Growth Of Adipose-derived Stem Cells Research Articles

Autophagy Modulates Cell Mineralization on Fluorapatite-Modified Scaffolds

As a major intracellular degradation and recycling machinery, autophagy plays an important role in maintaining cellular homeostasis and remodeling during normal development. Our previous study showed that fluorapatite (FA) crystal-coated electrospun polycaprolactone (PCL) was capable of inducing differentiation and mineralization of human dental pulp stem cells. However, how autophagy changes and whether autophagy plays a vital role during these processes is still unknown. In this study, we seeded STEMPRO human adipose-derived stem cells (ASCs) on both PCL+FA and PCL scaffolds to investigate the osteogenic inductive ability of FA crystals and we observed the autophagy changes of these cells. Scanning electron microscopy and fluorescence microscopy images, along with DNA quantitation, showed that both PCL+FA and PCL scaffolds could sustain ASC growth but only the PCL+FA scaffold could sustain cell mineralization. This was confirmed by alkaline phosphatase activity and Alizarin red and Von Kossa staining results. The autophagy RT2 Profiler polymerase chain reaction array analysis showed many autophagy-related genes changes during ASC differentiation. Western blot analysis indicated that several autophagy-related proteins fluctuated during the procedure. Among them, the microtubule-associated protein 1 light chain 3 (LC3)-II protein changes of the ASCs grown on the 2- or 3-dimensional environments at 6 h, 12 h, 1 d, 3 d, 7 d, 14 d, and 21 d reached a peak value at day 7 during osteogenesis. At earlier stages (from day 0 to day 3), the addition of autophagy inhibitors (3-mathyladenine, bafilomycin A1, and NH4Cl) attenuated the expression of osteogenic related markers (osteopontin, alkaline phosphatase activity, Alizarin red, and Von Kossa) compared with the control group. All data indicated that autophagy played an important role in ASC differentiation on the PCL+FA scaffold. Inhibition of autophagy before day 3 strongly inhibited osteogenic differentiation and mineralization of ASCs in the 3-dimensional model. This observation further elucidates the mechanism of autophagy in mesenchymal stem cell osteogenic differentiation.

Exendin-4 promotes proliferation of adipose-derived stem cells through ERK and JNK signaling pathways.

Adipose-derived stem cell (ADSC) transplantation has emerged as a potential tool for the treatment of cardiovascular disease. However, with a limited renewal capacity and the need for mass cells during the engraftment, strategies are needed to enhance ADSC proliferative capacity. In this study, we explored the effects of exendin-4 (Ex-4), a glucagon-like peptide-1 analog, on the growth of ADSCs, focusing in particular on c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. Firstly, ADSCs were isolated and cultured in vitro. Then, flow cytometry demonstrated that ADSCs were positive for CD90 and CD29 but negative for CD31, CD34, and CD45. Ex-4 (0-50nM) treatment increased ADSC proliferation in a dose-dependent manner but had no effects on stem cell markers of ADSCs. Moreover, we found that Ex-4 treatment elevated the phosphorylation levels of the JNK and ERK signaling pathways. Furthermore, utilization of Ex-4 also promoted cyclin D1 and cyclin E protein expression, which was accompanied by more Edu(+) cells and a higher percentage of cells in the S-phase of the cell cycle after Ex-4 treatment. In parallel, the application of inhibitors SP600125 and PD98059, inhibitors of the JNK and ERK signaling pathways, respectively, not only reversed such effects of Ex-4 on JNK and ERK but also resulted in lower percentages of S-phase cells and fewer numbers of Edu(+) cells. In summary, Ex-4 has no effects on stem cell markers in ADSCs but promotes ADSC growth via JNK and ERK signaling pathways.

