Rat Adipose-derived Stem Cells Research Articles

Corrigendum] Lentivirus vector-mediated gene transduction of CNGRC peptide in rat adipose stem cells.

Mol Med Rep 11: [Related article:] 2555–2561, 2015; DOI: 10.3892/mmr.2014.3043 Following the publication of this article, an interested reader drew to our attention some anomalies associated with the presentation of Fig. 1. Fig. 1B and C presented different views of the cells in the same culture flask. The images in the present Fig. 1B and C are both associated with cells in passage 1 cultured for 11 days. An error was made in the compilation of this Figure, and an incorrect image was selected for Fig. 1C. A corrected version of the Figure is presented below, featuring the correct data for Fig. 1C [pertaining to adipose stem cells at passage 3 cultured for 3 days (magnification, ×400)]. This error did not affect the overall conclusions reported in the present study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused.

Neural differentiation of rat adipose tissue-derived stem cells in vitro

Objective To probe into the potential of neural differentiation of rat adipose-derived stem cells (ADSCs) in vitroto provide foundation to restore erectile dysfunction (ED) caused by cavernous nerves (CNs) injury.Methods Adipose tissue from SD rat abdomen and caputepididymidis was digested with collagenase type Ⅰ,followed by filtering and centrifugation.The isolated adipose stromal cells were cultured in dishes.ADSCs markers were measured by flow cytometry.Cells at the third passage were used for in vitro differentiation. Adipogenic and neuronal differentiation was induced by incubation of ADSCs with different induction media.Oil red O staining was carried out to identify adipose cells,and immunofluorescence to detect protein of glial fibrillary acidic protein (GFAP) and beta tubulin Ⅲ.Results Flow cytometry demonstrated that the ADSCs expressed CD44,but CD45 and CD34 were weakly expressed,and their positive rate was 96.4％,1.7％,and 0.9％ respectively.Adipose cells as evidenced by the lipid droplet were dyed red by oil red O dyeing.Neuron-like cells were evidenced by neuronal morphology and the presence of neuronal markers including GFAP and β-tubulin Ⅲ.The induction rate of GFAP and β-tubulin Ⅲ by method 1 [epidermal growth factor (EGF),basic fibroblast growth factor (bFGF),brain-derived neurotrophic factor (BDNF) + indomethacin,insulin,and 3-isobutyl-1-methylxanthine (IBMX)]and method 2 ( EGF,bFGF + indomethacin,insulin,and IBMX) was (74.0 ± 3.3 ) ％ and (65.3 ±2.1 ) ％,and (51.0 ± 1.2) ％ and (41.0 ± 1.1 ) ％ respectively.The number of the positive cells exhibited significant difference after the induction ( P ＜ 0.01 ).Conclusion ADSCs can be easily obtained from a small amount fat tissue and developed in culture.By using the method 1,the induction rate is high and induction time is short in neural differentiation of ADSCs.ADSCs may be an alternative source of stem cell therapy for neuropathic ED. Key words: Adipose tissue-derived stem cells; Neural cells; Differentiation

Decorin promotes myogenic differentiation and mdx mice therapeutic effects after transplantation of rat adipose-derived stem cells

Adipose-derived stem cells (ADSC) have been considered as attractive candidates for the treatment of Duchenne muscular dystrophy (DMD), but the rate of ADSC myogenesis is very low. Myostatin (Mstn), a negative regulator of myogenesis, is known to be responsible for limiting skeletal muscle regeneration. Decorin could bind Mstn and deactivate it. Decorin has been shown to improve myogenic differentiation in mdx mice. We hypothesized that inhibition of Mstn by using decorin may ameliorate myogenic differentiation of ADSC. Rat ADSC were transfected with the lentivirus-containing green fluorescence protein (GFP) and human decorin gene. The transfected ADSC were induced by 5-azacytidine (5-AzaC). The rates of myogenic differentiation and adipogenesis were detected. The transfected ADSC were injected into mdx mice and the expression of Mstn and decorin detected by Western blot. Dystrophin was detected after transfected ADSC transplantation by immunofluorescence staining and Western blot. Serum creatine kinase (CK) and histologic changes were also evaluated. The optimal multiplicity of infection of ADSC was 10. Decorin improved muscle mass. In accordance with the increased muscle mass, dystrophin expression increased. Following the level of decorin increase, the Mstn expression decreased. Furthermore, serum CK and histologic changes in centrally nucleated fiber (CNF) decreased. Improved myogenic differentiation of ADSC was observed by using decorin. This process was probably the result of decorin inhibiting Mstn. A new method of DMD therapy combining Mstn inhibition (using decorin) and ADSC transplantation is probably feasible.

