Adipose-derived stem cells in diabetic foot care:Bridging clinical trials and practical application

Diabetic foot ulcers(DFUs)pose a critical medical challenge,significantly impairing the quality of life of patients.Adipose-derived stem cells(ADSCs)have been identified as a promising therapeutic approach for improving wound healing in DFUs.Despite extensive exploration of the mechanical aspects of ADSC therapy against DFU,its clinical applications remain elusive.In this review,we aimed to bridge this gap by evaluating the use and advancements of ADSCs in the clinical management of DFUs.The review begins with a discussion of the classification and clinical management of diabetic foot conditions.It then discusses the current landscape of clinical trials,focusing on their geographic distribution,reported efficacy,safety profiles,treatment timing,administration techniques,and dosing considerations.Finally,the review discusses the preclinical strategies to enhance ADSC efficacy.This review shows that many trials exhibit biases in study design,unclear inclusion criteria,and intervention protocols.In conclusion,this review underscores the potential of ADSCs in DFU treatment and emphasizes the critical need for further research and refinement of therapeutic approaches,with a focus on improving the quality of future clinical trials to enhance treatment outcomes and advance the field of diabetic wound care.

Non-enzymatic methods for isolation of stromal vascular fraction and adipose-derived stem cells:A systematic review

BACKGROUND Adipose-derived stem cells(ADSCs)and the stromal vascular fraction(SVF)have garnered substantial interest in regenerative medicine due to their potential to treat a wide range of conditions.Traditional enzymatic methods for isolating these cells face challenges such as high costs,lengthy processing time,and regulatory complexities.AIM This systematic review aimed to assess the efficacy and practicality of nonenzymatic,mechanical methods for isolating SVF and ADSCs,comparing these to conventional enzymatic approaches.METHODS Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines,a comprehensive literature search was conducted across multiple databases.Studies were selected based on inclusion criteria focused on non-enzymatic isolation methods for SVF and ADSCs from adipose tissue.The risk of bias was assessed,and a qualitative synthesis of findings was performed due to the methodological heterogeneity of the included studies.RESULTS Nineteen studies met the inclusion criteria,highlighting various mechanical techniques such as centrifugation,vortexing,and ultrasonic cavitation.The review identified significant variability in cell yield and viability,and the integrity of isolated cells across different non-enzymatic methods compared to enzymatic procedures.Despite some advantages of mechanical methods,including reduced processing time and avoidance of enzymatic reagents,the evidence suggests a need for optimization to match the cell quality and therapeutic efficacy achievable with enzymatic isolation.CONCLUSION Non-enzymatic,mechanical methods offer a promising alternative to enzymatic isolation of SVF and ADSCs,potentially simplifying the isolation process and reducing regulatory hurdles.However,further research is necessary to standardize these techniques and ensure consistent,high-quality cell yields for clinical applications.The development of efficient,safe,and reproducible non-enzymatic isolation methods could significantly advance the field of regenerative medicine.

Photobiomodulation:a novel approach to promote trans-differentiation of adipose-derived stem cells into neuronal-like cells

Photobiomodulation,originally used red and near-infrared lasers,can alter cellular metabolism.It has been demonstrated that the visible spectrum at 451-540 nm does not necessarily increase cell proliferation,near-infrared light promotes adipose stem cell proliferation and affects adipose stem cell migration,which is necessary for the cells homing to the site of injury.In this in vitro study,we explored the potential of adipose-derived stem cells to differentiate into neurons for future translational regenerative treatments in neurodegenerative disorders and brain injuries.We investigated the effects of various biological and chemical inducers on trans-differentiation and evaluated the impact of photobiomodulation using 825 nm near-infrared and 525 nm green laser light at 5 J/cm2.As adipose-derived stem cells can be used in autologous grafting and photobiomodulation has been shown to have biostimulatory effects.Our findings reveal that adipose-derived stem cells can indeed trans-differentiate into neuronal cells when exposed to inducers,with pre-induced cells exhibiting higher rates of proliferation and trans-differentiation compared with the control group.Interestingly,green laser light stimulation led to notable morphological changes indicative of enhanced trans-differentiation,while near-infrared photobiomodulation notably increased the expression of neuronal markers.Through biochemical analysis and enzyme-linked immunosorbent assays,we observed marked improvements in viability,proliferation,membrane permeability,and mitochondrial membrane potential,as well as increased protein levels of neuron-specific enolase and ciliary neurotrophic factor.Overall,our results demonstrate the efficacy of photobiomodulation in enhancing the trans-differentiation ability of adipose-derived stem cells,offering promising prospects for their use in regenerative medicine for neurodegenerative disorders and brain injuries.

