Adult Adipose Tissue-derived Stem Research Articles

Comparison between USPIOs and SPIOs for Multimodal Imaging of Extracellular Vesicles Extracted from Adipose Tissue-Derived Adult Stem Cells.

Adipose tissue-derived adult stem (ADAS) cells and extracellular vesicle (EV) therapy offer promising avenues for treating neurodegenerative diseases due to their accessibility and potential for autologous cell transplantation. However, the clinical application of ADAS cells or EVs is limited by the challenge of precisely identifying them in specific regions of interest. This study compares two superparamagnetic iron oxide nanoparticles, differing mainly in size, to determine their efficacy for allowing non-invasive ADAS tracking via MRI/MPI and indirect labeling of EVs. We compared a USPIO (about 5 nm) with an SPIO (Resovist®, about 70 nm). A physicochemical characterization of nanoparticles was conducted using DLS, TEM, MRI, and MPI. ADAS cells were labeled with the two nanoparticles, and their viability was assessed via MTT assay. MRI detected labeled cells, while TEM and Prussian Blue staining were employed to confirm cell uptake. The results revealed that Resovist® exhibited higher transversal relaxivity value than USPIO and, consequently, allows for detection with higher sensitivity by MRI. A 200 µgFe/mL concentration was identified as optimal for ADAS labeling. MPI detected only Resovist®. The findings suggest that Resovist® may offer enhanced detection of ADAS cells and EVs, making it suitable for multimodal imaging. Preliminary results obtained by extracting EVs from ADAS cells labeled with Resovist® indicate that EVs retain the nanoparticles, paving the way to an efficient and multimodal detection of EVs.

Long-Term Effects of Adipose-Derived Stem Cells for the Treatment of Bilateral Limbal Stem Cell Deficiency

Purpose To determine the safety and feasibility of human autologous adipose tissue-derived adult mesenchymal stem cells (ASCs) for ocular surface regeneration in patients with bilateral limbal stem-cell deficiency (LSCD). Methods A phase IIa clinical trial was designed (https://Clinicaltrials.gov, NCT01808378) with 8 patients, 3 of whom had aniridia, 2 meibomian glands diseases, 2 multiple surgeries and 1 chronic chemical injury. The therapeutic protocol was as follows: 6-mm of central corneal epithelium was removed, 400,000 ASCs were injected into each limboconjunctival quadrant, 400,000 ASCs were suspended over the cornea for 20 min, and finally the cornea was covered with an amniotic membrane patch. Results No adverse events were detected after a mean of 86,5 months of follow-up. One year after surgery, 6 of the 8 transplants were scored as successful, five patients had improved uncorrected visual acuity (mean of 12 letters), two patients presented epithelial defects (also present at baseline) and the mean percentage of corneal neovascularization was of 28.75% (36.98%, at baseline). Re-examination 24 months after treatment disclosed preserved efficacy in 4 patients. At the last visit (after a mean of 86,5 months of follow up) epithelial defects were absent in all patients although improvement in all of the variables was only maintained in patient 3 (meibomian glands agenesia). Conclusion ASCs are a feasible and conservative therapy for treating bilateral LSCD. The therapeutic effect differs between etiologies and diminishes over time.

Improvement of an Effective Protocol for Directed Differentiation of Human Adipose Tissue-Derived Adult Mesenchymal Stem Cells to Corneal Endothelial Cells.

Corneal disease affects 12.5 million individuals worldwide, with 2 million new cases each year. The standard treatment consists of a corneal transplantation from a human donor; however, the worldwide demand significantly exceeds the available supply. Lamellar endothelial keratoplasty, the replacement of only the endothelial layer of the cornea, can partially solve the problem. Progressive efforts have succeeded in expanding hCECs; however, the ability to expand hCECs is still limited, and new sources of CECs are being sought. Crucial advances have been achieved by the directed differentiation of embryonic or induced pluripotent stem cells, but these cells have disadvantages, such as the use of oncogenes, and are still difficult to establish. We aimed to transfer such knowledge to obtain hCECs from adipose tissue-derived adult mesenchymal stem cells (ADSC) by modifying four previously published procedures. We present several protocols capable of the directed differentiation of human ADSCs to hCECs. In our hands, the protocol by Ali et al. was the best adapted to such differentiation in terms of efficiency, time, and financial cost; however, the protocol by Wagoner et al. was the best for CEC marker expression. Our results broaden the type of cells of autologous extraocular origin that could be employed in the clinical setting for corneal endothelial deficiency.

