Adipose-derived Stem Cells Cells Research Articles

Osteogenic Differential Expression of Rabbit Adipose-Derived Mesenchymal Stem Cells by PPARγ Transfection Coated with Nanoparticles

Adipose-derived stem cells (ADSCs) have advantages in utilization. Nanoparticles (NP) are degradable, nontoxic and have good biocompatibility, besides, they can encapsulate and transport water-soluble, fat-soluble and deoxyribonucleic acid. This study explored the influence of NP-coated PPARγ transfection on osteogenic differentiation of rabbit ADSCs. After preparing PPARγ transfection system coated with nanoparticles, Petri dishes containing the same amount of cells were divided at the beginning of cell culture into blank group (not transfected), control group and observation group (transfected with PPARγ coated NP). Alkaline phosphatase (ALP) staining and Western Blot were done to detect the contents of lipoprteinlipase (LPL), Runx2 and serum osteocalcin (BGP) protein. The levels of Runx2, BGP and PPARγ mRNA were measured by RT-PCR. After ADSCs cells were stained, a small number of cells adhered to each other, and the cells were mainly fibroblast-like and spindle-shaped, and proliferated in the form of colonies. The levels of Runx2 and BGP in the observation group were significantly lower than those in the control group (p < 0.05) after the ADSCs cells were cultured for 7 and 14 days. The expression of LPL and PPARγ mRNA in the observation group was higher control group (p < 0.05). Transfection of PPARγ coated with nanoparticles inhibited the osteogenic differentiation of rabbit ADSCs by inhibiting the expression of Runx2 and BGP and promoting the expression of LPL and PPARγ.

Use of adipose derived stem cells accelerates the healing process in third-degree burns

IntroductionBurns are defined as a traumatic injury, usually of thermal origin, that affects the epithelial and adjacent tissue and is classified according to the depth reached. Tissue repair involved in this type of injury is often a challenge both due to its severity and the multiplicity of complications. Regenerative medicine has focused on the use of low-level laser photobiomodulation therapy (LLLT) and adipose-derived stem cells (ADSC), especially in the early stages of the process, to promote better healing and shorten repair time. Therefore, aim of this study was to evaluate the action of LLLT (660 nm) and ADSC in the repair process of burned skin tissue and investigate the association of the techniques (LLLT and ADSC). Materials and methodsAn in vivo study was carried out using 96 rats (Wister) with a scald burn model at a temperature of 95ºC, exposing the animal's back for 14 s. Animals were randomized into seven groups and three periods, five, 14 and 21 days. The groups included GC: Control group, ADSC-: Group treated with CD49d negative cells, ADSC+ : Group treated with positive CD49d cells, CULT: Group treated with conventional isolation cells, LLLT: Group treated only with LLLT Low Power Laser, ADSC-LLLT: Group treated with CD49d negative cells and LLLT. ADSC+LLLT: Group treated with positive CD49d cells and LLLT. The groups treated with LLLT (660 nm; 5 J/cm2) received irradiation three times a week, on alternate days for five, 14 and 21 days, according to the time of biopsy. ADSC-treated groups received one to three applications of the cells in a total volume of 1000 μL starting soon after the surgical debridement of the burn. Photographic monitoring was carried out at 5, 14 and 21 days after the beginning of the experiment to assess the degree of lesion contraction. Macroscopic, morphometric and histopathological analyzes were performed. ResultsWe showed significant re-epithelialization as well as an improvement in the healing process in the ADSC+, LLLT and ADSC+LLLT groups. We observed effects in the reduction of the inflammatory phase, increase in angiogenesis, decrease in oedema, greater collagen deposition, and better organization of the extracellular matrix compared to the other treatments. Moreover, the immunomagnetic separation of ADSC cells through the expression of the CD49d protein proved to be a useful means to obtain a more homogeneous population of cells with a role in tissue regeneration compared to the ADSC- and CULT groups. ConclusionIn conclusion, the association of ADSC+ with LLLT was effective in accelerating the burn repair process, stimulating cell proliferation and formation of more normal skin tissue.

Finite Element Analysis of Different Osseocartilaginous Reconstruction Techniques in Animal Model Knees.