Induction of adipose-derived stem cells on injectable nanofibrous spongy microspheres for vascularized adipose tissue regeneration in vivo

Event Abstract Back to Event Induction of adipose-derived stem cells on injectable nanofibrous spongy microspheres for vascularized adipose tissue regeneration in vivo Yasmine Doleyres1, Xiaobing Jin2 and Peter X. Ma1, 2, 3, 4 1 University of Michigan, Macromolecular Science and Engineering Program, United States 2 University of Michigan, Department of Biologic and Materials Sciences, United States 3 University of Michigan, Department of Biomedical Engineering, United States 4 University of Michigan, Department of Materials Science and Engineering, United States Soft tissue defects in the craniofacial region or the breast region often occur due to congenital defects, traumatic injury, or cancer occurring in the underlying tissue[1]. Correcting these defects in the case of facial reconstruction can be done by transplanting adipose (fat) tissue from another area of the body into the facial region or in the case of reconstruction following breast cancer can be done through autologous tissue transplantation, if available, or using silicone or saline-filled implants. The problem with these options is that between 20-90% of transplanted fat tissue often resorbs within a couple months after being transplanted due to lack of vascularization and implants often fail because of capsular constriction and become infected, leading to multiple surgeries in both cases[1]-[8]. The most ideal option would be to regenerate one’s own adipose tissue just as it is naturally produced by the body. Lasting tissue regeneration options for in vivo adipose tissue have yet to be developed, and this is the goal of this project. Novel nanofibrous spongy (porous) microspheres (NF-SMS) have been developed and appear to be a suitable scaffolding or cell-carrying material for the differentiation and growth of adipose-derived stem cells (ASCs). These microspheres made of poly-L-lactic acid (PLLA) have a nanofibrous architecture that well-mimics the cells’ natural extracellular matrix and also allows for cells to pack the interior of the microspheres[9]. A high number of cells are able to pack into the interior of the microspheres and the high cell attachment on the microspheres is critical for adipose tissue regeneration as high cell-cell contact is necessary for tissue growth. Adipogenesis of the isolated ASCs is induced for 3-4 days in growth medium containing Dexamethasone, Indomethacin, and 3-isobuytl-1-methylxanthine (IBMX)[10],[11]. The cells are then combined with the microspheres for a few hours and subsequently injected subcutaneously in a nude mouse model for testing. Early in vitro experiments comparing the growth and differentiation of the cells on the NF-SMS against enclosed nanofibrous hollow microspheres as a control show increased cell attachment and integration on the NF-SMS. The cells on the NF-SMS also showed greater Nile Red lipid staining and overall positive results in vitro. Current in vivo experiments are being done and are incorporating poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with insulin for sustained released of insulin from these particles during the early period of growth (1-2 weeks). Insulin appears to be very critical in the adipogenesis pathway and should be crucial in helping to produce substantial tissue in vivo[12]-[14]. These PLGA nanoparticles are injected along with the microsphere-cell complexes. Having a patient regenerate adipose tissue from their own pre-adipocyte cells will be advantageous because it eliminates the need for something foreign in the body and multiple invasive surgeries. Ensuring that the adipose tissue produced does not resorb as is the case in previous research attempts would be done by functionalizing the microsphere scaffolds with an angiogenic factor, which is the next goal of this project.

Histone H3K9 acetylation influences growth characteristics of goat adipose-derived stem cells in vitro.

Adipose-derived stem cells (ADSCs) show nearly unlimited potential in medical and animal science. Currently, understanding of the biological mechanisms regulating ADSC growth in vitro remains very limited. Histone acetylation, an epigenetic modification, plays a key role in maintaining stem cell properties. To further study its effect on ADSC growth characteristics in vitro, we treated goat ADSCs with the histone deacetylase inhibitors trichostatin A (TSA) and vorinostat (SAHA). This inhibited SIRT1 expression and increased histone H3K9 acetylation, leading to decreased cell viability, cell cycle arrest, and apoptosis. Quantitative real-time polymerase chain reaction revealed that H3K9 hyperacetylation stimulated transcription of NANOG, OCT4, SOX2, and TERT, but inhibited that of PCNA, P53, and BAX. Western blotting indicated that TSA and SAHA increased protein expression of NANOG, reduced that of SOX2, TERT, PCNA, P53, and BAX, and did not change that of OCT4. These findings provide new experimental evidence contributing to our understanding of the mechanisms underlying ADSC growth characteristics in vitro.

Development of an implantable synthetic membrane for the treatment of preterm premature rupture of fetal membranes.