Comparison of three sperm selection methods for ICSI-DGC, Cumulus column, and incubation with supernatant product of adipose tissue-derived adult stem cells: An experimental study.

BackgroundThe examination of sperm parameters and sperm DNA integrity are necessary for male fertility expression. These parameters can be affected by method of sperm separation.ObjectiveTo measure the damage of each sperm separation method on the sperm parameters and sperm DNA integrity.Materials and MethodsIn this experimental study, semen samples of 20 infertile men with asthenoteratozoospermia (Infertility Research Center, Qom, Iran, 2017) were processed in three ways: density gradient centrifugation (DGC), cumulus column, and incubation with supernatant products of adipose tissue-derived adult stem cells (SPAS). The results of sperm parameters and DNA fragmentation before and after the process were statistically analyzed.ResultsThe number of separated sperms by normal morphologies during the SPAS and the cumulus column was significantly more than the corresponding population in the DGC group. In addition, although all three methods have the same ability to increase total sperm motility and the number of recovered sperms, in the field of forwarding movement and DNA fragmentation, the SPAS method performed more efficiently (p = 0.021).ConclusionSperm parameters and DNA fragmentation in the SPAS group were better than those in the DGC and cumulus column groups. Furthermore, it has been shown that the sperm capacity was increased with the SPAS method. However, the rearrangement of sperm chromatin by reducing the disulfide bridges and providing the possibility of re-histone over capacity causes a significant reduction in DNA fragmentation.

Mouse Adipose Tissue-Derived Adult Stem Cells Expressed Osteogenic Specific Transcripts of Osteocalcin and Parathyroid Hormone Receptor During Osteogenesis

IntroductionAdult mesenchymal stem cells (MSCs) have potential to differentiate into various lineages, replacing cells during normal turnover and tissue regeneration to replace damaged or lost adult tissues during osteoporosis and arthritis, or traumatic injuries. We investigated the osteogenic signature in mouse adipose tissue (AD)- and bone marrow (BM)-derived MSCs. Materials and MethodsMSCs from adipose tissue and bone marrow were compared for osteogenic endogenous mRNA markers by reverse-transcription polymerase chain reaction (RT-PCR). Cellular proliferation and immunophenotype analyzed by flow cytometry revealed that mouse AD-MSCs and BM-MSCs shared similar characteristics. ResultIsolated AD-MSC and BM-MSC showed high proliferation rates and fibroblast morphology. Flow cytometry revealed positive markers for mesenchyme, but negative for primitive hematopoietic and endothelial cells. At day 21, Alizarin red S and Von-kossa staining of differentiated cells showed high calcium deposits compared with undifferentiated cells. After 21 days of osteogenic differentiation, AD-MSCs expressed osteocalcin and parathyroid hormone (PTH) compared with undifferentiated cells. Osteogenic-specific transcript of osteocalcin (OC), bone gamma carboxyglutamate protein, and PTH receptor (PTHr) were detected only in differentiated not undifferentiated cells. Undifferentiated BM-MSCs, expressed all markers at low intensity, which amplified during differentiation. ConclusionOur findings suggest that the OC and PTHr can be used as differentiation markers for osteogenesis of mouse AD-MSC.

In Vivo Tracking of Murine Adipose Tissue-Derived Multipotent Adult Stem Cells and Ex Vivo Cross-Validation

Stem cells are characterized by the ability to renew themselves and to differentiate into specialized cell types, while stem cell therapy is believed to treat a number of different human diseases through either cell regeneration or paracrine effects. Herein, an in vivo and ex vivo near infrared time domain (NIR TD) optical imaging study was undertaken to evaluate the migratory ability of murine adipose tissue-derived multipotent adult stem cells [mAT-MASC] after intramuscular injection in mice. In vivo NIR TD optical imaging data analysis showed a migration of DiD-labelled mAT-MASC in the leg opposite the injection site, which was confirmed by a fibered confocal microendoscopy system. Ex vivo NIR TD optical imaging results showed a systemic distribution of labelled cells. Considering a potential microenvironmental contamination, a cross-validation study by multimodality approaches was followed: mAT-MASC were isolated from male mice expressing constitutively eGFP, which was detectable using techniques of immunofluorescence and qPCR. Y-chromosome positive cells, injected into wild-type female recipients, were detected by FISH. Cross-validation confirmed the data obtained by in vivo/ex vivo TD optical imaging analysis. In summary, our data demonstrates the usefulness of NIR TD optical imaging in tracking delivered cells, giving insights into the migratory properties of the injected cells.