Lesions of the articular cartilage are frequent in all age populations and lead to functional impairment. Multiple surgical techniques have failed to provide an effective method for cartilage repair. The aim of our research was to evaluate the effect of two different compression forces on three types of cartilage repair using finite element analysis (FEA). Initially, an in vivo study was performed on sheep. The in vivo study was prepared as following: Case 0-control group, without cartilage lesion; Case 1-cartilage lesion treated with macro-porous collagen implants; Case 2-cartilage lesion treated with collagen implants impregnated with bone marrow concentrate (BMC); Case 3-cartilage lesion treated with collagen implants impregnated with adipose-derived stem cells (ASC). Using the computed tomography (CT) data, virtual femur-cartilage-tibia joints were created for each Case. The study showed better results in bone changes when using porous collagen implants impregnated with BMC or ASC stem cells for the treatment of osseocartilaginous defects compared with untreated macro-porous implant. After 7 months postoperative, the presence of un-resorbed collagen influences the von Mises stress distribution, total deformation, and displacement on the Z axis. The BMC treatment was superior to ASC cells in bone tissue morphology, resembling the biomechanics of the control group in all FEA simulations.

Influence of Tamoxifen on Different Biological Pathways in Tumorigenesis and Transformation in Adipose-Derived Stem Cells, Mammary Cells and Mammary Carcinoma Cell Lines-An In Vitro Study.

Breast carcinoma is one of the most common malignant tumors in women. In cases of hormone-sensitive cells, tamoxifen as an anti-estrogenic substance is a first line medication in the adjuvant setting. The spectrum of autologous breast reconstructions ranges from fat infiltrations to complex microsurgical procedures. The influence of adipose-derived stem cells (ASC) on the tumor bed and a possibly increased recurrence rate as a result are critically discussed. In addition, there is currently no conclusive recommendation regarding tamoxifen-treated patients and autologous fat infiltrations. The aim of the present study was to investigate the effect of tamoxifen on the gene expression of a variety of genes involved in tumorigenesis, cell growth and transformation. Mammary epithelial cell line and mammary carcinoma cell lines were treated with tamoxifen in vitro as well as co-cultured with ASC. Gene expression was quantified by PCR arrays and showed increased expression in the mammary carcinoma cell lines with increasing time of treatment and concentration of tamoxifen. The data presented can be considered as an addition to the controversial discussion on the relationship between ASC and breast carcinoma cells. Further studies are needed to quantify the in vivo interaction of ASC and mammary carcinoma cells and to conclusively assess the impact of tamoxifen in reconstructive cases with fat grafting.

Pomegranate seed extract enhances the inhibitory effect of adipose-derived mesenchymal stem cells on breast cancer cell line in co-culture conditions.

Background and purpose:Pomegranate seed extract (PSE) possesses anticancer activities and healing effects. Adipose-derived stem cells (ADSCs) are being considered a new candidate for cancer treatment. The purpose of this study was to investigate the effect of PSE on the cell cycle and apoptosis of the MCF-7 cell line in the co-culture condition with ADSCs.Experimental approach:MCF-7 and ADSC cells (ratio 1/1) were cultured in a transwell plate with and without PSE (PSE-co-culture and co-culture groups). MCF-7 cells were cultured in monolayer without and with PSE (mono-culture and PSE-mono-culture groups). MCF-7 cell line was harvested on day 5 and cell viability, apoptotic activity, cell cycle, and gene expression were evaluated.Findings / Results:The results of the MTT assay indicated that PSE at 100 μg/mL has the highest cytotoxicity on the MCF-7 in the PSE-co-culture group. The cell cycle analysis revealed that ADSCs in combination with PSE significantly increased the population of MCF-7 cells in the G1 phase, resulting in the arrest of MCF-7 cells cycle in the G0/G1 transition. In addition, the most apoptotic MCF-7 cells (41.5%) were detected in the same group. Expression of BAX and caspase3 genes were upregulated while anti-apoptotic (BCL-2) and angiogenesis inducer (VEGF) genes were downregulated in the PSE-co-culture group compared with the other groups.Conclusion and implications:ADSCs reduced cell viability and proliferation of MCF-7 cells in co-culture conditions and adding PSE to the medium increased the apoptosis of cancer cells. This study suggests that ADSCs with PSE can suppress tumor cells.