Exercise combined with administration of adipose-derived stem cells ameliorates neuropathic pain after spinal cord injury

Experimental studies have shown that exercise and human adipose-derived stem cells(ADSCs)play positive roles in spinal cord injury(SCI).However,whether ADSCs and/or exercise have a positive effect on SCI-induced neuropathic pain is still unclear.Thus,there is a need to explore the effects of exercise combined with administration of ADSCs on neuropathic pain after SCI.In this study,a thoracic 11(T11)SCI contusion model was established in adult C57BL/6 mice.Exercise was initiated from 7 days post-injury and continued to 28 days post-injury,and approximately 1×105 ADSCs were transplanted into the T11 spinal cord lesion site immediately after SCI.Motor function and neuropathic pain-related behaviors were assessed weekly using the Basso Mouse Scale,von Frey filament test,Hargreaves method,and cold plate test.Histological studies(Eriochrome cyanine staining and immunohistochemistry)were performed at the end of the experiment(28 days post-injury).Exercise combined with administration of ADSCs partially improved early motor function(7,14,and 21 days postinjury),mechanical allodynia,mechanical hypoalgesia,thermal hyperalgesia,and thermal hypoalgesia.Administration of ADSCs reduced white and gray matter loss at the lesion site.In addition,fewer microglia and astrocytes(as identified by expression of ionized calcium-binding adapter molecule 1 and glial fibrillary acidic protein,respectively)were present in the lumbar dorsal horn in the SCI+ADSCs and SCI+exercise+ADSCs groups compared with the sham group.Our findings suggest that exercise combined with administration of ADSCs is beneficial for the early recovery of motor function and could partially ameliorate SCIinduced neuropathic pain.

Assessment of the effects of intrastromal injection of adipose-derived stem cells in keratoconus patients

·AIM: To evaluate the efficacy and safety of intrastromal transplantation of adipose-derived stem cells(ASCs) in keratoconus patients.·METHODS: This study was conducted on 8 eyes of 8 patients with moderate to severe keratoconus. In the patients, ophthalmic assessments including visual acuity, refraction, slit lamp examination, fundoscopy, corneal topography, and confocal microscopy were performed. Autologous stem cells were used. The isolated stem cells were injected into the corneal stroma by using femtosecond laser. Surgical procedure was similar to intracorneal ring implantation. All patients were re-assessed 1, 3, and 6mo after surgery.·RESULTS: The baseline mean visual acuity was 0.48±0.18 and improved to 0.66±0.17 after surger y and final acuity increased by 1.85±0.80 lines(P=0.001).The mean spherical refraction of patients improved 0.34 ± 0.35 D(P=0.039), and the mean cylindrical refraction of patients improved 0.84±0.23 D(P=0.016). The mean flat keratometry decreased 0.78±0.71 D(P=0.017), and the mean steep keratometry decreased 0.59±0.68 D(P=0.023). The mean central corneal thickness of patients improved of 6.29±4.47 μm(P=0.03). The mean keratocyte density at the anterior and middle stroma of cornea increased(P<0.05) but remained stable at the posterior stroma after 6mo. All patients had no complications and their corneas remained transparent. ·CONCLUSION: Intrastromal transplantation of ASCs has positive effects on vision and refractive parameters in most patients with keratoconus. After six months, visual acuity improved moderately, corneal parameters reduced slightly, and stromal keratocytes density increased. This modality is safe, and patients do not have any complications.

Role of adipose-derived stem cells in breast cancer

The clinical use of fat grafts for breast reconstruction post-mastectomy or radiotherapy has the disadvantages of limited retention and survival rates.To solve this problem,adipose-derived stem cells(ADSCs)have been suggested as an alternative cell source for breast reconstruction,because they are simple to access,have low immunogenicity,and support the survival of mature adipose grafts.However,despite their outstanding properties,the use of ADSCs in patients with breast cancer is controversial,and the oncological safety of this method has been questioned.The biological effects of ADSCs on breast cancer are complex,and clinical research on ADSC-assisted fat grafting is limited.Here,we review the current experimental findings on the effects of ADSCs on breast cancer,mainly focusing on the role of ADSCs in breast cancer proliferation and growth processes,such as epithelial-mesenchymal transition(EMT)and angiogenesis.We also discuss the safety of ADSCs in clinical breast reconstruction.