Preterm premature rupture of fetal membranes is a very common condition leading to premature labour of a non viable fetus. Significant morbidities may occur when preterm premature rupture of fetal membranes management is attempted to prolong the pregnancy for fetal maturation. Reducing the rate of loss of amniotic fluid and providing a barrier to bacterial entry may allow the pregnancy to continue to term, avoiding complications. Our aim is to develop a synthetic biocompatible membrane to form a distensible barrier for cervical closure which acts to reduce fluid loss and provide a surface for epithelial ingrowth to help repair the damaged membranes. Therefore, a bilayer membrane was developed using an electrospinning technique of combining two FDA-approved polymers, poly-L-lactic acid (PLA) and polyurethane (Z3) polymer. This was compared to a plain electrospun Z3 membrane. The physical and mechanical properties were assessed using scanning electron microscope images and a BOSE tensiometer, respectively, and compared to native fetal membranes. The performance of the membranes in preventing fluid loss was assessed by measuring their ability to support a column of water. Finally the ability of the membranes to support cell ingrowth was assessed by culturing adipose-derived stem cells on the membranes for two weeks and assessing metabolic activity after 7 and 14 days. The physical properties of the bilayer were similar to that of the native fetal membranes and it was resistant to fluid penetration. This bilayer membrane presented mechanical properties close to those for fetal membranes and showed elastic distention, which may be crucial for progress of the pregnancy. The membrane was also able to retain surgical sutures. In addition, it also supported the attachment and growth of adipose-derived stem cells for two weeks. In conclusion, this membrane may prove a useful approach in the treatment of preterm premature rupture of fetal membranes and now merits further investigation.

New therapy of skin repair combining adipose-derived mesenchymal stem cells with sodium carboxymethylcellulose scaffold in a pre-clinical rat model.

Lesions with great loss of skin and extensive burns are usually treated with heterologous skin grafts, which may lead rejection. Cell therapy with mesenchymal stem cells is arising as a new proposal to accelerate the healing process. We tested a new therapy consisting of sodium carboxymethylcellulose (CMC) as a biomaterial, in combination with adipose-derived stem cells (ADSCs), to treat skin lesions in an in vivo rat model. This biomaterial did not affect membrane viability and induced a small and transient genotoxicity, only at the highest concentration tested (40 mg/mL). In a rat wound model, CMC at 10 mg/mL associated with ADSCs increased the rate of cell proliferation of the granulation tissue and epithelium thickness when compared to untreated lesions (Sham), but did not increase collagen fibers nor alter the overall speed of wound closure. Taken together, the results show that the CMC is capable to allow the growth of ADSCs and is safe for this biological application up to the concentration of 20 mg/mL. These findings suggest that CMC is a promising biomaterial to be used in cell therapy.

Expansion of Semi-Automatic Processed Human Adipose-Derived Stem Cells in Medium Supplemented with Autologous Serum and Antioxidants

Objective: Excised fat tissue (EFT) is an excellent source of adipose-derived stem cells (ADSCs) for autologous cell therapy. However, the current technique for excising fat tissues with conventional surgical scissors and using culture medium containing fetal bovine serum (FBS) may not comply with Good Tissue Practice (GTP) guidelines. We thus developed a semi-automated process complying with the abovementioned guideline to manufacture clinicalgrade ADSCs from small volumes of EFT. Methods: A semi-automated process was developed by using a disposable disperser tube operated on a Tube Disperser Workstation to obtain stromal vascular fraction (SVF) from EFT. A culture medium supplemented with human autologous serum (HAS) and mesenchymal stem cell culture adjuvant (MCA) consisted of basic fibroblast growth factor (bFGF/FGF-2) and antioxidants, was formulated to expand ADSCs from SVF in vitro. Results: The semi-automated process can markedly enhance cell yields and reduce the operation time for mincing tissues from an hour down to a few minutes. The growth of ADSCs was slower in the 10% HAS medium than that in medium supplemented with FBS, but their growth in the 10% HAS medium containing MCA (10% HAS+MCA) was superior to both media. The 10% HAS+MCA medium also promoted adipogenesis, osteogenesis, chondrogenesis and increased the expression of CD44, CD73 and the stemness genes in the ADSCs. Conclusion: Our novel semi-automated process provides an efficient isolation of SVF, and the 10% HAS+MCA medium accelerates the growth of ADSCs from small amounts of EFT without losing their differentiation ability.