Fluorapatite and fluorohydroxyapatite apatite surfaces drive adipose-derived stem cells to an osteogenic lineage

PurposeHydroxyapatite (HA) scaffolds are common replacement materials used in the clinical management of critical-sized bone defects. This study was undertaken to examine the potential benefits of fluoridated derivatives of hydroxyapatite, fluorapatite (FA), and fluorohydroxyapatite (FHA) as bone scaffolds in conjunction with adipose-derived stem cells (ADSCs). If FHA and FA surfaces could drive the differentiation of stem cells to an osteogenic phenotype, the combination of these ceramic scaffolds with ADSCs could produce materials with mechanical strength and remodeling potential comparable to autologous bone. This study was designed to investigate the ability of the apatite surfaces HA, FA, and FHA produced at different sintering temperatures to drive ADSCs toward osteogenic lineages. Methods: HA, FHA, and FA surfaces sintered at 1150 °C and 1250 °C were seeded with ADSCs and evaluated for cell growth and gene and protein expression of osteogenic markers at 2 and 10 days post-seeding. Results: In vitro, ADSC cells were viable on all surfaces; however, differentiation of these cells into osteoblastic lineage only observed in apatite surfaces. ADSCs seeded on FA and FHA expressed genes and proteins related to osteogenic differentiation markers to a greater extent by Day 2 when compared to HA and cell culture controls. By day 10, HA, FA, and FHA all expressed more bone differentiation markers compared to cell culture controls. Conclusion: FA and FHA apatite scaffolds may promote the differentiation of ADSCs at an earlier time point than HA surfaces. Combining apatite scaffolds with ADSCs has the potential to improve bone regeneration following bone injury.

The evaluation of a multiphasic 3D-bioplotted scaffold seeded with adipose derived stem cells to repair osteochondral defects in a porcine model.

There is a need for the development of effective treatments for focal articular cartilage injuries. We previously developed a multiphasic 3D-bioplotted osteochondral scaffold design that can drive site-specific tissue formation when seeded with adipose-derived stem cells (ASC). The objective of this study was to evaluate this scaffold in a large animal model. Osteochondral defects were generated in the trochlear groove of Yucatan minipigs and repaired with scaffolds that either contained or lacked an electrospun tidemark and were either unseeded or seeded with ASC. Implants were monitored via computed tomography (CT) over the course of 4months of in vivo implantation and compared to both open lesions and autologous explants. ICRS II evaluation indicated that defects with ASC-seeded scaffolds had healing that most closely resembled the aulogous explant. Scaffold-facilitated subchondral bone repair mimicked the structure of native bone tissue, but cartilage matrix staining was not apparent within the scaffold. The open lesions had the highest volumetric infill detected using CT analysis (p< 0.05), but the repair tissue was largely disorganized. The acellular scaffold without a tidemark had significantly more volumetric filling than either the acellular or ASC seeded groups containing a tidemark (p< 0.05), suggesting that the tidemark limited cell infiltration into the cartilage portion of the scaffold. Overall, scaffold groups repaired the defect more successfully than an open lesion but achieved limited repair in the cartilage region. With further optimization, this approach holds potential to treat focal cartilage lesions in a highly personalized manner using a human patient's own ASC cells.

Extracellular Vesicles Act as Nano-Transporters of Tyrosine Kinase Inhibitors to Revert Iodine Avidity in Thyroid Cancer.

