgeoning field of research. The plastic surgeon is uniquely positioned to harness this technology because of the relative frequency in which they perform procedures such as liposuction and autologous fat grafting. This review examines the current landscape of ADSC isolation and identification, summarizes the current applications of ADSCs in the field of plastic surgery, discusses the risks associated with their use, current barriers to universal clinical translatability, and surveys the latest research which may help to overcome these obstacles. PMID:25425096

  20. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    SciTech Connect

    Kakudo, Natsuko . E-mail: kakudon@takii.kmu.ac.jp; Shimotsuma, Ayuko; Kusumoto, Kenji

    2007-07-27

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration.

  1. Xeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells.

    PubMed

    Escobar, Carlos Hugo; Chaparro, Orlando

    2016-03-01

    Molecules of animal or bacterial origin, which pose a risk for zoonoses or immune rejection, are commonly used for extraction, culture, and cryopreservation of mesenchymal stem cells. There is no sequential and orderly protocol for producing human adipose-derived stem cells (hASCs) under xeno-free conditions. After standardizing a human platelet lysate (hPL) production protocol, four human adipose tissue samples were processed through explants with fetal bovine serum (FBS)-supplemented or hPL-supplemented media for extracting the adipose-derived stem cells. The cells were cultivated in cell culture medium + hPL (5%) or FBS (10%). The cellular replication rate, immunophenotype, and differentiation potential were evaluated at fourth passage. Cellular viability was evaluated before and after cryopreservation of the cells, with an hPL-based solution compared with an FBS-based solution. The explants cultured in hPL-supplemented media showed earlier and faster hASC proliferation than did those supplemented with FBS. Likewise, cells grown in hPL-supplemented media showed a greater proliferation rate, without losing the immunophenotype. Osteogenic differentiation of xeno-free hASC was higher than the hASC produced in standard conditions. However, adipogenic differentiation was reduced in xeno-free hASC. Finally, the cells cryopreserved in an hPL-based solution showed a higher cellular viability than the cells cryopreserved in an FBS-based. In conclusion, we have developed a complete xeno-free protocol for extracting, culturing, and cryopreserving hASCs that can be safely implemented in clinical studies. ©AlphaMed Press.

  2. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells.

    PubMed

    Barretto, Letícia Siqueira de Sá; Lessio, Camila; Sawaki e Nakamura, Ahy Natally; Lo Turco, Edson Guimarães; da Silva, Camila Gonzaga; Zambon, João Paulo; Gozzo, Fábio César; Pilau, Eduardo Jorge; de Almeida, Fernando Gonçalves

    2014-10-01

    Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.

  3. Effects of hypergravity on adipose-derived stem cell morphology, mechanical property and proliferation

    SciTech Connect

    Tavakolinejad, Alireza; Rabbani, Mohsen; Janmaleki, Mohsen

    2015-08-21

    Alteration in specific inertial conditions can lead to changes in morphology, proliferation, mechanical properties and cytoskeleton of cells. In this report, the effects of hypergravity on morphology of Adipose-Derived Stem Cells (ADSCs) are indicated. ADSCs were repeatedly exposed to discontinuous hypergravity conditions of 10 g, 20 g, 40 g and 60 g by utilizing centrifuge (three times of 20 min exposure, with an interval of 40 min at 1 g). Cell morphology in terms of length, width and cell elongation index and cytoskeleton of actin filaments and microtubules were analyzed by image processing. Consistent changes observed in cell elongation index as morphological change. Moreover, cell proliferation was assessed and mechanical properties of cells in case of elastic modulus of cells were evaluated by Atomic Force Microscopy. Increase in proliferation and decrease in elastic modulus of cells are further results of this study. Staining ADSC was done to show changes in cytoskeleton of the cells associated to hypergravity condition specifically in microfilament and microtubule components. After exposing to hypergravity, significant changes were observed in microfilaments and microtubule density as components of cytoskeleton. It was concluded that there could be a relationship between changes in morphology and MFs as the main component of the cells. - Highlights: • Hypergravity (10 g, 20 g, 40 g and 60 g) affects on adipose derived stem cells (ADSCs). • ADSCs after exposure to the hypergravity are more slender. • The height of ADSCs increases in all test groups comparing their control group. • Hypergravity decreases ADSCs modulus of elasticity and cell actin fiber content. • Hypergravity enhances proliferation rate of ADSCs.

  4. Characterization of adipose-derived stem cells of anatomical region from mice.

    PubMed

    Luna, Arthur C L; Madeira, Maria E P; Conceição, Thais O; Moreira, José A L C; Laiso, Rosa A N; Maria, Durvanei A

    2014-08-20

    Stem cells constitute a group of great capacity for self-renewal, long-term viability, and multi-lineage potential. Several studies have provided evidence that adipose tissue represents an alternative source of stem cells, with the main benefit of adipose-derived stem cells being that they can be easily harvested from patients by a simple minimally invasive method and can be easily cultured. The aim of this study was to establish a culture protocol for obtaining and characterizing adipose-derived stem cells (ADSCs) from C57BL/6 J mice. The results showed that the yield, viability, and cell morphology obtained differ according to the age of isolated anatomic regions of the adipose tissue from ovarian and epididymis. The results of determination of cyclin D1 showed uniformity in the expression between different populations of ADSCs. A significant increase in the expression of caspase-3 active, was also observed in large cell populations from mice after 120 days. ADSCs were positive for mesenchymal markers CD90 and CD105, Nanog, SSEA-1, CD106, and VEGFR-1, and negative for hematopoietic markers CD34 and CD45. A large number of cells in S + G2/M phases was also observed for both sexes, demonstrating high proliferative capacity of ADSCs. We observed that the adipose tissue of C57BL/6 J mice, isolated from the studied anatomic regions, is a promising source for obtaining pluripotent mesenchymal stem cells with high viability and proliferative response.

  5. Standardized Sophora pachycarpa Root Extract Enhances Osteogenic Differentiation in Adipose-derived Human Mesenchymal Stem Cells.

    PubMed

    Mollazadeh, Samaneh; Neshati, Vajiheh; Fazly Bazzaz, Bibi Sedigheh; Iranshahi, Mehrdad; Mojarrad, Majid; Naderi-Meshkin, Hojjat; Kerachian, Mohammad Amin

    2017-05-01

    Bone defect is an important topic in public health. Novel therapies are based on osteogenic induction by natural antiosteoporotic compounds including plant-derived estrogens. In the current study, the osteogenic potential of Sophora pachycarpa root extract (SPRE) was explored on human adipose-derived mesenchymal stem cells. Herein, adipose-derived mesenchymal stem cells were osteoinducted in the presence of increased concentrations of the extract for 21 days. Then, cell viability was evaluated by MTT assay, and the differentiated cells were stained by Alizarin Red S for calcium deposition and subjected to alkaline phosphatase (ALP) assay for enzymatic activity. To assess the expression of bone-related genes, treated cells were evaluated by real-time polymerase chain reaction. The MTT test demonstrated that SPRE had no toxic effects on the cell viability. Treating the cells with SPRE noticeably promoted ALP activity, mineralization, and mRNA expression of runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). Additionally, cells subjected to 0.1 μg/mL SPRE showed the highest osteogenic effects. According to high-performance liquid chromatography fingerprinting of SPRE, the osteoprotective effects of SPRE is probably due to presence of phytochemicals with estrogen-like activity in the extract. Thus, SPRE might be a suitable therapeutic agent for bone defects therapy in the future research. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Comparative analysis of adipose-derived mesenchymal stem cells isolated from abdominal and breast tissue.

    PubMed

    Hanson, Summer E; Kim, Jaehyup; Hematti, Peiman

    2013-08-01

    Adipose-derived mesenchymal stem cells (ADSC) may have a potential dual role in soft tissue augmentation by suppressing inflammation and promoting regeneration. Due to these properties, there is increasing interest in their potential use in autologous fat grafting, particularly to the breast. The authors isolate and compare ADSC derived from abdominal and breast tissues with a hypothesis that different adipose tissue sources may demonstrate different functional characteristics affecting outcomes in autologous cell transplantation in reconstructive and aesthetic surgery. Adipose-derived mesenchymal stem cells from abdominal and breast tissues were isolated and compared in terms of surface marker expression, differentiation capabilities, and both fibroblast growth factor (FGF) and receptor expression. Immunophenotype of macrophages was also investigated using cell surface markers following a 7-day co-culture period with ADSC. Results showed similar cell surface phenotype and multilineage differentiation capabilities of ADSC derived from abdominal and breast tissues. Variations of FGF expression were demonstrated on reverse transcription polymerase chain reaction, with a significantly higher expression of FGF2 seen in breast ADSC. Following the 7-day co-culture period, increased expression of the anti-inflammatory surface marker CD206 was identified, with decreased CD16 and human leukocyte antigen-DR on macrophages co-cultured with ADSC compared with controls. The data indicate similarities between ADSC derived from abdominal and breast tissues. Significant differences were seen, however, in the expression of FGF2, which is important in angiogenesis and wound healing. The results support the utility of ADSC in cell-based therapies such as autologous fat grafting.

  7. Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells.

    PubMed

    Swioklo, Stephen; Constantinescu, Andrei; Connon, Che J

    2016-03-01

    Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C-23°C) preservation of human adipose-derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature-dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 10(6) cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno-free, serum-free,current Good Manufacturing Practice-grade medium. We present a simple, low-cost system capable of enhancing the preservation of human adipose-derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state. ©AlphaMed Press.

  8. In vitro induction of human adipose-derived stem cells into lymphatic endothelial-like cells.

    PubMed

    Yang, Yi; Chen, Xiao-hu; Li, Fu-gui; Chen, Yun-xian; Gu, Li-qiang; Zhu, Jia-kai; Li, Ping

    2015-02-01

    Human adipose-derived stem cells (hADSCs) may provide a suitable number of progenitors for the treatment of lymphatic edema; however, to date the protocols for inducing hADSCs into this tissue type have not been standardized. We wished to investigate the induction of hADSCs into lymphatic endothelial-like cells using vascular endothelial growth factor-C156S (VEGF-C156S) and other growth factors in vitro. hADSCs from healthy adult adipose tissue were purified using enzyme digestion. Differentiation was induced using medium containing VEGF-C156S and bovine fibroblast growth factor (bFGF). Differentiation was confirmed using immunostaining for lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and fms-related tyrosine kinase 4 (FLT-4), two lymphatic endothelial cell markers. The expression levels of LYVE-1, prospero homeobox 1 (PROX-1), and FLT-4 throughout induction were assessed using reverse transcriptase quantitative polymerase chain reaction. hADSCs were successfully obtained by trypsin digest and purification. Flow cytometry showed these cells were similar to mesenchymal stem cells, with a high positive rate of CD13, CD29, CD44, and CD105, and a low positive rate of CD31, CD34, CD45, and HLA-DR. Induction to lymphatic endothelial-like cells was successful, with cells expressing high levels of LYVE-1, PROX-1, and FLT-4. Adipose-derived stem cells can be induced to differentiate into lymphatic endothelial-like cells using a medium containing VEGF-C156S, bFGF, and other growth factors. This population of lymphatic endothelial-like cells may be useful for lymphatic reconstruction in the future.

  9. Autologous adipose-derived stem cells: Basic science, technique, and rationale for application in ulcer and wound healing.

    PubMed

    Zollino, Ilaria; Zuolo, Michele; Gianesini, Sergio; Pedriali, Massimo; Sibilla, Maria Grazia; Tessari, Mirko; Carinci, Francesco; Occhionorelli, Savino; Zamboni, Paolo

    2017-04-01

    Objectives The present review represents a translational boundary between basic research and surgery, particularly focusing on the promising application of adipose-derived stem cells harvested intra-operatively during debridement of venous leg ulcers. Methods We reviewed 830 out of 5578 articles on MEDLINE starting from 1997 and sorted by the relevance option. Results The technique currently used for adipose-derived stem cells intra-operative harvesting is presented, including a safety evaluation on a cohort of 5089 revised patients who underwent plastic surgery and maxillo-facial surgical procedures. Complications were reported in 169 cases (3.3%). One hundred and forty-one (2.77%) patients were classified as having minor complications, specifically: nodularity/induration 93 (1.83%), dysesthesia 14 (0.26%), hematoma 12 (0.23%), superficial infection 11 (0.21%), pain 7 (0.13%), poor cosmesis 3 (0.06%), and abnormal breast secretion 1 (0.02%), while 28 patients (0.55%) were classified as having major complications, specifically: deep infection 22 (0.43%), sepsis 3 (0.06%), abdominal hematoma 2 (0.04%), and pneumothorax 1 (0.02%). Application of cell therapy in venous leg ulcer is currently used only for patients not responding to the standard treatment. The review shows the lack of randomized clinical trials for application of adipose-derived stem cells among treatments for venous leg ulcer. Finally, adipose-derived stem cells implantation at the wound site promotes a new tissue formation rich in vascular structures and remodeling collagen. Conclusion Adipose-derived stem cells strategy represents a great opportunity for the treatment of chronic wounds, due to the simplicity of the technique and the application of cell treatment in the operating room immediately following debridement. However, clinical studies and data from randomized trials are currently lacking.

  10. Sirtuins 1-7 expression in human adipose-derived stem cells from subcutaneous and visceral fat depots: influence of obesity and hypoxia.

    PubMed

    Mariani, Stefania; Di Rocco, Giuliana; Toietta, Gabriele; Russo, Matteo A; Petrangeli, Elisa; Salvatori, Luisa

    2016-11-14

    The sirtuin family comprises seven NAD(+)-dependent deacetylases which control the overall health of organisms through the regulation of pleiotropic metabolic pathways. Sirtuins are important modulators of adipose tissue metabolism and their expression is higher in lean than obese subjects. At present, the role of sirtuins in adipose-derived stem cells has not been investigated yet. Therefore, in this study, we evaluated the expression of the complete panel of sirtuins in adipose-derived stem cells isolated from both subcutaneous and visceral fat of non-obese and obese subjects. We aimed at investigating the influence of obesity on sirtuins' levels, their role in obesity-associated inflammation, and the relationship with the peroxisome proliferator-activated receptor delta, which also plays functions in adipose tissue metabolism. The mRNA levels in the four types of adipose-derived stem cells were evaluated by quantitative polymerase chain reaction, in untreated cells and also after 8 h of hypoxia exposure. Correlations among sirtuins' expression and clinical and molecular parameters were also analyzed. We found that sirtuin1-6 exhibited significant higher mRNA expression in visceral adipose-derived stem cells compared to subcutaneous adipose-derived stem cells of non-obese subjects. Sirtuin1-6 levels were markedly reduced in visceral adipose-derived stem cells of obese patients. Sirtuins' expression in visceral adipose-derived stem cells correlated negatively with body mass index and C-reactive protein and positively with peroxisome proliferator-activated receptor delta. Finally, only in the visceral adipose-derived stem cells of obese patients hypoxia-induced mRNA expression of all of the sirtuins. Our results highlight that sirtuins' levels in adipose-derived stem cells are consistent with protective effects against visceral obesity and inflammation, and suggest a transcriptional mechanism through which acute hypoxia up-regulates sirtuins in the visceral

  11. Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer.

    PubMed

    Feng, Y; Zhu, M; Dangelmajer, S; Lee, Y M; Wijesekera, O; Castellanos, C X; Denduluri, A; Chaichana, K L; Li, Q; Zhang, H; Levchenko, A; Guerrero-Cazares, H; Quiñones-Hinojosa, A

    2014-12-11

    Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM.

  12. Periostin enhances adipose-derived stem cell adhesion, migration, and therapeutic efficiency in Apo E deficient mice with hind limb ischemia.

    PubMed

    Qin, Jinbao; Yuan, Fukang; Peng, Zhiyou; Ye, Kaichuang; Yang, Xinrui; Huang, Lijia; Jiang, Mier; Lu, Xinwu

    2015-07-24

    Therapeutic angiogenesis by transplantation of autologous/allogeneic adipose-derived stem cells (ADSCs) is a potential approach for severe ischemic diseases. However, poor viability, adhesion, migration and differentiation limit the therapeutic efficiency after the cells were transplanted into the targeted area. Periostin, an extracellular matrix protein, exhibits a critical role in wound repair as well as promotes cell adhesion, survival, and angiogenesis. ADSCs were obtained and genetically engineered with periostin gene (P-ADSCs). The viability, proliferation, migration, and apoptosis of P-ADSCs under hypoxia were analyzed. Moreover, P-ADSCs were implanted into Apo E deficient mice with hind limb ischemia. The Laser Doppler perfusion index, immunofluorescence, and histological pathology assay were tested to evaluate the therapeutic effects. The associated molecular mechanism of periostin on the proliferation, adhesion, migration, and differentiation of ADSCs was also analyzed. The in vitro studies have shown that periostin-transfected ADSCs (P-ADSCs) promoted viability, proliferation, and migration of ADSCs. Apoptosis of ADSCs was inhibited under hypoxic conditions. The Laser Doppler perfusion index was significantly higher in the P-ADSCs group compared with that in the ADSC and control groups after 4 weeks. Immunofluorescence and histological pathology assay showed that the P-ADSCs were in and around the ischemic sites, and some cells differentiated into capillaries and endothelium. Microvessel densities were significantly improved in P-ADSCs group compared with those in the control group. The molecular mechanisms that provide the beneficial effects of periostin were connected with the upregulated expression of integrinβ1/FAK/PI3K/Akt/eNOS signal pathway and the increased secretion of growth factors. Overexpression of periostin by gene transfection on ADSCs promotes survival, migration, and therapeutic efficiency, which will bring new insights into the

  13. Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer

    PubMed Central

    Feng, Y; Zhu, M; Dangelmajer, S; Lee, Y M; Wijesekera, O; Castellanos, C X; Denduluri, A; Chaichana, K L; Li, Q; Zhang, H; Levchenko, A; Guerrero-Cazares, H; Quiñones-Hinojosa, A

    2014-01-01

    Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM. PMID:25501828

  14. Ultrasound-Assisted Liposuction Does Not Compromise the Regenerative Potential of Adipose-Derived Stem Cells.

    PubMed

    Duscher, Dominik; Atashroo, David; Maan, Zeshaan N; Luan, Anna; Brett, Elizabeth A; Barrera, Janos; Khong, Sacha M; Zielins, Elizabeth R; Whittam, Alexander J; Hu, Michael S; Walmsley, Graham G; Pollhammer, Michael S; Schmidt, Manfred; Schilling, Arndt F; Machens, Hans-Günther; Huemer, Georg M; Wan, Derrick C; Longaker, Michael T; Gurtner, Geoffrey C

    2016-02-01

    Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31-/CD45-), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance: Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency

  15. Ultrasound-Assisted Liposuction Does Not Compromise the Regenerative Potential of Adipose-Derived Stem Cells

    PubMed Central

    Duscher, Dominik; Atashroo, David; Maan, Zeshaan N.; Luan, Anna; Brett, Elizabeth A.; Barrera, Janos; Khong, Sacha M.; Zielins, Elizabeth R.; Whittam, Alexander J.; Hu, Michael S.; Walmsley, Graham G.; Pollhammer, Michael S.; Schmidt, Manfred; Schilling, Arndt F.; Machens, Hans-Günther; Huemer, Georg M.; Wan, Derrick C.; Longaker, Michael T.

    2016-01-01

    Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31−/CD45−), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency

  16. Comparative Analysis of Media and Supplements on Initiation and Expansion of Adipose-Derived Stem Cells

    PubMed Central

    Riis, Simone; Nielsen, Frederik Mølgaard; Pennisi, Cristian Pablo; Zachar, Vladimir

    2016-01-01

    Adipose-derived stem cells (ASCs) are being tested in clinical trials related to cell-based regenerative therapies. Although most of the current expansion protocols for ASCs use fetal calf serum (FCS), xenogeneic-free medium supplements are greatly desired. This study aims to compare the effect of FCS, human platelet lysate (hPL), and a fully defined medium on the initiation and maintenance of ASC cultures. ASCs obtained from five donors were cultured in five different media: StemPro, Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% hPL, or α-minimum essential medium (A-MEM) supplemented with 5% hPL, 10% hPL, or 10% FCS. The effect of media on proliferation, colony-forming units (CFUs), attachment, and morphology was assessed along with cell size, granularity, and immunophenotype. StemPro greatly compromised the initiation of ASC cultures, which could not survive more than a few passages. Cells cultured in A-MEM proliferated at a faster rate than in DMEM, and hPL significantly enhanced cell size, granularity, and proliferation compared with FCS. All media except StemPro supported CFUs equally well. Analysis of surface markers revealed higher levels of CD73 and CD105 in FCS-cultured ASCs, whereas increased levels of CD146 were found in hPL-cultured cells. Multiparametric flow cytometric analysis performed after seven passages revealed the existence of four distinct ASC subpopulations, all positive for CD73, CD90, and CD105, which mainly differed by their expression of CD146 and CD271. Analysis of the different subpopulations might represent an important biological measure when assessing different medium formulations for a particular clinical application. Significance In most clinical trials using adipose-derived stem cells (ASCs), the cells have been expanded in culture media supplemented with fetal calf serum. However, there is much interest in replacing fetal calf serum with human platelet lysate or using completely serum- and xenogeneic

  17. Human adipose-derived stem cells contribute to chondrogenesis in coculture with human articular chondrocytes.

    PubMed

    Hildner, Florian; Concaro, Sebastian; Peterbauer, Anja; Wolbank, Susanne; Danzer, Martin; Lindahl, Anders; Gatenholm, Paul; Redl, Heinz; van Griensven, Martijn

    2009-12-01

    Adipose tissue is easily available and contains high numbers of stem cells that are capable for chondrogenic differentiation. We hypothesize that a partial substitution of chondrocytes with autologous adipose-derived stem cells (ASC) might be a possible strategy to reduce the number of chondrocytes needed in matrix-associated autologous chondrocyte transplantation. To lay the ground, in vitro coculture experiments were performed using human chondrocytes and human ASC. Chondrocytes were obtained from donors undergoing matrix-associated autologous chondrocyte transplantation. ASC were isolated from liposuction material. Chondrocytes and ASC were seeded either in fibrin (Tisseel; Baxter, Vienna, Austria) or collagen matrix (Tissue Fleece; Baxter, Unterschleissheim, Germany). RNA for quantitative reverse transcriptase (RT)-polymerase chain reaction was isolated after 2 weeks of culture in chondrogenic medium, and after 4 weeks samples were processed for histology. Related to the number of chondrocytes used, coculture with ASC led to strong increase in collagen type IX mRNA expression, which is an indicator for long-term stability of cartilage. Moderate upregulation was shown for SOX9, aggrecan, melanoma inhibitory activity, cartilage link protein 1, and cartilage oligomeric matrix protein mRNA. However, expression of collagen I and collagen II indicates the synthesis of fibrous tissue, which might be due to the use of dedifferentiated chondrocytes. Tisseel provided slightly better chondrogenic conditions than Tissue Fleece. These data support the possibility to take advantage of ASC in cartilage regeneration in conjunction with autologous chondrocytes.

  18. Human Adipose-Derived Mesenchymal Progenitor Cells Engraft into Rabbit Articular Cartilage

    PubMed Central

    Wang, Wen; He, Na; Feng, Chenchen; Liu, Victor; Zhang, Luyi; Wang, Fei; He, Jiaping; Zhu, Tengfang; Wang, Shuyang; Qiao, Weiwei; Li, Suke; Zhou, Guangdong; Zhang, Li; Dai, Chengxiang; Cao, Wei

    2015-01-01

    Mesenchymal stem cells (MSCs) are known to have the potential for articular cartilage regeneration, and are suggested for the treatment of osteoarthritis (OA). Here, we investigated whether intra-articular injection of xenogeneic human adipose-derived mesenchymal progenitor cells (haMPCs) promoted articular cartilage repair in rabbit OA model and engrafted into rabbit articular cartilage. The haMPCs were cultured in vitro, and phenotypes and differentiation characteristics of cells were evaluated. OA was induced surgically by anterior cruciate ligament transection (ACLT) and medical meniscectomy of knee joints. At six weeks following surgery, hyaluronic acid (HA) or haMPCs was injected into the knee joints, the contralateral knee served as normal control. All animals were sacrificed at the 16th week post-surgery. Assessments were carried out by macroscopic examination, hematoxylin/eosin (HE) and Safranin-O/Fast green stainings and immunohistochemistry. The data showed that haMPC treatment promoted cartilage repair. Signals of human mitochondrial can be directly detected in haMPC treated cartilage. The haMPCs expressed human leukocyte antigen I (HLA-I) but not HLA-II-DR in vivo. These results suggest that intra-articular injection of haMPCs promotes regeneration of articular cartilage in rabbit OA model, and support the notion that MPCs are transplantable between HLA-incompatible individuals. PMID:26023716

  19. Adipose-Derived Stem Cells as a Tool in Cell-Based Therapies.

    PubMed

    Bajek, Anna; Gurtowska, Natalia; Olkowska, Joanna; Kazmierski, Lukasz; Maj, Malgorzata; Drewa, Tomasz

    2016-12-01

    Recent development in stem cell isolation methods and expansion under laboratory conditions create an opportunity to use those aforementioned cells in tissue engineering and regenerative medicine. Particular attention is drawn towards mesenchymal stem cells (MSCs) being multipotent progenitors exhibiting several unique characteristics, including high proliferation potential, self-renewal abilities and multilineage differentiation into cells of mesodermal and non-mesodermal origin. High abundance of MSCs found in adipose tissue makes it a very attractive source of adult stem cells for further use in regenerative medicine applications. Despite immunomodulating properties of adipose-derived stem cells (ASCs) and a secretion of a wide variety of paracrine factors that facilitate tissue regeneration, effectiveness of stem cell therapy was not supported by the results of clinical trials. Lack of a single, universal stem cell marker, patient-to-patient variability, heterogeneity of ASC population combined with multiple widely different protocols of cell isolation and expansion hinder the ability to precisely identify and analyze biological properties of stem cells. The above issues contribute to conflicting data reported in literature. We will review the comprehensive information concerning characteristic features of ASCs. We will also review the regenerative potential and clinical application based on various clinical trials.

  20. The current landscape of adipose-derived stem cells in clinical applications.

    PubMed

    Lim, Ming Hui; Ong, Wee Kiat; Sugii, Shigeki

    2014-05-07

    Adipose-derived stem cells (ASCs) are considered a great alternative source of mesenchymal stem cells (MSCs). Unlike bone marrow stem cells (BMSCs), ASCs can be retrieved in high numbers from lipoaspirate, a by-product of liposuction procedures. Given that ASCs represent an easily accessible and abundant source of multipotent cells, ASCs have garnered attention and curiosity from both scientific and clinical communities for their potential in clinical applications. Furthermore, their unique immunobiology and secretome are attractive therapeutic properties. A decade since the discovery of a stem cell reservoir residing within adipose tissue, ASC-based clinical trials have grown over the years around the world along with assessments made on their safety and efficacy. With the progress of ASCs into clinical applications, the aim towards producing clinical-grade ASCs becomes increasingly important. Several countries have recognised the growing industry of cell therapies and have developed regulatory frameworks to assure their safety. With more research efforts made to understand their effects in both scientific and clinical settings, ASCs hold great promise as a future therapeutic strategy in treating a wide variety of diseases. Therefore, this review seeks to highlight the clinical applicability of ASCs as well as their progress in clinical trials across various medical disciplines.