Secretory Factors From Rat Adipose Tissue Explants Promote Adipogenesis and Angiogenesis

Adipogenic differentiation of adipose-derived stem cells (ASCs) is known to be affected by many promoting and inhibiting factors, and correlated with surrounding cells and extracellular matrix. However, few studies have evaluated the effect of secreted biological molecules from adipose tissue explants on the growth of ASCs. In this study, ASCs were isolated and the secretory factors from adipose tissue explants (SFAE) were prepared from adipose tissue explants. The multilineage differentiation potential of ASCs was determined using different inductive media. The influence of SFAE on the colony formation and proliferation of ASCs was determined using colony-forming efficiency assay and Cell Counting Kit-8 assay, respectively. Real-time polymerase chain reaction and Western blot were performed to analyze the beneficial effect of SFAE on the adipogenesis and angiogenesis of ASCs. Utilizing gelatin scaffold, the influence of SFAE on ASCs was investigated in vivo. Subsequently, cell/gelatin constructs were cultured in a subcutaneous pocket on a rat dorsum for 2 and 9 weeks, and the resultant samples were histologically evaluated. Results showed that ASCs derived from adipose explants can differentiate along multiple lineages in vitro. Utilizing SFAE, the proliferation and colony-forming efficiency of ASCs were inhibited, while the expression of adipogenesis markers such as C/EBPβ, PPARγ2, and LPL, as well as angiogenesis factor VEGF-A were promoted. Moreover, the beneficial effect of SFAE on adipogenesis was revealed in vivo. In conclusion, our results suggested that SFAE has beneficial influence on adipogenesis and angiogenesis both in vitro and in vivo.

Novel macro-microporous gelatin scaffold fabricated by particulate leaching for soft tissue reconstruction with adipose-derived stem cells

The restoration of body contours as shaped by adipose tissue remains a clinical challenge specifically in patients who have experienced loss of contour due to trauma, surgical removal of tumours or congenital abnormalities. We have developed a novel macro-microporous biomaterial for use in soft tissue re-bulking and augmentation. Alginate beads provided the pore template for the construct. Incorporation, and subsequent dissolution, of the beads within a 7 % (w/v) gelatin matrix, produced a highly porous scaffold with an average pore size of 2.01 ± 0.08 mm. The ability of this scaffold to support the in vitro growth and differentiation of human adipose-derived stem cells (ADSCs) was then investigated. Histological analysis confirmed that the scaffold itself provided a suitable environment to support the growth of ADSCs on the scaffold walls. When delivered into the macropores in a fibrin hydrogel, ADSCs proliferated and filled the pores. In addition, ADSCs could readily be differentiated along the adipogenic lineage. These results therefore describe a novel scaffold that can support the proliferation and delivery of ADSCs. The scaffold is the first stage in developing a clinical alternative to current treatment methods for soft tissue reconstruction.

Harvest site influences the growth properties of adipose derived stem cells.

The therapeutic potential of adult stem cells may become a relevant option in clinical care in the future. In hand and plastic surgery, cell therapy might be used to enhance nerve regeneration and help surgeons and clinicians to repair debilitating nerve injuries. Adipose-derived stem cells (ASCs) are found in abundant quantities and can be harvested with a low morbidity. In order to define the optimal fat harvest location and detect any potential differences in ASC proliferation properties, we compared biopsies from different anatomical sites (inguinal, flank, pericardiac, omentum, neck) in Sprague-Dawley rats. ASCs were expanded from each biopsy and a proliferation assay using different mitogenic factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was performed. Our results show that when compared with the pericardiac region, cells isolated from the inguinal, flank, omental and neck regions grow significantly better in growth medium alone. bFGF significantly enhanced the growth rate of ASCs isolated from all regions except the omentum. PDGF had minimal effect on ASC proliferation rate but increases the growth of ASCs from the neck region. Analysis of all the data suggests that ASCs from the neck region may be the ideal stem cell sources for tissue engineering approaches for the regeneration of nervous tissue.