A new approach for using extracellular vesicles (EVs) to deliver tyrosine kinase inhibitors (TKIs) to enhance iodine avidity in radioactive iodine-refractory thyroid cancer is needed. We isolated and characterized primary human adipose-derived stem cells (ADSCs) and isolated their EVs. The EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. A new TKI was loaded into the EVs by incubation (37 °C; 10 min) or sonication (18 cycles; 4 s per cycle) with 2 s intervals and a 2 min ice bath every six cycles. TKI loading was confirmed and measured by mass spectrometry. EV uptake into radioactive iodine-refractory thyroid cancer cells (SW1736 cells) was confirmed by microscopy. We treated the SW1736 cells with vehicle, TKI, or TKI-loaded EVs (sonication TKI-loaded EVs [EVsTKI(S)]) and examined the expression of iodide-metabolizing proteins and radioiodine uptake in the SW1736 cells. ADSCs cells showed >99% of typical stem cell markers, such as CD90 and CD105. The EVs displayed a round morphology, had an average size of 211.4 ± 3.83 nm, and were positive for CD81 and Alix and negative for cytochrome c. The mass spectrometry results indicate that the sonication method loaded ~4 times more of the TKI than did the incubation method. The EVsTKI(S) were used for further experiments. Higher expression levels of iodide-metabolizing mRNA and proteins in the EVsTKI(S)-treated SW1736 cells than in TKI-treated SW1736 cells were confirmed. EVsTKI(S) treatment enhanced 125I uptake in the recipient SW1736 cells compared with free-TKI treatment. This is the first study that demonstrated successful delivery of a TKI to radioactive iodine-refractory thyroid cancer cells using EVs as the delivery vehicle. This approach can revert radioiodine-resistant thyroid cancer cells back to radioiodine-sensitive thyroid cancer cells.

MicroRNA-124-3p affects myogenic differentiation of adipose-derived stem cells by targeting Caveolin-1 during pelvic floor dysfunction in Sprague Dawley rats.

BackgroundThe aim of this study was to investigate using myogenic differentiation of adipose stem cells for the treatment of female pelvic floor dysfunction (PFD) and aimed to further study the influences of microRNA-124-3p (miR-124-3p) in the process of myogenic differentiation of adipose-derived stem cells (ADSCs) through targeting Caveolin-1 (Cav1) during PFD in Sprague Dawley (SD) rats.MethodsThe ADSCs were separated from 6–8-week-old female SD rats (n=25) and were cultivated. Then, we observed the cell status and conducted fat and osteogenic experiments. We then constructed an ADSC-green fluorescent protein (GFP) stable transfer strain. Flow cytometry was used to identify the positive rates of CD44, CD90, and CD45 in ADSCs and ADSC-GFP. Real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting were used to mRNA and protein expression levels. Myogenic differentiation of ADSCs was measured with immunofluorescence methods. A dual-luciferase reporter assay was executed to affirm whether Cav1 was a target of miR-124-3p.ResultsThe isolated ADSCs cells were in good condition under the microscope. The results of flow cytometry showed that the positive rate of CD44 and CD90 was high, and the positive rate of CD45 was low in ADSCs and ADSC-GFP. Under normal culture conditions, ADSCs-GFP cells can be massively adipated and osteogenic. After 5-Aza induced ADSC-GFP myogenic differentiation, the level of miR-124-3p was significantly increased. We found that MiR-124-3p mimics promoted the myogenic differentiation of ADSCs. Moreover, we discovered that Cav1 was a target gene of miR-124-3p and was negatively regulated by miR-124-3p. The results of leak point pressure (LPP), hematoxylin and eosin (HE), and Masson showed that the collagen fiber content of the PFD group was lower than that of the control group; the collagen fiber content of ADSC-GFP, 5-Aza, or miR-124-3p mimics were increased after intervention. Furthermore, the outcomes qRT-PCR, western blotting, and immunofluorescence suggested that miR-124-3p facilitated the survival ADSC-GFP fat transplantation by regulating many key factors in vivo.ConclusionsThese results proofed that miR-124-3p could accelerate myogenic differentiation of ADSCs by down-regulating Cav1 to improve PFD in SD rats, which will pave the way for therapeutic delivery of miRNA targeting PFD disease.

Mechanisms by Which Membrane and Nuclear ER Alpha Inhibit Adipogenesis in Cells Isolated From Female Mice

Mesenchymal stem cells can differentiate into mature chondrocytes, osteoblasts, and adipocytes. Excessive and dysfunctional visceral adipocytes increase upon menopause and importantly contribute to altered metabolism in postmenopausal women. We previously showed both plasma membrane and nuclear estrogen receptors alpha (ERα) with endogenous estrogen are required to suppress adipogenesis in vivo. Here we determined mechanisms by which these liganded ER pools collaborate to inhibit the peroxisome proliferator-activated gamma (PPARγ) gene and subsequent progenitor differentiation. In 3T3-L1 pre-adipocytes and adipose-derived stem cells (ADSC), membrane ERα signaled through phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) to enhance ERα nuclear localization, importantly at the PPARγ gene promoter. AKT also increased overall abundance and recruitment of co-repressors GATA3, β-catenin, and TCF4 to the PPARγ promoter. Membrane ERα signaling additionally enhanced wingless-integrated (Wnt)1 and 10b expression. The components of the repressor complex were required for estrogen to inhibit rosiglitazone-induced differentiation of ADSC and 3T3-L1 cells to mature adipocytes. These mechanisms whereby ER cellular pools collaborate to inhibit gene expression limit progenitor differentiation to mature adipocytes.

A Lipid-Nanosphere-Small MyoD Activating RNA-Bladder Acellular Matrix Graft Scaffold [NP(saMyoD)/BAMG] Facilitates Rat Injured Bladder Muscle Repair and Regeneration [NP(saMyoD)/BAMG

BackgroundBladder tissue engineering is an excellent alternative to conventional gastrointestinal bladder enlargement in the treatment of various acquired and congenital bladder abnormalities. We constructed a nanosphere-small MyoD activating RNA-bladder acellular matrix graft scaffold NP(saMyoD)/BAMG inoculated with adipose-derived stem cells (ADSC) to explore its effect on smooth muscle regeneration and bladder repair function in a rat augmentation model.MethodsWe performed many biotechniques, such as reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, MTT assay, HE staining, masson staining, and immunohistochemistry in our study. Lipid nanospheres were transfected into rat ADSCs after encapsulate saRNA-MyoD as an introduction vector. Lipid nanospheres encapsulated with saRNA-MyoD were transfected into rat ADSCs. The functional transfected rat ADSCs were called ADSC-NP(saMyoD). Then, Rat models were divided into four groups: sham group, ADSC-BAMG group, ADSC-NP(saMyoD)/BAMG group, and ADSC-NP(saMyoD)/SF(VEGF)/BAMG group. Finally, we compared the bladder function of different models by detecting the bladder histology, bladder capacity, smooth muscle function in each group.ResultsRT-PCR and Western blot results showed that ADSCs transfected with NP(saMyoD) could induce high expression of α-SMA, SM22α, and Desmin. At the same time, MTT analysis showed that NP(saMyoD) did not affect the activity of ADSC cells, suggesting little toxicity. HE staining and immunohistochemistry indicated that the rat bladder repair effect (smooth muscle function, bladder capacities) was better in the ADSC-NP(saMyoD)/BAMG group, ADSC-NP(saMyoD)/SF(VEGF)/BAMG group than in the control group.ConclusionsTaken together, our results demonstrate that the NP(saMyoD)/SF(VEGF)/BAMG scaffold seeded with ADSCs could promote bladder morphological regeneration and improved bladder urinary function. This strategy of ADSC-NP(saMyoD)/SF(VEGF)/BAMG may has a potential to repair bladder defects in the future.

Healing of Full-Thickness Wounds Using Platelet-Rich Fibrin Seeded with Adipose Stem Cells in a Diabetic Mouse Model

Diabetic wounds are refractory to spontaneous healing and treatment regimes due to their detrimental nature. Adipose derived stem cells (ASCs) hold great potential in stem cell-based therapies. However, insufficient cell activity and survival after transplantation dramatically reduce the therapeutic effects. Platelet-rich fibrin (PRF) is a great source of growth factors and has been widely used in clinical practice, but few studies explored its application in diabetic wounds. This study was designed to investigate therapeutic effect of PRF as a delivery scaffold of ASCs on promoting healing of diabetic wound in a mouse model. ASCs isolated from subcutaneous fat were cultured in the three-dimensional PRF scaffold for 3 and 7 days, respectively. H&amp;E staining, laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM) observation revealed that ASCs could survive and proliferate in PRF. Cell numbers in PRF increased dramatically in the duration of 7 days post-seeding. Meanwhile, it is revealed that conditioned medium of PRF enhanced ASCs proliferation and migration in an in vitro wound model. Furthermore, transplantation of PRF alone or PRF loaded with ASCs improved significantly diabetic wound repair, respectively. Capillary density in wounds received either PRF or PRF-ASCs was significantly higher than wounds without intervention. A in vivo survival assay was also conducted to find that PRF could maintain ASCs cells survive in diabetic wounds. Our study indicated that PRF, in particular that loaded with ASCs, can be considered as a promising approach in the treatment of diabetic wounds.