  1. Effect of hypoxia on human adipose-derived mesenchymal stem cells and its potential clinical applications.

    PubMed

    Choi, Jane Ru; Yong, Kar Wey; Wan Safwani, Wan Kamarul Zaman

    2017-02-21

    Human adipose-derived mesenchymal stem cells (hASCs) are an ideal cell source for regenerative medicine due to their capabilities of multipotency and the readily accessibility of adipose tissue. They have been found residing in a relatively low oxygen tension microenvironment in the body, but the physiological condition has been overlooked in most studies. In light of the escalating need for culturing hASCs under their physiological condition, this review summarizes the most recent advances in the hypoxia effect on hASCs. We first highlight the advantages of using hASCs in regenerative medicine and discuss the influence of hypoxia on the phenotype and functionality of hASCs in terms of viability, stemness, proliferation, differentiation, soluble factor secretion, and biosafety. We provide a glimpse of the possible cellular mechanism that involved under hypoxia and discuss the potential clinical applications. We then highlight the existing challenges and discuss the future perspective on the use of hypoxic-treated hASCs.

  2. Fat grafting to the breast and adipose-derived stem cells: recent scientific consensus and controversy.

    PubMed

    Mizuno, Hiroshi; Hyakusoku, Hiko

    2010-01-01

    Recent technical advances in fat grafting and the development of surgical devices such as liposuction cannulae have made fat grafting a relatively safe and effective procedure. However, new guidelines issued by the American Society of Plastic Surgeons in 2009 announced that fat grafting to the breast is not a strongly recommended procedure, as there are limited scientific data on the safety and efficacy of this particular type of fat transfer. Recent progress by several groups has revealed that multipotent adult stem cells are present in human adipose tissue. This cell population, termed adipose-derived stem cells (ADSC), represents a promising approach to future cell-based therapies, such as tissue engineering and regeneration. In fact, several reports have shown that ADSC play a pivotal role in graft survival through both adipogenesis and angiogenesis. Although tissue augmentation by fat grafting does have several advantages in that it is a noninvasive procedure and results in minimal scarring, it is essential that such a procedure be supported by evidence-based medicine and that further basic scientific and clinical research is conducted to ensure that fat grafting is a safe and effective procedure.

  3. PHB/PHBHHx scaffolds and human adipose-derived stem cells for cartilage tissue engineering.

    PubMed

    Ye, Chuan; Hu, Ping; Ma, Min-Xian; Xiang, Yang; Liu, Ri-Guang; Shang, Xian-Wen

    2009-09-01

    The goal of this study was to investigate the potential of polyhydroxybutyrate (PHB)/poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) (PHB/PHBHHx) to produce neocartilage upon seeding with differentiated human adipose-derived stem cells (hASCs). hASCs were grown on a three-dimensional PHB/PHBHHx scaffold in vitro with or without chondrogenic media for 14 days. Scanning electron microscopy showed that differentiated cells produced abundant extracellular matrices with increasing culture time. No cytotoxicity was observed by the live/dead cell viability assay. GAG and total collagen content in the differentiated cells increased significantly with in vitro culture time. After 14 days of in vitro culture, the differentiated cells grown on the (PHB/PHBHHx) scaffold (differentiated cells/(PHB/PHBHHx)) were implanted into the subcutaneous layer nude mice for 12 or 24 weeks, non-differentiated cells/(PHB/PHBHHx) were implanted as the control group. The differentiated cells/(PHB/PHBHHx) implants formed cartilage-like tissue after 24 weeks of implantation, and stained positive for collagen type II, safranin O, and toluidine blue. In addition, typical cartilage lacuna was observed, and there were no remnants of PHB/PHBHHx. Collagen type II was detected by Western blot at 12 and 24 weeks of implantation. In the control group, no cartilage formation was observed. This study demonstrated that PHB/PHBHHx is a suitable material for cartilage tissue engineering.

  4. [Induction of hepatic specification of human adipose-derived stem cells (hADSCs) in vitro].

    PubMed

    Wang, Min; Pei, Hai-yun; Guan, Li-dong; Nan, Xue; Bai, Ci-xian; Liu, Hui; Li, Bao-wei; Wang, Yun-fang; Pei, Xue-tao

    2009-07-01

    To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro. hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver specific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined. The number of hADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21 days (t=6.59, 8.69, 15.94 and 24.64, respectively, P<0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production. hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.

  5. Repair of critical size defects using bioactive glass seeded with adipose-derived mesenchymal stem cells.

    PubMed

    Saçak, Bülent; Certel, Furkan; Akdeniz, Zeynep D; Karademir, Betül; Ercan, Feriha; Özkan, Naziye; Akpinar, İhsan Nuri; Çelebiler, Özhan

    2016-02-17

    Bioactive glass has been demonstrated as a biocompatible bone substitute. However bone healing process can be prolonged due to late resorption of the material. Adipose derived stem cells (ASC) have osteogenic differentiation potential and hence can be a cell source for bone regeneration. The aim of this study was to test whether combination of bioactive glass with ASCs would enhance bone regeneration. Following creation of critical sized defects on the calvaria of 32 Wistar rats, the animals were randomly divided into four groups: Group C (control): Defects were left untreated; Group G: Defects were covered with autologous bone graft; Group BG: Defects were filled with bioactive glass; Group BG/ASC: Defects were filled with bioactive glass seeded with ASCs. The defect size was significantly greater in Group C compared to all other groups. Bone density was significantly lower in Group C compared to Group G and Group BG/ASC. Bone regeneration score of Group C was significantly lower than other groups. Group BG/ASC demonstrated lamellar bone and havers canal formation. The results of this study demonstrated that bioactive glass implanted with ASC is a biocompatible construct stimulating radiologically and histologically evident bone regeneration similar to autologous bone grafting. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

  6. In vivo imaging of human adipose-derived stem cells in Alzheimer's disease animal model.

    PubMed

    Ha, Sungji; Ahn, Sangzin; Kim, Saeromi; Joo, Yuyoung; Chong, Young Hae; Suh, Yoo-Hun; Chang, Keun-A

    2014-05-01

    Stem cell therapy is a promising tool for the treatment of diverse conditions, including neurodegenerative diseases such as Alzheimer's disease (AD). To understand transplanted stem cell biology, in vivo imaging is necessary. Nanomaterial has great potential for in vivo imaging and several noninvasive methods are used, such as magnetic resonance imaging, positron emission tomography, fluorescence imaging (FI) and near-infrared FI. However, each method has limitations for in vivo imaging. To overcome these limitations, multimodal nanoprobes have been developed. In the present study, we intravenously injected human adipose-derived stem cells (hASCs) that were labeled with a multimodal nanoparticle, LEO-LIVE™-Magnoxide 675 or 797 (BITERIALS, Seoul, Korea), into Tg2576 mice, an AD mouse model. After sequential in vivo tracking using Maestro Imaging System, we found fluorescence signals up to 10 days after injection. We also found strong signals in the brains extracted from hASC-transplanted Tg2576 mice up to 12 days after injection. With these results, we suggest that in vivo imaging with this multimodal nanoparticle may provide a useful tool for stem cell tracking and understanding stem cell biology in other neurodegenerative diseases.

  7. In vivo imaging of human adipose-derived stem cells in Alzheimer's disease animal model

    NASA Astrophysics Data System (ADS)

    Ha, Sungji; Ahn, Sangzin; Kim, Saeromi; Joo, Yuyoung; Chong, Young Hae; Suh, Yoo-Hun; Chang, Keun-A.

    2014-05-01

    Stem cell therapy is a promising tool for the treatment of diverse conditions, including neurodegenerative diseases such as Alzheimer's disease (AD). To understand transplanted stem cell biology, in vivo imaging is necessary. Nanomaterial has great potential for in vivo imaging and several noninvasive methods are used, such as magnetic resonance imaging, positron emission tomography, fluorescence imaging (FI) and near-infrared FI. However, each method has limitations for in vivo imaging. To overcome these limitations, multimodal nanoprobes have been developed. In the present study, we intravenously injected human adipose-derived stem cells (hASCs) that were labeled with a multimodal nanoparticle, LEO-LIVE™-Magnoxide 675 or 797 (BITERIALS, Seoul, Korea), into Tg2576 mice, an AD mouse model. After sequential in vivo tracking using Maestro Imaging System, we found fluorescence signals up to 10 days after injection. We also found strong signals in the brains extracted from hASC-transplanted Tg2576 mice up to 12 days after injection. With these results, we suggest that in vivo imaging with this multimodal nanoparticle may provide a useful tool for stem cell tracking and understanding stem cell biology in other neurodegenerative diseases.

  8. Sundew-Inspired Adhesive Hydrogels Combined with Adipose-Derived Stem Cells for Wound Healing

    PubMed Central

    Sun, Leming; Huang, Yujian; Bian, Zehua; Petrosino, Jennifer; Fan, Zhen; Wang, Yongzhong; Park, Ki Ho; Yue, Tao; Schmidt, Michael; Galster, Scott; Ma, Jianjie; Zhu, Hua; Zhang, Mingjun

    2016-01-01

    The potential to harness the unique physical, chemical, and biological properties of the sundew (Drosera) plant’s adhesive hydrogels has long intrigued researchers searching for novel wound-healing applications. However, the ability to collect sufficient quantities of the sundew plant’s adhesive hydrogels is problematic and has eclipsed their therapeutic promise. Inspired by these natural hydrogels, we asked if sundew-inspired adhesive hydrogels could overcome the drawbacks associated with natural sundew hydrogels and be used in combination with stem-cell-based therapy to enhance wound-healing therapeutics. Using a bioinspired approach, we synthesized adhesive hydrogels comprised of sodium alginate, gum arabic, and calcium ions to mimic the properties of the natural sundew-derived adhesive hydrogels. We then characterized and showed that these sundew-inspired hydrogels promote wound healing through their superior adhesive strength, nanostructure, and resistance to shearing when compared to other hydrogels in vitro. In vivo, sundew-inspired hydrogels promoted a “suturing” effect to wound sites, which was demonstrated by enhanced wound closure following topical application of the hydrogels. In combination with mouse adipose-derived stem cells (ADSCs) and compared to other therapeutic biomaterials, the sundew-inspired hydrogels demonstrated superior wound-healing capabilities. Collectively, our studies show that sundew-inspired hydrogels contain ideal properties that promote wound healing and suggest that sundew-inspired-ADSCs combination therapy is an efficacious approach for treating wounds without eliciting noticeable toxicity or inflammation. PMID:26731614

  9. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells.

    PubMed

    Sun, Bo Kyung; Kim, Ji Hye; Choi, Joon-Seok; Hwang, Sung-Joo; Sung, Jong-Hyuk

    2015-07-22

    Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs) or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA), significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

  10. Intrinsic mechanisms underlying the neurotrophic activity of adipose derived stem cells.

    PubMed

    Tse, Kai-Hei; Novikov, Lev N; Wiberg, Mikael; Kingham, Paul J

    2015-02-01

    Adipose derived stem cells (ADSC) can be differentiated into Schwann cell-like cells which enhance nerve function and regeneration. However, the signalling mechanisms underlying the neurotrophic potential of ADSC remain largely unknown. In this study, we hypothesised that ADSC, upon stimulation with a combination of growth factors, could rapidly produce brain derived neurotrophic factor (BDNF) with a similar molecular mechanism to that functioning in the nervous system. Within 48 h of stimulation, ADSC demonstrated potent neurotrophic effects on dorsal root ganglion neurons, at a magnitude equivalent to that of the longer term differentiated Schwann cell-like cells. Stimulated ADSC showed rapid up-regulation of the neuronal activity dependent promoter BDNF exon IV along with an augmented expression of full length protein encoding BDNF exon IX. BDNF protein was secreted at a concentration similar to that produced by differentiated Schwann cell-like cells. Stimulation also activated the BDNF expression gating transcription factor, cAMP responsive element binding (CREB) protein. However, blocking phosphorylation of CREB with the protein kinase A small molecule inhibitor H89 did not suppress secretion of BDNF protein. These results suggest rapid BDNF production in ADSC is mediated through multiple compensatory pathways independent of, or in addition to, the CREB neuronal activation cascade. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. The Role of Adipose-Derived Stem Cells in Breast Cancer Progression and Metastasis

    PubMed Central

    Gorantla, Vijay S.; Rubin, J. Peter

    2015-01-01

    Conventional breast cancer extirpation involves resection of parts of or the whole gland, resulting in asymmetry and disfiguration. Given the unsatisfactory aesthetic outcomes, patients often desire postmastectomy reconstructive procedures. Autologous fat grafting has been proposed for reconstructive purposes for decades to restore form and anatomy after mastectomy. Fat has the inherent advantage of being autologous tissue and the most natural-appearing filler, but given its inconsistent engraftment and retention rates, it lacks reliability. Implementation of autologous fat grafts with cellular adjuncts, such as multipotent adipose-derived stem cells (ADSCs), has shown promising results. However, it is pertinent and critical to question whether these cells could promote any residual tumor cells to proliferate, differentiate, or metastasize or even induce de novo carcinogenesis. Thus far, preclinical and clinical study findings are discordant. A trend towards potential promotion of both breast cancer growth and invasion by ADSCs found in basic science studies was indeed not confirmed in clinical trials. Whether experimental findings eventually correlate with or will be predictive of clinical outcomes remains unclear. Herein, we aimed to concisely review current experimental findings on the interaction of mesenchymal stem cells and breast cancer, mainly focusing on ADSCs as a promising tool for regenerative medicine, and discuss the implications in clinical translation. PMID:26000019

  12. Semitendinosus myopathy and treatment with adipose-derived stem cells in working German shepherd police dogs.

    PubMed

    Gibson, Melissa A; Brown, S Gary; Brown, Nancy O

    2017-03-01

    Semitendinosus myopathy has been treated with numerous surgical and non-surgical therapies resulting in recurrence of lameness within 2 to 9 months. Eleven cases of semitendinosus myopathy diagnosed in 8 working police dogs that were treated with adipose-derived mesenchymal stem cells were retrospectively evaluated. At short-term follow-up < 6 mo, ultrasound and gait evaluations revealed a mean reduction in the overall intramuscular lesion size of 54.82% (SD +/- 18.02; range: 30.5% to 82.7%) and reduction in the Visual Assessment Score (VAS) of 1 to 3 points. At long-term follow-up > 1 y, in 8 cases the dogs had a normal gait and in 3 cases the dogs had an improved gait compared with initial examination, and all 8 dogs returned to active police work. Fisher's exact test resulted in P = 0.000008 when comparing published historical reports and these 11 cases for resolution of lameness and return to active duty.

  13. Current progress in use of adipose derived stem cells in peripheral nerve regeneration

    PubMed Central

    Zack-Williams, Shomari DL; Butler, Peter E; Kalaskar, Deepak M

    2015-01-01

    Unlike central nervous system neurons; those in the peripheral nervous system have the potential for full regeneration after injury. Following injury, recovery is controlled by schwann cells which replicate and modulate the subsequent immune response. The level of nerve recovery is strongly linked to the severity of the initial injury despite the significant advancements in imaging and surgical techniques. Multiple experimental models have been used with varying successes to augment the natural regenerative processes which occur following nerve injury. Stem cell therapy in peripheral nerve injury may be an important future intervention to improve the best attainable clinical results. In particular adipose derived stem cells (ADSCs) are multipotent mesenchymal stem cells similar to bone marrow derived stem cells, which are thought to have neurotrophic properties and the ability to differentiate into multiple lineages. They are ubiquitous within adipose tissue; they can form many structures resembling the mature adult peripheral nervous system. Following early in vitro work; multiple small and large animal in vivo models have been used in conjunction with conduits, autografts and allografts to successfully bridge the peripheral nerve gap. Some of the ADSC related neuroprotective and regenerative properties have been elucidated however much work remains before a model can be used successfully in human peripheral nerve injury (PNI). This review aims to provide a detailed overview of progress made in the use of ADSC in PNI, with discussion on the role of a tissue engineered approach for PNI repair. PMID:25621105

  14. Adipose-derived stem cells sustain prolonged angiogenesis through leptin secretion.

    PubMed

    Delle Monache, Simona; Calgani, Alessia; Sanità, Patrizia; Zazzeroni, Francesca; Gentile Warschauer, Emilio; Giuliani, Antonio; Amicucci, Gianfranco; Angelucci, Adriano

    2016-08-01

    Recent studies suggest that adipose-derived stem cells (ASCs) play a role in tissue remodeling through the release of cytokines and growth factors. We compared the secreted cytokine profile of hypoxia-conditioned ASCs (hASCs) with normoxic ASCs (nASCs) and we analyzed the effect of ASCs conditioned medium (CM) on endothelial cells. We found that hypoxia induced a transient upregulation of VEGF in ASCs and a notable and enduring upregulation of leptin mRNA expression 30-fold greater than control after 24 h and up to 60-fold greater than control at day 7. CM from hASC stimulated EC tube formation to a significantly greater extent than CM from nASC. This might be due to leptin-secreted factor. Indeed, exogenous leptin stimulated the expression of HIF2-α, but not HIF1-α, and upregulated the expression of Flt-1 and Tie-1 proangiogenic receptors. In conclusion, hASCs may be particularly efficient in sustaining angiogenesis through the release of leptin.

  15. Melatonin and Vitamin D Interfere with the Adipogenic Fate of Adipose-Derived Stem Cells.

    PubMed

    Basoli, Valentina; Santaniello, Sara; Cruciani, Sara; Ginesu, Giorgio Carlo; Cossu, Maria Laura; Delitala, Alessandro Palmerio; Serra, Pier Andrea; Ventura, Carlo; Maioli, Margherita

    2017-05-05

    Adipose-derived stem cells (ADSCs) represent one of the cellular populations resident in adipose tissue. They can be recruited under certain stimuli and committed to become preadipocytes, and then mature adipocytes. Controlling stem cell differentiation towards the adipogenic phenotype could have a great impact on future drug development aimed at counteracting fat depots. Stem cell commitment can be influenced by different molecules, such as melatonin, which we have previously shown to be an osteogenic inducer. Here, we aimed at evaluating the effects elicited by melatonin, even in the presence of vitamin D, on ADSC adipogenesis assessed in a specific medium. The transcription of specific adipogenesis orchestrating genes, such as aP2, peroxisome proliferator-activated receptor γ (PPAR-γ), and that of adipocyte-specific genes, including lipoprotein lipase (LPL) and acyl-CoA thioesterase 2 (ACOT2), was significantly inhibited in cells that had been treated in the presence of melatonin and vitamin D, alone or in combination. Protein content and lipid accumulation confirmed a reduction in adipogenesis in ADSCs that had been grown in adipogenic conditions, but in the presence of melatonin and/or vitamin D. Our findings indicate the role of melatonin and vitamin D in deciding stem cell fate, and disclose novel therapeutic approaches against fat depots.

  16. Pretreatment of Adipose Derived Stem Cells with Curcumin Facilitates Myocardial Recovery via Antiapoptosis and Angiogenesis.

    PubMed

    Liu, Jianfeng; Zhu, Ping; Song, Peng; Xiong, Weiping; Chen, Haixu; Peng, Wenhui; Wang, Shuxia; Li, Shan; Fu, Zhiqing; Wang, Yutang; Wang, Haibin

    2015-01-01

    The poor survival rate of transplanted stem cells in ischemic myocardium has limited their therapeutic efficacy. Curcumin has potent antioxidant property. This study investigates whether prior curcumin treatment protects stem cells from oxidative stress injury and improves myocardial recovery following cells transplantation. Autologous Sprague-Dawley rat adipose derived mesenchymal stem cells (ADSCs) were pretreated with or without curcumin. The hydrogen peroxide/serum deprivation (H2O2/SD) medium was used to mimic the ischemic condition in vitro. Cytoprotective effects of curcumin on ADSCs were evaluated. Curcumin pretreatment significantly increased cell viability and VEGF secretion, and decreased cell injury and apoptosis via regulation of PTEN/Akt/p53 and HO-1 signal proteins expression. The therapeutic potential of ADSCs implantation was investigated in myocardial ischemia-reperfusion injury (IRI) model. Transplantation of curcumin pretreated ADSCs not only resulted in better heart function, higher cells retention, and smaller infarct size, but also decreased myocardial apoptosis, promoted neovascularization, and increased VEGF level in ischemic myocardium. Together, priming of ADSCs with curcumin improved tolerance to oxidative stress injury and resulted in enhancement of their therapeutic potential of ADSCs for myocardial repair. Curcumin pretreatment is a promising adjuvant strategy for stem cells transplantation in myocardial restoration.

  17. [Use of adipose-derived stem cells in an experimental rotator cuff fracture animal model].

    PubMed

    Barco, R; Encinas, C; Valencia, M; Carrascal, M T; García-Arranz, M; Antuña, S

    2015-01-01

    Rotator cuff repairs have shown a high level of re-ruptures. We hypothesized that the use of adipose-derived stem cells (ASC) could improve the biomechanical and histological properties of the repair. Controlled experimental study conducted on 44 BDIX rats with section and repair of the supraspinatus tendon and randomization to one of three groups: group A, no intervention (control); group B, local applications of a fibrin sealant; and group C, application of the fibrin sealant with 2 x 10(6) ASC. At 4 and 8 weeks a biomechanical and histological analysis was performed. There were no differences in load-to-failure at 4 and 8 weeks between groups. The load-to-failure did increase between week 4 and week 8. Histologically the tendon-to bone union showed a disorganized fibrovascular tissue. Group C showed a different inflammatory pattern, with less presence of neutrophils and more presence of plasma cells. The use of ASC does not improve the biomechanical or histological properties of the repair site. More studies are needed to improve techniques that enhance the healing site of the repair. Copyright © 2014 SECOT. Published by Elsevier Espana. All rights reserved.

  18. Adhesion, Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings

    PubMed Central

    Strauß, Sarah; Neumeister, Anne; Barcikowski, Stephan; Kracht, Dietmar; Kuhbier, Jörn W.; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M.

    2013-01-01

    Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells. PMID:23308190

  19. Improvement of Anal Function by Adipose-Derived Stem Cell Sheets.

    PubMed

    Inoue, Yusuke; Fujita, Fumihiko; Yamaguchi, Izumi; Kinoe, Hiroko; Kawahara, Daisuke; Sakai, Yusuke; Kuroki, Tamotsu; Eguchi, Susumu

    2017-05-12

    One of the most troublesome complications of anal preserving surgery is anal sphincter dysfunction. The aim of this study was to evaluate functional recovery after implantation of adipose-derived stem cell (ADSC) sheets, novel biotechnology, for an anal sphincter resection animal model. Eighteen female Sprague-Dawley rats underwent removal of the nearest half of the internal and external anal sphincter muscle. Nine rats received transplantation with ADSC sheets to the resected area while the remaining rats received no transplantation. The rats were evaluated for the anal function by measuring their resting pressure before surgery and on postoperative days 1, 7, 14, 28, and 56. In addition, the rats were examined for the presence of smooth muscle and also to determine its origin. The improvement of the anal pressure was significantly greater in the ADSC sheet transplantation group compared with the control group. Histologically, at the vicinity of the remaining smooth muscle, reproduction of smooth muscle was detected. Using in fluorescence in situ hybridization, the cells were shown to be from the recipient. Regenerative therapy using ADSC sheet has the potential to recover anal sphincter dysfunction due to anorectal surgery. © 2017 S. Karger AG, Basel.

  20. Exosomes from adipose-derived stem cells ameliorate phenotype of Huntington's disease in vitro model.

    PubMed

    Lee, Mijung; Liu, Tian; Im, Wooseok; Kim, Manho

    2016-08-01

    Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by the aggregation of mutant Huntingtin (mHtt). Adipose-derived stem cells (ASCs) have a potential for use in the treatment of incurable disorders, including HD. ASCs secrete various neurotrophic factors and microvesicles, and modulate hostile microenvironments affected by disease through paracrine mechanisms. Exosomes are small vesicles that transport nucleic acid and protein between cells. Here, we investigated the therapeutic role of exosomes from ASCs (ASC-exo) using in vitro HD model by examining pathological phenotypes of this model. Immunocytochemistry result showed that ASC-exo significantly decreases mHtt aggregates in R6/2 mice-derived neuronal cells. Western blot result further confirmed the reduction in mHtt aggregates level by ASC-exo treatment. ASC-exo up-regulates PGC-1, phospho-CREB and ameliorates abnormal apoptotic protein level in an in vitro HD model. In addition, MitoSOX Red, JC-1 and cell viability assay showed that ASC-exo reduces mitochondrial dysfunction and cell apoptosis of in vitro HD model. These findings suggest that ASC-exo has a therapeutic potential for treating HD by modulating representative cellular phenotypes of HD.

  1. Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes

    SciTech Connect

    Andersen, Jens Isak; Juhl, Morten; Nielsen, Thøger; Emmersen, Jeppe; Fink, Trine; Zachar, Vladimir; Pennisi, Cristian Pablo

    2014-07-25

    Highlights: • Uniaxial cyclic tensile strain (CTS) applied to ASCs alone or in coculture with myogenic precursors. • CTS promoted the formation of a highly ordered array of parallel ASCs. • Without biochemical supplements, CTS did not support advanced myogenic differentiation of ASCs. • Mechanical stimulation of cocultures boosted fusion of ASCs with skeletal myoblasts. - Abstract: Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48 h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.

  2. Effect of T3 hormone on neural differentiation of human adipose derived stem cells.

    PubMed

    Razavi, Shahnaz; Mostafavi, Fatemeh Sadat; Mardani, Mohammad; Zarkesh Esfahani, Hamid; Kazemi, Mohammad; Esfandiari, Ebrahim

    2014-12-01

    Human adult stem cells, which are capable of self-renewal and differentiation into other cell types, can be isolated from various tissues. There are no ethical and rejection problems as in the case of embryonic stem cells, so they are a promising source for cell therapy. The human body contains a great amount of adipose tissue that contains high numbers of mesenchymal stem cells. Human adipose-derived stem cells (hADSCs) could be easily induced to form neuron-like cells, and because of its availability and abundance, we can use it for clinical cell therapy. On the other hand, T3 hormone as a known neurotropic factor has important impressions on the nervous system. The aim of this study was to explore the effects of T3 treatment on neural differentiation of hADSCs. ADSCs were harvested from human adipose tissue, after neurosphere formation, and during final differentiation, treatment with T3 was performed. Immunocytochemistry, real-time RT-PCR, Western blotting techniques were used for detection of nestin, MAP2, and GFAP markers in order to confirm the effects of T3 on neural differentiation of hADSCs. Our results showed an increase in the number of glial cells but reduction in neuronal cells number fallowing T3 treatment.

  3. Adhesion, vitality and osteogenic differentiation capacity of adipose derived stem cells seeded on nitinol nanoparticle coatings.

    PubMed

    Strauss, Sarah; Neumeister, Anne; Barcikowski, Stephan; Kracht, Dietmar; Kuhbier, Jörn W; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M

    2013-01-01

    Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.

  4. Hyaluronic acid effect on adipose-derived stem cells. Biological in vitro evaluation.

    PubMed

    Moreno, A; Martínez, A; Olmedillas, S; Bello, S; de Miguel, F

    2015-01-01

    To evaluate the in vitro effects of hyaluronic acid (HA) on adipose-derived stem cells (ASC) in order to consider the possibility of their combined used in the treatment of knee arthrosis. The ASC cells were grown both in the presence and absence of AH, and several studies were carried out: proliferation (WST8) and cell viability studies (Alamar Blue® and Trypan Blue), possible chondrogenic differentiation (collagen type 2 expression) by RT-PCR, AH receptor expression (CD44) by flow cytometry and RT-QPCR, and expression of inflammatory and anti-inflammatory factors (IL-6, TGFß, IL-10) by RT-QPCR. The number of ASC significantly increased after 7 days with HA (158±39%, p <0.05). Additionally, the cell viability of the ASC treated with HA after 1, 3, 5 and 7 days was similar to that of the control cells, being considered non-toxic. There were no changes observed in the expression of CD44 and chondrogenic differentiation. TGFß expression was not modified after AH treatment, but there was a 4-fold decrease in IL-6 expression and IL-10 expression increased up to 2-fold compared to control cells. Hyaluronic acid favours ASC proliferation without causing cellular toxicity, and inducing an anti-inflammatory profile in these cells. Hyaluronic acid appears to be a suitable vehicle for the intra-articular administration of mesenchymal stem cells. Copyright © 2014 SECOT. Published by Elsevier Espana. All rights reserved.