In vitro differentiation of adipose-derived stem cells and bone marrow-derived stromal stem cells into neuronal-like cells

Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analysis by western blot and quantitative real-time PCR showed that protein levels of Nanog, Oct4, and Sox2, and mRNA levels of miR/125a/3p were decreased, while expression of insulin-like growth factor-2 and neuron specific enolase was increased. In comparison the generation of neuron specific enolase-positive cells was most successful when adipose-derived stem cells were co-cultured with Aβ1-40-treated PC12 cells. Our results demonstrate that adipose-derived stem cells and bone marrow-derived stromal stem cells exhibit trends of neuronal-like cell differentiation after co-culture with Aβ1-40-treated PC12 cells. This process may relate to a downregulation of miR-125a-3p mRNA expression and increased levels of insulin-like growth factor-2 expression.

The role of undifferentiated adipose-derived stem cells in peripheral nerve repair

Peripheral nerve injuries impose significant health and economic consequences, yet no surgical repair can deliver a complete recovery of sensory or motor function. Traditional methods of repair are less than ideal： direct coaptation can only be performed when tension-free repair is possible, and transplantation of nerve autograft can cause donor-site morbidity and neuroma formation. Cell-based therapy delivered via nerve conduits has thus been explored as an alternative method of nerve repair in recent years. Stem cells are promising sources of the regenerative core material in a nerve conduit because stem cells are multipotent in function, abundant in supply, and more accessible than the myelinating Schwann cells. Among different types of stem cells, undifferentiated adipose-derived stem cell（uASC）, which can be processed from adipose tissue in less than two hours, is a promising yet underexplored cell type. Studies of uASC have emerged in the past decade and have shown that autologous uASCs are non-immunogenic, easy to access, abundant in supply, and efficacious at promoting nerve regeneration. Two theories have been proposed as the primary regenerative mechanisms of uASC： in situ trans-differentiation towards Schwann cells, and secretion of trophic and anti-inflammatory factors. Future studies need to fully elucidate the mechanisms, side effects, and efficacy of uASC-based nerve regeneration so that uASCs can be utilized in clinical settings.

Combination of mild therapeutic hypothermia and adipose-derived stem cells for ischemic brain injury

Mild therapeutic hypothermia has been shown to mitigate cerebral ischemia, reduce cerebral edema, and improve the prognosis of patients with cerebral ischemia. Adipose-derived stem cell-based therapy can decrease neuronal death and infiltration of inflammatory cells, exerting a neuroprotective effect. We hypothesized that the combination of mild therapeutic hypothermia and adipose-derived stem cells would be neuroprotective for treatment of stroke. A rat model of transient middle cerebral artery occlusion was established using the nylon monofilament method. Mild therapeutic hypothermia（33°C） was induced after 2 hours of ischemia. Adipose-derived stem cells were administered through the femoral vein during reperfusion. The severity of neurological dysfunction was measured by a modified Neurological Severity Score Scaling System. The area of the infarct lesion was determined by 2,3,5-triphenyltetrazolium chloride staining. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling（TUNEL） staining. The regeneration of microvessels and changes in the glial scar were detected by immunofluorescence staining. The inflammatory responses after ischemic brain injury were evaluated by in situ staining using markers of inflammatory cells. The expression of inflammatory cytokines was measured by reverse transcription-polymerase chain reaction. Compared with mild therapeutic hypothermia or adipose-derived stem cell treatment alone, their combination substantially improved neurological deficits and decreased infarct size. They synergistically reduced the number of TUNEL-positive cells and glial fibrillary acidic protein expression, increased vascular endothelial growth factor levels, effectively reduced inflammatory cell infiltration and down-regulated the m RNA expression of the proinflammatory cytokines interleukin-1β, tumor necrosis factor-α and interleukin-6. Our findings indicate that combined treatment is a better approach for treating stroke compared with mild therapeutic hypothermia or adipose-derived stem cells alone.