The Impact of Long-Term In Vitro Expansion on the Senescence-Associated Markers of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P < 0.05). The data showed that growth of ASCs reduced in long-term culture and the ß-galactosidase activity was significantly increased at later passage (P20). The morphology of ASCs in long-term culture showed the manifestation of senescent feature at P15 and P20. Significant alteration in the senescence-associated genes expression levels was observed in MMP1, p21, Rb and Cyclin D1 at P15 and P20. Significant increase in CD45 and HLA DR DQ DP surface marker was observed at P20. While cell cycle analysis showed significant decrease in percentage of ASCs at S and G2/M phase at later passage (P15). Our data showed ASCs cultured beyond P10 favours the senescence pathway and its clinical usage in cell-based therapy may be limited.

Construction of scaffold with human extracellular matrix from adipose tissue

To investigate the feasibility of constructing scaffold for tissue engineering with human extracellular matrix from adipose tissue. Fresh human adipose tissue was obtained by liposuction in 7 women who undergone liposuction. One part of the fat was used to isolate the adipose-derived stem cells (ADSCs), the other part was used to extract human extracellular matrix powder. After removing blood and oil components, the tissue was homogenized, centrifuged, freeze-dried, and crushed to powder by instrument. The structure of human ECM powder was observed with electron microscopy. The ADSCs were seeded and attached to the human extracellular matrix powder before and after labeled with fluorescent DiI, respectively. The adhesion rate was detected. The adhesion and growth of ADSCs were observed with Fluorescence microscope. The adhesion rate before and after DiI labeling was analyzed statistically with two-sample test of SPSS 13.0. The ADSCs and human extracellular matrix powder were obtained successfully from adipose tissue. The ADSCs could be differentiated into adipose cells, bone cells and chondrocytes. SEM images showed that the power had both rugged and smooth surface with a porous structure characteristics. ADSCs could adhere to the scaffold easily, and the adhesion rate was (88.81 +/- 4.81)% and (86.48 +/- 4.58)% before and after DiI labeling. There was no difference between two groups. DiI labeled ADSCs were adhered to extracellular matrix scaffold and could grow in good condition. Human adipose tissue extracellular matrix powder was easy to obtain, with diversity in size and shape which provided excellent substrates for cell adhesion and growth. It could be an ideal adipose tissue engineering scaffold.

Low Doses of Bone Morphogenetic Protein 4 Increase the Survival of Human Adipose-Derived Stem Cells Maintaining Their Stemness and Multipotency

Mesenchymal stem cells (MSCs) have emerged as important tools for cell therapy; therefore, identification of factors capable of governing their ex vivo expansion become essential. In this study we demonstrate that human adipose-derived stem cells (ASCs) express all components of the bone morphogenetic protein (BMP)/BMP receptor signaling pathway and respond to BMP4 inducing upregulated expression of its specific target genes Id1-Id4. Moreover, ASCs grown in a medium reduced in serum produce endogenous BMP4 that could affect autocrinely ASC growth. On the contrary, dorsomorphin, an inhibitor of BMP signaling pathway, decreases cell numbers yielded from ASC cultures in correlation with increased apoptosis and decreased cycling cells. Therefore, BMP4 emerges as a possible factor for ex vivo expanding human ASCs. Our results demonstrate that, as other morphogens, BMP4 effects on human MSCs are dose dependent. High doses significantly increased apoptosis and drastically reduced cell proliferation, whereas low doses of BMP4 (0.01-0.1 ng/mL) significantly increase culture cell content, reduce the number of apoptotic cells, and increase that of cycling cells. Further, treatment of human ASCs with low doses of BMP4 does not modify expression of Nanog and Oct4, two transcription factors involved in self-renewal and pluripotency of stem cells or avoid their osteogenic or osteoblastic differentiation capacities when cultured in adequate inducing media, as shown by the induction of specific gene expression (CEBP, PPARγ, and RUNX2). Our results therefore support BMP4 as a promising factor for expanding human adipose tissue-derived MSCs maintaining their properties of stemness and multipotency.