  5. Effect of sertraline on proliferation and neurogenic differentiation of human adipose-derived stem cells

    PubMed Central

    Razavi, Shahnaz; Jahromi, Maliheh; Amirpour, Nushin; Khosravizadeh, Zahra

    2014-01-01

    Background: Antidepressant drugs are commonly employed for anxiety and mood disorders. Sertraline is extensively used as antidepressant in clinic. In addition, adipose tissue represents an abundant and accessible source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human adipose-derived stem cells (hADSCs) may be useful for autologous transplantation. Materials and Methods: In the present study, we assessed the effect of antidepressant drug Sertraline on the proliferation and neurogenic differentiation of hADSCs using MTT assay and immunofluorescence technique respectively. Results: MTT assay analysis showed that 0.5 μM Sertraline significantly increased the proliferation rate of hADSCs induced cells (P < 0.05), while immunofluorescent staining indicated that Sertraline treatment during neurogenic differentiation could be decreased the percentage of glial fibrillary acidic protein and Nestin-positive cells, but did not significantly effect on the percentage of MAP2 positive cells. Conclusion: Overall, our data show that Sertraline can be promoting proliferation rate during neurogenic differentiation of hADSCs after 6 days post-induction, while Sertraline inhibits gliogenesis of induced hADSCs. PMID:24800186

  6. Changes of neural markers expression during late neurogenic differentiation of human adipose-derived stem cells

    PubMed Central

    Razavi, Shahnaz; Khosravizadeh, Zahra; Bahramian, Hamid; Kazemi, Mohammad

    2015-01-01

    Background: Different studies have been done to obtain sufficient number of neural cells for treatment of neurodegenerative diseases, spinal cord, and traumatic brain injury because neural stem cells are limited in central nerves system. Recently, several studies have shown that adipose-derived stem cells (ADSCs) are the appropriate source of multipotent stem cells. Furthermore, these cells are found in large quantities. The aim of this study was an assessment of proliferation and potential of neurogenic differentiation of ADSCs with passing time. Materials and Methods: Neurosphere formation was used for neural induction in isolated human ADSCs (hADSCs). The rate of proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and potential of neural differentiation of induced hADSCs was evaluated by immunocytochemical and real-time reverse transcription polymerase chain reaction analysis after 10 and 14 days post-induction. Results: The rate of proliferation of induced hADSCs increased after 14 days while the expression of nestin, glial fibrillary acidic protein, and microtubule-associated protein 2 was decreased with passing time during neurogenic differentiation. Conclusion: These findings showed that the proliferation of induced cells increased with passing time, but in early neurogenic differentiation of hADSCs, neural expression was higher than late of differentiation. Thus, using of induced cells in early differentiation may be suggested for in vivo application. PMID:26605238

  7. PHBV and predifferentiated human adipose-derived stem cells for cartilage tissue engineering.

    PubMed

    Liu, Jiong; Zhao, Bin; Zhang, Yunqiang; Lin, Yunfeng; Hu, Ping; Ye, Chuan

    2010-08-01

    This study was conducted to investigate whether in vitro chondrogenic differentiated human adipose-derived stem cells (hASCs) can maintain the chondrogenic phenotype in (3-hydroxybutrate-co-3-hydroxyvalerate) (PHBV) scaffolds and whether differentiated hASCs/PHBV construct can produce neocartilage in a heterotopic animal model. hASCs were cultured with or without chondrogenic media in vitro and then seeded on PHBV foams. Differentiated cell/PHBV constructs were subcutaneously implanted in nude mice for 8 or 16 weeks; nondifferentiated cell/PHBV constructs were implanted in the control group. The results in the control group showed no cartilage formation and the disappearance of the scaffold at 8 weeks. Conversely, all differentiated hASCs/PHBV implants kept their original shape throughout 16 weeks. These implants at 16 weeks had stronger chondrocytes-specific histochemical staining than those at 8 weeks, with GAG, total collagen, and compressive moduli increased with implantation time. Cartilage lacunae were observed in all retrieved implants at 16 weeks. The chondrocytes-specific genes were detected by RT-PCR at 16 weeks. The remnants of PHBV were observed in the implants throughout 16 weeks. This study demonstrates that chondrogenic predifferentiated hASCs have the ability to maintain a chondrogenic phenotype in PHBV and that cell/PHBV constructs can produce neocartilage in a heterotopic site, but the degradation rates of PHBV in different environments needs more investigation.

  8. Influence of smartphone Wi-Fi signals on adipose-derived stem cells.

    PubMed

    Lee, Sang-Soon; Kim, Hyung-Rok; Kim, Min-Sook; Park, Sanghoon; Yoon, Eul-Sik; Park, Seung-Ha; Kim, Deok-Woo

    2014-09-01

    The use of smartphones is expanding rapidly around the world, thus raising the concern of possible harmful effects of radiofrequency generated by smartphones. We hypothesized that Wi-Fi signals from smartphones may have harmful influence on adipose-derived stem cells (ASCs). An in vitro study was performed to assess the influence of Wi-Fi signals from smartphones. The ASCs were incubated under a smartphone connected to a Wi-Fi network, which was uploading files at a speed of 4.8 Mbps for 10 hours a day, for a total of 5 days. We constructed 2 kinds of control cells, one grown in 37°C and the other grown in 39°C. After 5 days of Wi-Fi exposure from the smartphone, the cells underwent cell proliferation assay, apoptosis assay, and flow cytometry analysis. Three growth factors, vascular endothelial growth factor, hepatocyte growth factor, and transforming growth factor-β, were measured from ASC-conditioned media. Cell proliferation rate was higher in Wi-Fi-exposed cells and 39°C control cells compared with 37°C control cells. Apoptosis assay, flow cytometry analysis, and growth factor concentrations showed no remarkable differences among the 3 groups. We could not find any harmful effects of Wi-Fi electromagnetic signals from smartphones. The increased proliferation of ASCs under the smartphone, however, might be attributable to the thermal effect.

  9. From bench to bedside: use of human adipose-derived stem cells

    PubMed Central

    Feisst, Vaughan; Meidinger, Sarah; Locke, Michelle B

    2015-01-01

    Since the discovery of adipose-derived stem cells (ASC) in human adipose tissue nearly 15 years ago, significant advances have been made in progressing this promising cell therapy tool from the laboratory bench to bedside usage. Standardization of nomenclature around the different cell types used is finally being adopted, which facilitates comparison of results between research groups. In vitro studies have assessed the ability of ASC to undergo mesenchymal differentiation as well as differentiation along alternate lineages (transdifferentiation). Recently, focus has shifted to the immune modulatory and paracrine effects of transplanted ASC, with growing interest in the ASC secretome as a source of clinical effect. Bedside use of ASC is advancing alongside basic research. An increasing number of safety-focused Phase I and Phase IIb trials have been published without identifying any significant risks or adverse events in the short term. Phase III trials to assess efficacy are currently underway. In many countries, regulatory frameworks are being developed to monitor their use and assure their safety. As many trials rely on ASC injected at a distant site from the area of clinical need, strategies to improve the homing and efficacy of transplanted cells are also being explored. This review highlights each of these aspects of the bench-to-bedside use of ASC and summarizes their clinical utility across a variety of medical specialties. PMID:26586955

  10. Biocompatibility of polypropylene mesh scaffold with adipose-derived stem cells.

    PubMed

    Cheng, Hui; Zhang, Yanling; Zhang, Bei; Cheng, Jie; Wang, Weiqi; Tang, Xin; Teng, Peng; Li, Yanyu

    2017-06-01

    In this study, we investigated the rejection of the synthetic patch and human tissues in the host. We observed the growth of adipose-derived stem cells (ADSCs) cultured with polypropylene mesh in vitro. The results of flow cytometry showed that the expression of CD44, CD73, CD90, CD45, CD14 and CD34 was 98.54, 95.32, 98.49, 1.21, 3.01 and 2.14%, respectively. ADSCs were isolated from rabbit subcutaneous adipose tissue after collagenase digestion, filtration and centrifugation. The ADSCs of passage 3 were seeded onto the polypropylene mesh scaffolds. New Zealand White female breeder rabbits were implanted with polypropylene mesh, ADSC-fixed polypropylene mesh in the abdomen. After 4 weeks, adhesion was performed and the erosion of the mesh was evaluated. It was found that polypropylene mesh, ADSC-fixed polypropylene mesh all had different degrees of corrosion, and adhesion, but polypropylene mesh was more corroded. ADSC-fixed polypropylene mesh induced a milder chronic inflammation response compared with polypropylene, had significantly lower scores for inflammation (t=11.083), and had significantly higher scores for neovascularization (t=14.362) and fibroblastic proliferation (t=15.979). The relative amount of VEGF mRNA was significantly lower for ADSC-fixed polypropylene compared with the other polypropylene meshes (t=94.6). In conclusion, polypropylene mesh scaffold with ADSCs exhibit excellent cellular compatibility and are promising in clinical practice.

  11. Measurement of the biophysical properties of porcine adipose-derived stem cells by a microperfusion system.

    PubMed

    Wang, Jianye; Zhao, Gang; Zhang, Pengfei; Wang, Zhen; Zhang, Yunhai; Gao, Dayong; Zhou, Ping; Cao, Yunxia

    2014-12-01

    Adipose-derived stem cells (ADSCs), which are an accessible source of adult stem cells with capacities for self-renewal and differentiation into various cell types, have a promising potential in tissue engineering and regenerative medicine strategies. To meet the clinical demand for ADSCs, cryopreservation has been applied for long-term ADSC preservation. To optimize the addition, removal, freezing, and thawing of cryoprotective agents (CPAs) applied to ADSCs, we measured the transport properties of porcine ADSCs (pADSCs). The cell responses of pADSCs to hypertonic phosphate-buffered saline and common CPAs, dimethyl sulfoxide, ethylene glycol, and glycerol were measured by a microperfusion system at temperatures of 28, 18, 8, and -2°C. We determined the osmotically inactive cell volume (Vb), hydraulic conductivity (Lp), and CPA permeability (Ps) at various temperatures in a two-parameter model. Then, we quantitatively analyzed the effect of temperature on the transport properties of the pADSC membrane. Biophysical parameters were used to optimize CPA addition, removal, and freezing processes to minimize excessive shrinkage of pADSCs during cryopreservation. The biophysical properties of pADSCs have a great potential for effective optimization of cryopreservation procedures.

  12. Label-free protein profiling of adipose-derived human stem cells under hyperosmotic treatment.

    PubMed

    Oswald, Elizabeth S; Brown, Lewis M; Bulinski, J Chloë; Hung, Clark T

    2011-07-01

    Our previous work suggested that treatment of cells with hyperosmotic media during 2D passaging primes cells for cartilage tissue engineering applications. Here, we used label-free proteomic profiling to evaluate the effects of control and hyperosmotic treatment environments on the phenotype of multipotent adipose-derived stem cells (ASCs) cultivated with a chondrogenic growth factor cocktail. Spectra were recorded in a data-independent fashion at alternate low (precursor) and high (product) fragmentation voltages (MS(E)). This method was supplemented with data mining of accurate mass and retention time matches in precursor ion spectra across the experiment. The results indicated a complex cellular response to osmotic treatment, with a number of proteins differentially expressed between control and treated cell groups. The roles of some of these proteins have been documented in the literature as characteristic of the physiological states studied, especially aldose reductase (osmotic stress). This protein acted as a positive control in this work, providing independent corroborative validation. Other proteins, including 5'-nucleotidase and transgelin, have been previously linked to cell differentiation state. This study demonstrates that label-free profiling can serve as a useful tool in characterizing cellular responses to chondrogenic treatment regimes, recommending its use in optimization of cell priming protocols for cartilage tissue engineering.

  13. The Influence of Aging on the Regenerative Potential of Human Adipose Derived Mesenchymal Stem Cells

    PubMed Central

    Marycz, Krzysztof; Henry, Brandon Michael

    2016-01-01

    Tissue regeneration using human adipose derived mesenchymal stem cells (hASCs) has significant potential as a novel treatment for many degenerative bone and joint diseases. Previous studies have established that age negatively affects the proliferation status and the osteogenic and chondrogenic differentiation potential of mesenchymal stem cells. The aim of this study was to assess the age-related maintenance of physiological function and differentiation potential of hASCs in vitro. hASCs were isolated from patients of four different age groups: (1) >20 years (n = 7), (2) >50 years (n = 7), (3) >60 years (n = 7), and (4) >70 years (n = 7). The hASCs were characterized according to the number of fibroblasts colony forming unit (CFU-F), proliferation rate, population doubling time (PDT), and quantified parameters of adipogenic, chondrogenic, and osteogenic differentiation. Compared to younger cells, aged hASCs had decreased proliferation rates, decreased chondrogenic and osteogenic potential, and increased senescent features. A shift in favor of adipogenic differentiation with increased age was also observed. As many bone and joint diseases increase in prevalence with age, it is important to consider the negative influence of age on hASCs viability, proliferation status, and multilineage differentiation potential when considering the potential therapeutic applications of hASCs. PMID:26941800

  14. Endothelial Differentiation of Adipose-Derived Stem Cells from Elderly Patients with Cardiovascular Disease

    PubMed Central

    Zhang, Ping; Moudgill, Neil; Hager, Eric; Tarola, Nicolas; DiMatteo, Christopher; McIlhenny, Stephen; Tulenko, Thomas

    2011-01-01

    Adipose-derived stem cells (ASCs) possess significant therapeutic potential for tissue engineering and regeneration. This study investigates the endothelial differentiation and functional capacity of ASCs isolated from elderly patients. Isolation of ASCs from 53 patients (50–89 years) revealed that advanced age or comorbidity did not negatively impact stem cell harvest; rather, higher numbers were observed in older donors (>70 years) than in younger. ASCs cultured in endothelial growth medium-2 for up to 3 weeks formed cords upon Matrigel and demonstrated acetylated-low-density lipoprotein and lectin uptake. Further stimulation with vascular endothelial growth factor and shear stress upregulated endothelial cell-specific markers (CD31, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin). Inhibition of the PI3K but not mitogen-activated protein kinase pathway blocked the observed endothelial differentiation. Shear stress promoted an anti-thrombogenic phenotype as demonstrated by production of tissue-plasminogen activator and nitric oxide, and inhibition of plasminogen activator inhibitor-1. Shear stress augmented integrin α5β1 expression and subsequently increased attachment of differentiated ASCs to basement membrane components. Finally, ASCs seeded onto a decellularized vein graft resisted detachment despite application of shear force up to 9 dynes. These results suggest that (1) advanced age and comorbidity do not negatively impact isolation of ASCs, and (2) these stem cells retain significant capacity to acquire key endothelial cell traits throughout life. As such, adipose tissue is a practical source of autologous stem cells for vascular tissue engineering. PMID:20879833

  15. Promotion of human adipose-derived stem cell proliferation mediated by exogenous nucleosides.

    PubMed

    Rodríguez-Serrano, Fernando; Alvarez, Pablo; Caba, Octavio; Picón, Manuel; Marchal, Juan A; Perán, Macarena; Prados, José; Melguizo, Consolación; Rama, Ana R; Boulaiz, Houria; Aránega, Antonia

    2010-09-01

    Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose-derived stem cells), with or without co-treatment with 5-aza (5-azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5-aza induces a dose-dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 microM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5-Aza-induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.

  16. Proliferation and Differentiation Potential of Human Adipose-Derived Stem Cells Grown on Chitosan Hydrogel

    PubMed Central

    Debnath, Tanya; Ghosh, Sutapa; Potlapuvu, Usha Shalini; Kona, Lakshmi; Kamaraju, Suguna Ratnakar; Sarkar, Suprabhat; Gaddam, Sumanlatha; Chelluri, Lakshmi Kiran

    2015-01-01

    Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate. PMID:25746846

  17. Multilineage co-culture of adipose-derived stem cells for tissue engineering.

    PubMed

    Zhao, Yimu; Waldman, Stephen D; Flynn, Lauren E

    2015-07-01

    Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue-engineering strategies with adipose-derived stem cells (ASCs), the focus of this study was on developing indirect co-culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co-culture with each of the mature cell populations was shown to successfully induce or enhance lineage-specific differentiation of the ASCs. In general, a more homogeneous but lower-level differentiation response was observed in co-culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non-canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF-1 and DKK-1 on multilineage differentiation in co-culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co-culture with the mature cell populations. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Uninduced adipose-derived stem cells repair the defect of full-thickness hyaline cartilage.

    PubMed

    Zhang, Hai-Ning; Li, Lei; Leng, Ping; Wang, Ying-Zhen; Lv, Cheng-Yu

    2009-04-01

    To testify the effect of the stem cells derived from the widely distributed fat tissue on repairing full-thickness hyaline cartilage defects. Adipose-derived stem cells (ADSCs) were derived from adipose tissue and cultured in vitro. Twenty-seven New Zealand white rabbits were divided into three groups randomly. The cultured ADSCs mixed with calcium alginate gel were used to fill the full-thickness hyaline cartilage defects created at the patellafemoral joint, and the defects repaired with gel or without treatment served as control groups. After 4, 8 and 12 weeks, the reconstructed tissue was evaluated macroscopically and microscopically. Histological analysis and qualitative scoring were also performed to detect the outcome. Full thickness hyaline cartilage defects were repaired completely with ADSCs-derived tissue. The result was better in ADSCs group than the control ones. The microstructure of reconstructed tissue with ADSCs was similar to that of hyaline cartilage and contained more cells and regular matrix fibers, being better than other groups. Plenty of collagen fibers around cells could be seen under transmission electron microscopy. Statistical analysis revealed a significant difference in comparison with other groups at each time point (t equal to 4.360, P less than 0.01). These results indicate that stem cells derived from mature adipose without induction possess the ability to repair cartilage defects.

  19. Metabolic Rescue of Obese Adipose-Derived Stem Cells by Lin28/Let7 Pathway

    PubMed Central

    Pérez, Laura M.; Bernal, Aurora; San Martín, Nuria; Lorenzo, Margarita; Fernández-Veledo, Sonia; Gálvez, Beatriz G.

    2013-01-01

    Adipose-derived stem cells (ASCs) are promising candidates for autologous cell-based regeneration therapies by virtue of their multilineage differentiation potential and immunogenicity; however, relatively little is known about their role in adipose tissue physiology and dysfunction. Here we evaluated whether ASCs isolated from nonobese and obese tissue differed in their metabolic characteristics and differentiation potential. During differentiation to mature adipocytes, mouse and human ASCs derived from nonobese tissues both increased their insulin sensitivity and inhibition of lipolysis, whereas obese-derived ASCs were insulin-resistant, showing impaired insulin-stimulated glucose uptake and resistance to the antilipolytic effect of insulin. Furthermore, obese-derived ASCs showed enhanced release of proinflammatory cytokines and impaired production of adiponectin. Interestingly, the delivery of cytosol from control ASCs into obese-derived ASCs using a lipid-based, protein-capture methodology restored insulin sensitivity on glucose and lipid metabolism and reversed the proinflammatory cytokine profile, in part due to the restoration of Lin28 protein levels. In conclusion, glucose and lipid metabolism as well as maturation of ASCs is truncated in an obese environment. The reversal of the altered pathways in obese cells by delivery of normal subcellular fractions offers a potential new tool for cell therapy. PMID:23423565

  20. Effects of hypergravity on adipose-derived stem cell morphology, mechanical property and proliferation.

    PubMed

    Tavakolinejad, Alireza; Rabbani, Mohsen; Janmaleki, Mohsen

    2015-08-21

    Alteration in specific inertial conditions can lead to changes in morphology, proliferation, mechanical properties and cytoskeleton of cells. In this report, the effects of hypergravity on morphology of Adipose-Derived Stem Cells (ADSCs) are indicated. ADSCs were repeatedly exposed to discontinuous hypergravity conditions of 10 g, 20 g, 40 g and 60 g by utilizing centrifuge (three times of 20 min exposure, with an interval of 40 min at 1 g). Cell morphology in terms of length, width and cell elongation index and cytoskeleton of actin filaments and microtubules were analyzed by image processing. Consistent changes observed in cell elongation index as morphological change. Moreover, cell proliferation was assessed and mechanical properties of cells in case of elastic modulus of cells were evaluated by Atomic Force Microscopy. Increase in proliferation and decrease in elastic modulus of cells are further results of this study. Staining ADSC was done to show changes in cytoskeleton of the cells associated to hypergravity condition specifically in microfilament and microtubule components. After exposing to hypergravity, significant changes were observed in microfilaments and microtubule density as components of cytoskeleton. It was concluded that there could be a relationship between changes in morphology and MFs as the main component of the cells.

  1. Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression.

    PubMed

    Irioda, Ana Carolina; Cassilha, Rafael; Zocche, Larissa; Francisco, Julio Cesar; Cunha, Ricardo Correa; Ferreira, Priscila Elias; Guarita-Souza, Luiz Cesar; Ferreira, Reginaldo Justino; Mogharbel, Bassam Felipe; Garikipati, Venkata Naga Srikanth; Souza, Daiany; Beltrame, Mirian Perlingeiro; de Carvalho, Katherine Athayde Teixeira

    2016-01-01

    Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

  2. Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression

    PubMed Central

    Irioda, Ana Carolina; Cassilha, Rafael; Zocche, Larissa; Francisco, Julio Cesar; Cunha, Ricardo Correa; Ferreira, Priscila Elias; Guarita-Souza, Luiz Cesar; Ferreira, Reginaldo Justino; Mogharbel, Bassam Felipe; Garikipati, Venkata Naga Srikanth; Souza, Daiany; Beltrame, Mirian Perlingeiro; de Carvalho, Katherine Athayde Teixeira

    2016-01-01

    Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy. PMID:26981129

  3. Adipogenic and osteogenic differentiation of Lin(-)CD271(+)Sca-1(+) adipose-derived stem cells.

    PubMed

    Xiao, Jingang; Yang, Xiaojuan; Jing, Wei; Guo, Weihua; Sun, Qince; Lin, Yunfeng; Liu, Lei; Meng, Wentong; Tian, Weidong

    2013-05-01

    Adipose-derived stem cells (ASCs) have been defined as cells that undergo sustained in vitro growth and have multilineage differentiation potential. However, the identity and purification of ASCs has proved elusive due to the lack of specific markers and poor understanding of their physiological roles. Here, we prospectively isolated and identified a restricted homogeneous subpopulation of ASCs (Lin(-)CD271(+)Sca-1(+)) from mouse adipose tissues on the basis of cell-surface markers. Individual ASCs generated colony-forming unit-fibroblast at a high frequency and could differentiate into adipocytes, osteoblasts, and chondrocytes in vitro. Expansion of ASCs in a large quantity was feasible in medium supplemented with fibroblast growth factor-2 and leukemia inhibitory factor, without loss of adipogenic and osteogenic differentiation capacity. Moreover, we found that the transplanted ASCs can differentiate into adipocytes in adipogenic microenvironment in vivo and osteoblasts in osteogenic microenvironment in vivo. Thus we proved that Lin, CD271, and Sca-1 could be used as the specific markers to purify ASCs from adipose tissue. The method we established to identify ASCs as defined in vivo entities will help develop ASCs transplantation as a new therapeutic strategy for bone regeneration and adipose tissue regeneration in clinic.

  4. Biochanin a promotes osteogenic but inhibits adipogenic differentiation: evidence with primary adipose-derived stem cells.

    PubMed

    Su, Shu-Jem; Yeh, Yao-Tsung; Su, Shu-Hui; Chang, Kee-Lung; Shyu, Huey-Wen; Chen, Kuan-Ming; Yeh, Hua

    2013-01-01

    Biochanin A has promising effects on bone formation in vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1-1 μM) were assessed in vitro using various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPAR γ ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 μM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.

  5. Regulation of osteogenic differentiation of human adipose-derived stem cells by controlling electromagnetic field conditions

    PubMed Central

    Kang, Kyung Shin; Hong, Jung Min; Kang, Jo A; Rhie, Jong-Won; Jeong, Young Hun; Cho, Dong-Woo

    2013-01-01

    Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation. PMID:23306704

  6. The pivotal role of PDGF and its receptor isoforms in adipose-derived stem cells.

    PubMed

    Kim, Won-Serk; Park, Hyung-Sook; Sung, Jong-Hyuk

    2015-07-01

    Platelet-derived growth factor (PDGF) is one of the growth factors that reportedly regulates cell growth and division of mesenchymal cells. Although PDGF isoforms and their receptors reportedly play a pivotal role in mesenchymal stem cell regulation, there is a paucity of literature reviewing the role of PDGF in adipose-derived stem cells (ASCs). Therefore, we summarized previous reports on the expression and functional roles of PDGF and its receptor isoforms in this review. In addition, we examined findings pertaining to underlying molecular mechanisms and signaling pathways with special focus on PDGF-D/PDGFRβ. ASCs only express PDGF-A, -C, -D, PDGFRα, and PDGFRβ. PDGFRα expression decreases with adipocyte lineage, while PDGFRβ inhibits white adipocyte differentiation. In addition, PDGFRβ induces proliferation, migration, and angiogenesis and up-regulates the expression of paracrine factors in ASCs. Although PDGF-B and -D mediate their functions mainly by PDGFRβ and ROS generation, there are many differences between them in terms of regulating ASCs. PDGF-D is endogenous, generates ROS via the mitochondrial electron transport system, and regulates the autocrine loop of ASCs in vivo. Furthermore, PDGF-D has stronger mitogenic effects than PDGF-B.

  7. Small Diameter Blood Vessels Bioengineered From Human Adipose-derived Stem Cells

    PubMed Central

    Zhou, Renpeng; Zhu, Lei; Fu, Shibo; Qian, Yunliang; Wang, Danru; Wang, Chen

    2016-01-01

    Bioengineering of small-diameter blood vessels offers a promising approach to reduce the morbidity associated with coronary artery and peripheral vascular disease. The aim of this study was to construct a two-layered small-diameter blood vessel using smooth muscle cells (SMCs) and endothelial cells (ECs) differentiated from human adipose-derived stem cells (hASCs). The outer layer was constructed with biodegradable polycaprolactone (PCL)-gelatin mesh seeded with SMCs, and this complex was then rolled around a silicone tube under pulsatile stimulation. After incubation for 6 to 8 weeks, the PCL-gelatin degraded and the luminal supporting silicone tube was removed. The smooth muscle layer was subsequently lined with ECs differentiated from hASCs after stimulation with VEGF and BMP4 in combination hypoxia. The phenotype of differentiated SMCs and ECs, and the cytotoxicity of the scaffold and biomechanical assessment were analyzed. Our results demonstrated that the two-layered bioengineered vessels exhibited biomechanical properties similar to normal human saphenous veins (HSV). Therefore, hASCs provide SMCs and ECs for bioengineering of small-diameter blood vessels. PMID:27739487

  8. Live-Cell, Temporal Gene Expression Analysis of Osteogenic Differentiation in Adipose-Derived Stem Cells

    PubMed Central

    Desai, Hetal V.; Voruganti, Indu S.; Jayasuriya, Chathuraka; Chen, Qian

    2014-01-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3–5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs. PMID:24367991

  9. Mitogenic and chondrogenic effects of fibroblast growth factor-2 in adipose-derived mesenchymal cells

    SciTech Connect

    Chiou, Michael; Xu Yue; Longaker, Michael T. . E-mail: Longaker@stanford.edu

    2006-05-05

    Adipose-derived mesenchymal cells (AMCs) have demonstrated a great capacity for differentiating into bone, cartilage, and fat. Studies using bone marrow-derived mesenchymal cells (BMSCs) have shown that fibroblast growth factor (FGF)-2, a potent mitogenic factor, plays an important role in tissue engineering due to its effects in proliferation and differentiation for mesenchymal cells. The aim of this study was to investigate the function of FGF-2 in AMC chondrogenic differentiation and its possible contributions to cell-based therapeutics in skeletal tissue regeneration. Data demonstrated that FGF-2 significantly promoted the proliferation of AMCs and enhanced chondrogenesis in three-dimensional micromass culture. Moreover, priming AMCs with treatment of FGF-2 at 10 ng/ml demonstrated that cells underwent chondrogenic phenotypic differentiation, possibly by inducing N-Cadherin, FGF-receptor 2, and transcription factor Sox9. Our results indicated that FGF-2 potentiates chondrogenesis in AMCs, similar to its functions in BMSCs, suggesting the versatile potential applications of FGF-2 in skeletal regeneration and cartilage repair.