In vivo tracking of human adipose-derived stem cells labeled with ferumoxytol in rats with middle cerebral artery occlusion by magnetic resonance imaging

Ferumoxytol, an iron replacement product, is a new type of superparamagnetic iron oxide ap- proved by the US Food and Drug Administration. Herein, we assessed the feasibility of tracking transplanted human adipose-derived stem cells labeled with ferumoxytol in middle cerebral artery occlusion-injured rats by 3.0 T MRI in vivo. 1 × 104 human adipose-derived stem cells labeled with ferumoxytol-heparin-protamine were transplanted into the brains of rats with middle cerebral artery occlusion. Neurologic impairment was scored at 1, 7, 14, and 28 days after transplantation. T2-weighted imaging and enhanced susceptibility-weighted angiography were used to observe transplanted cells. Results of imaging tests were compared with results of Prussian blue staining. The modified neurologic impairment scores were significantly lower in rats transplanted with cells at all time points except I day post-transplantation compared with rats without transplantation. Regions with hypointense signals on T2-weighted and enhanced susceptibility-weighted angiography images corresponded with areas stained by Prussian blue, suggesting the presence of superparamagnetic iron oxide particles within the engrafted cells. Enhanced susceptibility-weighted angiography image exhibited better sensitivity and contrast in tracing ferumoxytol-heparin-protamine-labeled human adipose-derived stem ceils compared with T2-weighted imaging in routine MRI.

Drilling Combined with Adipose-derived Stem Cells and Bone Morphogenetic Protein-2 to Treat Femoral Head Epiphyseal Necrosis in Juvenile Rabbits

This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells （ADSCs） and bone morphogenetic protein-2 （BMP-2） to treat femoral head epiphyseal ischemic necrosis, which can be done in juvenile rabbits. Passagefour bromodeoxyuridine （BrdU）-labeled ADSCs were cultured, assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability. Twomonth-old, healthy male rabbits （1.2 to 1.4 kg, n=45） underwent ischemic induction and were randomly divided into five groups （group A： animal model control; group B： drilling; group C： drilling ＆ ADSCs; group D： drilling ＆ BMP-2; and group E： drilling ＆ ADSCs ＆ BMP-2）. Magnetic resonance imaging （MRI）, X-ray imaging, hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4, 6 and 10 weeks after treatment. Approximately 90% of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability. Similar results were observed in the rabbits in groups C and E at weeks 6 and 10. The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B （P〈0.01）. Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B （P〈0.05）. In summary, drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.

Adipose-derived stem cells enhance myogenic differentiation in the mdx mouse model of muscular dystrophy via paracrine signaling

Adipose-derived stem cells have been shown to promote peripheral nerve regeneration through the paracrine secretion of neurotrophic factors. However, it is unclear whether these cells can promote myogenic differentiation in muscular dystrophy. Adipose-derived stem cells （6 × 106） were injected into the gastrocnemius muscle of mdx mice at various sites. Dystrophin expression was found in the muscle fibers. Phosphorylation levels of Akt, mammalian target of rapamycin （mTOR）, eIF-4E binding protein 1 and $6 kinase 1 were increased, and the Akt/mTOR pathway was activated. Simultaneously, myogenin levels were increased, whereas cleaved caspase 3 and vimentin levels were decreased. Necrosis and fibrosis were reduced in the muscle fibers. These findings suggest that adipose-derived stem cells promote the re- generation and survival of muscle cells by inhibiting apoptosis and fibrosis, thereby alleviating muscle damage in muscular dystrophy.

Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis

Human adipose tissues are an ideal source of stem cells. It is important to find inducers that can safely and effectively differentiate stem cells into functional neurons for clinical use. In this study, we investigate the use of Radix Angelicae Sinensis as an inducer of neuronal differentiation. Primary human adipose-derived stem cells were obtained from adult subcutaneous fatty tissue, then pre-induced with 10% Radix Angelicae Sinensis injection for 24 hours, and incubated in serum-free Dulbecco＇s modified Eagle＇s medium/Nutrient Mixture F-12 containing 40% Radix Angelicae Sinensis to induce its differentiation into neuron-like cells. Butylated hydroxyanisole, a common in- ducer for neuronal differentiation, was used as the control. After human adipose-derived stem cells differentiated into neuron-like cells under the induction of Radix Angelicae Sinensis for 24 hours, the positive expression of neuron-specific enolase was lower than that of the butylated hydroxyani- sole-induced group, and the expression of glial fibrillary acidic protein was negative. Alter they were induced for 48 hours, the positive expression of neuron specific enolase in human adipose-derived stem cells was significantly higher than that of the butylated hydroxyanisole-induced group. Our experimental findings indicate that Radix Angelicae Sinensis can induce human adipose-derived stem cell differentiation into neuron-like cells and produce less cytotoxicity.