Adipose tissue engineering with human adipose tissue‐derived adult stem cells and a novel porous scaffold

We investigated the effect of a novel porous scaffold composed with water-soluble poly(L-glutamic acid) (PLGA) and chitosan (CS) on the attachment, proliferation, and adipogenic differentiation of human adipose tissue-derived adult stem cells (ADSCs) in vitro and in vivo. Scanning electron microscope and fluorescent Dil labeling were used to reveal the attachment and growth of ADSCs on scaffolds; cell proliferation was detected by DNA assay. The adipogenic differentiation potential of ADSCs on the scaffolds was assayed by Oil-red O staining and further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) for adipogenic gene markers (peroxisome proliferator-activated receptor γ2, lipoprotein lipase, fatty acid-binding protein, adiponectin). Cell-seeded constructs exposed to adipogenic medium for 2 weeks in vitro were implanted in severe combined immunodeficient (SCID) mice for 6 weeks. It was shown that ADSCs attached and spread well on scaffolds with good proliferation behaviors and abundance of extracellular matrix deposition. Oil-red O staining and RT-PCR showed adipogenic differentiation potential of ADSCs on scaffolds. Newly formed adipose-like tissue was confirmed in vivo in SCID mice by Oil-red O staining. PLGA/CS porous scaffolds exhibit good compatibility to ADSCs and can be promising biomaterials for adipose tissue engineering.

Evaluation of Polyvinylpyrrolidone as a Cryoprotectant for Adipose Tissue-Derived Adult Stem Cells

The objective of this study was to test the hypothesis that human adipose tissue-derived adult stem cells (ASCs) can be effectively cryopreserved and stored in liquid nitrogen using a freezing medium containing a high-molecular-weight polymer, polyvinylpyrrolidone (PVP), as the cryoprotective agent (CPA) instead of dimethylsulfoxide (DMSO). To this end we investigated the postfreeze/thaw viability and apoptotic behavior of passage 1 ASCs cryopreserved in 15 different media: (i) the traditional media containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% DMSO; (ii) DMEM with 80% human serum (HS) and 10% DMSO; (iii) DMEM with various concentrations (1%, 5%, 10%, 20%, and 40%) of PVP as the sole CPA; (iv) DMEM with PVP (5%, 10%, and 20%) and HS (10%); (v) DMEM with PVP (5%, 10%, and 20%) and FCS (10%); and (vi) DMEM with PVP (10%) and FCS (40% and 80%). Approximately 1 mL (10(6) cells/mL) of passage 1 ASCs were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees C water bath (1-2 min of agitation), resuspended in culture media, and seeded in separate wells of a six-well plate for a 24-h incubation period at 37 degrees C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 10% PVP and DMEM was comparable with that obtained in freezing media with DMSO and serum (HS or FCS), that is, approximately 70% + or - 8% and approximately 83% + or - 8%, respectively. Slightly enhanced cell viability was observed with the addition of serum (either HS or FCS) to the freezing media containing PVP as the CPA. Adipogenic and osteogenic differentiation behaviors of the frozen thawed cells were also assessed using histochemical staining and optical density measurements and the expression of adipogenic-associated genes was analyzed using reverse transcription-polymerase chain reaction. Our results suggest that after thawing, ASC viability and adipogenic and osteogenic differentiation abilities can be maintained even when ASCs are frozen in the absence of serum but with 10% PVP in DMEM.

Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering.

400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.

Hemangioblastic Characteristics of Human Adipose Tissue-derived Adult Stem Cells In Vivo

Recent studies have demonstrated the existence of a population of adipose tissue-derived adult stem (ADAS) cells that can undergo multilineage differentiation in vitro. However, it remains unclear whether these cells maintain their multilineage potential in vivo. The aim of this study was to investigate whether Flk1(+)CD31(-)CD34(-) ADAS cells have characteristics of hemangioblasts. These human ADAS (hADAS) cells were able to differentiate into endothelial and hematopoietic cells at the single-cell level in vivo. These postnatal Flk1(+)CD31(-)CD34(-) hADAS cells bear characteristics of hemangioblast and may have potential application for the hematopoietic and vascular diseases.

Harvesting human adipose tissue-derived adult stem cells: resection versus liposuction

Adipose tissue is an abundant source of mesenchymal stem cells (MSC), which can be used for tissue-engineering purposes. The aim of our study was to determine the more suitable procedure, surgical resection or liposuction, for harvesting human adipose tissue-derived stem cells (hASC) with regard to viability, cell count and differentiation potential. After harvesting hASC, trypan blue staining and cell counting were carried out. Subsequently, hASC were cultured, analyzed by fluorescence-activated cell sorting (FACS) and differentiated under adipogenic, osteogenic and chondrogenic conditions. Histologic and functional analyzes were performed at the end of the differentiation period. No significant difference was found with regard to the cell counts of hASC from liposuction and surgically resected material (P=0.086). The percentage of viable cells was significantly higher for liposuction aspirates than for resection material (P=0.002). No significant difference was found in the adipogenic differentiation potential (P=0.179). A significantly lower number of cultures obtained from liposuction material than from resection material could be differentiated into osteocytes (P=0.049) and chondrocytes (P=0.012). Even though some lineages from lipoaspirated hASC can not be differentiated as frequently as those from surgically resected material, liposuction may be superior for some tissue-engineering purposes, particularly because of the less invasive harvesting procedure, the higher percentage of viable cells and the fact that there is no significant difference between lipoaspirated and resected hASC with regard to adipogenic differentiation potential.

288 CHARACTERIZATION OF PORCINE ADIPOSE TISSUE-DERIVED ADULT STEM CELL SURFACE PROTEINS

Human adipose tissue-derived adult stem (ADAS) cells are a self-renewing population of cells with a multilineage plasticity similar to bone marrow-derived mesenchymal stem cells. Human ADAS have promise for use in combination with various biomaterials for reconstructive tissue engineering. The phenotypic profile of human ADAS cell surface proteins has been partially characterized for stem cell-associated cluster differentiation molecules including CD29, CD44, and CD90. Porcine ADAS cells, an animal model for tissue engineering, also have the ability to self-renew and differentiate into multiple tissue lineages. However, the surface protein phenotype has not been described. Because porcine ADAS are isolated from fat depots likely different from human ADAS liposuction aspirates, it is important to characterize these cells. In this study, we have partially characterized the surface protein phenotype of undifferentiated porcine ADAS cells in comparison with the immunophenotype of undifferentiated human ADAS cells as reported in the literature. Flow cytometry and enhanced chemiluminescence Western blot analysis of early passage (passages 0–4) porcine ADAS cell populations demonstrated that the profiles are not similar to the human ADAS cell surface. Immunoblot detection paired with an enhanced chemiluminescence kit revealed a positive expression for CD44 and CD90 in human ADAS cells as indicated by bands present at the expected sizes and a negative expression for CD44 and CD90 in porcine ADAS cells. Flow cytometric analysis also indicated differences between human and early passage porcine ADAS cell surfaces with a relatively low expression of CD29 (5 cell lines with a mean percent positive of 4.5 ± 1.7 and a range of 2.5–7.2%) and CD44 (5 cell lines with a mean percent positive of 0.66 ± 0.67 and a range of 0.0–1.8%) compared with human ADAS values of 98 ± 1 and 60 ± 15, respectively (Gronthos et al. 2001). Other cell surface proteins analyzed at early passages include CD3 (3 cell lines; 0.07 ± 0.06% positive and 0.0–0.1 range), CD8 (3 cell lines; 0.10 ± 0.10% positive and 0–0.2 range), and CD90 [5 cell lines; 12.7 ± 11.9% positive and 2.4–33 range; human ADAS geometric mean 25.96% (Zuk et al. 2002)]. Analysis of late passage (passages 5–11) porcine ADAS cell populations revealed an increased expression of CD29 (3 cell lines; 26.4 ± 7.2% positive and 21.2–34.6 range). The expression level of CD90 at late passages were 21.3 and 26.9% positive for 2 cell lines and CD44 remained low (3 cell lines; 4.1 ± 3.5% positive and 0.2–7.0 range). Later passages were also analyzed for c-Kit (CD117), which was expressed at low levels (2 cell lines; 0.3 and 0.4% positive). The characterization of adipose tissue-derived adult stem cell surface proteins present at different stages of in vitro culture from a model animal, such as the pig, could have valuable impacts on tissue engineering research. These results suggest that care should be taken when interpreting results from animal models of somatic stem cells.

Isolation and Characterization of Porcine Adipose Tissue-Derived Adult Stem Cells

Background: Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Methods: Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. Results: An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Conclusion: Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34.

Muscle regeneration by adipose tissue-derived adult stem cells attached to injectable PLGA spheres

The use of adult stem cells for cell-based tissue engineering and regeneration strategies represents a promising approach for skeletal muscle repair. We have evaluated the combination of adipose tissue-derived adult stem cells (ADSCs) obtained from autologous liposuction and injectable poly(lactic-co-glycolic acid) (PLGA) spheres for muscle regeneration. ADSCs attached to PLGA spheres and PLGA spheres alone were cultured in myogenic medium for 21 days and injected subcutaneously into the necks of nude mice. After 30 and 60 days, the mice were sacrificed, and newly formed tissues were analyzed by immunostaining, H and E staining, and RT-PCR. We found that ADSCs attached to PLGA spheres, but not PLGA spheres alone, were able to generate muscle tissue. These findings suggest that ADSCs and PLGA spheres are useful materials for muscle tissue engineering and that their combination can be used in clinical settings for muscle regeneration.

Transport phenomena during freezing of adipose tissue derived adult stem cells

In the present study a well-established differential scanning calorimeter (DSC) technique is used to measure the water transport phenomena during freezing of stromal vascular fraction (SVF) and adipose tissue derived adult stem (ADAS) cells at different passages (Passages 0 and 2). Volumetric shrinkage during freezing of adipose derived cells was obtained at a cooling rate of 20 degrees C/min in the presence of extracellular ice and two different, commonly used, cryoprotective agents, CPAs (10% DMSO and 10% Glycerol). The adipose derived cells were modeled as spheres of 50 microm diameter with an osmotically inactive volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the "best-fit" membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined. The "best-fit" membrane permeability parameters for adipose derived cells in the absence and presence of CPAs ranged from: Lpg=23.1-111.5x10(-15) m3/Ns (0.135-0.652 microm/min-atm) and ELp=43.1-168.8 kJ/mol (9.7-40.4 kcal/mol). Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degrees C/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing adipose derived cells in the presence and absence of CPAs.

Immunomodulatory effect of human adipose tissue‐derived adult stem cells: comparison with bone marrow mesenchymal stem cells

Like mesenchymal stem cells from bone marrow (BM-MSCs), adipose tissue-derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well-characterized immunosuppressive properties of BM-MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM-MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin-10 and transforming growth factor-beta (TGF-beta), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM-MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM-MSCs. Therefore, ADAS cell-based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM-MSCs to treat allogenic conflicts.

Epithelial differentiation of human adipose tissue-derived adult stem cells

Adult human stem cells are employed in novel treatments and bio-artificial devices. Recent studies have identified an abundant source of stem cells termed adipose-derived adult stem (ADAS)-cells in the subcutaneous adipose tissue. Under appropriate culture conditions ADAS-cells differentiate to various cell types, including chondrocytes, adipocytes, and smooth muscle cells. Aiming at epithelial differentiation this study investigated the effect of all- trans retinoic acid (ATRA) on human ADAS-cells. ATRA-induced cytokeratin 18 expression in ADAS-cells and nearly abolished vimentin expression as shown by Western blot. In immunofluorescence, the formation of keratin fibers in ATRA-treated ADAS-cells could be observed. The percentage of ADAS-cells being able to undergo epithelial differentiation as quantified by FACS-analysis was above 80%. Inhibition of cell growth by ATRA was shown using DAPI- and MTT-assays. ATRA can differentiate ADAS-cells toward the epithelial lineage. This finding, along with a previously described neural differentiation, shows that ADAS-cells have epithelial potential.

Effect of Various Freezing Parameters on the Immediate Post‐Thaw Membrane Integrity of Adipose Tissue Derived Adult Stem Cells

The effect of four thermal parameters on post-thaw membrane integrity of adipose tissue derived adult stem (ADAS) cells after controlled-rate freezing was investigated with the help of a two-level four-parameter (2(4)) experimental design. The four thermal parameters studied were cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Several passages, including Passage-0 (P0), Passage-1 (P1), Passage-2 (P2), Passage-3 (P3), and Passage-4 (P4), obtained from the suspended culture of stromal vascular fraction (SVF) of the ADAS cells were used for this study. The two levels (low and high) of the four parameters [CR (1 and 40 degrees C/min); ET (-80 and -20 degrees C); HT (1 and 15 min); and TR (10 and 200 degrees C/min)] are chosen in such a way that they enclosed all parameter values possible using commercially available controlled-rate freezing equipment. Individual effect of each parameter on the immediate post-thaw membrane integrity was determined through the calculation of parameter effect values (E), and any synergy among the parameters on post-thaw membrane integrity was assessed through the calculation of two or more parameter interaction effect values (I). Nonlinearity in the experimental results was represented through the calculation of curvature value (CV). The results suggest that for 99% confidence level the parameters CR and ET have considerable effect on post-thaw membrane integrity of all passages of ADAS cells. A significant individual effect of TR was observed with P3 and P4 cells and a significant two-parameter interaction was observed between CR-ET for all passages. These observed results will be used as a basis to further develop freezing storage protocols of ADAS cells.