  10. Therapeutic potentials of human adipose-derived stem cells on the mouse model of Parkinson's disease.

    PubMed

    Choi, Hee Soon; Kim, Hee Jin; Oh, Jin-Hwan; Park, Hyeong-Geun; Ra, Jeong Chan; Chang, Keun-A; Suh, Yoo-Hun

    2015-10-01

    The treatment of Parkinson's disease (PD) using stem cells has long been the focus of many researchers, but the ideal therapeutic strategy has not yet been developed. The consistency and high reliability of the experimental results confirmed by animal models are considered to be a critical factor in the stability of stem cell transplantation for PD. Therefore, the aim of this study was to investigate the preventive and therapeutic potential of human adipose-derived stem cells (hASC) for PD and was to identify the related factors to this therapeutic effect. The hASC were intravenously injected into the tail vein of a PD mouse model induced by 6-hydroxydopamine. Consequently, the behavioral performances were significantly improved at 3 weeks after the injection of hASC. Additionally, dopaminergic neurons were rescued, the number of structure-modified mitochondria was decreased, and mitochondrial complex I activity was restored in the brains of the hASC-injected PD mouse model. Overall, this study underscores that intravenously transplanted hASC may have therapeutic potential for PD by recovering mitochondrial functions. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Canine Adipose Derived Mesenchymal Stem Cells Transcriptome Composition Alterations: A Step towards Standardizing Therapeutic

    PubMed Central

    Šimić, Ivana; Lojkić, Ivana; Bedeković, Tomislav

    2017-01-01

    Although canine adipose derived stem cells (cASCs) morphology characteristics and differentiation ability are well documented, transcriptome alterations of undifferentiated cASCs during ex vivo cultivation remain unknown. Here we demonstrate, for the first time, the transcriptome composition of isolated cASCs in undifferentiated state originating from six donors. Transcriptome changes were monitored during ex vivo cultivation between passage 3 (P3) and P5, which are mostly used in therapy. Influence of donors' age in given passage number on transcriptome composition was also investigated. Cultivation from P3 to P5 resulted in 16 differentially expressed genes with cooverexpression of pluripotency and self-renewal transcription factors genes SOX2 and POU5F1 dominant in old donors' cells. Furthermore, cASCs demonstrated upregulation of IL-6 in young and old donors' cells. In addition, ex vivo cultivation of cASCs revealed well-known morphological alterations accompanied with decrease in expression of CD90 and CD44 markers in P4 and higher monitored by flow cytometry and successful osteo- and chondrodifferentiation but inefficient adipodifferentiation in P3. Our results revealed the impact of ex vivo cultivation on nature of cells. Correlation of transcriptome changes with secretome composition is needed and its further impact on therapeutic potential of cASCs remains to be evaluated in clinical trials. PMID:28246532

  12. The Therapeutic Effect of Adipose-Derived Mesenchymal Stem Cells for Radiation-Induced Bladder Injury

    PubMed Central

    Qiu, Xuefeng; Zhang, Shiwei; Zhao, Xiaozhi; Fu, Kai; Guo, Hongqian

    2016-01-01

    This study was designed to investigate the protective effect of adipose derived mesenchymal stem cells (AdMSCs) against radiation-induced bladder injury (RIBI). Female rats were divided into 4 groups: (a) controls, consisting of nontreated rats; (b) radiation-treated rats; (c) radiation-treated rats receiving AdMSCs; and (d) radiation-treated rats receiving AdMSCs conditioned medium. AdMSCs or AdMSCs conditioned medium was injected into the muscular layer of bladder 24 h after radiation. Twelve weeks after radiation, urinary bladder tissue was collected for histological assessment and enzyme-linked immunosorbent assay (ELISA) after metabolic cage investigation. At the 1 w, 4 w, and 8 w time points following cells injection, 3 randomly selected rats in RC group and AdMSCs group were sacrificed to track injected AdMSCs. Metabolic cage investigation revealed that AdMSCs showed protective effect for radiation-induced bladder dysfunction. The histological and ELISA results indicated that the fibrosis and inflammation within the bladder were ameliorated by AdMSCs. AdMSCs conditioned medium showed similar effects in preventing radiation-induced bladder dysfunction. In addition, histological data indicated a time-dependent decrease in the number of AdMSCs in the bladder following injection. AdMSCs prevented radiation induced bladder dysfunction and histological changes. Paracrine effect might be involved in the protective effects of AdMSCs for RIBI. PMID:27051426

  13. Effect of autologous adipose-derived stem cells in renal cold ischemia and reperfusion injury.

    PubMed

    Wang, Y-L; Li, G; Zou, X-F; Chen, X-B; Liu, T; Shen, Z-Y

    2013-11-01

    The aim of this study was to investigate the effect of autologous adipose-derived stem cells (ADSCs) on renal cold ischemia and reperfusion (I/R) injury via intravenous infusion on rats. A renal cold I/R injury rat model was established. Rats were equally randomized into Sham group, Cold I/R group (cold I/R plus culture medium only), and ADSC-treated group (cold I/R plus immediate intrarenal administration of 2 × 10(6) autologous ADSCs, followed by intravenous autologous ADSCs 6 hours after reperfusion). All rats were killed 24 hours after the I/R procedure. Serum creatinine levels were significantly reduced in the ADSC-treated group compared with the Cold I/R group (P < .01). The renal tissue in the ADSC-treated group had well conserved renal architecture compared with the Cold I/R group. The mRNA expression of tumor necrosis factor α was significantly lower and Bcl-2 was higher in the ADSC-treated group than in the Cold I/R group (P < .05). Autologous ADSC infusions ameliorated renal damage undergoing cold I/R injury and improved the renal function, partly through inhibiting inflammatory reactions and reducing apoptosis. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Whitening effect of adipose-derived stem cells: a critical role of TGF-beta 1.

    PubMed

    Kim, Won-Serk; Park, So-Hyun; Ahn, Se-Jin; Kim, Hyung-Ki; Park, Jeong-Soo; Lee, Ga-Young; Kim, Kea-Jeung; Whang, Kyu-Kwang; Kang, Seung-Hee; Park, Byung-Soon; Sung, Jong-Hyuk

    2008-04-01

    It has been demonstrated that adipose-derived stem cells (ADSCs) secrete cytokines and exhibit diverse pharmacological actions. The present study examined the unknown pharmacological action of ADSCs regarding whitening effects. A conditioned medium of ADSCs (ADSC-CM) was harvested and the whitening effect of ADSC-CM was studied in melanoma B16 cells. ADSC-CM treatment inhibited the synthesis of melanin and the activity of tyrosinase in a dose dependent manner. To clarify the underlying mechanisms of the whitening action of ADSCs, protein levels of melanogenic proteins were measured by Western blot. Although expressions of microphthalmia-associated transcription factor and tyrosinase-related protein 2 (TRP2) remained unchanged, those of tyrosinase and TRP1 were down-regulated. Transforming growth factor-beta1 (TGF-beta 1), a potent regulator of melanogenic proteins, was neutralized by the addition of a blocking antibody to ADSC-CM, and down-regulated expression of tyrosinase and TRP1 was almost reversed. Collectively, these results indicate that secretary factors of ADSC inhibit melanin synthesis by down-regulating the expression of tyrosinase and TRP1, which are mainly mediated by TGF-beta1.

  15. Efficient generation of functional Schwann cells from adipose-derived stem cells in defined conditions.

    PubMed

    Xie, Songtao; Lu, Fan; Han, Juntao; Tao, Ke; Wang, Hongtao; Simental, Alfred; Hu, Dahai; Yang, Hao

    2017-03-15

    Schwann cells (SCs) are hitherto regarded as the most promising candidates for viable cell-based therapy to peripheral nervous system (PNS) injuries or degenerative diseases. However, the extreme drawbacks of transplanting autologous SCs for clinical applications still represent a significant bottleneck in neural regenerative medicine, mainly owing to the need of sacrificing a functional nerve to generate autologous SCs and the nature of slow expansion of the SCs. Thus, it is of great importance to establish an alternative cell system for the generation of sufficient SCs. Here, we demonstrated that adipose-derived stem cells (ADSCs) of rat robustly give rise to morphological, phenotypic and functional SCs using an optimized protocol. After undergoing a 3-week in vitro differentiation, almost all of treated ADSCs exhibited spindle shaped morphology similar to genuine SCs and expressed SC markers GFAP and S100. Most importantly, apart from acquisition of SC antigenic and biochemical features, the ADSC-derived SCs were functionally identical to native SCs as they possess a potential ability to form myelin, and secret nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF). The current study may provide an ideal strategy for harvesting sufficient SCs for cell-based treatment of various peripheral nerve injuries or disorders.

  16. Adipose-derived mesenchymal cells for bone regereneration: state of the art.

    PubMed

    Barba, Marta; Cicione, Claudia; Bernardini, Camilla; Michetti, Fabrizio; Lattanzi, Wanda

    2013-01-01

    Adipose tissue represents a hot topic in regenerative medicine because of the tissue source abundance, the relatively easy retrieval, and the inherent biological properties of mesenchymal stem cells residing in its stroma. Adipose-derived mesenchymal stem cells (ASCs) are indeed multipotent somatic stem cells exhibiting growth kinetics and plasticity, proved to induce efficient tissue regeneration in several biomedical applications. A defined consensus for their isolation, classification, and characterization has been very recently achieved. In particular, bone tissue reconstruction and regeneration based on ASCs has emerged as a promising approach to restore structure and function of bone compromised by injury or disease. ASCs have been used in combination with osteoinductive biomaterial and/or osteogenic molecules, in either static or dynamic culture systems, to improve bone regeneration in several animal models. To date, few clinical trials on ASC-based bone reconstruction have been concluded and proved effective. The aim of this review is to dissect the state of the art on ASC use in bone regenerative applications in the attempt to provide a comprehensive coverage of the topics, from the basic laboratory to recent clinical applications.

  17. Investigation of coculture of human adipose-derived stem cells and mature adipocytes.

    PubMed

    Song, Kedong; Li, Wenfang; Wang, Hong; Wang, Hai; Liu, Tianqing; Ning, Ruiming; Wang, Ling

    2012-08-01

    The purpose of this study was to evaluate the differentiation potential of human adipose-derived stem cells (hADSCs) into adipocytes by coculturing them with human mature adipocytes. The transwell culture system was utilized for indirect coculture of hADSCs and human mature adipocytes at four different hADSCs-to-mature adipocytes ratios, i.e., 1:5, 1:1, 2:1, and 5:1. After 8 days of coculture, the Oil Red O and Trypan Blue stainings were performed for the evaluation of adipogenic differentiation of hADSCs. In addition, flow cytometric analysis and Hoechst 33342/PI double staining were performed after 20 days of coculture. The Oil Red O and Trypan Blue stainings showed that hADSCs with high viability could not differentiate into mature adipocytes after 8 or 20 days of coculture. However, flow cytometric analysis indicated that CD105 expression of hADSCs decreased after 20 days of coculture. These results indicated that hADSCs cocultured with human adult adipocytes could not successfully differentiate into adipocytes.

  18. Moderate Hypoxia Influences Potassium Outward Currents in Adipose-Derived Stem Cells

    PubMed Central

    Prasad, Mayuri; Zachar, Vladimir; Fink, Trine; Pennisi, Cristian Pablo

    2014-01-01

    Moderate hypoxic preconditioning of adipose-derived stem cells (ASCs) enhances properties such as proliferation and secretion of growth factors, representing a valuable strategy to increase the efficiency of cell-based therapies. In a wide variety of cells potassium (K+) channels are key elements involved in the cellular responses to hypoxia, suggesting that ASCs cultured under low oxygen conditions may display altered electrophysiological properties. Here, the effects of moderate hypoxic culture on proliferation, whole-cell currents, and ion channel expression were investigated using human ASCs cultured at 5% and 20% oxygen. Although cell proliferation was greatly enhanced, the dose-dependent growth inhibition by the K+ channel blocker tetraethylammonium (TEA) was not significantly affected by hypoxia. Under both normoxic and hypoxic conditions, ASCs displayed outward K+ currents composed by Ca2+-activated, delayed rectifier, and transient components. Hypoxic culture reduced the slope of the current-voltage curves and caused a negative shift in the voltage activation threshold of the whole-cell currents. However, the TEA-mediated shift of voltage activation threshold was not affected by hypoxia. Semiquantitative real-time RT-PCR revealed that expression of genes encoding for various ion channels subunits related to oxygen sensing and proliferation remained unchanged after hypoxic culture. In conclusion, outward currents are influenced by moderate hypoxia in ASCs through a mechanism that is not likely the result of modulation of TEA-sensitive K+ channels. PMID:25115627

  19. Human adipose derived stroma/stem cells grow in serum-free medium as floating spheres.

    PubMed

    Dromard, C; Bourin, P; André, M; De Barros, S; Casteilla, L; Planat-Benard, V

    2011-04-01

    With the goal of obtaining clinically safe human adipose-derived stroma/stem cells (ASC) and eliminating the use of serum, we have developed a new culture system that allows the expansion of ASC as spheres in a defined medium. These spheres can be passaged several times. They are not only aggregated cells but rather originate from single cells as clonal spheres can be obtained after seeding at very low density and reform clonal spheres after dissociation. These spheres can also revert to monolayer growth when plated in medium containing human plasma and even generate fibroblast-like colonies (CFU-f). Under several differentiation-specific media, spheres-derived ASC maintain their capacity to differentiate into osteoblasts, endothelial cells and adipocytes. These results indicate that human ASC can be maintained in a serum-free 3D culture system, which is of great interest for the expansion in bioreactors of autologous ASC and their use in clinical trials. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Stimulating the neurotrophic and angiogenic properties of human adipose-derived stem cells enhances nerve repair.

    PubMed

    Kingham, Paul J; Kolar, Mallappa K; Novikova, Liudmila N; Novikov, Lev N; Wiberg, Mikael

    2014-04-01

    In future, adipose-derived stem cells (ASC) might be used to treat neurological disorders. In this study, the neurotrophic and angiogenic properties of human ASC were evaluated, and their effects in a peripheral nerve injury model were determined. In vitro growth factor stimulation of the cells resulted in increased secretion of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor-A (VEGF-A), and angiopoietin-1 proteins. Conditioned medium from stimulated cells increased neurite outgrowth of dorsal root ganglia (DRG) neurons. Similarly, stimulated cells showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. ASC were seeded into a fibrin conduit that was used to bridge a 10 mm rat nerve gap. After 2 weeks, the animals treated with control or stimulated ASC showed an enhanced axon regeneration distance. Stimulated cells evoked more total axon growth. Analysis of regeneration and apoptosis-related gene expression showed that both ASC and stimulated ASC enhanced GAP-43 and activating transcription factor 3 (ATF-3) expression in the spinal cord and reduced c-jun expression in the DRG. Caspase-3 expression in the DRG was reduced by stimulated ASC. Both ASC and stimulated ASC also increased the vascularity of the fibrin nerve conduits. Thus, ASC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for nerve regeneration.

  1. The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells

    PubMed Central

    Fan, Jun; Sun, Zhongjie

    2017-01-01

    Klotho was originally discovered as an aging-suppressor gene. The purpose of this study was to investigate whether secreted Klotho (SKL) affects the proliferation and differentiation of adipose-derived stem cells (ADSCs). RT-PCR and Western blot analysis showed that short-form Klotho was expressed in mouse ADSCs. The Klotho gene mutation KL(−/−) significantly decreased proliferation of ADSCs and expression of pluripotent transcription factors (Nanog, Sox-2, and Oct-4) in mice. The adipogenic differentiation of ADSCs was also decreased in KL(−/−) mice. Incubation with Klotho-deficient medium decreased ADSC proliferation, pluripotent transcription factor levels, and adipogenic differentiation, which is similar to what was found in KL(−/−) mice. These results indicate that Klotho deficiency suppresses ADSC proliferation and differentiation. Interestingly, treatment with recombinant SKL protein rescued the Klotho deficiency-induced impairment in ADSC proliferation and adipogenic differentiation. SKL also regulated ADSCs’ differentiation to other cell lineages (osteoblasts, myofibroblasts), indicating that SKL maintains stemness of ADSCs. It is intriguing that overexpression of SKL significantly increased PPAR-γ expression and lipid formation in ADSCs following adipogenic induction, indicating enhanced adipogenic differentiation. Overexpression of SKL inhibited expression of TGFβ1 and its downstream signaling mediator Smad2/3. This study demonstrates, for the first time, that SKL is essential to the maintenance of normal proliferation and differentiation in ADSCs. Klotho regulates adipogenic differentiation in ADSCs, likely via inhibition of TGFβ1 and activation of PPAR-γ. PMID:26865060

  2. Trophic effects of adipose derived stem cells on Langerhans islets viability--Review.

    PubMed

    Dietrich, Isa; Crescenzi, Alessandra; Chaib, Elezar; D'Albuquerque, Luiz Augusto Carneiro

    2015-07-01

    Langerhans islets transplantation has been proposed to provide an endogenous source of insulin in Type I diabetes. However, the hypoxic stress and the receptor's immune reaction suffered by the implants cause them to fail in sustaining the insulin production along the time. Experimental studies have shown that adipose derived stem cells (ADSCs) can secrete cytokines that activate free radical scavengers, antioxidants and chaperone heat/shock proteins leading to reduction of apoptosis in damaged tissues. Therefore, using the PubMed database, we reviewed the experimental studies that investigated the trophic effects of ADSCs on Langerhans islets viability, in vitro and in vivo, from 2009 to 2014. We excluded articles that investigated the effects of other types of mesenchymal stem cells on β-cell survival as well articles that worked in the differentiation of ADSCs into insulin producing cells. The analysis of the experiments revealed that exposure of islets to ADSCs in vitro, even for a short period of time, can enhance islet cell viability and function. In vivo studies also corroborated the trophic effects of ADSCs leading to the improvement of islet function and reduction of the number of the islets required for controlling the receptor's glucose levels. This review can contribute to guide future experiments looking for a long term diabetes treatment employing ADSC trophic effects for the enhancement of transplanted Langerhans islet viability and functioning. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Sundew-Inspired Adhesive Hydrogels Combined with Adipose-Derived Stem Cells for Wound Healing.

    PubMed

    Sun, Leming; Huang, Yujian; Bian, Zehua; Petrosino, Jennifer; Fan, Zhen; Wang, Yongzhong; Park, Ki Ho; Yue, Tao; Schmidt, Michael; Galster, Scott; Ma, Jianjie; Zhu, Hua; Zhang, Mingjun

    2016-01-27

    The potential to harness the unique physical, chemical, and biological properties of the sundew (Drosera) plant's adhesive hydrogels has long intrigued researchers searching for novel wound-healing applications. However, the ability to collect sufficient quantities of the sundew plant's adhesive hydrogels is problematic and has eclipsed their therapeutic promise. Inspired by these natural hydrogels, we asked if sundew-inspired adhesive hydrogels could overcome the drawbacks associated with natural sundew hydrogels and be used in combination with stem-cell-based therapy to enhance wound-healing therapeutics. Using a bioinspired approach, we synthesized adhesive hydrogels comprised of sodium alginate, gum arabic, and calcium ions to mimic the properties of the natural sundew-derived adhesive hydrogels. We then characterized and showed that these sundew-inspired hydrogels promote wound healing through their superior adhesive strength, nanostructure, and resistance to shearing when compared to other hydrogels in vitro. In vivo, sundew-inspired hydrogels promoted a "suturing" effect to wound sites, which was demonstrated by enhanced wound closure following topical application of the hydrogels. In combination with mouse adipose-derived stem cells (ADSCs) and compared to other therapeutic biomaterials, the sundew-inspired hydrogels demonstrated superior wound-healing capabilities. Collectively, our studies show that sundew-inspired hydrogels contain ideal properties that promote wound healing and suggest that sundew-inspired-ADSCs combination therapy is an efficacious approach for treating wounds without eliciting noticeable toxicity or inflammation.

  4. Adipose-derived stem cell differentiation as a basic tool for vascularized adipose tissue engineering.

    PubMed

    Volz, Ann-Cathrin; Huber, Birgit; Kluger, Petra J

    2016-01-01

    The development of in vitro adipose tissue constructs is highly desired to cope with the increased demand for substitutes to replace damaged soft tissue after high graded burns, deformities or tumor removal. To achieve clinically relevant dimensions, vascularization of soft tissue constructs becomes inevitable but still poses a challenge. Adipose-derived stem cells (ASCs) represent a promising cell source for the setup of vascularized fatty tissue constructs as they can be differentiated into adipocytes and endothelial cells in vitro and are thereby available in sufficiently high cell numbers. This review summarizes the currently known characteristics of ASCs and achievements in adipogenic and endothelial differentiation in vitro. Further, the interdependency of adipogenesis and angiogenesis based on the crosstalk of endothelial cells, stem cells and adipocytes is addressed at the molecular level. Finally, achievements and limitations of current co-culture conditions for the construction of vascularized adipose tissue are evaluated. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  5. Biological and Clinical Availability of Adipose-Derived Stem Cells for Pelvic Dead Space Repair

    PubMed Central

    Takahashi, Hidekazu; Haraguchi, Naotsugu; Nishikawa, Shimpei; Miyazaki, Susumu; Suzuki, Yozou; Mizushima, Tsunekazu; Nishimura, Junichi; Takemasa, Ichirou; Yamamoto, Hirofumi; Mimori, Koshi; Ishii, Hideshi; Doki, Yuichiro

    2012-01-01

    Adipose-derived stem cells (ADSCs) are a very attractive cell source for regenerative and reconstructive medicine. Although ADSCs have already been used in cardiovascular disease and cosmetic surgery, they have not yet been used in gastroenterological surgery. In this study, we clarified the utility of the combined application of ADSCs and resected intraperitoneal fatty tissues as a sealant for the pelvic dead space that sometimes causes severe and fatal complications in colorectal and gynecological surgeries. In pelvic dead space model mice, mouse ADSCs efficiently maintained transplanted intraperitoneal fatty tissues without any incidence of adhesion to surrounding organs. In vivo and in vitro analyses revealed that transplanted ADSCs differentiated into endothelial cells by expressing the angiogenic factors vascular endothelial growth factor and hepatocyte growth factor. Mouse and human ADSCs contained a CD45−CD34+ subset possessing high colony formation and sphere formation abilities. In addition, the CD45−CD34+ subset consisted of two characteristic subsets: the CD34+CD90+ angiogenic subset and the CD34+CD90− adipogenic subset. Grafts of human ADSCs with fat transplanted into mice were efficiently maintained for more than 12 months without volume reductions. A comparative study of graft maintenance efficacy between cultured human ADSCs and freshly isolated ADSCs indicated that the cultivation of ADSCs decreased their graft maintenance ability. These findings suggested that the angiogenic and adipogenic subsets act in coordination with each other and are essential for efficient graft maintenance. PMID:23197692

  6. Human adipose-derived mesenchymal progenitor cells engraft into rabbit articular cartilage.

    PubMed

    Wang, Wen; He, Na; Feng, Chenchen; Liu, Victor; Zhang, Luyi; Wang, Fei; He, Jiaping; Zhu, Tengfang; Wang, Shuyang; Qiao, Weiwei; Li, Suke; Zhou, Guangdong; Zhang, Li; Dai, Chengxiang; Cao, Wei

    2015-05-27

    Mesenchymal stem cells (MSCs) are known to have the potential for articular cartilage regeneration, and are suggested for the treatment of osteoarthritis (OA). Here, we investigated whether intra-articular injection of xenogeneic human adipose-derived mesenchymal progenitor cells (haMPCs) promoted articular cartilage repair in rabbit OA model and engrafted into rabbit articular cartilage. The haMPCs were cultured in vitro, and phenotypes and differentiation characteristics of cells were evaluated. OA was induced surgically by anterior cruciate ligament transection (ACLT) and medical meniscectomy of knee joints. At six weeks following surgery, hyaluronic acid (HA) or haMPCs was injected into the knee joints, the contralateral knee served as normal control. All animals were sacrificed at the 16th week post-surgery. Assessments were carried out by macroscopic examination, hematoxylin/eosin (HE) and Safranin-O/Fast green stainings and immunohistochemistry. The data showed that haMPC treatment promoted cartilage repair. Signals of human mitochondrial can be directly detected in haMPC treated cartilage. The haMPCs expressed human leukocyte antigen I (HLA-I) but not HLA-II-DR in vivo. These results suggest that intra-articular injection of haMPCs promotes regeneration of articular cartilage in rabbit OA model, and support the notion that MPCs are transplantable between HLA-incompatible individuals.

  7. Osteogenic differentiation of human adipose-derived mesenchymal stem cells on gum tragacanth hydrogel.

    PubMed

    Haeri, Seyed Mohammad Jafar; Sadeghi, Yousef; Salehi, Mohammad; Farahani, Reza Masteri; Mohsen, Nourozian

    2016-05-01

    Currently, natural polymer based hydrogels has attracted great attention of orthopedic surgeons for application in bone tissue engineering. With this aim, osteoinductive capacity of Gum Tragacanth (GT) based hydrogel was compared to collagen hydrogel and tissue culture plate (TCPS). For this purpose, adipose-derived mesenchymal stem cells (AT-MSCs) was cultured on the hydrogels and TCPS and after investigating the biocompatibility of hydrogels using MTT assay, osteoinductivity of hydrogels were evaluated using pan osteogenic markers such as Alizarin red staining, alkaline phosphatase (ALP) activity, calcium content and osteo-related genes. Increasing proliferation trend of AT-MSCs on GT hydrogel demonstrated that TG has no-cytotoxicity and can even be better than the other groups i.e., highest proliferation at day 5. GT hydrogel displayed highest ALP activity and mineralization when compared to the collagen hydrogel and TCPS. Relative gene expression levels have demonstrated that highest expression of Runx2, osteonectin and osteocalcin in the cells cultured GT hydrogel but the expression of collagen type-1 remains constant in hydrogels. Above results demonstrate that GT hydrogel could be an appropriate scaffold for accelerating and supporting the adhesion, proliferation and osteogenic differentiation of stem cells which further can be used for orthopedic applications.

  8. Concise review: Adipose-derived stem cells as a novel tool for future regenerative medicine.

    PubMed

    Mizuno, Hiroshi; Tobita, Morikuni; Uysal, A Cagri

    2012-05-01

    The potential use of stem cell-based therapies for the repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions for a number of diseases. The use of either embryonic stem cells (ESCs) or induced pluripotent stem cells in clinical situations is limited due to cell regulations and to technical and ethical considerations involved in the genetic manipulation of human ESCs, even though these cells are, theoretically, highly beneficial. Mesenchymal stem cells seem to be an ideal population of stem cells for practical regenerative medicine, because they are not subjected to the same restrictions. In particular, large number of adipose-derived stem cells (ASCs) can be easily harvested from adipose tissue. Furthermore, recent basic research and preclinical studies have revealed that the use of ASCs in regenerative medicine is not limited to mesodermal tissue but extends to both ectodermal and endodermal tissues and organs, although ASCs originate from mesodermal lineages. Based on this background knowledge, the primary purpose of this concise review is to summarize and describe the underlying biology of ASCs and their proliferation and differentiation capacities, together with current preclinical and clinical data from a variety of medical fields regarding the use of ASCs in regenerative medicine. In addition, future directions for ASCs in terms of cell-based therapies and regenerative medicine are discussed. Copyright © 2012 AlphaMed Press.

  9. The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test.

    PubMed

    Abud, Ana Paula Ressetti; Zych, Jaiesa; Reus, Thamile Luciane; Kuligovski, Crisciele; de Moraes, Elizabeth; Dallagiovanna, Bruno; de Aguiar, Alessandra Melo

    2015-12-01

    Human adipose-derived stem cells (ADSC) were evaluated as cell culture model for cytotoxicity assay and toxicity prediction by using the neutral red uptake assay (NRU). In this study, we compared ADSC and the murine cell line BALB/c 3T3 clone A31 to predict the toxicity of 12 reference substances as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods. We predicted the LD50 for RC-rat-only weight and RC-rat-only millimole regressions for both cell culture models. For RC rat-only weight regression, both cells had the same accordance (50%), while for RC rat-only millimole regression, the accordance was 50% for ADSC and 42% for 3T3s. Thus, ADSC have similar capability for GHS class prediction as the 3T3 cell line for the evaluated reference substances. Therefore, ADSCs showed the potential to be considered a novel model for use in evaluating cytotoxicity in drug development and industry as well as for regulatory purposes to reduce or replace the use of laboratory animals with acceptable sensitivity for toxicity prediction in humans. These cells can be used to complete the results from other models, mainly because of its human origin. Moreover, it is less expensive in comparison with other existing models. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Characterization of adipose tissue macrophages and adipose-derived stem cells in critical wounds

    PubMed Central

    Tilstam, Pathricia V.; Springenberg-Jung, Katrin; Boecker, Arne Hendrick; Schmitz, Corinna; Heinrichs, Daniel; Hwang, Soo Seok; Stromps, Jan Philipp; Ganse, Bergita; Kopp, Ruedger; Knobe, Matthias; Bernhagen, Juergen

    2017-01-01

    Background Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers. Methods In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis. Results Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confirmed. Furthermore, a decrease in the mesenchymal stem cell markers CD29, CD34, and CD105 was observed whereas CD73 and CD90 remained unchanged. Discussion This is the first report describing the predominance of M1 adipose tissue macrophages and the reduction of stem cell marker expression in acutely inflamed, non-healing wounds. PMID:28070458

  11. Assembling Composite Dermal Papilla Spheres with Adipose-derived Stem Cells to Enhance Hair Follicle Induction

    PubMed Central

    Huang, Chin-Fu; Chang, Ya-Ju; Hsueh, Yuan-Yu; Huang, Chia-Wei; Wang, Duo-Hsiang; Huang, Tzu-Chieh; Wu, Yi-Ting; Su, Fong-Chin; Hughes, Michael; Chuong, Cheng-Ming; Wu, Chia-Ching

    2016-01-01

    Intradermal adipose tissue plays an essential role for hair follicles (HFs) regeneration by regulating hair cycles. However, the effect of reconstruction of HFs and the involvement of adipose-related cells are poorly understood. We investigated assembly strategies for the interactions of dermal papilla (DP) cells with adipose-derived stem cells (ASCs) in promoting hair formation. DP cells lose DP traits during adherent culture, but preserved DP markers with a unified sphere diameter by seeding on chitosan-coated microenvironments. Next, ASCs isolated from rats were co-cultured with DP spheres by different assembling approaches to determine their interactions; a mixed sphere of ASCs with DP cells (MA-DPS), or a core-shell structure, outer ASCs shell and an inner DP core (CSA-DPS). CSA-DPS exhibited superior DP characteristics compared to MA-DPS. Conditional medium from ASCs, but not differentiated adipocytes, promoted DP markers and functional alkaline phosphatase activity from the DP cells. In vivo patch assay showed the core-shell assembling of CSA-DPS can reconstruct cellular arrangements and microenvironmental niches as dominated by PPARα signal in ASCs to induce the greater hair induction than MA-DPS or DP spheres alone. Therefore, the assembling of a core-shell sphere for DP with ASCs could reconstruct the HF cellular arrangement for hair formation. This paper set the groundwork for further evaluation of the input of other cell types. PMID:27210831

  12. Human adipose-derived stem cells promote vascularization of collagen-based scaffolds transplanted into nude mice

    PubMed Central

    Cherubino, Mario; Valdatta, Luigi; Balzaretti, Riccardo; Pellegatta, Igor; Rossi, Federica; Protasoni, Marina; Tedeschi, Alessandra; Accolla, Roberto S; Bernardini, Giovanni; Gornati, Rosalba

    2016-01-01

    Aim: After in vivo implantation of cell-loaded devices, only the cells close to the capillaries can obtain nutrients to maintain their functions. It is known that factors secreted by stem cells, rather than stem cells themselves, are fundamental to guarantee new vascularization in the area of implant. Materials & methods: To investigate this possibility, we have grafted mice with Bilayer and Flowable Integra® scaffolds, loaded or not with human adipose-derived stem cells. Results: Our results support the therapeutic potential of human adipose-derived stem cells to induce new vascular networks of engineered organs and tissues. Conclusion: This finding suggests that our approach can help to form new vascular networks that allow sufficient vascularization of engineered organs and tissues in cases of difficult wound healing due to ischemic conditions. PMID:26965659

  13. Comparison of Markers and Functional Attributes of Human Adipose-Derived Stem Cells and Dedifferentiated Adipocyte Cells from Subcutaneous Fat of an Obese Diabetic Donor.

    PubMed

    Watson, James E; Patel, Niketa A; Carter, Gay; Moor, Andrea; Patel, Rekha; Ghansah, Tomar; Mathur, Abhishek; Murr, Michel M; Bickford, Paula; Gould, Lisa J; Cooper, Denise R

    2014-03-01

    Objective: Adipose tissue is a robust source of adipose-derived stem cells (ADSCs) that may be able to provide secreted factors that promote the ability of wounded tissue to heal. However, adipocytes also have the potential to dedifferentiate in culture to cells with stem cell-like properties that may improve their behavior and functionality for certain applications. Approach: ADSCs are adult mesenchymal stem cells that are cultured from the stromal vascular fraction of adipose tissue. However, adipocytes are capable of dedifferentiating into cells with stem cell properties. In this case study, we compare ADSC and dedifferentiated fat (DFAT) cells from the same patient and fat depot for mesenchymal cell markers, embryonic stem cell markers, ability to differentiate to adipocytes and osteoblasts, senescence and telomerase levels, and ability of conditioned media (CM) to stimulate migration of human dermal fibroblasts (HDFs). Innovation and Conclusions: ADSCs and DFAT cells displayed identical levels of CD90, CD44, CD105, and were CD34- and CD45-negative. They also expressed similar levels of Oct4, BMI1, KLF4, and SALL4. DFAT cells, however, showed higher efficiency in adipogenic and osteogenic capacity. Telomerase levels of DFAT cells were double those of ADSCs, and senescence declined in DFAT cells. CM from both cell types altered the migration of fibroblasts. Despite reports of ADSCs from a number of human depots, there have been no comparisons of the ability of dedifferentiated DFAT cells from the same donor and depot to differentiate or modulate migration of HDFs. Since ADSCs were from an obese diabetic donor, reprogramming of DFAT cells may help improve a patient's cells for regenerative medicine applications.

  14. Impact of Hyperglycemia and Low Oxygen Tension on Adipose-Derived Stem Cells Compared with Dermal Fibroblasts and Keratinocytes: Importance for Wound Healing in Type 2 Diabetes

    PubMed Central

    Lafosse, Aurore; Dufeys, Cécile; Beauloye, Christophe; Horman, Sandrine; Dufrane, Denis

    2016-01-01

    Aim Adipose-derived stem cells (ASC) are currently proposed for wound healing in those with type 2 diabetes mellitus (T2DM). Therefore, this study investigated the impact of diabetes on adipose tissue in relation to ASC isolation, proliferation, and growth factor release and the impact of hyperglycemia and low oxygen tension (found in diabetic wounds) on dermal fibroblasts, keratinocytes, and ASC in vitro. Methods Different sequences of hypoxia and hyperglycemia were applied in vitro to ASC from nondiabetic (n = 8) or T2DM patients (n = 4) to study cell survival, proliferation, and growth factor release. Comparisons of dermal fibroblasts (n = 8) and keratinocytes (primary lineage) were made. Results No significant difference of isolation and proliferation capacities was found in ASC from nondiabetic and diabetic humans. Hypoxia and hyperglycemia did not impact cell viability and proliferation. Keratinocyte Growth Factor release was significantly lower in diabetic ASC than in nondiabetic ASC group in each condition, while Vascular Endothelial Growth Factor release was not affected by the diabetic origin. Nondiabetic ASC exposition to hypoxia (0.1% oxygen) combined with hyperglycemia (25mM glucose), resulted in a significant increase in VEGF secretion (+64%, p<0.05) with no deleterious impact on KGF release in comparison to physiological conditions (5% oxygen and 5 mM glucose). Stromal cell-Derived Factor-1α (-93%, p<0.001) and KGF (-20%, p<0.05) secretion by DF decreased in these conditions. Conclusions A better profile of growth factor secretion (regarding wound healing) was found in vitro for ASC in hyperglycemia coupled with hypoxia in comparison to dermal fibroblasts and keratinocytes. Interestingly, ASC from T2DM donors demonstrated cellular growth rates and survival (in hypoxia and hyperglycemic conditions) similar to those of healthy ASC (from normoglycemic donors); however, KGF secretion was significantly depleted in ASC obtained from T2DM patients. This

  15. Comparison of Markers and Functional Attributes of Human Adipose-Derived Stem Cells and Dedifferentiated Adipocyte Cells from Subcutaneous Fat of an Obese Diabetic Donor

    PubMed Central

    Watson, James E.; Patel, Niketa A.; Carter, Gay; Moor, Andrea; Patel, Rekha; Ghansah, Tomar; Mathur, Abhishek; Murr, Michel M.; Bickford, Paula; Gould, Lisa J.; Cooper, Denise R.

    2014-01-01

    Objective: Adipose tissue is a robust source of adipose-derived stem cells (ADSCs) that may be able to provide secreted factors that promote the ability of wounded tissue to heal. However, adipocytes also have the potential to dedifferentiate in culture to cells with stem cell-like properties that may improve their behavior and functionality for certain applications. Approach: ADSCs are adult mesenchymal stem cells that are cultured from the stromal vascular fraction of adipose tissue. However, adipocytes are capable of dedifferentiating into cells with stem cell properties. In this case study, we compare ADSC and dedifferentiated fat (DFAT) cells from the same patient and fat depot for mesenchymal cell markers, embryonic stem cell markers, ability to differentiate to adipocytes and osteoblasts, senescence and telomerase levels, and ability of conditioned media (CM) to stimulate migration of human dermal fibroblasts (HDFs). Innovation and Conclusions: ADSCs and DFAT cells displayed identical levels of CD90, CD44, CD105, and were CD34- and CD45-negative. They also expressed similar levels of Oct4, BMI1, KLF4, and SALL4. DFAT cells, however, showed higher efficiency in adipogenic and osteogenic capacity. Telomerase levels of DFAT cells were double those of ADSCs, and senescence declined in DFAT cells. CM from both cell types altered the migration of fibroblasts. Despite reports of ADSCs from a number of human depots, there have been no comparisons of the ability of dedifferentiated DFAT cells from the same donor and depot to differentiate or modulate migration of HDFs. Since ADSCs were from an obese diabetic donor, reprogramming of DFAT cells may help improve a patient's cells for regenerative medicine applications. PMID:24669358

  16. Icariin promotes proliferation and osteogenic differentiation of rat adipose-derived stem cells by activating the RhoA-TAZ signaling pathway.

    PubMed

    Ye, Yaping; Jing, Xingzhi; Li, Na; Wu, Yingxing; Li, Bingbing; Xu, Tao

    2017-04-01

    Icariin, the main active flavonoid glucoside isolated from Herba epimedii, has been demonstrated to be a potential alternative therapy to prevent postmenopausal osteoporosis. Icariin has also been shown to regulate the proliferation and osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). However, the detailed molecular mechanism of icariin has remained largely unknown. Besides, no investigation has focused on the relevance of icariin in the regulation of rat adipose-derived stem cells (rASCs) proliferation and osteogenic differentiation. In the present study, we detected that icariin promotes proliferation and osteogenic differentiation of rASCs in a concentration range from 10(-8)M to 10(-6)M, with 10(-7)M to be the optimal concentration. We found that 10(-7)M icariin significantly increased active RhoA protein expression and ROCK substrate molecule p-MYPT1 expression in rASCs. C3 (the RhoA inhibitor) treatment abrogated the increased proliferation and osteogenic differentiation of rASCs induced by icariin. Interestingly, we also found that C3 abrogated the activation of TAZ induced by icariin. Depletion of TAZ by siRNA transfection significantly blocked icariin promoted proliferation and osteogenic differentiation of rASCs. However, icariin induced active RhoA protein expression was not affected by TAZ specific siRNA transfection, suggesting that RhoA lies upstream of TAZ. Taken together, our data indicate that icariin promotes proliferation and osteogenic differentiation of rASCs by activating the RhoA-TAZ signaling pathway.

  17. Mitochondrial Functional Changes Characterization in Young and Senescent Human Adipose Derived MSCs

    PubMed Central

    Stab, Bernd R.; Martinez, Laura; Grismaldo, Adriana; Lerma, Alejandra; Gutiérrez, María L.; Barrera, Luis A.; Sutachan, Jhon J.; Albarracín, Sonia L.

    2016-01-01

    Mitochondria are highly dynamic organelles that in response to the cell's bio-energetic state continuously undergo structural remodeling fission and fusion processes. This mitochondrial dynamic activity has been implicated in cell cycle, autophagy, and age-related diseases. Adult tissue-derived mesenchymal stromal/stem cells present a therapeutic potential. However, to obtain an adequate mesenchymal stromal/stem cell number for clinical use, extensive in vitro expansion is required. Unfortunately, these cells undergo replicative senescence rapidly by mechanisms that are not well understood. Senescence has been associated with metabolic changes in the oxidative state of the cell, a process that has been also linked to mitochondrial fission and fusion events, suggesting an association between mitochondrial dynamics and senescence. In the present work, we studied the mitochondrial structural remodeling process of mesenchymal stromal/stem cells isolated from adipose tissue in vitro to determine if mitochondrial phenotypic changes were associated with mesenchymal stromal/stem cell senescence. For this purpose, mitochondrial dynamics and oxidative state of stromal/stem cell were compared between young and old cells. With increased cell passage, we observed a significant change in cell morphology that was associated with an increase in β-galactosidase activity. In addition, old cells (population doubling seven) also showed increased mitochondrial mass, augmented superoxide production, and decreased mitochondrial membrane potential. These changes in morphology were related to slightly levels increases in mitochondrial fusion proteins, Mitofusion 1 (MFN1), and Dynamin-related GTPase (OPA1). Collectively, our results showed that adipose tissue-derived MSCs at population doubling seven developed a senescent phenotype that was characterized by metabolic cell changes that can lead to mitochondrial fusion. PMID:28018212

  18. Mitochondrial Functional Changes Characterization in Young and Senescent Human Adipose Derived MSCs.

    PubMed

    Stab, Bernd R; Martinez, Laura; Grismaldo, Adriana; Lerma, Alejandra; Gutiérrez, María L; Barrera, Luis A; Sutachan, Jhon J; Albarracín, Sonia L

    2016-01-01

    Mitochondria are highly dynamic organelles that in response to the cell's bio-energetic state continuously undergo structural remodeling fission and fusion processes. This mitochondrial dynamic activity has been implicated in cell cycle, autophagy, and age-related diseases. Adult tissue-derived mesenchymal stromal/stem cells present a therapeutic potential. However, to obtain an adequate mesenchymal stromal/stem cell number for clinical use, extensive in vitro expansion is required. Unfortunately, these cells undergo replicative senescence rapidly by mechanisms that are not well understood. Senescence has been associated with metabolic changes in the oxidative state of the cell, a process that has been also linked to mitochondrial fission and fusion events, suggesting an association between mitochondrial dynamics and senescence. In the present work, we studied the mitochondrial structural remodeling process of mesenchymal stromal/stem cells isolated from adipose tissue in vitro to determine if mitochondrial phenotypic changes were associated with mesenchymal stromal/stem cell senescence. For this purpose, mitochondrial dynamics and oxidative state of stromal/stem cell were compared between young and old cells. With increased cell passage, we observed a significant change in cell morphology that was associated with an increase in β-galactosidase activity. In addition, old cells (population doubling seven) also showed increased mitochondrial mass, augmented superoxide production, and decreased mitochondrial membrane potential. These changes in morphology were related to slightly levels increases in mitochondrial fusion proteins, Mitofusion 1 (MFN1), and Dynamin-related GTPase (OPA1). Collectively, our results showed that adipose tissue-derived MSCs at population doubling seven developed a senescent phenotype that was characterized by metabolic cell changes that can lead to mitochondrial fusion.

  19. Fat on sale: role of adipose-derived stem cells as anti-fibrosis agent in regenerative medicine.

    PubMed

    Gupta, Manoj K; Ajay, Amrendra Kumar

    2015-12-01

    The potential use of stem cells for cell-based tissue repair and regeneration offers alternative therapeutic strategies for various diseases. Adipose-derived stem cells (ADSCs) have emerged as a promising source of stem cells suitable for transplantation in regenerative medicine and wound repair. A recent publication in Stem Cell Research & Therapy by Zhang and colleagues reports a new finding about the anti-fibrosis role of ADSCs and conditioned media derived from them on hypertrophic scar formation in vivo.

  20. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

    PubMed

    Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

    2008-11-01

    This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

  1. Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells.

    PubMed

    Wall, Michelle E; Bernacki, Susan H; Loboa, Elizabeth G

    2007-06-01

    Adipose-derived human mesenchymal stem cells (hMSCs) will be more valuable for tissue engineering applications if they can be extensively subcultured without loss of phenotype and multilineage differentiation ability. This study examined the effects of serial passaging on growth rate, gene expression, and differentiation potential of adipose-derived hMSCs. Differentiation was assessed by analyzing changes in messenger RNA (mRNA) expression of osteogenic and adipogenic marker genes and by determining production of calcium deposits and lipid vacuoles. Cells cultured in osteogenic medium for 2 weeks upregulated expression of alkaline phosphatase mRNA relative to cells in growth medium, and deposited calcium. Calcium deposition decreased in cells from passages 4 to 6 but returned to levels near or above those of primary cells by passage 10. Cells cultured in adipogenic medium upregulated expression of lipoprotein lipase and peroxisome proliferator activated receptor-gamma mRNA relative to cells in growth medium, and formed lipid vacuoles at all passages. By passage 8, however, cells in adipogenic medium also deposited calcium. Growth rate was stable through passage 5, then decreased. The results of this study indicate that adipose-derived hMSCs are capable of both adipogenic and osteogenic differentiation through 10 passages (34 population doublings) but that osteogenic differentiation may start to dominate at later passages.

  2. Effectiveness of autologous serum as an alternative to fetal bovine serum in adipose-derived stem cell engineering.

    PubMed

    Choi, Jaehoon; Chung, Jee-Hyeok; Kwon, Geun-Yong; Kim, Ki-Wan; Kim, Sukwha; Chang, Hak

    2013-09-01

    In cell culture, medium supplemented with fetal bovine serum is commonly used, and it is widely known that fetal bovine serum supplies an adequate environment for culture and differentiation of stem cells. Nevertheless, the use of xenogeneic serum can cause several problems. We compared the effects of four different concentrations of autologous serum (1, 2, 5, and 10%) on expansion and adipogenic differentiation of adipose-derived stem cells using 10% fetal bovine serum as a control. The stem cells were grafted on nude mice and the in vivo differentiation capacity was evaluated. The isolation of adipose-derived stem cells was successful irrespective of the culture medium. The proliferation potential was statistically significant at passage 2, as follows: 10% autologous serum > 10% fetal bovine serum = 5% autologous serum > 2% autologous serum = 1% autologous serum. The differentiation capacity appeared statistically significant at passage 4, as follows: 10% fetal bovine serum > 10% autologous serum = 5% autologous serum > 2% autologous serum = 1% autologous serum. Ten percent autologous serum and 10% fetal bovine serum had greater differentiation capacity than 1 and 2% autologous serum in vivo, and no significant difference was observed between the groups at ≥ 5% concentration at 14 weeks. In conclusion, 10% autologous serum was at least as effective as 10% fetal bovine serum with respect to the number of adipose-derived stem cells at the end of both isolation and expansion, whereas 1 and 2% autologous serum was inferior.

  3. Protein deubiquitinase USP7 is required for osteogenic differentiation of human adipose-derived stem cells.

    PubMed

    Tang, Yiman; Lv, Longwei; Li, Wenyue; Zhang, Xiao; Jiang, Yong; Ge, Wenshu; Zhou, Yongsheng

    2017-08-14

    Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with self-renewal capabilities and multilineage differentiation potential, including osteogenesis. Although protein deubiquitinases have been linked to stem cell fate determination, whether protein deubiquitination contributes to lineage commitment during osteogenic differentiation of hASCs remains to be investigated. The objective of this study was to evaluate the effects of the ubiquitin specific protease 7 (USP7) on osteogenic differentiation of hASCs. An osteocalcin promoter driven luciferase reporter system was established to initially discover the potential association between USP7 and hASC osteogenesis. To further characterize the function of USP7 in osteogenic differentiation of hASCs, a combination of in vitro and in vivo experiments were carried out through genetic depletion or overexpression of USP7 using a lentiviral strategy. Moreover, HBX 41,108, a cyanoindenopyrazine-derived deubiquitinase inhibitor of USP7, was utilized at different doses to further examine whether USP7 regulated osteogenic differentiation of hASCs through its enzymatic activity. We demonstrated that USP7 depletion was associated with remarkable downregulation of the reporter gene activity. Genetic depletion of USP7 by lentiviral RNAi markedly suppressed hASC osteogenesis both in vitro and in vivo, while overexpression of USP7 enhanced the osteogenic differentiation of hASCs. Notably, chemical blockade via the small molecular inhibitor HBX 41,108 could efficiently mimic the effects of USP7 genetic depletion in a dose-dependent manner. Taken together, our study revealed that protein deubiquitinase USP7 is an essential player in osteogenic differentiation of hASCs through its catalytic activity, and supported the pursuit of USP7 as a potential target for modulation of hASC-based stem cell therapy and bone tissue engineering.

  4. Adipose-derived stem cells combined with neuregulin-1 delivery systems for heart tissue engineering.

    PubMed

    Díaz-Herráez, P; Garbayo, E; Simón-Yarza, T; Formiga, F R; Prosper, F; Blanco-Prieto, M J

    2013-09-01

    Myocardial infarction (MI) is the leading cause of death worldwide, and extensive research has therefore been performed to find a cure. Neuregulin-1 (NRG) is a growth factor involved in cardiac repair after MI. We previously described how biocompatible and biodegradable microparticles, which are able to release NRG in a sustained manner, represent a valuable approach to avoid problems related to the short half-life after systemic administration of proteins. The effectiveness of this strategy could be improved by combining NRG with several cytokines involved in cardiac regeneration. The present study investigates the potential feasibility of using NRG-releasing particle scaffold combined with adipose-derived stem cells (ADSC) as a multiple growth factor delivery-based tissue engineering strategy for implantation in the infarcted myocardium. NRG-releasing particle scaffolds with a suitable size for intramyocardial implantation were prepared by TROMS. Next, ADSC were adhered to particle scaffolds and their potential for heart administration was assessed in a MI rat model. NRG was successfully encapsulated reaching encapsulation efficiencies of 92.58 ± 3.84%. NRG maintained its biological activity after the microencapsulation process. ADSCs adhered efficiently to particle scaffolds within a few hours. The ADSC-cytokine delivery system developed proved to be compatible with intramyocardial administration in terms of injectability through a 23-gauge needle and tissue response. Interestingly, ADSC-scaffolds were present in the peri-infarted tissue 2 weeks after implantation. This proof of concept study provides important evidence required for future effectiveness studies and for the translation of this approach.

  5. Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

    PubMed

    Mendel, Thomas A; Clabough, Erin B D; Kao, David S; Demidova-Rice, Tatiana N; Durham, Jennifer T; Zotter, Brendan C; Seaman, Scott A; Cronk, Stephen M; Rakoczy, Elizabeth P; Katz, Adam J; Herman, Ira M; Peirce, Shayn M; Yates, Paul A

    2013-01-01

    Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is

  6. Transdifferentiation of Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Engineering a Stratified Epidermis

    PubMed Central

    Chavez-Munoz, Claudia; Nguyen, Khang T.; Xu, Wei; Hong, Seok-Jong; Mustoe, Thomas A.; Galiano, Robert D.

    2013-01-01

    Skin regeneration is an important area of research in the field of tissue-engineering, especially for cases involving loss of massive areas of skin, where current treatments are not capable of inducing permanent satisfying replacements. Human adipose-derived stem cells (ASC) have been shown to differentiate in-vitro into both mesenchymal lineages and non-mesenchymal lineages, confirming their transdifferentiation ability. This versatile differentiation potential, coupled with their ease of harvest, places ASC at the advancing front of stem cell-based therapies. In this study, we hypothesized that ASC also have the capacity to transdifferentiate into keratinocyte-like cells and furthermore are able to engineer a stratified epidermis. ASC were successfully isolated from lipoaspirates and cell sorted (FACS). After sorting, ASC were either co-cultured with human keratinocytes or with keratinocyte conditioned media. After a 14-day incubation period, ASC developed a polygonal cobblestone shape characteristic of human keratinocytes. Western blot and q-PCR analysis showed the presence of specific keratinocyte markers including cytokeratin-5, involucrin, filaggrin and stratifin in these keratinocyte-like cells (KLC); these markers were absent in ASC. To further evaluate if KLC were capable of stratification akin to human keratinocytes, ASC were seeded on top of human decellularized dermis and cultured in the presence or absence of EGF and high Ca2+ concentrations. Histological analysis demonstrated a stratified structure similar to that observed in normal skin when cultured in the presence of EGF and high Ca2+. Furthermore, immunohistochemical analysis revealed the presence of keratinocyte markers such as involucrin, cytokeratin-5 and cytokeratin-10. In conclusion this study demonstrates for the first time that ASC have the capacity to transdifferentiate into KLC and engineer a stratified epidermis. This study suggests that adipose tissue is potentially a readily available

  7. Alginate microencapsulation technology for the percutaneous delivery of adipose-derived stem cells.

    PubMed

    Moyer, Hunter R; Kinney, Ramsey C; Singh, Kimberly A; Williams, Joseph K; Schwartz, Zvi; Boyan, Barbara D

    2010-11-01

    Autologous fat is the ideal soft-tissue filler; however, its widespread application is limited because of variable clinical results and poor survival. Engineered fillers have the potential to maximize survival. Alginate is a hydrogel copolymer that can be engineered into spheres of <200 μm, thus facilitating mass transfer, allowing for subcutaneous injection, and protecting cells from shearing forces. Alginate powder was dissolved in saline, and adipose-derived stem cells (ADSCs) were encapsulated (1 million cells/mL) in alginate using an electrostatic bead generator. To assess effects of injection on cell viability, microspheres containing ADSCs were separated into 2 groups: the control group was decanted into culture wells and the injection group was mixed with basal media and injected through a 21-gauge needle into culture wells. Microbeads were cultured for 3 weeks, and cell number and viability were measured weekly using electron and confocal microscopy. To assess effects of percutaneous injection in vivo, twenty-four male nude mice were randomly separated into 2 groups and injected with either empty microcapsules or ADSC-laden microcapsules. Mice were harvested at 1 and 3 months, and the implants were examined microscopically to assess bead and cell viability. A flow rate of 5 mL/h and an electrostatic potential of 7 kV produced viable ADSC-laden microbeads of <200 μm. There were no differences in bead morphology and ADSC viability between microcapsules placed versus injected into tissue culture plates for up to 3 weeks. Microspheres implanted in a nude mouse model show durability up to 3 months with a host response around each individual sphere. ADSCs remained viable and showed signs of mitosis. ADSCs can be readily cultured, encapsulated, and injected in alginate microspheres. Stem cells suspended in alginate microspheres survive in vivo and are seen to replicate in vitro.

  8. The osteogenic response of undifferentiated human adipose-derived stem cells under mechanical stimulation.

    PubMed

    Du, Hong-ming; Zheng, Xiao-hui; Wang, Li-ya; Tang, Wei; Liu, Lei; Jing, Wei; Lin, Yun-feng; Tian, Wei-dong; Long, Jie

    2012-01-01

    The purpose of this study was to investigate the osteogenic response of human adipose-derived stem cells (hASCs) under mechanical and/or chemical stimulation. hASCs were divided into three groups. In group A, the cells were cultured without any stimulation, in group B, the cells were induced with chemical stimulation, and in group C, the cells were induced with a combination of chemical stimulation and stretch loading. Stretch loading and chemical stimulation were applied using a four-point bending apparatus (0.5 Hz, 2,000 µε, 2 h/day) and osteogenic differentiation medium, respectively. At the 1st, 2nd, 3rd, 5th and 7th day following initiation of stretch loading, we detected alkaline phosphatase activity, mRNA expression (RUNX2, ALPL, osteonectin, osteopontin and type I collagen) and protein expression (RUNX2 and osteopontin) by colorimetric assay, real-time PCR and Western blot methods, respectively. Alkaline phosphatase activity, mRNA expression and protein expression all increased in groups B and C along with the culture time, but were observed to be downregulated by the 7th day in group C (p < 0.05). Compared to group A, most of the above markers were significantly higher in groups B and C (p < 0.05). All of the above markers in group C were higher than those in group B before the 5th day (p < 0.05), except at the 1st day. These results indicated that stretch loading promoted osteogenic differentiation of hASCs and that the combination of mechanical and chemical stimulation could enhance the osteogenic capability up to the 5th day relative to chemical stimulation alone. Copyright © 2012 S. Karger AG, Basel.

  9. Effect of varied ionic calcium on human adipose-derived stem cell mineralization.

    PubMed

    McCullen, Seth D; Zhan, Jackie; Onorato, Maureen L; Bernacki, Susan H; Loboa, Elizabeth G

    2010-06-01

    Human adipose-derived stem cells (hASCs) are a relatively abundant and accessible stem cell source with multilineage differentiation capability and have great potential for bone tissue engineering applications. The success of bone tissue engineering is intimately linked with the production of a mineralized matrix that mimics the natural mineral present within native bone. In this study, we examined the effects of ionic calcium levels of 1.8 (normal concentration in cell culture medium), 8, and 16 mM on hASCs seeded in both two-dimensional monolayer and three-dimensional electrospun scaffolds and cultured in either complete growth medium (CGM) or osteogenic differentiation medium (ODM). The impact of calcium supplementation on hASC viability, proliferation, and mineral deposition was determined. hASCs remained viable for all experimental treatments. hASC proliferation increased with the addition of 8 mM Ca(2+) CGM, but decreased for the 16 mM Ca(2+) CGM treatment. Materials deposited by hASCs were analyzed using four techniques: (1) histological staining with Alizarin Red S, (2) calcium quantification, (3) Fourier transform infrared spectroscopy, and (4) wide-angle X-ray diffraction. Mineral deposition was significantly enhanced under both growth and osteogenic medium conditions by increasing extracellular Ca(2+). The greatest mineral deposition occurred in the ODM 8 mM Ca(2+) treatment group. Fourier transform infrared spectroscopy analysis indicated that elevated calcium concentrations of 8 mM Ca(2+) significantly increased both PO(4) amount and PO(4) to protein ratio for ODM. X-ray diffraction indicated that mineral produced with elevated Ca(2+) in both CGM and ODM had a crystalline structure characteristic of hydroxyapatite. Ionic calcium should be considered a potent regulator in hASC mineralization and could serve as a potential treatment for inducing prompt ossification of hASC-seeded scaffolds for bone tissue engineering prior to implantation.

  10. Cytotoxic and Genotoxic effects of Arsenic and Lead on Human Adipose Derived Mesenchymal Stem Cells (AMSCs).

    PubMed

    Shakoori, Ar; Ahmad, A

    2013-01-01

    Arsenic and lead, known to have genotoxic and mutagenic effects, are ubiquitously distributed in the environment. The presence of arsenic in drinking water has been a serious health problem in many countries. Human exposure to these metals has also increased due to rapid industrialization and their use in formulation of many products. Liposuction material is a rich source of stem cells. In the present study cytotoxic and genotoxic effects of these metals were tested on adipose derived mesenchymal stem cells (AMSCs). Cells were exposed to 1-10 μg/ml and 10-100 μg/ml concentration of arsenic and lead, respectively, for 6, 12, 24 and 48 h. The cytotoxic effects were measured by neutral red uptake assay, while the genotoxic effects were tested by comet assay. The growth of cells decreased with increasing concentration and the duration of exposure to arsenic. Even the morphology of cells was changed; they became round at 10 μg /ml of arsenic. The cell growth was also decreased after exposure to lead, though it proved to be less toxic when cells were exposed for longer duration. The cell morphology remained unchanged. DNA damage was observed in the metal treated cells. Different parameters of comet assay were investigated for control and treated cells which indicated more DNA damage in arsenic treated cells compared to that of lead. Intact nuclei were observed in control cells. Present study clearly demonstrates that both arsenic and lead have cytotoxic and genotoxic effects on AMSCs, though arsenic compared to lead has more deleterious effects on AMSCs.

  11. Cytotoxic and Genotoxic effects of Arsenic and Lead on Human Adipose Derived Mesenchymal Stem Cells (AMSCs)

    PubMed Central

    Shakoori, AR; Ahmad, A

    2013-01-01

    Arsenic and lead, known to have genotoxic and mutagenic effects, are ubiquitously distributed in the environment. The presence of arsenic in drinking water has been a serious health problem in many countries. Human exposure to these metals has also increased due to rapid industrialization and their use in formulation of many products. Liposuction material is a rich source of stem cells. In the present study cytotoxic and genotoxic effects of these metals were tested on adipose derived mesenchymal stem cells (AMSCs). Cells were exposed to 1-10 μg/ml and 10-100 μg/ml concentration of arsenic and lead, respectively, for 6, 12, 24 and 48 h. The cytotoxic effects were measured by neutral red uptake assay, while the genotoxic effects were tested by comet assay. The growth of cells decreased with increasing concentration and the duration of exposure to arsenic. Even the morphology of cells was changed; they became round at 10 μg /ml of arsenic. The cell growth was also decreased after exposure to lead, though it proved to be less toxic when cells were exposed for longer duration. The cell morphology remained unchanged. DNA damage was observed in the metal treated cells. Different parameters of comet assay were investigated for control and treated cells which indicated more DNA damage in arsenic treated cells compared to that of lead. Intact nuclei were observed in control cells. Present study clearly demonstrates that both arsenic and lead have cytotoxic and genotoxic effects on AMSCs, though arsenic compared to lead has more deleterious effects on AMSCs. PMID:24693207

  12. Gibberellic acid induces α-amylase expression in adipose-derived stem cells.

    PubMed

    Kasamatsu, Atsushi; Iyoda, Manabu; Usukura, Katsuya; Sakamoto, Yosuke; Ogawara, Katsunori; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

    2012-08-01

    Salivary α-amylase is the most important enzyme for oral digestion of dietary starch. Therefore, regeneration of the salivary glands via a tissue engineering approach is clearly required for patients with salivary gland dysfunction. Early during seed germination, the embryo synthesizes gibberellic acid (GA3), a plant hormone that activates the synthesis and secretion of α-amylase. The purpose of this study was to explore an approach for differentiation of stem cells into salivary glands using GA3. We isolated adipose-derived stem cells (ASCs), which are positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and possess pluripotency to osteoblasts, adipocytes and neural cells, from human buccal fat pads, which are a readily available source for dentists and oral surgeons. In addition, we investigated the cytotoxicity of GA3 for human ASCs. GA3 neither affects cell morphology nor cell viability in a dose- or time-dependent manner. ASCs were incubated with GA3 to assess mRNA and protein expression of α-amylase by reverse transcriptase-polymerase chain reaction and western blot analyses. α-amylase mRNA expression on 21 days after treatment with GA3 (1 mM) was seven times greater than that in resting condition (Day 0). While we did not detect α-amylase bands on Day 0, α-amylase protein was detectable 7 days after treatment with GA3, reaching a maximal level on Day 21. Our results indicated that GA3 can increase cellular α-amylase expression and that our induction method would be useful for therapeutic application for salivary gland regeneration.

  13. Adipose-Derived Mesenchymal Stem Cells Restore Impaired Mucosal Immune Responses in Aged Mice

    PubMed Central

    Aso, Kazuyoshi; Tsuruhara, Akitoshi; Takagaki, Kentaro; Oki, Katsuyuki; Ota, Megumi; Nose, Yasuhiro; Tanemura, Hideki; Urushihata, Naoki; Sasanuma, Jinichi; Sano, Masayuki; Hirano, Atsuyuki; Aso, Rio; McGhee, Jerry R.; Fujihashi, Kohtaro

    2016-01-01

    It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer’s patch CD4+ T cells produced increased levels of IL-4. Further, CD4+ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice. PMID:26840058

  14. Paroxetine Can Enhance Neurogenesis during Neurogenic Differentiation of Human Adipose-derived Stem Cells

    PubMed Central

    Jahromi, Maliheh; Razavi, Shahnaz; Amirpour, Nushin; Khosravizadeh, Zahra

    2016-01-01

    Background: Some antidepressant drugs can promote neuronal cell proliferation in vitro as well as hippocampal neurogenesis in human and animal models. Furthermore, adipose tissue is an available source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human Adipose-Derived Stem Cells (hAD-SCs) may be a suitable source for regenerative medical applications. Since there is no evidence for the effect of Paroxetine as the most commonly prescribed antidepressant drug for neurogenic potential of hADSCs, an attempt was made to determine the effect of Paroxetine on proliferation and neural differentiation of hADSCs. Methods: ADSCs were isolated from human abdominal fat. These cells differentiated to neuron-like cells and were treated with Paroxetine. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and immunofluorescence technique were used for assessment of cell proliferation and neurogenic differentiation potential of induced cells, respectively. Results: MTT assay analysis showed that Paroxetine significantly increased the proliferation rate of induced hADSCs (p<0.05), while immunofluorescent staining indicated that Paroxetine treatment during neurogenic differentiation could enhance the mean percentage of Nestin and MAP2 (Microtubule-associated protein-2) positive cells but the mean percentage of GFAP (Glial acidic fibrillary protein) positive cells significantly decreased relative to control group (p<0.05). Conclusion: Our results provide evidence that Paroxetine can promote proliferation and differentiation rate during neurogenic differentiation of hADSCs. Moreover, Paroxetine can reduce gliogenesis of induced hADSCs during neurogenic differentiation. PMID:27920882

  15. Electrical conditioning of adipose-derived stem cells in a multi-chamber culture platform.

    PubMed

    Pavesi, A; Soncini, M; Zamperone, A; Pietronave, S; Medico, E; Redaelli, A; Prat, M; Fiore, G B

    2014-07-01

    In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and -4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration. © 2014 Wiley Periodicals, Inc.

  16. Lipokines and oxysterols: novel adipose-derived lipid hormones linking adipose dysfunction and insulin resistance.

    PubMed

    Murdolo, Giuseppe; Bartolini, Desirée; Tortoioli, Cristina; Piroddi, Marta; Iuliano, Luigi; Galli, Francesco

    2013-12-01

    The expansion of adipose tissue (AT) is, by definition, a hallmark of obesity. However, not all increases in fat mass are associated with pathophysiological cues. Indeed, whereas a "healthy" fat mass accrual, mainly in the subcutaneous depots, preserves metabolic homeostasis, explaining the occurrence of the metabolically healthy obese phenotype, "unhealthy" AT expansion is importantly associated with insulin resistance/type 2 diabetes and the metabolic syndrome. The development of a dysfunctional adipose organ may find mechanistic explanation in a reduced ability to recruit new and functional (pre)adipocytes from undifferentiated precursor cells. Such a failure of the adipogenic process underlies the "AT expandability" paradigm. The inability of AT to expand further to store excess nutrients, rather than obesity per se, induces a diabetogenic milieu by promoting the overflow and the ectopic deposition of fatty acids in insulin-dependent organs (i.e., lipotoxicity), the secretion of various metabolically detrimental adipose-derived hormones (i.e., adipokines and lipokines), and the occurrence of local and systemic inflammation and oxidative stress. Hitherto, fatty acids (i.e., lipokines) and the oxidation by-products of cholesterol and polyunsaturated fatty acids, such as nonenzymatic oxysterols and reactive aldehyde species, respectively, emerge as key modulators of (pre)adipocyte signaling through Wnt/β-catenin and MAPK pathways and potential regulators of glucose homeostasis. These and other mechanistic insights linking adipose dysfunction, oxidative stress, and impairment of glucose homeostasis are discussed in this review article, which focuses on adipose peroxidation as a potential instigator of, and a putative therapeutic target for, obesity-associated metabolic dysfunctions.

  17. Migration of Adipose-derived Mesenchymal Stem Cells Stably Expressing Chondroitinase ABC In vitro

    PubMed Central

    Wu, Jian-Huang; Li, Miao; Liang, Yan; Lu, Tao; Duan, Chun-Yue

    2016-01-01

    Background: Several studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro. Methods: ADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay. Results: Secondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05). Conclusions: Secondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs. PMID:27364797

  18. Early Intervention with Highly Condensed Adipose-Derived Stem Cells for Complicated Wounds Following Filler Injections.

    PubMed

    Kim, Joo Hyun; Park, Seong Hoon; Lee, Byeong Ho; Jeong, Hii Sun; Yang, Hyun Jin; Suh, In Suck

    2016-06-01

    A rise in cosmetic procedures has seen the use of fillers become more prevalent. Complications resulting from use of fillers have prompted introduction of various medical and surgical interventions. Recently, stem cell therapies have become more widely used as a new treatment option for tissue repair and regeneration. We utilized adipose-derived stem cells (ASCs) for tissue regeneration in patients with filler-related complications such as necrosis. All 12 patients were treated with ASCs and some patients had additional treatment. After relief of symptoms, wound surface area was compared in terms of pixel numbers and scar condition was evaluated using the Vancouver Scar Scale (VSS). In general, we achieved satisfactory resolution of filler-related complications in a short period of time without serious side effects. The average number of days from stem cell treatment to symptom relief was 7.3 days. The proportion of wound surface area from photographic record was 4.39 % before treatment, decreasing considerably to 1.01 % following treatment. Last, the VSS showed almost all patients scored below 3, with two patients receiving scores of 7 and 8; the average score was 2.78 (range from 0 to 8). ASCs are a new treatment option for post-filler injection wounds such as necrosis. Using stem cells, we were able to obtain satisfactory results in a short period of time without complications requiring surgical procedures. We suggest stem cell injections could be used as the first option for treatment of complications from filler injections. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

  19. Intradermal injection of human adipose-derived stem cells accelerates skin wound healing in nude mice.

    PubMed

    Rodriguez, Jonathan; Boucher, Fabien; Lequeux, Charlotte; Josset-Lamaugarny, Audrey; Rouyer, Ondine; Ardisson, Orianne; Rutschi, Héléna; Sigaudo-Roussel, Dominique; Damour, Odile; Mojallal, Ali

    2015-12-08

    The use of stem cells from adipose tissue or adipose-derived stem cells (ASCs) in regenerative medicine could be an interesting alternative to bone marrow stem cells because they are easily accessible and available in large quantities. The aim of this study was to evaluate the potential effect of ASCs on the healing of 12 mm diameter-excisional wounds (around 110 mm(2)) in nude mice. Thirty nude mice underwent surgery to create one 12-mm excisional wound per mouse (spontaneous healing, n = 6; Cytocare® 532, n = 12; ASCs, n = 12). The Galiano wound model was chosen to avoid shrinkage and thus slow the spontaneous healing (SH) of mouse skin, making it closer to the physiology of human skin healing. Transparent dressings were used to enable daily healing time measurements to be taken. Immunohistochemistry, histological and blood perfusion analysis were carried out on the healed skin. The in vivo results showed the effectiveness of using ASCs on reducing the time needed for complete healing to 21.2 days for SH, 17.4 days for vehicle alone (Cytocare® 532) and 14.6 days with the addition of ASCs (p < 0.001). Moreover, cutaneous perfusion of the healed wound was significantly improved in ASC-treated mice compared to SH group, as shown by laser Doppler flowmetry and the quantitation of blood vessels using immunohistochemistry of αsmooth muscle actin. The tolerance and efficacy of cryopreserved ASCs to accelerate the complete closure of the wound by increasing the maturation of the skin and its blood perfusion, shows their therapeutic benefit in the wound healing context.

  20. Hepatogenic differentiation from human adipose-derived stem cells and application for mouse acute liver injury.

    PubMed

    Guo, De-Liang; Wang, Zhi-Gang; Xiong, Liang-Kun; Pan, Le-Yu; Zhu, Qian; Yuan, Yu-Feng; Liu, Zhi-Su

    2017-03-01

    Adipose-derived stem cells (ADSCs) derived from adipose tissue have the capacity to differentiate into endodermal, mesoderm, and ectodermal cell lineages in vitro, which are an ideal engraft in tissue-engineered repair. In this study, human ADSCs were isolated from subcutaneous fat. The markers of ADSCs, CD13, CD71, CD73, CD90, CD105, CD166, CYP3A4, and ALB were detected by immunofluorescence assays. Human ADSCs were cultured in a specific hepatogenesis differentiation medium containing HGF, bFGF, nicotinamide, ITS, and oncostatin M for hepatogenic differentiation. The hepatocyte markers were analyzed using immunofluorescence and real-time PCR after dramatic changes in morphology. Hepatocytes derived from ADSCs or ADSCs were transplanted into the mice of liver injury for observation cells colonization and therapy in liver tissue. The result demonstrated that human ADSCs were positive for the CD13, CD71, CD73, CD90, CD105, and CD166 but negative for hepatocyte markers, ALB and CYP3A4. After hepatogenic differentiation, the hepatocytes were positive for liver special markers, gene expression level showed a time-lapse increase with induction time. Human ADSCs or ADSCs-derived hepatocyte injected into the vein could improve liver function repair and functionally rescue the CCl4-treated mice with liver injury, but the ADSCs transplantation was better than ADSCs-derived hepatocyte transplantation. In conclusion, our research shows that a population of hepatocyte can be specifically generated from human ADSCs and that cells may allow for participation in tissue-repair.

  1. Capillary Force Seeding of Hydrogels for Adipose-Derived Stem Cell Delivery in Wounds

    PubMed Central

    Garg, Ravi K.; Rennert, Robert C.; Duscher, Dominik; Sorkin, Michael; Kosaraju, Revanth; Auerbach, Lauren J.; Lennon, James; Chung, Michael T.; Paik, Kevin; Nimpf, Johannes; Rajadas, Jayakumar; Longaker, Michael T.

    2014-01-01

    Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a “stem cell niche” that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting. PMID:25038246

  2. The suitability of human adipose-derived stem cells for the engineering of ligament tissue.

    PubMed

    Eagan, Michael J; Zuk, Patricia A; Zhao, Ke-Wei; Bluth, Benjamin E; Brinkmann, Elyse J; Wu, Benjamin M; McAllister, David R

    2012-10-01

    Rupture of the anterior cruciate ligament (ACL) is the one of the most common sports-related injuries. With its poor healing capacity, surgical reconstruction using either autografts or allografts is currently required to restore function. However, serious complications are associated with graft reconstructions and the number of such reconstructions has steadily risen over the years, necessitating the search for an alternative approach to ACL repair. Such an approach may likely be tissue engineering. Recent engineering approaches using ligament-derived fibroblasts have been promising, but the slow growth rate of such fibroblasts in vitro may limit their practical application. More promising results are being achieved using bone marrow mesenchymal stem cells (MSCs). The adipose-derived stem cell (ASC) is often proposed as an alternative choice to the MSC and, as such, may be a suitable stem cell for ligament engineering. However, the use of ASCs in ligament engineering still remains relatively unexplored. Therefore, in this study, the potential use of human ASCs in ligament tissue engineering was initially explored by examining their ability to express several ligament markers under growth factor treatment. ASC populations treated for up to 4 weeks with TGFβ1 or IGF1 did not show any significant and consistent upregulation in the expression of collagen types 1 and 3, tenascin C and scleraxis. While treatment with EGF or bFGF resulted in increased tenascin C expression, increased expression of collagens 1 and 3 were never observed. Therefore, simple in vitro treatment of human ASC populations with growth factors may not stimulate their ligament differentiative potential. Copyright © 2011 John Wiley & Sons, Ltd.

  3. Adipose-derived stem cell sheet transplantation therapy in a porcine model of chronic heart failure.

    PubMed

    Ishida, Osamu; Hagino, Ikuo; Nagaya, Noritoshi; Shimizu, Tatsuya; Okano, Teruo; Sawa, Yoshiki; Mori, Hidezo; Yagihara, Toshikatsu

    2015-05-01

    Adipose-derived stem cells (ASCs) are a promising resource for cell transplantation therapy for damaged heart tissue. Cell death in the graft early after transplantation represents the main cause of unsatisfactory therapeutic efficacy, but tissue-engineered cell sheets grown in temperature-responsive cell culture dishes may enable improved engraftment of transplanted cells. We investigated the therapeutic potential of this method in chronic myocardial ischemia in swine. We created a porcine model of chronic heart failure by implanting an ameroid constrictor around the main trunk of the left anterior descending artery, just distal to the circumflex branch. Simultaneously, ASCs were obtained from a piece of subcutaneous adipose tissue and expanded to form ASC sheets using temperature-responsive dishes. Four weeks after ameroid constrictor placement, triple-layered ASC sheets were transplanted onto the area of the ischemic myocardium (sheet group, n = 7). Controls (n = 7) received no sheet. Just before and 4 weeks after transplantation, left ventriculography (LVG) and coronary angiography (CAG) were performed. LVG revealed a significant improvement in the left ventricular ejection fraction of the sheet group compared with controls (47.6 ± 2.9% vs 41.4 ± 2.8%, P < 0.05). Furthermore, development of collateral vessels was only detected in the sheet group with right CAG. Histologic analysis demonstrated that engrafted ASC sheets grew to form a thickened layer that included newly formed vessels. ASC sheet transplantation therapy is an intriguing therapeutic method for ischemic heart failure. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. A new method for Schwann-like cell differentiation of adipose derived stem cells.

    PubMed

    Liu, Yumei; Zhang, Ziqiang; Qin, Yan; Wu, Hua; Lv, Qiongxia; Chen, Xiaoguang; Deng, Wen

    2013-09-13

    Peripheral nerve repair can be enhanced by Schwann cell transplantation, but the clinical application of this procedure is limited by donor site morbidity and the inability to quickly generate a sufficient number of cells. Thus, alternative cell systems for the generation of Schwann cells are desired. Schwann-like cell induced from adipose-derived stem cells (ADSCs) may be one of the ideal alternative cell systems for Schwann cell generation. Although co-culture with Schwann cells or chemicals combined with a mixture of glial growth factors are often utilized for Schwann cell-like differentiation of ADSCs, these methods are usually complicated or expensive. In this experiment, the rat sciatic nerve was cut, and then soaked in culture medium for two days. The treated culture medium was used as an induction agent after filtering. The obtained ADSCs were incubated with the above induction culture medium for five days. Then, expression of the typical Schwann cell markers, S-100 and GFAP proteins was determined by immunocytochemical staining and Western blotting. The results showed that almost all of the treated ADSCs displayed a spindle shape like morphology after being incubated with induction culture medium for 24h and expressed S-100 and GFAP proteins after five days. All of these characteristics of differentiated rat ADSCs were similar to genuine Schwann cells. Thus, this new method, which utilized trophic factors secreted from sciatic nerve leachate, was capable of inducing ADSC differentiation into Schwann-like cell. Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  5. Effect of nano-structured bioceramic surface on osteogenic differentiation of adipose derived stem cells.

    PubMed

    Xia, Lunguo; Lin, Kaili; Jiang, Xinquan; Fang, Bing; Xu, Yuanjin; Liu, Jiaqiang; Zeng, Deliang; Zhang, Maolin; Zhang, Xiuli; Chang, Jiang; Zhang, Zhiyuan

    2014-10-01

    Tissue engineering strategies to construct vascularized bone grafts potentially revolutionize the treatment of massive bone loss. The surface topography of the grafts plays critical roles on bone regeneration, while adipose derived stem cells (ASCs) are known for their capability to promote osteogenesis and angiogenesis when applied to bone defects. In the present study, the effects of hydroxyapatite (HAp) bioceramic scaffolds with nanosheet, nanorod, and micro-nano-hybrid (the hybrid of nanorod and microrod) surface topographies on attachment, proliferation and osteogenic differentiation, as well as the expression of angiogenic factors of rat ASCs were systematically investigated. The results showed that the HAp bioceramic scaffolds with the micro-/nano-topography surfaces significantly enhanced cell attachment and viability, alkaline phosphatase (ALP) activity, and mRNA expression levels of osteogenic markers and angiogenic factors of ASCs. More importantly, the biomimetic feature of the hierarchical micro-nano-hybrid surface topography showed the highest stimulatory effect. The activation in Akt signaling pathway was observed in ASCs cultured on HAp bioceramics with nanorod, and micro-nano-hybrid surface topographies. Moreover, these induction effects could be repressed by Akt signaling pathway inhibitor LY294002. Finally, the in vivo bone regeneration results of rat critical-sized calvarial defect models confirmed that the combination of the micro-nano-hybrid surface and ASCs could significantly enhance both osteogenesis and angiogenesis as compared with the control HAp bioceramic scaffold with traditional smooth surface. Our results suggest that HAp bioceramic scaffolds with micro-nano-hybrid surface can act as cell carrier for ASCs, and consequently combine with ASCs to construct vascularized tissue-engineered bone.