Schwann cells induced neuronal differentiation of adipose-derived stem cells (ADSCs)

Objective To investigate the function of SD rats Schwann cells on the growth and differentiation of adipose-derived stem cells(ADSCs) when they are co-cultured in Transwell culture system. Methods The ADSCs harvested from SD rats were divided into 3 groups. Group A: ADSCs and Schwann cells were cultured in Transwell indirect co-culture system. Group B: ADSCs were induced by β-BME and BHA. Group C: a contrast group. The morphology of ADSCs was observed and immunohistology was pedormed. Results ADSCs of group A and group B were partly differentiated to the cells which had long neurite,and had a positive result in the NF staining, but negative in the GFAP staining. Group A shows no great difference in neurite length compared with group B(P < 0.05), and no great difference in cell quantity compared with group C (P < 0.05). Conclusion Schwann cells of SD rat have a growth supportive and neuronal differentiation inducing effect on ADSCs. Key words: Schwann cell; Adipose-derived stem cells; Neural differentiation

Effect of growth media and serum replacements on the proliferation and differentiation of adipose-derived stem cells

Studies of mesenchymal stromal cells (MSC) from BM and adipose tissue have demonstrated similar differentiation potentials along the adipo-, osteo- and chondrogenic lineages. While most clinical trials have been performed using BM-derived MSC, the focus is shifting toward the use of stem cells derived from fat tissue. The aim of the current investigation was to define optimal culture conditions that would facilitate clinical use of adipose-derived stem cells (ASC). Different types and concentrations of serum replacers and basal media were tested with respect to the optimal expansion and subsequent differentiation of primary human ASC. The effect of initial seeding density on the growth of ASC was also determined. While several of the serum replacements proved to be clearly inferior to fetal calf serum (FCS) in promoting ASC growth, the knockout serum replacement (KOSR) had expansion properties similar to those of FCS. However, with respect to the capacity to support adipo-, osteo- and chondrogenic differentiation, KOSR proved to be less consistent than FCS. Among the media formulations, modified Eagle medium alpha supported a significantly faster cell expansion than the other basal media while still maintaining the full differentiation potential of ASC. Regarding the plating density most favorable for rapid expansion, we found that initial plating densities ranging from 100 to 200 cell/cm(2) resulted in significantly shorter doubling times than plating densities both below and above that range. Identification of the optimal basal medium and serum replacer, together with the most favorable plating density, will facilitate cell-based and tissue-engineering applications employing ASC in pre-clinical and clinical settings.

200 TRANSPLANTATION AND IN VIVO DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN SWINE

A primary concern in stem cell biology is that observations made in vitro may be an artifact of the in vitro culture environment. In vitro derived stem cells can be implanted into the environment from which they are derived so that their response to physiological conditions may be observed. Several important cellular characteristics need to be examined following the cell's reintroduction to the in vivo environment, including the potential for differentiation, proliferative ability, and life span. Studying implanted stem cells will assist in determining the potential for stem cell use in clinical therapies and provide further understanding of the role adult stem cells have in the adult body. Currently, the scientific literature is lacking a detailed description of the cellular response of adipose-derived stem cells (ADSCs) reintroduced to their exact tissue of origin. Thus, the aim of this study was to evaluate porcine ADSC growth in vivo and to analyze cell differentiation in vivo following injection of undifferentiated ADSCs into subcutaneous fat. Subcutaneous adipose tissue was isolated from the back fat of male pigs (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 ADSCs were labeled with fluorescent dye (PKH26; Sigma, St. Louis, MO, USA) and sorted by flow cytometry. After sorting, positive cells were washed and re-suspended in culture medium. For transplantation, 100 �L of cell suspension in DMEM containing one of four cell concentrations (0 (control); 30 000; 300 000; and 900 000 cells) were placed in a 1-mL syringe and injected into the subcutaneous back fat of recipient pigs (n = 2). Each pig had previously been tattooed with 12 13 � 13 squares to mark injection sites. The treatments were replicated three times within each animal. Two and three weeks after transplantation, animals were euthanized, the back fat containing the transplantation site was harvested, and the cells were disaggregated as described above. The buoyant adipocytes and pelleted ADSCs cells were then analyzed by flow cytometry. The results indicated that there were dose- and time-dependent increases in labeled ADSCs and labeled adipocytes in the fat samples with increasing cell number (from 0 to 300 000 cells). There was, however, a decrease in labeled ADSCs at the 900 000-cell dose, which is likely due to excess cells being transplanted or an immune reaction. Both of these aspects are currently being evaluated. In conclusion, undifferentiated ADSCs from swine can be isolated from and returned to the subcutaneous adipose layer and differentiate into mature adipocytes. This work was supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois.