Markers Of Adipose-derived Stem Cells Research Articles

Depot-specific differences and heterogeneity of adipose-derived stem cells in diet-induced obesity.

Obesity is a global health concern. Studying the heterogeneity of adipose-derived stem cells (ADSCs) plays a pivotal role in understanding metabolic disorders, such as obesity. Mass cytometry was used to determine the depot-specific differences and heterogeneity of ADSCs and their alterations at the single-cell level in a diet-induced-obesity (DIO) model in which mice were treated with liraglutide. We characterized the relationship among ADSC markers and found that CD26 and CD142 could identify the most representative heterogeneous ADSCs in subcutaneous adipose tissue and visceral adipose tissue. Specifically, CD26+CD142- and CD26+CD142+ ADSCs were exclusive to subcutaneous adipose tissue and visceral adipose tissue, respectively, whereas CD26-CD142+ ADSCs were present in both. RNA analysis explored the potential functions of these three subgroups. In the visceral adipose tissue of DIO mice, we observed a substantial downregulation of CD26+CD142+ ADSCs and upregulation of CD26-CD142+ ADSCs, both of which were mitigated by liraglutide treatment. Our study highlights the depot-specific differences and heterogeneity of ADSCs and their alterations under DIO conditions, which can potentially be reversed by liraglutide treatment. This study provides new insights into the identification of more specific ADSC subgroups to explore the etiology of metabolism-related diseases.

Differentiation of Adipose-Derived Stem Cells into Vascular Smooth Muscle Cells for Tissue Engineering Applications.

Synthetic grafts have been developed for vascular bypass surgery, however, the risks of thrombosis and neointimal hyperplasia still limit their use. Tissue engineering with the use of adipose-derived stem cells (ASCs) has shown promise in addressing these limitations. Here we further characterized and optimized the ASC differentiation into smooth muscle cells (VSMCs) induced by TGF-β and BMP-4. TGF-β and BMP-4 induced a time-dependent expression of SMC markers in ASC. Shortening the differentiation period from 7 to 4 days did not impair the functional property of contraction in these cells. Stability of the process was demonstrated by switching cells to regular growth media for up to 14 days. The role of IGFBP7, a downstream effector of TGF-β, was also examined. Finally, topographic and surface patterning of a substrate is recognized as a powerful tool for regulating cell differentiation. Here we provide evidence that a non-woven PET structure does not affect the differentiation of ASC. Taken together, our results indicate that VSMCs differentiated from ASCs are a suitable candidate to populate a PET-based vascular scaffolds. By employing an autologous source of cells we provide a novel alternative to address major issues that reduces long-term patency of currently vascular grafts.

Bladder muscle regeneration enhanced by sustainable delivery of heparin from bilayer scaffolds carrying stem cells in a rat bladder partial cystectomy model

In bladder tissue engineering, regeneration of muscle is of equal importance to epithelial regeneration. However, as yet there is no effective strategy for promoting bladder muscle regeneration. In this study we aim to promote bladder muscle regeneration by sustainably delivering heparin from a bilayer scaffold carrying stem cells. The bilayer scaffold [heparin–polycaprolactone (PCL)/bladder decellularized matrix (BAM) Hep-PB/PCL] comprises an electrospun layer (Hep-PB electrospun membrane) and a three-dimensional (3D) printed layer (PCL scaffold), fabricated via coaxial-electrospinning and 3D printing, respectively. Heparin was encapsulated into the core of the Hep-PB fibers with a core–shell structure to sustain its release. The morphology of the bilayer scaffold and the microstructure of the electrospun fibers were characterized. The release behavior of heparin from various electrospun membranes was evaluated. The role of Hep-PB in promoting myogenic differentiation of the adipose-derived stem cells (ADSCs) through sustainable release of heparin was also evaluated. After 7 d culture, Hep-PB/PCL scaffolds carrying ADSCs (defined as ASHP) were used for bladder reconstruction in a rat partial cystotomy model. The result shows that the PCL printed scaffold has ordered macropores (∼370 μm), unlike the compact microstructure of electrospun films. The Hep-PB membrane exhibits a sustained release behavior for heparin. This membrane also shows better growth and proliferation of ADSCs than the other membranes. The polymerase chain reaction results show that the expression of smooth muscle cell markers in ADSCs is enhanced by the Hep-PB scaffold. The results of retrograde urethrography and histological staining indicate that the bladder volume in the ASHP group recovers better, and the regenerated bladder muscle bundles are arranged in a more orderly fashion compared with the direct suture and bladder decellularized matrix groups. Therefore, findings from this study show that bladder muscle regeneration could be enhanced by bilayer scaffolds delivering heparin and carrying stem cells, which may provide a new strategy for bladder tissue engineering.

Homing of Super Paramagnetic Iron Oxide Nanoparticles (SPIONs) Labeled Adipose-Derived Stem Cells by Magnetic Attraction in a Rat Model of Parkinson's Disease.

IntroductionStem cell therapies for neurodegenerative diseases such as Parkinson’s disease (PD) are intended to replace lost dopaminergic neurons. The basis of this treatment is to guide the migration of transplanted cells into the target tissue or injury site. The aim of this study is an evaluation of the homing of superparamagnetic iron oxide nanoparticles (SPIONs) labeled adipose-derived stem cells (ADSC) by an external magnetic field in a rat model of PD.MethodsADSCs were obtained from perinephric regions of male adult rats and cultured in a DMEM medium. ADSC markers were assessed by immunostaining with CD90, CD105, CD49d, and CD45. The SPION was coated using poly-L-lysine hydrobromide and transfection was determined in rat ADSC using the GFP reporter gene. For this in vivo study, rats with PD were divided into five groups: a positive control group, a control group with PD (lesion with 6-HD injection), and three treatment groups: the PD/ADSC group (PD transplant with ADSCs transfected by BrdU), PD/ADSC/SPION group (PD transplant with ADSCs labeled with SPION and transfected by GFP), and the PD/ADSC/SPION/EM group (PD transplant with ADSCs labeled with SPION and transfected by GFP induced with external magnet).ResultsADSCs were immunoreactive to fat markers CD90 (90.73±1.7), CD105 (87.4±2.9) and CD49d (79.6±2.6), with negative immunostaining at the hematopoietic stem cell marker (CD45: 1.4±0.4). The efficiency of cells with SPION/PLL was about 96% of ADSC. The highest number of GFP-positive cells was in the ADSC/SPION/EM group (54.5±1.3), which was significantly different from that in ADSC/SPION group (30.83±3 and P<0.01).ConclusionTransfection of ADSC by SPION/PLL is an appropriate protocol for cell therapy. External magnets can be used for the delivery and homing of transplanted stem cells in the target tissue.

Induction of Tendon-Specific Markers in Adipose-Derived Stem Cells in Serum-Free Culture Conditions.

Herein, we describe the tenogenic effect of bone morphogenetic protein-12 and transforming growth factor-β1 in cultured adipose-derived stem cells (ADSCs) in serum-free conditions. This culture system provides an insight into serum-free culture conditions in stem cell differentiation protocols. A positive response of the ADSCs to the tenogenic induction was observed. In particular, the different growth factors used in this study displayed notable differences both on the gene and on the protein expression of the tendon-specific markers. The results underline the positive outcome of the serum removal in tenogenic differentiation protocols, contributing to the development of future cell-based therapies for tendon regeneration and repair.

351 Phenotypic Modulation of Adipose-Derived Stem Cells Within 3D Scaffolds; Applications in Skin Bioengineering

Large burn injuries and wounds contribute to increased patient morbidity and mortality and impose a significant financial burden on healthcare systems. The main goal of wound treatment is to achieve a rapid closure of the lesion and promote healing with minimal scarring. Extracellular matrix- based biomaterials such as acellular dermal matrices (ADM) or in situ-forming scaffolds are advantageous for treatment of large wounds due to their mechanical strength and retained biological activity when compared with synthetic polymer materials. Further, recellularization of ADM with adipose-derived stem cells (ASCs) significantly increases the healing capacity. The aim of this study was to develop an ASC-populated ADM and assess its characteristics in vitro with the ultimate goal of engineering an optimized wound healing composite. To this end, we developed a novel method for de-cellularization of skin and used the acquired ADM as a 3D scaffold to seed human ASCs. We compared this 3D model with traditional 2D ASC cultures at different time points post-culture. A panel of cell surface markers was used for phenotypic characterization of ASCs, post-culture. Combinations of positive (CD146, CD44, CD90, and CD73) and negative (CD31, CD34, CD45) cell surface proteins were used as stem cell markers. Morphology and myofibroblast differentiation capacity of ASCs were also evaluated. The results showed a significant reduction in expression of CD73 and CD44 markers in ASCs when embedded within a 3D ADM, compared to the cells that were cultured in a 2D culture condition. We found that ASCs cultured under regular 2D conditions mainly differentiated towards a myofibroblastic phenotype with increased myofibroblast marker α-smooth muscle actin (α-SMA) and type I pro-collagen. In contrast, ASCs cultured on ADM showed a more balanced differentiation pattern with maintenance of important stem cell markers such as CD146. Taken together, these findings suggest that ASC differentiation can be regulated by a 3D scaffold. Embedding ASCs within 3D scaffolds prevents universal differentiation to stromal cells and maintain stemness features associated with ASCs. The ASC-ADM combination shows a promising potential as a novel therapeutic approach for treatment of large burn injuries and wounds. This bioengineered composite can reduce risk of fibrosis while maintaining the regenerative capacity of stem cells.

Lithium ions (Li+) and nanohydroxyapatite (nHAp) doped with Li+ enhance expression of late osteogenic markers in adipose-derived stem cells. Potential theranostic application of nHAp doped with Li+ and co-doped with europium (III) and samarium (III) ions

Lithium (Li+) ion due to its excellent bioactivity is one of the most well-studied element in bone-tissue engineering. In this study, we fabricated nanohydroxyapatite (nHAp) doped with Li+ ions (5 mol% Li+:nHAp) and co-doped with lanthanide ions. We investigated the effects of nHAp, 5 mol% Li+:nHAp or Li+ alone, on osteogenic differentiation of human Adipose Tissue-derived Stem Cells (hASCs), their proliferation, mitochondrial dynamics and apoptosis. Moreover, we monitored cell proliferation after treatment with samarium (III) (Sm3+) and europium (III) (Eu3+) ions co-doped 5 mol% Li+:nHAp as well as their luminescent property. The hASCs treated with 5 mol% Li+:nHAp and Li+ ions proliferated more rapidly and differentiated effectively than control cells without undergoing apoptosis. Both, 5 mol% Li+:nHAp and Li+ ions improved osteogenic differentiation of hASCs. Moreover they decreased expression of glycogen synthase kinase 3β (GSK3β) while increased β-catenin mRNA level. In addition, Li+, nHAp and 5 mol% Li+:nHAp improved mitochondrial dynamics and enhanced expression of neural differentiation marker genes. Collectively, the study indicates on pro-osteogenic and anti-apoptotic properties of nHAp doped with Li+ and Li+ alone. Moreover, unique properties of 5 mol% Li+:nHAp and 5 mol% Li+:nHAp co-doped with rare earth ions, such as Sm3+ and Eu3+ have shed a promising light on their potential application in theranostics.

Characterization of Sesquiterpene Dimers from Resina Commiphora That Promote Adipose-Derived Stem Cell Proliferation and Differentiation.

The new sesquiterpene dimers commiphoroids A-D (1-4) were isolated from Resina Commiphora, and their structures were assigned by spectroscopic methods and X-ray diffraction analysis. Compounds 1 and 2 are stereoisomers of putative [2 + 4]-cycloaddition reactions, and 3 is a trinorsesquiterpene dimer containing a 6/6/5/6/6/6 hexacyclic framework, while 4 possesses a 8-oxabicyclo[3.2.1]oct-6-ene skeletal core. Plausible biosynthetic pathways for 1-4 are proposed. Biochemical studies show that compound 1 promotes ca. 60% expression of keratinocyte-specific markers in adipose-derived stem cells at 10 μM.

Comparison of the effects of progesterone and 17 β-estradiol on Schwann cell markers expression in rat adipose-derived stem cells.

Steroids promote the myelination and regeneration in the peripheral nervous system. Whereas, little is known about the inducing effects by which the hormones exert their effects on Schwann cells differentiation. This could be revealed by the expression of Schwann cell markers in adipose-derived stem cells (ADSCs). The purpose of this study was to present the effects of progesterone and 17 β-estradiol on the Schwann cell markers in rat ADSCs. The mesenchymal stem cell markers (CD73, and CD90) were assayed by flow cytometry. Rat ADSCs were sequentially treated with β-mercaptoethanol, and all-trans-retinoic acid, followed by a mixture of basic fibrobroblast growth factor, platelet-derived growth factor, forskolin and heregulin. In experimental groups, forskolin and heregulin were substituted by progesterone and 17 β-estradiol. After induction, the expression of Schwann cell markers P0, and S-100 and the cellular immunocytochemical staining positive rate of anti-S100 and anti-glial fibrillary acidic protein (GFAP) antibodies were compared in the experimental and control groups. Progesterone and 17 β-estradiol triggered P0 and S-100 genes expression and induced a cellular immunocytochemical staining positive rate of S-100 and GFAP in rats ADSCs. Progesterone induced these changes stronger than 17 β-estradiol. Thus, progesterone may induce rat ADSCs toward Schwann-like cells by expression of Schwann cell markers and is more potent than 17 β-estradiol in the expression of these markers.

Hepatogenic differentiation from human adipose-derived stem cells and application for mouse acute liver injury

Adipose-derived stem cells (ADSCs) derived from adipose tissue have the capacity to differentiate into endodermal, mesoderm, and ectodermal cell lineages in vitro, which are an ideal engraft in tissue-engineered repair. In this study, human ADSCs were isolated from subcutaneous fat. The markers of ADSCs, CD13, CD71, CD73, CD90, CD105, CD166, CYP3A4, and ALB were detected by immunofluorescence assays. Human ADSCs were cultured in a specific hepatogenesis differentiation medium containing HGF, bFGF, nicotinamide, ITS, and oncostatin M for hepatogenic differentiation. The hepatocyte markers were analyzed using immunofluorescence and real-time PCR after dramatic changes in morphology. Hepatocytes derived from ADSCs or ADSCs were transplanted into the mice of liver injury for observation cells colonization and therapy in liver tissue. The result demonstrated that human ADSCs were positive for the CD13, CD71, CD73, CD90, CD105, and CD166 but negative for hepatocyte markers, ALB and CYP3A4. After hepatogenic differentiation, the hepatocytes were positive for liver special markers, gene expression level showed a time-lapse increase with induction time. Human ADSCs or ADSCs-derived hepatocyte injected into the vein could improve liver function repair and functionally rescue the CCl4-treated mice with liver injury, but the ADSCs transplantation was better than ADSCs-derived hepatocyte transplantation. In conclusion, our research shows that a population of hepatocyte can be specifically generated from human ADSCs and that cells may allow for participation in tissue-repair.

Enhanced Osteogenic Behavior of ADSCs Produced by Deproteinized Antler Cancellous Bone and Evidence for Involvement of ERK Signaling Pathway.

Calcinated antler cancellous bone (CACB) is useful in repair of bone defects, as its composition and architecture is analogous to natural extracellular bone matrix. The use of CACB scaffolds with adipose-derived stem cells (ADSCs) in repair of rabbit mandibular bone defects was investigated along with the underlying molecular signaling pathways involved. CACB promoted the adhesion, spreading, and viability of ADSCs. Increased extracellular matrix production and expression of osteogenic markers in ADSCs were observed when seeded in CACB. The temporal kinetics of mRNA expression of ADSCs cultured in CACB lagged in comparison with that observed in cells grown in medium with osteogenic supplements. Activation of the extracellular signal-related kinases (ERK) 1/2 and RUNX-2 in CACB-cultured ADSCs was observed, and this activation was attenuated by the MeK inhibitor U0126. Microcomputed tomography scanning analysis and histological evaluations showed that loading the CACB with ADSCs resulted in enhanced new bone formation and angiogenesis when the composites were implanted in rabbit mandibular defects. These results indicated that the osteogenic behavior of ADSCs might be driven by the microenvironment formed by CACB via the ERK signaling pathway. These CACB/ADSCs composites have promising therapeutic potential for large bone defect repairs.

Neurogenic differentiation from adipose-derived stem cells and application for autologous transplantation in spinal cord injury

Mesenchymal stem cells derived from adipose tissue have the capacity to differentiate into endodermal, mesoderm and ectodermal cell lineages in vitro, which are an ideal engraft in tissue-engineered repair. In this study, mouse adipose-derived stem cells (ADSCs) were isolated from subcutaneous fat. The markers of ADSCs, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, Nestin, GFAP and MAP-2 were detected by immunofluorescence assays. The ADSCs were cultured in cocktail factors (including ATRA, GGF-2, bFGF, PDGF and forskolin) for neurogenic differentiation. The neurogenic cells markers, Nestin, GFAP and MAP-2 were analyzed using immunofluorescence and real-time PCR after dramatic changes in morphology. Neurogenic cells from ADSCs were autologous transplanted into the mouse of spinal cord injury for observation neurogenic cells colonization in spinal cord. The result demonstrated that the mouse ADSCs were positive for the CD13, CD29, CD44, CD71, CD73, CD90, CD105 and CD166 but negative for neurogenic cell markers, MAP-2, GFAP and Nestin. After neurogenic differentiation, the neurogenic cells were positive for neurogenic cell special markers, gene expression level showed a time-lapse increase, and the cells were successful colonized into spinal cord. In conclusion, our research shows that a population of neuronal cells can be specifically generated from ADSCs and that induced cells may allow for participation in tissue-repair.

Identification of Specific Cell-Surface Markers of Adipose-Derived Stem Cells from Subcutaneous and Visceral Fat Depots

SummaryAdipose-derived stem/stromal cells (ASCs) from the anatomically distinct subcutaneous and visceral depots of white adipose tissue (WAT) differ in their inherent properties. However, little is known about the molecular identity and definitive markers of ASCs from these depots. In this study, ASCs from subcutaneous fat (SC-ASCs) and visceral fat (VS-ASCs) of omental region were isolated and studied. High-content image screening of over 240 cell-surface markers identified several potential depot-specific markers of ASCs. Subsequent studies revealed consistent predominant expression of CD10 in SC-ASCs and CD200 in VS-ASCs across 12 human subjects and in mice. CD10-high-expressing cells sorted from SC-ASCs differentiated better than their CD10-low-expressing counterparts, whereas CD200-low VS-ASCs differentiated better than CD200-high VS-ASCs. The expression of CD10 and CD200 is thus depot-dependent and associates with adipogenic capacities. These markers will offer a valuable tool for tracking and screening of depot-specific stem cell populations.

Sox9 Modulates Proliferation and Expression of Osteogenic Markers of Adipose-Derived Stem Cells (ASC)

Background: Mesenchymal stem cells (MSC) are promising tools for tissue-engineering and musculoskeletal regeneration. They reside within various tissues, like adipose tissue, periosteum, synovia, muscle, dermis, blood and bone marrow, latter being the most common tissue used for MSC isolation. A promising alternative source for MSC is adipose tissue due to better availability and higher yield of MSC in comparison to bone marrow. A drawback is the yet fragmentary knowledge of adipose-derived stem cell (ASC) physiology in order to make them a safe tool for in vivo application. Methods/Results: Here, we identified Sox9 as a highly expressed and crucial transcription factor in undifferentiated rat ASC (rASC). In comparison to rat bone marrow-derived stem cells (rBMSC), mRNA and protein levels of Sox9 were significantly higher in rASC. To study the role of Sox9 in detail, we silenced Sox9 with shRNA in rASC and examined proliferation, apoptosis and the expression of osteogenic differentiation markers. Our results clearly point to a difference in the expression profile of osteogenic marker genes between undifferentiated rASC and rBMSC in early passages. Sox9 silencing induced the expression of osteocalcin, Vegfα and Mmp13, and decreased rASC proliferation accompanied with an induction of p21 and cyclin D1 expression and delayed S-phase entry. Conclusions: We suggest a pro-proliferative role for Sox9 in undifferentiated rASC which may explain the higher proliferation rate of rASC compared to rBMSC. Moreover, we propose an osteogenic differentiation delaying role of Sox9 in rASC which suggests that Sox9 expression is needed to maintain rASC in an undifferentiated, proliferative state.

Suppression of zinc finger protein 467 alleviates osteoporosis through promoting differentiation of adipose derived stem cells to osteoblasts

Osteoblast and adipocyte are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. In this study, a comparative analysis of gene expression profiling using cDNA microarray and realtime-PCR indicated that Zinc finger protein 467 (Zfp467) involved in adipocyte and osteoblast differentiation of cultured adipose derived stem cells (ADSCs). Our results showed that RNA interference for Zfp467 in ADSCs inhibited adipocyte formation and stimulated osteoblast commitment. The mRNA levels of osteogenic and adipogenic markers in ADSCs were regulated by si-Zfp467. Zfp467 RNAi in ADSCs could restore bone function and structure in an ovariectomized (OVX)-induced osteoporotic mouse model. Thus Zfp467 play an important role in ADSCs differentiation to adipocyte and osteoblast. This has relevance to therapeutic interventions in osteoporosis, including si-Zfp467-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering.

Changes in the expression pattern of mesenchymal and pluripotent markers in human adipose‐derived stem cells

Pluripotent marker genes encode transcription factors that suppress differentiation and allow stem cells to continue proliferation while remaining poised to develop into cells from multiple lineages. Mesenchymal marker proteins are widely used to identify and characterize multipotent stem cells that are capable of differentiation into cells of the mesenchymal lineage. We examine here the expression pattern of mesenchymal and pluripotent markers in ADSCs (human adipose-derived stem cells) during culture. Flow cytometry indicates that a large fraction of ADSCs were positive for multiple mesenchymal markers, such as CD29, CD44 and CD90. The expression levels of these markers increase and become more uniform in cells that are cultured for long periods. Alternatively, mRNAs for several pluripotent genes, such as Nanog, Oct-4 and Rex-1, are detected only in cells from early passages. In addition, ADSCs cultured over long periods express several cardiac genes in response to serum deprivation and treatment with phorbol ester. Thus, these data demonstrate that ADSCs expanded in culture over multiple passages consistently express mesenchymal markers with the ability to differentiate towards the cardiac lineage.

Effects of Culturing on the Stability of the Putative Murine Adipose Derived Stem Cells Markers

Mesenchymal stem cells have generated much interest because of their potential use in regenerative medicine. The major draw back in the application of these cells is that there is no single marker or markers that have been established to identify and aid in isolating the cells from a variety of other cell types. The commonly expressed mesenchymal stem cell surface antigens include CD44, CD73, CD90.2, CD105, and CD146. In the present study we examined the stability of these surface antigens in culture and their potential application in identifying and isolating murine derived adipose derived stem cells. The data showed that the expression of these markers increased with culturing and appeared to stabilize by passage 8; the cells were sorted positively for the surface markers at this passage. Each subset was maintained in culture and evaluated for differentiation toward osteogenic lineage in vitro and in vivo. The CD73 and CD105 positive cell subsets demonstrated robust differentiation toward osteogenic lineage in vitro; the CD90.2+ cell subset exhibited the least differentiation toward osteogenic lineage. Assessment of the cell subpopulations for in vivo differentiation demonstrated that all the cell subsets exhibited potential to differentiate into osteoblasts. Taken together, these data suggest that this panel of markers although useful in identifying cells with potential to differentiate toward osteogenic lineage, cannot prospectively be used for enriching for ADSC from a variety of other cell types.