  6. Therapeutic effects of adipose-derived stem cells pretreated with pioglitazone in an emphysema mouse model

    PubMed Central

    Hong, Yoonki; Kim, You-Sun; Hong, Seok-Ho; Oh, Yeon-Mok

    2016-01-01

    There is no therapy currently available that influences the natural history of disease progression in patients with chronic obstructive pulmonary disease (COPD). Although stem cell therapy is considered a potential therapeutic option in COPD, there are no clinical trials proving definitive therapeutic effects in patients with COPD. Recently, it was reported that pioglitazone might potentiate the therapeutic effects of stem cells in patients with heart or liver disease. To test the capacity of pioglitazone pretreatment of stem cells for emphysema repair, we evaluated the therapeutic effects of pioglitazone-pretreated human adipose-derived mesenchymal stem cells (ASCs) on elastase-induced or cigarette smoke-induced emphysema in mice. We also investigated the mechanisms of action of pioglitazone-pretreated ASCs. Pioglitazone-pretreated ASCs had a more potent therapeutic effect than non-pretreated ASCs in the repair of both elastase-induced and smoke-induced emphysema models (mean linear intercept, 78.1±2.5 μm vs 83.2±2.6 μm in elastase models and 75.6±1.4 μm vs 80.5±3.2 μm in smoke models, P<0.05). Furthermore, we showed that pioglitazone-pretreated ASCs increased vascular endothelial growth factor (VEGF) production both in vitro and in mouse lungs in the smoke-induced emphysema model. Pioglitazone-pretreated ASCs may have more potent therapeutic effects than non-pretreated ASCs in emphysema mouse models. PMID:27765950

  7. Migration of Adipose-derived Mesenchymal Stem Cells Stably Expressing Chondroitinase ABC In vitro.

    PubMed

    Wu, Jian-Huang; Li, Miao; Liang, Yan; Lu, Tao; Duan, Chun-Yue

    2016-07-05

    Several studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro. ADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay. Secondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05). Secondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs.

  8. Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

    PubMed

    Li, Jin; Zeng, Guofang; Qi, Yawei; Tang, Xudong; Zhang, Jingjing; Wu, Zeyong; Liang, Jie; Shi, Lei; Liu, Hongwei; Zhang, Peihua

    2015-01-01

    Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs) after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3-4.3 hour post-fertilization (hpf). Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.

  9. Human adipose-derived stem cells for the treatment of chemically burned rat cornea: preliminary results.

    PubMed

    Zeppieri, Marco; Salvetat, Maria Letizia; Beltrami, Antonio Paolo; Cesselli, Daniela; Bergamin, Natascha; Russo, Rossella; Cavaliere, Federica; Varano, Giuseppe Pasquale; Alcalde, Ignacio; Merayo, Jesús; Brusini, Paolo; Beltrami, Carlo Alberto; Parodi, Pier Camillo

    2013-04-01

    Adipose-derived stem cells (ADSC) are multipotent, safe, non-immunogenic and can differentiate into functional keratocytes in situ. The topical use of ADSC derived from human processed lipoaspirate was investigated for treating injured rat cornea. A total of 19 rats were used. Six animals initially underwent corneal lesion experiments with 0.5 N NaOH (right eye) and 0.2 N (left). The 0.2 NaOH protocol was then used in 13 rats. All 26 eyes of 13 rats eyes received topical azythromycin bid for 3 d and divided into five treatment groups (n = 5 eyes/group), which included: control, stem cells, serum, stem + serum and adipose (raw human lipoaspirate). The four treatment groups received topical treatment three times daily for 3 d. Stem cells were isolated and harvested from human lipoaspirate. Topical eye drops were prepared daily with 1 × 10(5) cells/treatment. Fluorescein positive defect area and light microscope assessment was performed at 20, 28, 45, 50 and 74 h. Animals were sacrificed at 74 h for histological evaluation. Data were statistically analyzed for differences amongst groups. The stem cell-treated eyes had significantly smaller epithelial defects at each time point compared to control- and adipose-treated eyes (p < 0.05). This group showed slightly better epithelium healing than the serum and combined group, yet not significantly different. Histology showed that stem cell-treated corneas had complete re-epithelization, with less inflammatory cells and limited fibroblast activation structure compared with the control eyes. Our preliminary results show that topical treatment with ADSC seems to improve corneal wound healing.

  10. Adipose-Derived Regenerative Cell Injection Therapy for Postprostatectomy Incontinence: A Phase I Clinical Study

    PubMed Central

    Choi, Jae Young; Kim, Tae-Hwan; Yang, Jung Dug; Suh, Jang Soo

    2016-01-01

    Purpose We report our initial experience with transurethral injection of autologous adipose-derived regenerative cells (ADRCs) for the treatment of urinary incontinence after radical prostatectomy. Materials and Methods After providing written informed consent, six men with persistent urinary incontinence after radical prostatectomy were enrolled in the study. Under general anesthesia, about 50 mL of adipose tissue was obtained from the patients by liposuction. ADRCs were obtained by separation with centrifugation using the Celution cell-processing device. A mixture of ADRCs and adipose tissue were transurethrally injected into the submucosal space of the membranous urethra. Functional and anatomical improvement was assessed using a 24-h pad test, validated patient questionnaire, urethral pressure profile, and magnetic resonance imaging (MRI) during 12-week follow-up. Results Urine leakage volume was improved with time in all patients in the 24-h pad test, with the exemption of temporal deterioration at the first 2 weeks post-injection in 2 patients. Subjective symptoms and quality of life assessed on the basis of questionnaire results showed similar improvement. The mean maximum urethral closing pressure increased from 44.0 to 63.5 cm H2O at 12 weeks after injection. MRI showed an increase in functional urethral length (from 6.1 to 8.3 mm) between the lower rim of the pubic bone and the bladder neck. Adverse events, such as pelvic pain, inflammation, or de novo urgency, were not observed in any case during follow-up. Conclusion This study demonstrated that transurethral injection of autologous ADRCs can be a safe and effective treatment modality for postprostatectomy incontinence. PMID:27401646

  11. Transdifferentiation of adipose-derived stem cells into keratinocyte-like cells: engineering a stratified epidermis.

    PubMed

    Chavez-Munoz, Claudia; Nguyen, Khang T; Xu, Wei; Hong, Seok-Jong; Mustoe, Thomas A; Galiano, Robert D

    2013-01-01

    Skin regeneration is an important area of research in the field of tissue-engineering, especially for cases involving loss of massive areas of skin, where current treatments are not capable of inducing permanent satisfying replacements. Human adipose-derived stem cells (ASC) have been shown to differentiate in-vitro into both mesenchymal lineages and non-mesenchymal lineages, confirming their transdifferentiation ability. This versatile differentiation potential, coupled with their ease of harvest, places ASC at the advancing front of stem cell-based therapies. In this study, we hypothesized that ASC also have the capacity to transdifferentiate into keratinocyte-like cells and furthermore are able to engineer a stratified epidermis. ASC were successfully isolated from lipoaspirates and cell sorted (FACS). After sorting, ASC were either co-cultured with human keratinocytes or with keratinocyte conditioned media. After a 14-day incubation period, ASC developed a polygonal cobblestone shape characteristic of human keratinocytes. Western blot and q-PCR analysis showed the presence of specific keratinocyte markers including cytokeratin-5, involucrin, filaggrin and stratifin in these keratinocyte-like cells (KLC); these markers were absent in ASC. To further evaluate if KLC were capable of stratification akin to human keratinocytes, ASC were seeded on top of human decellularized dermis and cultured in the presence or absence of EGF and high Ca(2+) concentrations. Histological analysis demonstrated a stratified structure similar to that observed in normal skin when cultured in the presence of EGF and high Ca(2+). Furthermore, immunohistochemical analysis revealed the presence of keratinocyte markers such as involucrin, cytokeratin-5 and cytokeratin-10. In conclusion this study demonstrates for the first time that ASC have the capacity to transdifferentiate into KLC and engineer a stratified epidermis. This study suggests that adipose tissue is potentially a readily

  12. Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy

    PubMed Central

    Mendel, Thomas A.; Clabough, Erin B. D.; Kao, David S.; Demidova-Rice, Tatiana N.; Durham, Jennifer T.; Zotter, Brendan C.; Seaman, Scott A.; Cronk, Stephen M.; Rakoczy, Elizabeth P.; Katz, Adam J.; Herman, Ira M.; Peirce, Shayn M.; Yates, Paul A.

    2013-01-01

    Background Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. Methodology/Principal Findings We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). Conclusions/Significance ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of

  13. Enhanced Autophagy of Adipose-Derived Stem Cells Grown on Chitosan Substrates

    PubMed Central

    Yang, Ching-Ming; Huang, Yen-Jang; Hsu, Shan-hui

    2015-01-01

    Abstract Autophagy is an important protein quality control mechanism for cells under stress conditions to promote cell survival. Modulation of autophagy on biomaterial substrates is rarely reported. In this study, the autophagy of adipose-derived stem cells (ADSCs) cultured on chitosan (CS) substrates was examined. Compared to the traditional monolayer culture, ADSCs cultured on CS substrates showed spheroid formation as well as a prolonged upregulation of autophagosomal marker-microtubule-associated protein 1 light chain 3 (LC3) II protein expression. In addition, the green fluorescent protein tagged-LC3 (GFP-LC3) expressing ADSCs also revealed more GFP-LC3 puncta on CS substrates. The enhanced autophagy on CS substrates was associated with Ca2+, while ethylene glycol tetraacetic acid (EGTA), a Ca2+ chelator, repressed the autophagy in a dose-dependent manner. Moreover, ADSC spheroids on CS substrates demonstrated a higher survival rate and autophagy response upon H2O2 treatment. The upstream components of autophagy signal pathway-UNC51-like kinase 1 (Ulk1), autophagy-related protein 13 (Atg13), and autophagy/beclin-1 regulator 1 (Ambra1) genes were more highly expressed in ADSC spheroids before and after adding H2O2 than those in the conventional culture. EGTA also decreased the cell viability and autophagy-associated gene expression for ADSC spheroids on CS substrates after H2O2 treatment. Therefore, we suggest that three-dimensional (3D) cell culture on CS may confer ADSCs the ability to increase the autophagic flux in response to stimulations in a Ca2+-dependent manner. PMID:26309785

  14. Differentiation of Human Adipose-derived Stem Cells along the Keratocyte Lineage In vitro

    PubMed Central

    Zhang, Shijia; Espandar, Ladan; Imhof, Kathleen M.P.; Bunnell, Bruce A.

    2013-01-01

    Purpose To evaluate differentiation of human adipose-derived stem cells (hASCs) to the keratocyte lineage by co-culture with primary keratocytes in vitro. Materials and Methods A co-culture system using transwell inserts to grow hASCs on bottom and keratocytes on top in keratocyte differentiating medium (KDM) was developed. hASCs that were cultured in complete culture medium (CCM) and KDM were used as control. After 16 days, hASCs were examined for morphologic changes and proliferation by cell count. qRT-PCR and flow cytometry were used to detect the expression of aldehyde dehydrogenase 3 family, member A1 (ALDH3A1) and keratocan. Results hASCs became more dendritic and elongated in co-culture system relative to CCM and KDM. The doubling time of the cells was longer as differentiation progressed. qRT-PCR showed a definite trend towards increased expression of both ALDH3A1 and keratocan in co-culture system despite statistically non-significant p-values. Flow cytometry showed significantly increased protein levels of ALDH3A1 and keratocan in co-culture system relative to CCM group (p < 0.001) and even relative to KDM group (p < 0.001 for ALDH3A1 and p < 0.01 for keratocan). Conclusion The co-culture method is a promising approach to induce differentiation of stem cell populations prior to in vivo applications. This study reveals an important potential for bioengineering of corneal tissue using autologous multi-potential stem cells. PMID:23936748

  15. Adipose-Derived Mesenchymal Stem Cells Prevent Systemic Bone Loss in Collagen-Induced Arthritis

    PubMed Central

    Garimella, Manasa G.; Kour, Supinder; Piprode, Vikrant; Mittal, Monika; Kumar, Anil; Rani, Lekha; Pote, Satish T.; Mishra, Gyan C.; Chattopadhyay, Naibedya

    2015-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammatory synovitis leading to joint destruction and systemic bone loss. The inflammation-induced bone loss is mediated by increased osteoclast formation and function. Current antirheumatic therapies primarily target suppression of inflammatory cascade with limited or no success in controlling progression of bone destruction. Mesenchymal stem cells (MSCs) by virtue of their tissue repair and immunomodulatory properties have shown promising results in various autoimmune and degenerative diseases. However, the role of MSCs in prevention of bone destruction in RA is not yet understood. In this study, we investigated the effect of adipose-derived MSCs (ASCs) on in vitro formation of bone-resorbing osteoclasts and pathological bone loss in the mouse collagen-induced arthritis (CIA) model of RA. We observed that ASCs significantly inhibited receptor activator of NF-κB ligand (RANKL)–induced osteoclastogenesis in both a contact-dependent and -independent manner. Additionally, ASCs inhibited RANKL-induced osteoclastogenesis in the presence of proinflammatory cytokines such as TNF-α, IL-17, and IL-1β. Furthermore, treatment with ASCs at the onset of CIA significantly reduced clinical symptoms and joint pathology. Interestingly, ASCs protected periarticular and systemic bone loss in CIA mice by maintaining trabecular bone structure. We further observed that treatment with ASCs reduced osteoclast precursors in bone marrow, resulting in decreased osteoclastogenesis. Moreover, ASCs suppressed autoimmune T cell responses and increased the percentages of peripheral regulatory T and B cells. Thus, we provide strong evidence that ASCs ameliorate inflammation-induced systemic bone loss in CIA mice by reducing osteoclast precursors and promoting immune tolerance. PMID:26538398

  16. Therapeutic effects of adipose-derived stem cells pretreated with pioglitazone in an emphysema mouse model.

    PubMed

    Hong, Yoonki; Kim, You-Sun; Hong, Seok-Ho; Oh, Yeon-Mok

    2016-10-21

    There is no therapy currently available that influences the natural history of disease progression in patients with chronic obstructive pulmonary disease (COPD). Although stem cell therapy is considered a potential therapeutic option in COPD, there are no clinical trials proving definitive therapeutic effects in patients with COPD. Recently, it was reported that pioglitazone might potentiate the therapeutic effects of stem cells in patients with heart or liver disease. To test the capacity of pioglitazone pretreatment of stem cells for emphysema repair, we evaluated the therapeutic effects of pioglitazone-pretreated human adipose-derived mesenchymal stem cells (ASCs) on elastase-induced or cigarette smoke-induced emphysema in mice. We also investigated the mechanisms of action of pioglitazone-pretreated ASCs. Pioglitazone-pretreated ASCs had a more potent therapeutic effect than non-pretreated ASCs in the repair of both elastase-induced and smoke-induced emphysema models (mean linear intercept, 78.1±2.5 μm vs 83.2±2.6 μm in elastase models and 75.6±1.4 μm vs 80.5±3.2 μm in smoke models, P<0.05). Furthermore, we showed that pioglitazone-pretreated ASCs increased vascular endothelial growth factor (VEGF) production both in vitro and in mouse lungs in the smoke-induced emphysema model. Pioglitazone-pretreated ASCs may have more potent therapeutic effects than non-pretreated ASCs in emphysema mouse models.

  17. Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis.

    PubMed

    ter Huurne, Menno; Schelbergen, Rik; Blattes, Roxane; Blom, Arjen; de Munter, Wouter; Grevers, Lilyanne C; Jeanson, Jannik; Noël, Danièle; Casteilla, Louis; Jorgensen, Christian; van den Berg, Wim; van Lent, Peter L E M

    2012-11-01

    In experimental collagenase-induced osteoarthritis (OA) in the mouse, synovial lining macrophages are crucial in mediating joint destruction. It was recently shown that adipose-derived stem cells (ASCs) express immunosuppressive characteristics. This study was undertaken to explore the effect of intraarticular injection of ASCs on synovial lining thickness and its relation to joint pathology in experimental mouse OA. ASCs were isolated from fat surrounding the inguinal lymph nodes and cultured for 2 weeks. Experimental OA was induced by injection of collagenase into the knee joints of C57BL/6 mice. OA phenotypes were measured within 8 weeks after induction. Histologic analysis was performed, and synovial thickening, enthesophyte formation, and cartilage destruction were measured in the knee joint. ASCs were injected into the knee joints of mice 7 days after the induction of collagenase-induced OA. On day 1, green fluorescent protein-labeled ASCs were attached to the lining layer in close contact with macrophages. Thickening of the synovial lining, formation of enthesophytes associated with medial collateral ligaments, and formation of enthesophytes associated with cruciate ligaments were significantly inhibited on day 42 after ASC treatment, by 31%, 89%, and 44%, respectively. Destruction of cartilage was inhibited on day 14 (65%) and day 42 (35%). In contrast to early treatment, injection of ASCs on day 14 after OA induction showed no significant effect on synovial activation or joint pathology on day 42. These findings indicate that a single injection of ASCs into the knee joints of mice with early-stage collagenase-induced OA inhibits synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction. Copyright © 2012 by the American College of Rheumatology.

  18. Adipose-Derived Stem Cells Improve Efficacy of Melanocyte Transplantation in Animal Skin

    PubMed Central

    Lim, Won-Suk; Kim, Chang-Hyun; Kim, Ji-Young; Do, Byung-Rok; Kim, Eo Jin; Lee, Ai-Young

    2014-01-01

    Vitiligo is a pigmentary disorder induced by a loss of melanocytes. In addition to replacement of pure melanocytes, cocultures of melanocytes with keratinocytes have been used to improve the repigmentation outcome in vitiligo treatment. We previously identified by in vitro studies, that adipose-derived stem cells (ADSCs) could be a potential substitute for keratinocytes in cocultures with melanocytes. In this study, the efficacy of pigmentation including durability of grafted melanocytes and short-term safety was examined in the nude mouse and Sprague-Dawley rat after grafting of primary cultured human melanocytes, with or without different ratios of primary cultured human ADSCs. Simultaneous grafting of melanocytes and ADSCs, which were separately cultured and mixed on grafting at the ratios of 1:1, 1:2, or 1:3, showed better efficacy than that of pure melanocytes. Grafting of melanocytes cocultured with ADSCs resulted in a similar outcome as the grafting of cell mixtures. Skin pigmentation by melanocytes : ADSCs at the ratios of 1:1 and 1:2 was better than at 1:3. No significant difference was observed between the 1-week and 2-week durations in coculturing. Time-course microscopic examination showed that the grafted melanocytes remained a little longer than 6-week post-grafting. No inflammatory cell infiltration was observed in the grafted skin and no melanocytes were detectable in other organs. Collectively, grafting of melanocytes and ADSCs was equally safe and more effective than grafting of melanocytes alone. Despite the absence of significant differences in efficacy between the group of 1:1 and that of 1:2 ratio, 1:2 ratio for 1-week coculturing may be better for clinical use from the cost-benefit viewpoint. PMID:25143812

  19. Adipose-Derived Stem Cell Delivery into Collagen Gels Using Chitosan Microspheres

    PubMed Central

    Natesan, Shanmugasundaram; Baer, David G.; Walters, Thomas J.; Babu, Mary

    2010-01-01

    Integration of stem cells to injured tissues requires an appropriate delivery device and scaffolding system. In the present study we have developed an in vitro strategy to load and release adipose-derived mesenchymal stem cells (ASC) from chitosan microspheres (CSM) into a collagen gel scaffold. Porous CSM of uniform size and composition were prepared and used as a stem cell carrier. ASC were allowed to attach to the microspheres and infiltrate through the microsphere pores. The number of viable cells was counted in vitro, using MTT and Calcein acetoxymethyl ester (AM) assays, and it showed a proportional increase with seeding density and reached a maximum cell number by 24 h. The cells inside the microspheres remained metabolically active and viable, could be retrieved from the spheres, and maintained expression of stem-cell-specific markers. Electron microscopic evaluation of the cell–microsphere complex showed that the CSM were able to support cell attachment and that the cells had infiltrated into the pores of the microspheres. The ability of the cells to proliferate and differentiate into adipogenic- and osteogenic-like precursors indicates that the cells have maintained their multipotency after migration out of the microspheres. To mimic cell delivery into a tissue, ASC-loaded CSM were embedded in type-1 collagen scaffold by mixing them with type-1 collagen solution while inducing gelation. By 14 days the cells released into the collagen gel and were able to populate the entire scaffold. When observed through transmission electron microscopy, the cells align along the collagen fibrils with a characteristic fibroblast-like morphology. This study provides a model to capture pluripotent stem cells, expand their cell number within a biomaterial scaffold in vitro, and deliver within an appropriate matrix to repair damaged tissue. PMID:19916819

  20. Human Adipose-derived Stem Cells Ameliorate Cigarette Smoke-induced Murine Myelosuppression via TSG-6

    PubMed Central

    Xie, Jie; Broxmeyer, Hal E.; Feng, Dongni; Schweitzer, Kelly S.; Yi, Ru; Cook, Todd G.; Chitteti, Brahmananda R.; Barwinska, Daria; Traktuev, Dmitry O.; Van Demark, Mary J.; Justice, Matthew J.; Ou, Xuan; Srour, Edward F.; Prockop, Darwin J.; Petrache, Irina; March, Keith L.

    2015-01-01

    Objective Bone marrow-derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose-derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. Methods C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA-transfected hASC. For in vitro experiments, cigarette smoke extract (CSE) was used to mimic the toxicity of CS exposure. Analysis of bone marrow hematopoietic progenitor cells (HPC) were performed both by flow cytometry and colony forming unit assays. Results In this study, we demonstrate that as few as three days of CS exposure result in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically-defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS-induced myelosuppression. This effect was specifically dependent on the anti-inflammatory factor TSG-6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. Conclusion Our results suggest that systemic administration of hASC or TSG-6 may be novel approaches to reverse cigarette smoking-induced myelosuppression. PMID:25329668

  1. Boiling Method-Based Zinc Oxide Nanorods for Enhancement of Adipose-Derived Stem Cell Proliferation.

    PubMed

    Jin, Su-Eon; Ahn, Hyo-Sun; Kim, Ji Hye; Arai, Yoshie; Lee, Soo-Hong; Yoon, Tae-Jong; Hwang, Sung-Joo; Sung, Jong-Hyuk

    2016-09-01

    Adipose-derived stem cells (ASCs) are typically expanded to acquire large numbers of cells for therapeutic applications. Diverse stimuli such as sphingosylphosphocholine and vitamin C have been used to increase the production yield and regenerative potential of ASCs. In the present study, we hypothesized that ZnO nanorods have promising potential for the enhancement of ASC proliferation. ZnO nanorods were prepared using three different methods: grinding and boiling at low temperature with and without surfactant. The physicochemical properties of the nanorods such as their crystallinity, morphology, size, and solvent compatibility were evaluated, and then, the ability of the synthesized ZnO nanorods to enhance ASC proliferation was investigated. Scanning electron microscopy images of all of the ZnO powders showed rod-shaped nanoflakes with lengths of 200-500 nm. Notably, although ZnO-G produced by the grinding method was well dispersed in ethanol, atomic force microscopy images of dispersions of both ZnO-B from boiling methods and ZnO-G indicated the presence of clusters of ZnO nanorods. In contrast, ZnO-B was freely dispersible in 5% dextrose of water and dimethyl sulfoxide, whereas ZnO-G and ZnO-M, produced by boiling with ethanolamine, were not. All three types of ZnO nanorods increased the proliferation of ASCs in a dose-dependent manner. These results collectively suggest that ZnO nanorods have promising potential for use as an agent for the enhancement of ASC proliferation.

  2. Functional regulation of adipose-derived stem cells by PDGF-D.

    PubMed

    Hye Kim, Ji; Gyu Park, Sang; Kim, Wang-Kyun; Song, Sun U; Sung, Jong-Hyuk

    2015-02-01

    Platelet-derived growth factor-D (PDGF-D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF-D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF-D in adipose-derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF-D in this study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF-D-induced stimulation of ASCs. It is of interest that PDGF-B is not expressed, but PDGF-D and PDGF receptor-β are expressed in ASCs. PDGF-D showed the strongest mitogenic effect on ASCs, and PDGF-D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF-D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2-related protein A1 and Serpine peptidase inhibitor, clade E, member 1 mediated the proliferation and migration of ASCs. In addition, PDGF-D upregulated the mRNA expression of diverse growth factors such as vascular endothelial growth factor A, fibroblast growth factor 1 (FGF1), FGF5, leukemia inhibitory factor, inhibin, beta A, interleukin 11, and heparin-binding EGF-like growth factor. Therefore, the preconditioning of PDGF-D enhanced the hair-regenerative potential of ASCs. PDGF-D-induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen-activated protein kinase pathway. In summary, PDGF-D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF-D also upregulates growth factor expression in ASCs. Therefore, PDGF-D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation. © 2014 AlphaMed Press.

  3. lncRNAs are associated with polysomes during adipose-derived stem cell differentiation.

    PubMed

    Dallagiovanna, Bruno; Pereira, Isabela T; Origa-Alves, Ana Carolina; Shigunov, Patricia; Naya, Hugo; Spangenberg, Lucía

    2017-04-30

    Over the past few years, an increasing number of long noncoding RNAs (lncRNAs) have been identified in mammalian genomes. Most of these lncRNAs are expressed at low levels in different human cell types. lncRNAs are found not only in the nucleus but are also enriched in the cytosolic fraction and are associated with translating polysomes. Expression of lncRNAs that have putative roles in cell differentiation has been identified in embryonic and adult stem cells. Nevertheless, the mechanisms by which lncRNAs operate in the cell are still poorly understood.Here, we studied the expression of the subpopulation of lncRNAs that are associated with polysomes in adipose-derived stem cells (hASCs) during their commitment to adipocytes. We established that lncRNAs and protein coding genes have similar expression levels. The relatively comparable expression of these transcripts could be a particular feature of hASCs. We then show that lncRNAs are associated with polysomes in undifferentiated and early differentiating cells, which was confirmed by quantitative RT-PCR. The association of lncRNAs with polysomes was also comparable to that of mRNAs. Our results suggest that the presence of lncRNAs in the polysomal RNA fraction is not the result of random association. We observed that a high percentage of lncRNAs are actively mobilized to or from polysomes during early stages of adipogenesis. Moreover, we found several lncRNAs that can potentially target miRNAs relevant to adipogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Autologous fat injection therapy including a high concentration of adipose-derived regenerative cells in a vocal fold paralysis model: animal pilot study.

    PubMed

    Nishio, N; Fujimoto, Y; Suga, K; Iwata, Y; Toriyama, K; Takanari, K; Kamei, Y; Yamamoto, T; Gotoh, M

    2016-10-01

    To verify the effectiveness and safety of the addition of adipose-derived regenerative cells to autologous fat injection therapy. Unilateral vocal fold paralysis models were made by cutting the right recurrent laryngeal nerve in two pigs. At day 30, 0.5 ml adipose-derived regenerative cells mixed with 1 ml autologous fat was injected into the right vocal fold of one pig, with the other receiving 0.5 ml Ringer's solution mixed with 1 ml autologous fat. At day 120, fibrescopy, laser Doppler flowmeter, computed tomography, vocal function evaluation and histological assessment were conducted. Although histological assessment revealed atrophy of the thyroarytenoid muscle fibre in both pigs, there was remarkable hypertrophy of the thyroarytenoid muscle fibre in the area surrounding the adipose-derived regenerative cells injection site. The addition of a high concentration of adipose-derived regenerative cells to autologous fat injection therapy has the potential to improve the treatment outcome for unilateral vocal fold paralysis.