Wnt3a expression during the differentiation of adipose-derived stem cells into cholinergic neurons

The present study analyzed changes in Wnt3a expression during differentiation of adipose-derived stern cells into cholinergic neurons. Immunocytochemistry and immunofluorescence revealed significantly increased nestin, neuron-specific enolase, microtubule-associated protein 2, and choline acetyltransferase expression in adipose-derived stem cells isolated from Sprague-Dawley rats and cultured in vitro in neural-induced medium. These expressions increased with prolonged induction time. Real-time reverse transcription-PCR and western blot assay results demonstrated significantly increased choline acetyltransferase and Wnt3a protein and mRNA expressions, respectively, in adipose-derived stem cells following induction. Choline acetyltransferase expression positively correlated with Wnt3a protein and mRNA expressions. These results demonstrated that neural-induced medium induced differentiation of adipose-derived stem cells into cholinergic neuronal-like cells, with subsequent increased Wnt3a expression.

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 adi-pose-derived stem cells (ADSCs) was investigated.Rat ADSCs underwent LIPUS (intensity=100 mW/cm2) or sham exposure for 8 min per treatment once everyday in vitro,and then the alkaline phos-phatase (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 con-trols,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.

Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells

BACKGROUND A major problem in the healing of bone defects is insufficient or absent blood supply within the defect.To overcome this challenging problem,a plethora of approaches within bone tissue engineering have been developed recently.Bearing in mind that the interplay of various diffusible factors released by endothelial cells(ECs)and osteoblasts(OBs)have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions,we set the focus on the simultaneous application of these cell types together with platelet-rich plasma(PRP)as a growth factor reservoir within ectopic bone tissue engineering constructs.AIM To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells(ASCs)induced into ECs and OBs.METHODS ASCs isolated from adipose tissue,induced in vitro into ECs,OBs or just expanded were used for implant construction as followed:BPEO,endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix;BPUI,uninduced ASCs with PRP and bone mineral matrix;BC(control),only bone mineral matrix.At 1,2,4 and 8 wk after subcutaneous implantation in mice,implants were extracted and endothelial-related and bone-related gene expression were analyzed,while histological analyses were performed after 2 and 8 wk.RESULTS The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation.BC had the lowest endothelial-related gene expression,weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO.Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO.BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs.Except Vwf,endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk.Tissue regression was noticed in BPEO constructs after 8 wk.CONCLUSION Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis,but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.

Neuronal differentiation of adipose-derived stem cells and their transplantation for cerebral ischemia

OBJECTIVE： To review published data on the biological characteristics, differentiation and applications of adipose-derived stem cells in ischemic diseases. DATA RETRIEVAL： A computer-based online search of reports published from January 2005 to June 2012 related to the development of adipose-derived stem cells and their transplantation for treatment of cerebral ischemia was performed in Web of Science using the key words ＂adipose-derived stem cells＂, ＂neural-like cells＂, ＂transplantation＂, ＂stroke＂, and ＂cerebral ischemia＂. SELECTION CRITERIA： The documents associated with the development of adipose-derived stem cells and their transplantation for treatment of cerebral ischemia were selected, and those published in the last 3-5 years or in authoritative journals were preferred in the same field. Totally 89 articles were obtained in the initial retrieval, of which 53 were chosen based on the inclusion criteria. MAIN OUTCOME MEASURES： Biological characteristics and induced differentiation of adipose-derived stem cells and cell transplantation for disease treatment as well as the underlying mechanism of clinical application. RESULTS： The advantages of adipose-derived stem cells include their ease of procurement, wide availability, rapid expansion, low tumorigenesis, low immunogenicity, and absence of ethical constraints. Preclinical experiments have demonstrated that transplanted adipose-derived stem cells can improve neurological functions, reduce small regions of cerebral infarction, promote angiogenesis, and express neuron-specific markers. The improvement of neurological functions was demonstrated in experiments using different methods and time courses of adipose-derived stem cell transplantation, but the mechanisms remain unclear. CONCLUSION： Further research into the treatment of ischemic disease by adipose-derived stem cell transplantation is needed to determine their mechanism of action.