Fabrication of a Microfluidic Device to Study the Interactions between Human Chordoma UCH-1 and Human Adipose-Derived Stem Cells

According to the National Institute of Health, 80 percent of adults suffer low back pain at some point in their lifetime. The loss of intervertebral disc elements, such as the nucleus pulposus, leads to abnormal spine mechanics, inflammatory signaling and compression of neural structures that lead to pain. Recently, undifferentiated stem cells have been used to rescue specialized cells via transfer of mitochondria and growth factors. However, it is not known if this method could be used to aid in the regeneration of human nucleus pulposus. In this work, we used a microfluidic device to co-culture human adipose-derived stem cells (ADSCs) next to human chordoma UCH-1 cells. Human chordoma derived UCH-1 cells have the same genetical and morphological properties as nucleus pulposus cells and serve as a model for in vitro studies. We made a microfluidic device that consisted of two wells separated by a channel, each well allows for culture of a specific cell type. When the channel is opened, it mixes the media from each cell line. The microfluidic device was fabricated using SLA resin 3D printing and polydimethylsiloxane (PDMS). On average we saw about a 50% increase in cell number when UCH-1 cells where cultured with ADSC cells than just UCH-1 cells alone. These results indicate that even at a distance stem cells (ADSC) are able to influence the growth of UCH-1 cells. We observed ADSCs migrate towards the chamber with UCH-1 cells, suggesting that there is a chemotactic signal attracting ADSCs to the chordoma chamber. In the future, we want to check for the growth factors that affect cell proliferation and the maximum distance between cell groups at which effects can be seen.

β-Adrenergic Blocker, Carvedilol, Abolishes Ameliorating Actions of Adipose-Derived Stem Cell Sheets on Cardiac Dysfunction and Remodeling After Myocardial Infarction.

Treatment of myocardial infarction (MI) includes inhibition of the sympathetic nervous system (SNS). Cell-based therapy using adipose-derived stem cells (ASCs) has emerged as a novel therapeutic approach to treat heart failure in MI. The purpose of this study was to determine whether a combination of ASC transplantation and SNS inhibition synergistically improves cardiac functions after MI.Methods and Results:ASCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ASC cells, mRNA levels of angiogenic factors under normoxia or hypoxia, and the effects of norepinephrine and a β-blocker, carvedilol, on the mRNA levels were determined. Hypoxia increased vascular endothelial growth factor (VEGF) mRNA in ASCs. Norepinephrine further increased VEGF mRNA; this effect was unaffected by carvedilol. VEGF promoted VEGF receptor phosphorylation and tube formation of human umbilical vein endothelial cells, which were inhibited by carvedilol. In in vivo studies using a rat MI model, transplanted ASC sheets improved contractile functions of MI hearts; they also facilitated neovascularization and suppressed fibrosis after MI. These beneficial effects of ASC sheets were abolished by carvedilol. The effects of ASC sheets and carvedilol on MI heart functions were confirmed by Langendorff perfusion experiments using isolated hearts. ASC sheets prevented cardiac dysfunctions and remodeling after MI in a rat model via VEGF secretion. Inhibition of VEGF effects by carvedilol abolished their beneficial effects.

Interacting adipose-derived stem cells and microvascular endothelial cells provide a beneficial milieu for soft tissue healing.

There is growing evidence suggesting that healing of chronic soft tissue wounds profits from the presence of adipose-derived stem cells (ADSC). Among the large spectrum of mechanisms by which ADSC might act, especially the interaction with the microvascular endothelial cell, a main player during angiogenesis, is of special interest. In the present 2D model on the basis of endothelial cell ADSC co-cultures, we focused on the identification of characteristics of both cell types in response to a typical condition in acute and chronic wounds: hypoxia. Parameters like proliferation capacity, migration, myofibroblastoid differentiation of ADSC and the quantification of important paracrine factors related to angiogenesis and inflammation were used to correlate our experimental model with the in vivo situation of soft tissue healing. ADSC were not negatively affected by hypoxia in terms of proliferation, referring to their excellent hypoxia tolerance. Myofibroblastoid differentiation among ADSC was enhanced by hypoxia in mono- but not in co-culture. Furthermore, co-cultures were able to migrate under hypoxia. These effects might be caused to some extent by the distinct milieu created by interacting ADSC and endothelial cells, which was characterized by modulated levels of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor. The identification of these cell characteristics in the present 2D in vitro model provide new insights into the process of human soft tissue healing, and underpin a beneficial role of ADSC by regulating inflammation and angiogenesis.