  5. Osteogenic potential: Comparison between bone marrow and adipose-derived mesenchymal stem cells.

    PubMed

    Liao, Han-Tsung; Chen, Chien-Tzung

    2014-07-26

    Bone tissue engineering (BTE) is now a promising research issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self renewal and multi-lineage differentiation. Unlike embryonic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells (BMSCs) are the earliest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical application. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stem cells (ASCs), is found to be more suitable in clinical application because of high stem cells yield from lipoaspirates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated because most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation potential. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in

  6. Adipose derived mesenchymal stem cells express keratinocyte lineage markers in a co-culture model.

    PubMed

    Irfan-Maqsood, M; Matin, M M; Heirani-Tabasi, A; Bahrami, M; Naderi-Meshkin, H; Mirahmadi, M; Hassanzadeh, H; Sanjar Moussavi, N; Raza-Shah, H; Raeesolmohaddeseen, M; Bidkhori, H; Bahrami, A R

    2016-04-30

    Cutaneous wound healing is a complex type of biological event involving proliferation, differentiation, reprograming, trans/de-differentiation, recruitment, migration, and apoptosis of a number of cells (keratinocytes, fibroblasts, endothelial cells, nerve cells and stem cells) to regenerate a multi-layered tissue that is damaged by either internal or external factors. The exact regeneration mechanism of damaged skin is still unknown but the epithelial and other kinds of stem cells located in skin play crucial roles in the healing process. In this work, a co-culture model composed of adipose derived mesenchymal stem cells and keratinocytes was developed to understand the cellular differentiation behaviour in wound healing. Human mesenchymal stem cells were isolated from waste lipoaspirates. Keratinocytes were isolated from neonatal rats skin as well from human adult skin. Both types of cells were cultured and their culturing behaviour was observed microscopically under regular intervals of time. The identity of both cells was confirmed by flow cytometry and qRT-PCR. Cells were co-cultured under the proposed co-culturing model and the model was observed for 7, 14 and 21 days. The cellular behaviour was studied based on change in morphology, colonization, stratification, migration and expression of molecular markers. Expression of molecular markers was studied at transcriptional level and change in cellular morphology and migration capabilities was observed under the invert microscope regularly. Successfully isolated and characterized mesenchymal stem cells were found to express keratinocyte lineage markers i.e. K5, K10, K14, K18, K19 and Involucrin when co-cultured with keratinocytes after 14 and 21 days. Their expression was found to increase by increasing the time span of cell culturing. The keratinocyte colonies started to disappear after 10 days of culturing which might be due to stratification process initiated by possibly transdifferentiated stem cells. It can

  7. Tendon Tissue Engineering: Mechanism and Effects of Human Tenocyte Coculture With Adipose-Derived Stem Cells.

    PubMed

    Long, Chao; Wang, Zhen; Legrand, Anais; Chattopadhyay, Arhana; Chang, James; Fox, Paige M

    2017-09-06

    Adipose-derived stem cells (ASCs) are a potential candidate for cell-based therapy targeting tendon injury; however, their therapeutic benefit relies on their ability to interact with native tenocytes. This study examines the mechanism and effects of coculturing human tenocytes and ASCs. Tenocytes (T) were directly cocultured with either ASCs (A) or fibroblasts (F) (negative control) in the following ratios: 50% T/50% A or F; 25% T/75% A or F; and 75% T/25% A or F. Cells were indirectly cocultured using a transwell insert that allowed for exchange of soluble factors only. Proliferation and collagen I production were measured and compared with monoculture controls. Synergy was quantified using the interaction index (II), which normalizes measured values by the expected values assuming no interaction (no synergy when II = 1). The ability of ASCs to elicit tenocyte migration was examined in vitro using a transwell migration assay and ex vivo using decellularized human flexor tendon explants. Compared with monoculture controls, II of proliferation was greater than 1 for all tenocyte and ASC direct coculture ratios, but not for tenocyte and fibroblast direct coculture ratios or for tenocyte and ASC indirect coculture. The ASCs elicited greater tenocyte migration in vitro and ex vivo. The II of collagen I production was greater than 1 for direct coculture groups with 25% T/75% A and 75% T/25% A. Direct coculture of ASCs and tenocytes demonstrated synergistic proliferation and collagen I production, and ASCs elicited tenocyte migration in vitro and ex vivo. These interactions play a key role in tendon healing and were absent when ASCs were replaced with fibroblasts, supporting the use of ASCs for cell-based therapy targeting tendon injuries. When ASCs are delivered for cell-based therapy, they directly interact with native tenocytes to increase cell proliferation, collagen I production, and tenocyte migration, which may enhance tendon healing. Copyright © 2017

  8. Therapeutic Potential of Adipose-Derived Therapeutic Factor Concentrate for Treating Critical Limb Ischemia.

    PubMed

    Procházka, Václav; Jurčíková, Jana; Laššák, Ondrej; Vítková, Kateřina; Pavliska, Lubomír; Porubová, Ludmila; Buszman, Piotr P; Krauze, Agata; Fernandez, Carlos; Jalůvka, František; Špačková, Iveta; Lochman, Ivo; Jana, Dvořáčková; Merfeld-Clauss, Stephanie; March, Keith L; Traktuev, Dmitry O; Johnstone, Brian H

    2016-01-01

    Transplantation of adipose-derived stem cells (ADSCs) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ADSCs are primarily mediated through paracrine mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) composed of a cocktail of beneficial growth factors and cytokines that individually and in combination demonstrate disease-modifying effects. The ability of a TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 27 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. An additional five rabbits served as sham controls. One week after surgery, the ischemic limbs received intramuscular injections of either (1) placebo (control medium), (2) a low dose of TFC, or (3) a high dose of TFC. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem on day 35 and assessed for capillary density by immunohistochemistry. At 1 month after treatment, tissue perfusion in ischemic limbs treated with a high dose of TFC was almost double (p < 0.05) that of the placebo group [58.8 ± 23 relative perfusion units (RPU) vs. 30.7 ± 13.6 RPU; mean ± SD]. This effect was correlated with greater capillary density in the affected tissues and with transiently higher serum levels of the angiogenic and prosurvival factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conclusions from this study are that a single bolus administration of TFC demonstrated robust effects for promoting tissue reperfusion in a rabbit model of CLI and that a possible mechanism of revascularization was promotion of angiogenesis by TFC. Results of this study demonstrate that TFC represents a potent

  9. Human and feline adipose-derived mesenchymal stem cells have comparable phenotype, immunomodulatory functions, and transcriptome.

    PubMed

    Clark, Kaitlin C; Fierro, Fernando A; Ko, Emily Mills; Walker, Naomi J; Arzi, Boaz; Tepper, Clifford G; Dahlenburg, Heather; Cicchetto, Andrew; Kol, Amir; Marsh, Lyndsey; Murphy, William J; Fazel, Nasim; Borjesson, Dori L

    2017-03-20

    Adipose-derived mesenchymal stem cells (ASCs) are a promising cell therapy to treat inflammatory and immune-mediated diseases. Development of appropriate pre-clinical animal models is critical to determine safety and attain early efficacy data for the most promising therapeutic candidates. Naturally occurring diseases in cats already serve as valuable models to inform human clinical trials in oncologic, cardiovascular, and genetic diseases. The objective of this study was to complete a comprehensive side-by-side comparison of human and feline ASCs, with an emphasis on their immunomodulatory capacity and transcriptome. Human and feline ASCs were evaluated for phenotype, immunomodulatory profile, and transcriptome. Additionally, transwells were used to determine the role of cell-cell contact in ASC-mediated inhibition of lymphocyte proliferation in both humans and cats. Similar to human ASCs, feline ASCs were highly proliferative at low passages and fit the minimal criteria of multipotent stem cells including a compatible surface protein phenotype, osteogenic capacity, and normal karyotype. Like ASCs from all species, feline ASCs inhibited mitogen-activated lymphocyte proliferation in vitro, with or without direct ASC-lymphocyte contact. Feline ASCs mimic human ASCs in their mediator secretion pattern, including prostaglandin E2, indoleamine 2,3 dioxygenase, transforming growth factor beta, and interleukin-6, all augmented by interferon gamma secretion by lymphocytes. The transcriptome of three unactivated feline ASC lines were highly similar. Functional analysis of the most highly expressed genes highlighted processes including: 1) the regulation of apoptosis; 2) cell adhesion; 3) response to oxidative stress; and 4) regulation of cell differentiation. Finally, feline ASCs had a similar gene expression profile to noninduced human ASCs. Findings suggest that feline ASCs modulate lymphocyte proliferation using soluble mediators that mirror the human ASC secretion

  10. [Experiment of adipose derived stem cells induced into smooth muscle cells].

    PubMed

    Yang, Ping; Yin, Shuo; Cui, Lei; Li, Hong; Wu, Yingchen; Liu, Wei; Cao, Yilin

    2008-04-01

    To study the feasibility of human adipose derived stem cells (ADSCs) in monolayer culture induced into smooth muscle cells in vitro as seeding cells in vascular tissue engineering. The mononuclear cells in human adipose were separated by collagenase treatment and seeded on culture dishes with the density of 5 x 10(5)/cm2. Cells were cultured in M-199 plus 10% FBS. When reaching confluence, the cells were subcultured by 0.1% trypsin and 0.02% EDTA treatment, PDGF-BB (50 ng/mL) and TGF-beta1 (5 ng/mL) were added at the passage 1 to enhance the smooth muscle cells' phenotype. Cells were cultured under the inducing medium for 14 days. The morphology of induced cells was observed under the microscope. Cellular immunofluorescence and RT-PCR were used to determine the expression of smooth muscle cell markers of the post-induced cells. Flow cytometry (FACs) was used to examine the positive rate of induced team. Cocultured in M-199 media including TGF-beta1 and PDGF-BB, the proliferating capability of the induced cells was significantly downregulated compared with the uninduced cells (P < 0.01). The induced cells exhibited "Hill and Valley" morphology, while the uninduced cells were similar to ADSCs of P0 which had the fibroblast-like morphology. The results of immunofluorescence indicated that the induced cells expressed smoothmuscle (SM) cell-specific markers including a-smooth muscle actin (alpha-SMA), SM-myosin heavy chain (SM-MHC) and Calponin. The results of RT-PCR revealed that the induced cells also expressed alpha-SMA, SM-MHC, Calponin and SM-22alpha. The positive rates of alpha-SMA, SM-MHC and Calponin in FACs were 3.26% +/- 1.31%, 3.55% +/- 1.6% and 4.02% +/- 1.81%, respectively, before the cells were induced. However, 14 days after the cell induction, the positive rates were 48.13% +/- 8.31%, 45.33% +/- 10.68% and 39.13% +/- 9.42%, respectively. The positive rates in induced cells were remarkably higher than those in uninduced cells (P < 0.01). The human ADSCs

  11. Resveratrol Effect on Osteogenic Differentiation of Rat and Human Adipose Derived Stem Cells in a 3-D Culture Environment

    PubMed Central

    Dosier, Christopher R.; Erdman, Christopher P.; Park, Jung Hwa; Schwartz, Zvi; Boyan, Barbara D.; Guldberg, Robert E.

    2011-01-01

    The goal of this study was to investigate the effect of resveratrol treatment on the osteogenic potential of human and rat adipose derived stem cells in a 3-D culture environment. Adipose derived stem cells (ADSCs) have been widely studied and have shown promise as a potential source of osteogenic progenitor cells. Previous work had investigated the effect of 25 μM resveratrol on the osteogenic differentiation of rat ADSCs in a 3-D environment and found that pre-treating cells for one passage prior to seeding on the scaffold yielded significantly more mineralization than untreated cells. We first sought to investigate whether this result was also observable with human ADSCs and found that the human cells did not respond to 25 μM resveratrol in a positive manner suggesting a species specific difference in resveratrol dosage. Therefore we next investigated multiple doses at or below 25 μM resveratrol for both rat and human ADSCs. We found that doses below 25 μM caused significantly more mineralization than 0 (untreated) and 25 μM treated cells in a 3-D culture environment. Further, we observed species differences in the total amount of mineralized matrix, as well as the mean mineral density suggesting that the nature of mineralization of the extracellular matrix was different between species. Histological examination of the scaffolds showed that the human cell constructs remain highly cellular in nature with small pockets of mineralization; while rat cell constructs showed much larger and more mature mineralized nodules. Taken together we demonstrate dose dependent differences in the mineralization response of human and rat ADSCs to resveratrol treatment, suggesting that in vitro pre-conditioning of 3D adipose-derived stem cell constructs may be an effective strategy to promote osteogenic differentiation prior to implantation. PMID:22658160

  12. Human adipose-derived mesenchymal stem cells as a new model of spinal and bulbar muscular atrophy.

    PubMed

    Dossena, Marta; Bedini, Gloria; Rusmini, Paola; Giorgetti, Elisa; Canazza, Alessandra; Tosetti, Valentina; Salsano, Ettore; Sagnelli, Anna; Mariotti, Caterina; Gellera, Cinzia; Navone, Stefania Elena; Marfia, Giovanni; Alessandri, Giulio; Corsi, Fabio; Parati, Eugenio Agostino; Pareyson, Davide; Poletti, Angelo

    2014-01-01

    Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked CAG/polyglutamine expansion motoneuron disease, in which an elongated polyglutamine tract (polyQ) in the N-terminal androgen receptor (ARpolyQ) confers toxicity to this protein. Typical markers of SBMA disease are ARpolyQ intranuclear inclusions. These are generated after the ARpolyQ binds to its endogenous ligands, which promotes AR release from chaperones, activation and nuclear translocation, but also cell toxicity. The SBMA mouse models developed so far, and used in preclinical studies, all contain an expanded CAG repeat significantly longer than that of SBMA patients. Here, we propose the use of SBMA patients adipose-derived mesenchymal stem cells (MSCs) as a new human in vitro model to study ARpolyQ toxicity. These cells have the advantage to express only ARpolyQ, and not the wild type AR allele. Therefore, we isolated and characterized adipose-derived MSCs from three SBMA patients (ADSC from Kennedy's patients, ADSCK) and three control volunteers (ADSCs). We found that both ADSCs and ADSCKs express mesenchymal antigens, even if only ADSCs can differentiate into the three typical cell lineages (adipocytes, chondrocytes and osteocytes), whereas ADSCKs, from SBMA patients, showed a lower growth potential and differentiated only into adipocyte. Moreover, analysing AR expression on our mesenchymal cultures we found lower levels in all ADSCKs than ADSCs, possibly related to negative pressures exerted by toxic ARpolyQ in ADSCKs. In addition, with proteasome inhibition the ARpolyQ levels increased specifically in ADSCKs, inducing the formation of HSP70 and ubiquitin positive nuclear ARpolyQ inclusions. Considering all of this evidence, SBMA patients adipose-derived MSCs cultures should be considered an innovative in vitro human model to understand the molecular mechanisms of ARpolyQ toxicity and to test novel therapeutic approaches in SBMA.

  13. Healing of grafted adipose tissue: current clinical applications of adipose-derived stem cells for breast and face reconstruction.

    PubMed

    Philips, Brian J; Marra, Kacey G; Rubin, J Peter

    2014-05-01

    Since their isolation and characterization nearly a decade ago, adipose-derived stem cells (ASCs) have become one of the most popular adult stem cell populations for soft tissue engineering and regenerative medicine applications. Compared with other stem cell sources, ASCs offer several advantages including abundant autologous source, minor invasive harvesting (liposuction), significant proliferative capacity in culture, and multilineage potential. In this mini review, we focus on some of the more salient published clinical and preclinical data to date regarding ASC treatment for breast and facial soft tissue reconstruction. © 2014 by the Wound Healing Society.

  14. Transplantation of adipose derived mesenchymal stem cells for acute thoracolumbar disc disease with no deep pain perception in dogs

    PubMed Central

    Kim, Yongsun; Lee, Seung Hoon; Kim, Wan Hee

    2016-01-01

    Thirty-four dogs with no deep pain perception due to acute thoracolumbar intervertebral disc disease underwent decompression surgery within 1 week of diagnosis. All dogs underwent hemilaminectomy. Adipose derived mesenchymal stem cells (AD-MSCs) were transplanted into the injured spinal cord parenchyma for the AD-MSCs transplant dogs. Long-term outcome was evaluated at the end of the follow-up period (> 6 months). AD-MSCs combination treatment showed better recovery outcomes compared to decompression surgery alone. These results indicate that this stem cell therapy is a potential therapeutic strategy to overcome the limitations of treatment for spinal cord injury in clinical medicine. PMID:27051350

  15. The potential of chondrogenic pre-differentiation of adipose-derived mesenchymal stem cells for regeneration in harsh nucleus pulposus microenvironment.

    PubMed

    Wang, Jingkai; Tao, Yiqing; Zhou, Xiaopeng; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qi-Xin

    2016-12-01

    Recent studies indicated that cell-based therapy could be a promising approach to treat intervertebral disc degeneration. Though the harsh microenvironment in disc is still challenging to implanted cells, it could be overcome by pre-conditioning graft cells before transplantation, suggested by previous literatures. Therefore, we designed this study to identify the potential effect of chondrogenic pre-differentiation on adipose-derived mesenchymal stem cells in intervertebral disc-like microenvironment, characterized by limited nutrition, acidic, and high osmosis in vitro. Adipose-derived mesenchymal stem cells of rat were divided into five groups, embedded in type II collagen scaffold, and cultured in chondrogenic differentiation medium for 0, 3, 7, 10, and 14 days. Then, the adipose-derived mesenchymal stem cells were implanted and cultured in intervertebral disc-like condition. The proliferation and differentiation of adipose-derived mesenchymal stem cells were evaluated by cell counting kit-8 test, real-time quantitative polymerase chain reaction, and Western blotting and immunofluorescence analysis. Analyzed by the first week in intervertebral disc-like condition, the results showed relatively greater proliferative capability and extracellular matrix synthesis ability of the adipose-derived mesenchymal stem cells pre-differentiated for 7 and 10 days than the control. We concluded that pre-differentiation of rat adipose-derived mesenchymal stem cells in chondrogenic culture medium for 7 to 10 days could promote the regeneration effect of adipose-derived mesenchymal stem cells in intervertebral disc-like condition, and the pre-differentiated cells could be a promising cell source for disc regeneration medicine.

  16. Estrogen as a novel agent for induction of adipose-derived mesenchymal stem cells for osteogenic differentiation: in vivo bone tissue-engineering study.

    PubMed

    Calis, Mert; Demirtas, Tugrul Tolga; Atilla, Pergin; Tatar, Ilkan; Ersoy, Orkun; Irmak, Gulseren; Celik, Hakan Hamdi; Cakar, Ayse Nur; Gumusderelioglu, Menemse; Ozgur, Figen

    2014-04-01

    This study investigated whether the in vivo osteogenic differentiation potential of adipose-derived mesenchymal stem cells is enhanced by 17β-estradiol. Thirty Sprague-Dawley rats were randomized and divided into five experimental groups. For the surgical procedure, biparietal full-thickness bone defects (7 mm in diameter) were created. A chitosan-hydroxyapatite scaffold was used as the vehicle system for 17β-estradiol-loaded nanoparticles and adipose-derived mesenchymal stem cells. The first group, the blank defect group, was the control group. The defects were filled with either scaffold, estradiol, and scaffold; scaffold and adipose-derived mesenchymal stem cells; or estradiol, scaffold, and adipose-derived mesenchymal stem cells as experimental groups. The rats were killed at the end of weeks 4 and 12, and their calvariae were harvested for histologic and microtomographic evaluation. Micro-computed tomographic evaluation of estradiol, scaffold, and adipose-derived mesenchymal stem cells revealed the highest median value (82.59 ± 17.17), and the difference was significant compared with the blank defect group (p = 0.004). Histologic samples demonstrated a significant difference between experimental groups for bone defect repair at the end of weeks 4 and 12 (p = 0.003 and p < 0.001). The estradiol, scaffold, and adipose-derived mesenchymal stem cell group had the highest median score (3.00 ± 0.0) at week 12, which was significantly higher than scores for the scaffold and adipose-derived mesenchymal stem cell group and the blank defect group. 17β-Estradiol appears to be a novel and promising agent for future cell-based bone tissue-engineering studies.

  17. The antisenescence effect of trans-cinnamaldehyde on adipose-derived stem cells.

    PubMed

    Rajamani, Karthyayani; Lin, Yi-Chun; Wen, Tung-Chou; Hsieh, Jeanne; Subeq, Yi-Maun; Liu, Jen-Wei; Lin, Po-Cheng; Harn, Horng-Jyh; Lin, Shinn-Zong; Chiou, Tzyy-Wen

    2015-01-01

    As assuring cell quality is an essential parameter for the success of stem cell therapy, the impact of various senescence-inducing stress signals, and strategies to circumvent them, has been an important area of focus in stem cell research. The aim of this study was to demonstrate the capacity of Trans-cinnamaldehyde (TC) in reversing stress-induced senescence and maintaining the quality of stem cells in a chemically (H2O2)-induced cell senescence model. Because of the availability and the promising application potential in regenerative medicine, adipose-derived stem cells (ADSCs) were chosen for the study. We found that H2O2 treatment resulted in the expression of senescence characteristics in the ADSCs, including decreased proliferation rate, increased senescence-associated β-galactosidase (SA-β-gal) activity, decreased silent mating type information regulation 2 homolog (SIRT1) expression, and decreased telomerase activity. However, TC treatment was sufficient to rescue or reduce the effects of H2O2 induction, ultimately leading to an increased proliferation rate, a decrease in the percentage of SA-β-gal-positive cells, upregulation of SIRT1 expression, and increased telomerase activity of the senescent ADSCs at the cellular level. Moreover, a chemically induced liver fibrosis animal model was used to evaluate the functionality of these rescued cells in vivo. Liver dysfunction was established by injecting 200 mg/kg thioacetamide (TAA) intraperitoneally into Wistar rats every third day for 60 days. The experimental rats were separated into groups: normal group (rats without TAA induction), sham group (without ADSC transplantation), positive control group (transplanted with normal ADSCs), H2O2 group (transplanted with H2O2-induced senescent ADSCs), and H2O2 + TC group (transplanted with ADSCs pretreated with H2O2 and then further treated with TC). In the transplantation group, 1 × 10(6) human ADSCs were introduced into each rat via direct liver injection

  18. 197 THE EFFECT OF ZINC ON THE DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO OSTEOBLASTS.

    PubMed

    Bertels, J C; Rubessa, M; Schreiber, S R; Wheeler, M B

    2016-01-01

    The aim of this project was to evaluate the effects of zinc in osteogenic media and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Zinc has a stimulatory effect on bone formation and mineralization in vivo and vitro (Seo et al. 2010 Nutr. Res. Pract. 4, 356-361). Our hypothesis was that the presence of zinc in the osteogenic media would positively influence both the speed of formation and the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 8 different treatments: 6 different concentrations of zinc in the osteogenic medium (8, 4, 0.8, 0.4, 0.08, and 0.04mM) plus 2 control treatments (osteogenic medium without zinc and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 4 times. At the end of the culture period, cells were stained with Alizarin Red S. In each well, we counted the nodules and divided them in 2 categories: formed and forming nodules. The second evaluation that we did was to evaluate the diameter of the largest nodules (2/well) in each group. Data were analysed by ANOVA using the Generalized Linear Model procedure (SPSS, IBM Corp., Armonk, NY, USA). Bonferroni's post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that the doses of zinc of both 4 and 8mM were toxic to the whole cell populations in this treatment, which was indicated by cell death, whereas the concentrations of 0.8 and 0.4mM were not cytotoxic but no nodules formed. Here we report the results that are greater than zero in Table 1. There is a positive effect on nodule formation when the zinc is added to the media. It is clear that the total number of nodules is different between the 0.08mM zinc group and the control (P<0.003). When we evaluated nodule diameter we found a direct correlation between the zinc concentration

  19. Use of Adipose-Derived Mesenchymal Stem Cells to Accelerate Neovascularization in Interpolation Flaps.

    PubMed

    Izmirli, Hakki Hayrettin; Alagoz, Murat Sahin; Gercek, Huseyin; Eren, Guler Gamze; Yucel, Ergin; Subasi, Cansu; Isgoren, Serkan; Muezzinoglu, Bahar; Karaoz, Erdal

    2016-01-01

    Interpolation flaps are commonly used in plastic surgery to cover wide and deep defects. The need to, wait for 2 to 3 weeks until the division of the pedicle still, however, poses a serious challenge, not only extending treatment and hospital stay, but also increasing hospital expenses. To solve this problem, we have aimed to use the angiogenic potential of stem cells to selectively accelerate neovascularization with a view to increasing the viability of interpolation flaps and achieving early pedicle removal. A total of 32 rats were allocated to 2 groups as control (N = 16) and experiment (N = 16). The cranial flaps 6 × 5 cm in size located on the back of the rats were raised. Then, a total suspension containing 3 × 10(6) adipose-derived mesenchymal stem cells (ADSC) tagged with a green fluorescent protein (GFP) was injected diffusely into the distal part of the flap, receiving bed, and wound edges. In the control group, only a medium solution was injected into the same sites. After covering the 3 × 5 cm region in the proximal part of the area where the flap was removed, the distal part of the flap was adapted to the uncovered distal area. The pedicles of 4 rats in each group were divided on postoperative days 5, 8, 11, and 14. The areas were photographed 7 days after the pedicles were released. The photographs were processed using Adobe Acrobat 9 Pro software (San Jose, CA) to measure the flap survival area in millimeters and to compare groups. Seven days after the flap pedicle was divided, the rats were injected with 250 mCi Tc-99 mm (methoxy-isobutyl-isonitrie) from the penile vein, and scintigraphic images were obtained. The images obtained from each group were subjected to a numerical evaluation, which was then used in the comparison between groups. The flaps were then examined by histology to numerically compare the number of newly formed vessels. Neovascularization was also assessed by microangiography. In addition, radiographic images were obtained by

  20. Use of Adipose-Derived Mesenchymal Stem Cells to Increase Viability of Composite Grafts.

    PubMed

    Yucel, Ergin; Alagoz, Murat Sahin; Eren, Guler Gamze; Yasar, Emrah Kagan; Izmirli, Hakki Hayrettin; Duruksu, Gokhan; Isgoren, Serkan; Muezzinoglu, Bahar; Karaoz, Erdal

    2016-07-01

    Chondrocutaneous composite grafts figure among the reconstruction alternatives for alar rim defects resulting from tumor resection and trauma. The major problem with composite grafts is the limited graft survival area. In the present study, the authors aimed to increase the survival area of composite grafts by utilizing the ability of stem cells to promote neovascularization which is crucial in composite graft viability. The study included 36 adult Wistar Albino rats, which were allocated to 6 groups. Groups 1, 2, and 3 were the groups in which the grafts were implanted immediately after the defect was formed, and Groups 4, 5, and 6 were those in which grafts were adapted 4 days after the defect was formed. Composite grafts of 1 × 1 cm containing both the cartilage and the skin were prepared from 1 ear, and after forming punctures and incisions on the cartilage, the grafts were adapted to the 1 × 1 cm defects on the back. The backs of the rats in groups 1 and 4 were injected with adipose-derived stem cell (ADSC), those in groups 2 and 5 with medium solution, while the rats in Groups 3 and 6 did not receive any injection. The procedures were followed by histopathological and scintigraphic evaluations. An evaluation of the statistical results showed that composite graft survival areas of the group treated with stem cells increased significantly, in comparison with control and medium groups. When scintigraphic evaluations were considered, it was seen that the group treated with stem cells had significantly higher radioactive substance retention than the control group. Histopathological examination demonstrated that microscopic survival rates in the stem cell group were higher than those in the control group. Green fluorescent protein (GFP) was used in the experiment to tag adipose tissue-derived stem cells. Immunofluorescence staining studies showed less apoptosis and fewer GFP (+) stem cells in the composite grafts of the stem cell group. However