Influence of donor age on the differentiation and division capacity of human adipose-derived stem cells

BACKGROUND Human adipose-derived stromal/stem cells(hASCs)are one of the most useful types of mesenchymal stromal/stem cells,which are adult multipotent cells with great therapeutic potential for the treatment of several diseases.However,for successful clinical application,it is critical that high-quality cells can be obtained.Diverse factors seem to be able to influence cell quality and performance,especially factors related to donors’intrinsic characteristics,such as age.Nevertheless,there is no consensus regarding this characteristic,and there is conflicting information in the literature.AIM To investigate the growth kinetics and differentiation potential of adipose-derived stem cells isolated from the lipoaspirates of elderly and young donors.METHODS hASCs were harvested from liposuctioned adipose tissue obtained from female donors(aged 20-70 years).Cells were distributed into two groups according to age range:old hASCs(oASCs,≥55 years,n=9)and young hASCs(yASCs,≤35 years,n=9).For each group,immunophenotypic characterization was performed by flow cytometry.Population doubling time was assessed over seven days.For adipogenic potential evaluation,lipid deposits were assessed after 7 d,14 d and 21 d of adipogenic induction.Osteogenic potential was verified by analyzing cell mineralization after 14 d,21 d and 28 d of osteogenic induction.mRNA expression of PPARγ2,CEBPA and Runx2 were detected by quantitative reverse transcription polymerase chain reaction.RESULTS hASCs were successfully obtained,cultured,and grouped according to their age:yASCs(26.33±4.66 years old)and oASCs(64.78±4.58 years old).After maintenance of the cells in culture,there were no differences in morphology between cells from the young and old donors.Additionally,both groups showed classical immunophenotypic characteristics of mesenchymal stem/stromal cells.The average doubling time indicated that yASCs(4.09±0.94 d)did not significantly differ from oASCs(4.19±1.29 d).Concerning differentiation potential,after adipogenic and osteogenic induction,yASCs and oASCs were able to differentiate to greater levels than the noninduced control cells.However,no differences were found in the differentiation efficiency of yASCs and oASCs in adipogenesis or osteogenesis.Additionally,the mRNA expression of PPARγ2,CEBPA and Runx2 were similar in yASCs and oASCs.CONCLUSION Our findings suggest that age does not seem to significantly affect the cell division or adipogenic or osteogenic differentiation ability of adipose-derived stem cells isolated from lipoaspirates.

Transgene expression and differentiation of baculovirus-transduced adipose-derived stem cells from dystrophin-utrophin double knock-out mouse

In this study, recombinant baculovirus carrying the microdystrophin and β-catenin genes was used to infect adipose-derived stem cells from a dystrophin-utrophin double knock-out mouse. Results showed that, after baculovirus transgene infection, microdystrophin and β-catenin genes were effectively expressed in adipose-derived stem cells from the dystrophin-utrophin double knock-out mouse. Furthermore, this transgenic expression promoted adipose-derived stem cell differentiation into muscle cells, but inhibited adipogenic differentiation. In addition, protein expression related to the microdystrophin and Wnt/β-catenin signaling pathway was upregulated. Our experimental findings indicate that baculovirus can successfully deliver the microdystrophin and β-catenin genes into adipose-derived stem cells, and the microdystrophin and Wnt/β-catenin signaling pathway plays an important role in myogenesis of adipose-derived stem cells in the dystrophin-utrophin double knock-out mouse.

Tissue Inhibitor of Metalloprotease-1(TIMP-1)Regulates Adipogenesis of Adipose-derived Stem Cells(ASCs)via the Wnt Signaling Pathway in an MMP-independent Manner

Tissue inhibitor of m etalloprotease-1(TIM P-1)is a tissue inhibitor o f matrix metalloproteinases(MMPs).It however exerts multiple effects on biological processes,such as cell growth,proliferation,differentiation and apoptosis,in an MMP-independent manner.This study aimed to examine the role of TIMP-1 in adipogenesis of adipose-derived stem cells(ASCs)and the underlying mechanism.We knocked down the TIMP-1 gene in ASCs through lentiviral vectors encoding TIMP-1 small interfering RNA(siRNA),and then found that the knockdown of TIMP-1 in ASCs promoted the adipogenic differentiation of stem cells and inhibited the Wnt/β-catenin signaling pathway in ASCs.We also noted that mutant TIMP-1 without the inhibitory activity on MMPs promoted the activation of Wnt/β-catenin pathway as well as the recombinant wild type TIMP-1 did,which indicated that the effect of TIMP-1 on Wnt/β-catenin pathway was MMPindependent.Our study suggested that TIMP-1 negatively regulated the adipogenesis of ASCs via the Wnt/β-catenin signaling pathway in an MMP-independent manner.