Neural differentiation of rat adipose-derived stem cells in vitro.

It is reported that adipose-derived stem cells (ADSCs) had multilineage differentiation potential, and could differentiate into neuron-like cells induced by special induction media, which may provide a new idea for restoration of erectile dysfunction (ED) after cavernous nerve injury. The aim of this research was to explore the neuronal differentiation potential of ADSCs in vitro. ADSCs isolated from inguinal adipose tissue of rat were characterized by flow cytometry, and results showed that ADSCs were positive for mesenchymal stem cell markers CD90 and CD44, but negative for hematopoietic stem cell markers. ADSCs maintained self-renewing capacity and could differentiate into adipocytes and neurocytes under special culture condition. In this research, two methods were used to induce ADSCs. In method 1, ADSCs were treated with the preinduction medium including epithelium growth factor, basic fibroblast growth factor, and brain derived neurotrophic factor (BDNF) for 3 days, then with the neurogenic induction medium containing isobutylmethylxanthine, indomethacin, and insulin. While in method 2, BDNF was not used to treat ADSCs. After induction, neuronal differentiation of ADSCs was evaluated. Neuronal markers, glial fibrillary acidic protein (GFAP), and β-tubulin III (Tuj-1) were detected by immunofluorescence and Western Blot analyses. The expressions of GFAP and Tuj-1 in method 1 were obviously higher then those in method 2. In addition, the positive rate of the neuron-like cells was higher in method 1. It suggested that ADSCs are able to differentiate into neural-like cells in vitro, and the administration of BDNF in the preinduction medium may provide a new way to modify the culture method for getting more neuron-like cells in vitro.

Adipose tissue engineering using adipose-derived stem cells enclosed within an injectable carboxymethylcellulose-based hydrogel

In situ gelation of an aqueous solution of carboxymethylcellulose derivative bearing phenolic hydroxyl groups (CMC-Ph) that contained suspended adipose-derived stem cells (ASCs) was studied in vitro and in vivo for evaluating feasibility in adipose tissue-engineering strategies. The rat ASCs that were enclosed in the CMC-Ph gels through a horseradish peroxidase-catalysed reaction showed 92.8% viability, good proliferation and adipogenic differentiation in vitro. Ten weeks after the subcutaneous injection of ASCs-suspending CMC-Ph for in situ gelation, clearly visible new vascularized adipose tissue formed at the injection site. The number of blood vessels and the area occupied by adipose tissues were five and eight times larger, respectively, than those found in the implanted acellular gel. The adipogenesis and neovascularization were further enhanced by incorporation of fibroblast growth factor into the CMC-Ph gel containing ASCs.

198 TRANSITION OF ADIPOSE DERIVED STEM CELLS INTO CONTRACTILE SMOOTH-MUSCLE-CELLS FOR BLADDER ENGINEERING

You have accessJournal of UrologyStem Cell Research1 Apr 2012198 TRANSITION OF ADIPOSE DERIVED STEM CELLS INTO CONTRACTILE SMOOTH-MUSCLE-CELLS FOR BLADDER ENGINEERING Souzan Salemi, Rita Gobet, Tullio Sulser, and Daniel Eberli Souzan SalemiSouzan Salemi Zürich, Switzerland More articles by this author , Rita GobetRita Gobet Zürich, Switzerland More articles by this author , Tullio SulserTullio Sulser Zürich, Switzerland More articles by this author , and Daniel EberliDaniel Eberli Zürich, Switzerland More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.251AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Smooth muscle cells (SMCs) are essential cell types of many organs including gastrointestinal and urogenital tract. Tissue engineering using SMCs may provide alternative treatments for diseases with smooth muscle pathologies such as bladder voiding dysfunction, urinary incontinence and erectile dysfunction. However, autologous SMC cannot be harvested from organs with end stage disease or from organs with cancer development. An alternative cell source would be adipose derived stem cells (ADSCs) which can be differentiated into a variety of cell types. However, ADSC differentiated to SMCs loose their expansion capacity and their contractile phenotype followed by a switch to a synthetic phenotype. Since little is known with regard to the baseline functional characteristics of differentiated SMCs, we have investigated the gene and protein expression and the cell contractility during induction of ADSCs into SMCs. METHODS Primary rat ADSCs were characterized by morphological analysis, immunocytology and FACS. The ADSCs were induced towards SMCs using induction medium. The changes in gene and protein expression levels for lineage specific markers were investigated by Real time PCR, FACS, and western blot (WB). Using gel contraction assay the degree of spontaneous and carbochol stimulated contraction of differentiated cells were investigated. RESULTS ADSCs showed positive expression of lineage specific markers CD29, CD44, stro-1 and negative for CD34. Upon induction, Real- time PCR demonstrated gradual increase in mRNA expression of calponin, myosin heavy chain 11 (MyH) and smoothelin during 1 to 4 weeks and decrease after 5 and 6 weeks of differentiation. At protein level, FACS data showed an increase of alfa-smooth muscle and calponin but gradual decrease of smoothelin and MyH11 after 3 weeks. The same was observed using immunocytology. Smoothelin, the key contractile protein was reduced after 5 and 6 weeks. WB analysis revealed increased levels of smoothelin, calponin and MyH11 protein during 1-4 weeks and reduction after 5 to 6 weeks of differentiation. The gel contraction assay showed a maximal contraction after 2-4 weeks of differentiation. Furthermore, ADSCs differentiated for 2-4 weeks showed a response to carbochol whereas undifferentiated ADSCs did not contract. CONCLUSIONS Our study demonstrates that ADCSs are suitable cell source for engineering of tissues requiring functional and contractile SMCs. The induction time of 3 weeks seems sufficient to differentiate the cells into SMCs with a contractile phenotype suitable for bladder engineering. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e83 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Souzan Salemi Zürich, Switzerland More articles by this author Rita Gobet Zürich, Switzerland More articles by this author Tullio Sulser Zürich, Switzerland More articles by this author Daniel Eberli Zürich, Switzerland More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Transplantation of rat adipose-derived stem cells (ASCs) to improve the survival of autologous aspirated fat grafts

Although autologous lipoaspirated fat grafting is now the most commonly used technique for soft tissue augmentation, problems, such as, unpredictability and a low rate of graft survival remain. The purpose of this study was to compare the long-term viabilities of aspirated fat grafts mixed with adipose-derived stem cells (ASCs; the experimental group) and aspirated fat grafts alone (the control group). Autologous aspirated fats were taken from the interscapular area and transplanted under scalp skin with or without ASCs. Remaining fat volumes were measured at 8 and 12 weeks after transplantation. Numbers of fat cells were randomly measured by hematoxylin and eosin staining. Mean autologous fat volumes in the experimental and control groups at week 8 were 0.410±0.040 ml and 0.418±0.043 ml, respectively, and at week 12 were 0.227±0.025 ml and 0.363±0.047 ml, respectively. Adding ASCs increased the survival rate of transplanted autologous fats at week 12 (p=0.006), and many new blood vessels formed in the outer layer of transplanted tissues by gross examination. H&E staining showed that the density of adipocytes in the experimental group was significantly higher than in the control group (p=0.033). The present study suggest that ASCs improve the survival of autologous fat grafts at 12 weeks after transplantation, which supports the notion that adding ASCs at time of lipotransfer helps maintain soft tissue augmentation.

The performance of decellularized adipose tissue microcarriers as an inductive substrate for human adipose-derived stem cells

With the aim of developing a clinically-translatable cell expansion and delivery vehicle for adipose tissue engineering, the adipogenic differentiation of human adipose-derived stem cells (ASCs) was investigated on microcarriers fabricated from human decellularized adipose tissue (DAT). ASCs seeded on the DAT microcarriers and cultured in adipogenic differentiation medium within a low-shear spinner culture system demonstrated high levels of adipogenic differentiation, as measured by the expression of adipogenic genes, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and intracellular lipid accumulation. In contrast, gelatin microcarrier controls did not demonstrate significant adipogenesis, emphasizing the role of the native matrix in mediating ASC differentiation. Interestingly, ASCs cultured on the DAT microcarriers in proliferation medium expressed elevated levels of the adipogenic markers, suggesting that the DAT provided an adipo-inductive substrate for the human ASCs. In vivo testing of the DAT and gelatin microcarriers in a subcutaneous Wistar rat model confirmed injectability and demonstrated stable volume retention over 28 days. Under histological analysis, the DAT microcarriers demonstrated no evidence of immunogenicity or cytotoxicity, with the DAT supporting cellular infiltration and tissue remodeling. Pre-seeding the DAT microcarriers with allogenic rat ASCs enhanced cellularity and angiogenesis within the implant region.

Differentiated adipose‐derived stem cells promote myelination and enhance functional recovery in a rat model of chronic denervation

Transplantation of autologous Schwann cells (SCs) is a promising approach for treating various peripheral nerve disorders, including chronic denervation. However, given their drawbacks, such as invasive biopsy and lengthy culture in vitro, alternative cell sources would be needed. Adipose-derived stem cells (ASCs) are a candidate, and in this study rat ASCs transdifferentiated into a SC phenotype (dASC) cocultured with dorsal root ganglion neurons were shown to associate with neurites and to express myelin basic protein (MBP)-positive myelin protein. Furthermore, dASCs transplanted into a chronically denervated rat common peroneal nerve survived for at least for 10 weeks, maintaining their differentiated state. Immunohistochemical analysis revealed that transplanted dASCs associated with regenerating axons, forming MBP-/protein zero-positive myelin sheaths. The cell survival and myelin expression assessed by double labelling with S100 and glial fibrillary acidic protein were similar between the dASC- and SC-transplanted nerves. Importantly, transplantation of dASCs resulted in dramatically improved motor functional recovery and nerve regeneration, with a level comparable to, or even superior to, transplantation of SCs. In conclusion, dASCs are capable of myelinating axons in vivo and enhancing functional outcome after chronic denervation.

Adipogenesis on biphasic calcium phosphate using rat adipose‐derived mesenchymal stem cells: In vitro and in vivo

Developing adipose tissue-engineered construct to mend soft tissue defects arising from traumatic injury, tumor resections, and maxillofacial abnormalities is of prime importance in plastic and reconstructive surgical procedures. It is apparent that the clinical outcome of classic techniques like adipose tissue transplantation is unpredictable, with graft resorption, lack of vascularization, and impaired functionality. In this prospective, the concept of tissue engineering was adopted to fabricate a combination product with biphasic calcium phosphate (BCP) and rat adipose-derived mesenchymal stem cells (ASCs) toward the development of an adipose tissue construct. BCP, a combination of hydroxyapatite and α-tricalcium phosphate, was characterized for its physiochemical properties, and ASCs were characterized for their stemness. The cell-ceramic interactions were demonstrated in vitro, whereas adipogenesis was picturesquely depicted by Nile red-stained multilocular adipocyte-like cells. Subsequently, the three-dimensional cell-ceramic-engineered construct was implanted in the rat dorsal muscle for a period of 3 weeks to demonstrate the efficacy of the tissue construct in vivo. Interestingly, the histology of the postimplanted tissue construct revealed the distribution of chicken wire net-like fat cells within the vicinity of the construct. The efficacy of cell transplantation via the scaffold was traced using fluorescent in situ hybridization by labeling the Y chromosome. Thus, the ceramic-based construct may be a good option for reconstruction therapies.

Osteogenic effect of low intensity pulsed ultrasound on rat adipose-derived stem cells in vitro

The osteogenic in vitro effect of low intensity pulsed ultrasound (LIPUS) on SD rat adipose-derived stem cells (ADSCs) was investigated. Rat ADSCs underwent LIPUS (intensity=100 mW/cm(2)) or sham exposure for 8 min per treatment once everyday in vitro, and then the alkaline phosphatase (ALP) activity and mineralized nodule formation were assessed to evaluate the osteogenic effect of LIPUS on ADSCs. To further explore the underlying mechanism, the osteogenic-related gene mRNA expression was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR) at 1st, 3rd, 5th, 7th day after exposure repectively. Westen blot was used to evaluate the protein expression levels of two osteogenic differentiation associated genes at 7th and 14th day repectively. It was found that ALP activity was increased after LIPUS exposure and LIPUS resulted in mineralized nodule formation of ADSCs in vitro. LIPUS-treated ADSCs displayed higher mRNA expression levels of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), ALP and bone sialoprotein (BSP) genes than controls, and the protein levels of Runx2 and BSP were also increased. The results suggested that LIPUS may induce the osteogenic differentiation of ADSCs in vitro.

Mechanical stretch inhibits adipogenesis and stimulates osteogenesis of adipose stem cells

A reciprocal relationships between osteogenesis and adipogenesis has been observed in vitro and in vivo, and mechanical stretch has been believed to be a regulating factor of osteo-adipogenic axis differentiation of mesenchymal stem cells. In this study, rat adipose stem cells (ASCs) were isolated and cultured in adipogenic or normal medium. Their exposure to cyclic mechanical stretch (2000 με, 1 Hz) in the presence of adipogenic medium decreased mRNA and protein level of PPAR-γ, and increased Runx2 mRNA and protein levels as well as Pref-1 mRNA level, compared to static samples. ASCs cultured in normal medium without adipogenic induction did not show any significant change in mRNA expression of PPAR-γ, Runx2, nor Pref-1 irrespective of mechanical loading. Stretching induced phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) during the induction period. It was concluded that mechanical stretch inhibited adipogenesis and stimulated osteogenesis of these ASCs in the presence of adipogenic medium and that ERK1/2 activation may be involved in the mechanical stress-induced trans-differentiation.

Fibrin-Embedded Adipose Derived Stem Cells Enhance Skin Flap Survival

Surgical skin flaps are frequently used procedures in plastic and reconstructive surgery to cover acquired or congenital defects. Either partial or total skin flap loss is a common complication, as survival of the skin flaps is determined by tissue ischemia because of insufficient vascularity. To address this issue, a number of strategies have been described to enhance blood supply and to increase skin flap survival [1–3]. Among these, stem cell-based therapies play an increasing role, due to their capacity to self-renew and differentiate into a variety of specific cell lines. Especially adipose -derived stem cells (ADSCs)—therapies have raised tremendous interest in the field of soft tissue reconstruction as they offer distinct advantages over bone marrow-derived stem cells [4]. ADSCs can be easily harvested in a minimal invasive procedure, found in abundant quantities and have the ability to differentiate into osteoblasts, chondrocytes and adipocytes in controllable and reproducible manner [5–7]. With regard to plastic and reconstructive surgery perspectives, ADSCstherapy has recently been demonstrated to improve skin flap survival [8–14]. Further investigations documented a positive effect of rescuing ischemic skin flaps by increasing tissue perfusion, a significant rise of growth factors such as vascular endothelial growth factor (VEGF) as well as fibroblast growth factor (FGF) after ADSCtherapy in rodent model [8, 10–13]. However, in all these studies ADSCs were injected into the subcutaneous tissue which may result in an uncertain distribution of the ADSCs. Furthermore the purity of the applied stem cells was not clearly analyzed. In light of these recent results this study was designed to firstly isolate and cultivate rat adipose derived stem cells and secondly determine the potential of locally applied fibrinembedded ADSCs to enhance skin flap survival in an animal epigastric skin flap model.

The Osteogenic Study of Tissue Engineering Bone with BMP2 and BMP7 Gene-Modified Rat Adipose-Derived Stem Cell

To evaluate the feasibility and advantages of constructing a novel tissue engineering bone, using β-tricalcium phosphate (β-TCP) and rat adipose-derived stem cells (ADSCs), modified with BMP2 and BMP7 by lentivirus. In the present study, ADSCs transfected with Lv-BMP2 and Lv-BMP7, alone or together, were seeded on β-TCP scaffold and cultured in vitro. Based on the results of DNA assay, alkaline phosphatase (ALP) activity, alizarin red staining and osteogenic marker genes expression analysis, the BMP2 and BMP7 genes cotransfection group exhibited a higher degree of osteogenic differentiation in vitro. To investigate the in vivo osteogenesis of the tissue engineering bone, the ADSCs/β-TCP constructs were implanted in rat femurs defects for 6 weeks and studied histomorphology and radiography. The results showed that BMP2 and BMP7 genes cotransfection group dramatically enhanced the efficiency of new bone formation than BMP2 group and BMP7 group in vivo. These results demonstrated that it was advantageous to construct tissue engineering bone using ADSCs cotransfected with BMP2 and BMP7 on β-TCP, providing a potential way for treating bone defects.

Resveratrol effect on osteogenic differentiation of rat and human adipose derived stem cells in a 3-D culture environment

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 25μM 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 25μM resveratrol in a positive manner suggesting a species specific difference in resveratrol dosage. Therefore, we next investigated multiple doses at or below 25μM resveratrol for both rat and human ADSCs. We found that doses below 25μM caused significantly more mineralization than 0 (untreated) and 25μM 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.

Improvement of skin-graft survival after autologous transplantation of adipose-derived stem cells in rats

Skin grafts are frequently used for a variety of indications in plastic and reconstructive surgery. Their necrosis is a common complication, while different therapies have been proposed. Currently, adipose-derived stem cells (ASCs) hold great promise for their angiogenic potential and role during tissue repair. In this study, autologous transplantation of ASCs was used in skin grafts in rats to determine if it increases angiogenesis, skin-graft survival and wound healing. ASCs were isolated, cultured, labelled with fluorescent dye and injected under full-thickness skin grafts in 10 rats (group 1), while 10 others served as controls (group 2). Skin grafts were analysed after 1 week. Collagen's framework was assessed with Masson's trichrome stain and angiogenesis with von Willebrand factor (vWF) immunohistochemistry. In addition, immunohistochemical staining intensity of vascular endothelial growth factor (VEGF) and transforming growth factor b3 (TGFb3) was assessed in all grafts. Mean area of graft necrosis was significantly less in group 1 than in group 2 (6.12% vs. 32.62%, p<0.01). Statistically significant increase of microvessel density, collagen density, VEGF and TGFb3 expression was noted in group 1 compared with group 2 (all: p<0.01). These findings suggest that autologous ASCs transplantation increases full-thickness skin-graft survival and shows promise for use in skin-graft surgery. This might be both due to in situ differentiation of ASCs into endothelial cells and increased secretion by ASCs of growth factors, such as VEGF and TGFb3 that enhance angiogenesis and wound healing.

Reduction of infarct size by intravenous injection of uncultured adipose derived stromal cells in a rat model is dependent on the time point of application

Stem cell therapy is a promising tool to improve outcome after acute myocardial infarction (AMI), but needs to be optimized since results from clinical applications remain ambiguous. A potent source of stem cells is the stromal vascular fraction of adipose tissue (SVF), which contains high numbers of adipose derived stem cells (ASC). We hypothesized that: 1) intravenous injection can be used to apply stem cells to the heart. 2) Uncultured SVF cells are easier and safer when cultured ASCs. 3) Transplantation after the acute inflammation period of AMI is favorable over early injection. For this, AMI was induced in rats by 40 min of coronary occlusion. One or seven days after AMI, rats were intravenously injected with vehicle, 5 × 10 6 uncultured rat SVF cells or 1 × 10 6 rat ASCs. Rats were analyzed 35 days after AMI. Intravenous delivery of both fresh SVF cells and cultured ASCs 7 days after AMI significantly reduced infarct size compared to vehicle. Similar numbers of stem cells were found in the heart, after treatment with fresh SVF cells and cultured ASCs. Importantly, no adverse effects were found after injection of SVF cells. Using cultured ASCs, however, 3 animals had shortness of breath, and one animal died during injection. In contrast to application at 7 days post AMI, injection of SVF cells 1 day post AMI resulted in a small but non-significant infarct reduction (p = 0.35). Taken together, intravenous injection of uncultured SVF cells subsequent to the acute inflammation period, is a promising stem cell therapy for AMI.

Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems

Tendon tissue engineering with a biomaterial scaffold that mimics the tendon extracellular matrix (ECM) and is biomechanically suitable, and when combined with readily available autologous cells, may provide successful regeneration of defects in tendon. Current repair strategies using suitable autografts and freeze-dried allografts lead to a slow repair process that is sub-optimal and fails to restore function, particularly in difficult clinical situations such as zone II flexor tendon injuries of the hand. We have investigated the effect of GDF-5 on cell proliferation and gene expression by primary rat adipose-derived stem cells (ADSCs) that were cultured on a poly(dl-lactide-co-glycolide) PLAGA fiber scaffold and compared to a PLAGA 2D film scaffold. The electrospun scaffold mimics the collagen fiber bundles present in native tendon tissue, and supports the adhesion and proliferation of multipotent ADSCs. Gene expression of scleraxis, the neotendon marker, was upregulated seven- to eightfold at 1 week with GDF-5 treatment when cultured on a 3D electrospun scaffold, and was significantly higher at 2 weeks compared to 2D films with or without GDF-5 treatment. Expression of the genes that encode the major tendon ECM protein, collagen type I, was increased by fourfold starting at 1 week on treatment with 100 ng mL−1 GDF-5, and at all time points the expression was significantly higher compared to 2D films irrespective of GDF-5 treatment. Thus stimulation with GDF-5 can modulate primary ADSCs on a PLAGA fiber scaffold to produce a soft, collagenous musculoskeletal tissue that fulfills the need for tendon regeneration.

Viability and proliferation potential of adipose-derived stem cells following labeling with a positron-emitting radiotracer

Adipose-derived stem cells (ASCs) have promising potential in regenerative medicine and cell therapy. Our objective is to examine the biological function of the labeled stem cells following labeling with a readily available positron emission tomography (PET) tracer, (18)F-fluoro-2-deoxy-D: -glucose (FDG). In this work we characterize labeling efficiency through assessment of FDG uptake and retention by the ASCs and the effect of FDG on cell viability, proliferation, transdifferentiation, and cell function in vitro using rat ASCs. Samples of 10(5) ASCs (from visceral fat tissue) were labeled with concentrations of FDG (1-55 Bq/cell) in 0.75 ml culture medium. Label uptake and retention, as a function of labeling time, FDG concentration, and efflux period were measured to determine optimum cell labeling conditions. Cell viability, proliferation, DNA structure damage, cell differentiation, and other cell functions were examined. Non-labeled ASC samples were used as a control for all experimental groups. Labeled ASCs were injected via tail vein in several healthy rats and initial cell biodistribution was assessed. Our results showed that FDG uptake and retention by the stem cells did not depend on FDG concentration but on labeling and efflux periods and glucose content of the labeling and efflux media. Cell viability, transdifferentiation, and cell function were not greatly affected. DNA damage due to FDG radioactivity was acute, but reversible; cells managed to repair the damage and continue with cell cycles. Over all, FDG (up to 25 Bq/cell) did not impose severe cytotoxicity in rat ASCs. Initial biodistribution of the FDG-labeled ASCs was 80% + retention in the lungs. In the delayed whole-body images (2-3 h postinjection) there was some activity distribution resembling typical FDG uptake patterns. For in vivo cell tracking studies with PET tracers, the parameter of interest is the amount of radiotracer that is present in the cells being labeled and consequent biological effects. From our study we developed a labeling protocol for labeling ASCs with a readily available PET tracer, FDG. Our results indicate that ASCs can be safely labeled with FDG concentration up to 25 Bq/cell, without compromising their biological function. A labeling period of 90 min in glucose-free medium and efflux of 60 min in complete media resulted in optimum label retention, i.e., 60% + by the stem cells. The initial biodistribution of the implanted FDG-labeled stem cells can be monitored using microPET imaging.

A novel method for the isolation of subpopulations of rat adipose stem cells with different proliferation and osteogenic differentiation potentials

Bone marrow has been the elected cell source of studies published so far concerning bone and cartilage tissue-engineering approaches. Recent studies indicate that adipose tissue presents significant advantages over bone marrow as a cell source for tissue engineering. Most of these studies report the use of adipose stem cells (ASCs) isolated by a method based on the enzymatic digestion of the adipose tissue and on the ability of stem cells to adhere to a cell culture plastic surface. Using this method, a heterogeneous population was obtained containing different cell types that have been shown to compromise the proliferation and differentiation potential of the stem cells. This paper reports the development and optimization of a new isolation method that enables purified cell populations to be obtained that exhibit higher osteogenic differentiation and/or proliferation potential. This method is based on the use of immunomagnetic beads coated with specific antibodies and it is compared with other methods described in the literature for the selection of stem cell populations, e.g. methods based on a gradient solution and enzymatic digestion. The results showed that the isolation method based on immunomagnetic beads allows distinct subpopulations of rat ASCs to be isolated, showing different stem cells marker expressions and different osteogenic differentiation potentials. Therefore, this method can be used to study niches in ASC populations and/or also allow adipose tissue to be used as a stem cell source in a more efficient manner, increasing the potential of this cell source in future clinical applications.