Differentiated adipose-derived stem cell cocultures for bone regeneration in RADA16-I in vitro.

Craniofacial defects can cause morbidness. Adipose-derived stem cells (ADSCs) have shown great promise for osteogeneration and vascularization; therefore cocultures of differentiated ADSCs are explored to increase bone and vessel formation. In this study, ADSCs were induced into osteogenic ADSCs (os-ADSCs) and endothelial ADSCs (endo-ADSCs) cells, which were then cocultured in variable proportions (os-ADSCs/endo-ADSCs = 2:1, 1:1, 1:2). The os-ADSCs in a ratio of 1:1 expressed more ALP, RUNX2 and COL-I, whereas VEGF, vWF and CD31 were upregulated in the endo-ADSCs of this group. Next generation RNA sequencing (RNA-seq) was performed to evaluate the molecular mechanisms of cocultured ADSCs. The os-ADSCs and endo-ADSCs interacted with each other during osteogenic and angiogenic differentiation, especially at the ratio of 1:1, and were regulated by vascular-related genes, cell-mediated genes, bone-related genes and the transforming growth factor β signaling pathway (TGF-β), mitogen-activated protein kinase signaling pathway (MAPK) and wnt signaling pathway (Wnt). Angptl4, apoe, mmp3, bmp6, mmp13 and fgf18 were detected to be up-regulated, and cxcl12 and wnt5a were down-regulated. The results showed that the gene expression levels were consistent with that in RNA-seq. The cells were then seeded into self-assembling peptide RADA16-I scaffolds as cocultures (1:1) and monocultures (ADSCs, os-ADSCs, endo-ADSCs). The results showed that the cells of all groups grew and proliferated well on the scaffolds, and the cocultured group exhibited better osteogeneration and vascularization. In conclusion, cocultured os-ADSCs and endo-ADSCs at the ratio of 1:1 showed strong osteogenic and angiogenic differentiation. There is a great potential for osteogenesis and vascularization by 3D culturing cells in a 1:1 ratio in self-assembling peptide RADA16-I scaffolds, which requires evaluation for bone regeneration in vivo.

2057 L1 expression analysis in adipose-derived stem cells

OBJECTIVES/SPECIFIC AIMS: Long interspersed element-1s (L1s) are autonomous, mobile elements that are able to copy and insert themselves throughout the genome with their own reverse transcriptase and endonuclease. These elements make up 17% of the human genome with over 500,000 copies, though the vast majority of L1s are defective with only a few dozen potentially responsible for L1 activity. Full-length L1s have the potential to contribute to mutagenesis through random insertion and increased genetic instability. Here we set out to study L1 expression at the specific loci level in bone marrow-derived stem cells (bmSCs) and adipose-derived stem cells (ASCs) and compare the levels of expression from ASCs from donor patients who are young and lean, obese, and old. Our hypothesis is that L1-related damage may contribute to mutation and inflammation that alters the function of these stem cells throughout the life of an individual. METHODS/STUDY POPULATION: ASCs and bmSCs were isolated from patient donors. The following samples were collected: ASCs from 3 young (under the age of 59) and lean (BMI&lt;30) patients, ASCs from 3 older patients (over the age of 59), ASCs from 3 patients with BMI&gt;30, and bmSCs from 4 young and lean patients. Cytoplasmic RNA from the cell populations was isolated and sequenced by RNA-Seq from the cell populations. Using our recently developed bioinformatics pipeline, we set out to quantify L1 expression and identify the few culprit L1s at specific loci that are actively transcribing to RNA in the ASC and bmSC samples. RESULTS/ANTICIPATED RESULTS: Here we provide proof of concept with the application of this novel method in characterizing full-length expressed L1s at the specific loci level in ASCs and bmSCs. We identified L1 loci that are commonly expressed in these cell types and observed an increase in L1 expression in the obese and old ASC cells compared with the young, lean ASCs and bmSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: ASCs hold the promise of broad application in the biomedical field including regenerative treatment. There are reports that ASCs cultivated from older and obese donors are less effective in regenerative treatments. By demonstrating an increased expression of the mutagenic L1 element in ASCs from obese and old donors, this study provides further evidence suggesting the preferable use of ASCs from young and lean donors for regenerative therapies. These studies will also help us to understand the potential contribution of L1 expression to loss of stem cell function during the aging process.

The expression of growth factor signaling genes in co-culture IVM

The objective of this study was to determine the expression of growth factor signaling genes in human adiposederived stem cells (ASCs), porcine oocytes, and cumulus during in vitro maturation (IVM). The human ASCs (from 2 young and 2 old donors) were used for the co-culture IVM system. The maturation rate was examined based on polar body extrusion. The expression of the growth factor signaling genes from ASCs, oocytes, and cumulus were measured using qPCR. All data were analyzed using ANOVA followed by Tukey’s test. The expression of the h-IGF1 signaling genes from human ASCs cells showed similar values in all groups and the h-FGF2 expressions were higher in the young donors than the old ones. The p-FGF2, p-FGFR2, and p-TGFβ1 expressions in the oocytes as well as p-IGFR in the cumulus that were co-cultured from the young donors showed higher values than the old and control groups. The apoptotic ratio (p-BAX/p-BCL2) from the oocytes and cumulus in both co-culture groups also showed lower levels than the control (P&lt;0.05). Oocyte maturation rates were significantly increased in all co-cultured groups (Y1 (85.9 ± 2.2%), Y2 (91.2 ± 1.1%), O1 (86.3 ± 1.5%), and O2 (86.5 ± 2.3%)) compared with the control (76.7 ± 1.1%; P&lt;0.05). Although the expression of growth factor signaling genes was varied, young donors’ ASCs might support in vitro maturation beħer than those from old donors.

Platelet-Derived Growth Factor Receptor-Positive Pericytic Cells of White Adipose Tissue from Critical Limb Ischemia Patients Display Mesenchymal Stem Cell-Like Properties.

BackgroundThe pericytes in the blood vessel wall have recently been identified to be important in regulating vascular formation, stabilization, remodeling, and function. We isolated and identified pericyte-like platelet-derived growth factor receptor beta-positive (PDGFRβ+) cells from the stromal vascular fraction (SVF) of adipose tissue from critical limb ischemia (CLI) patients and investigated their potential as a reliable source of stem cells for cell-based therapy.MethodsDe-identified subcutaneous fat tissues were harvested after amputation in CLI patients. Freshly isolated SVF cells and culture-expanded adipose-derived stem cells (ADSCs) were quantified using flow cytometry. A matrigel tube formation assay and multi-lineage differentiation were performed to assess pericytic and mesenchymal stem cell (MSC)-like characteristics of PDGFRβ+ ADSCs.ResultsPDGFRβ+ cells were located in the pericytic area of various sizes of blood vessels and coexpressed mesenchymal stem cell markers. PDGFRβ+ cells in freshly isolated SVF cells expressed a higher level of stem cell markers (CD34 and CXCR4) and mesenchymal markers (CD13, CD44, CD54, and CD90) than PDGFRβ– cells. In vitro expansion of PDGFRβ+ cells resulted in enrichment of the perivascular mesenchymal stem-like (PDGFRβ+/CD90+/CD45–/CD31–) cell fractions. The Matrigel tube formation assay revealed that PDGFRβ+ cells were located in the peritubular area.ConclusionsPDGFRβ+ ADSCs cells demonstrated a good multilineage differentiation potential. Pericyte-like PDGFRβ+ cells from the SVF of adipose tissue from CLI patients had MSC-like characteristics and could be amplified by in vitro culture with preservation of their cell characteristics. We believe PDGFRβ+ cells in the SVF of adipose tissue can be used as a reliable source of stem cells even in CLI patients.

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

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. 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. 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.