Adipogenic Capacity Research Articles

Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells

BackgroundCritical tissues that undergo regeneration in periodontal tissue are of mesenchymal origin; thus, investigating the regulatory mechanisms underlying the fate of periodontal ligament stem cells could be beneficial for application in periodontal tissue regeneration. Nitric oxide (NO) regulates many biological processes in developing embryos and adult stem cells. The present study was designed to investigate the effects of NO on the function of human periodontal ligament stem cells (PDLSCs) as well as to elucidate the underlying molecular mechanisms.MethodsImmunofluorescent staining and flow cytometry were used for stem cell identification. Western blot, reverse transcription polymerase chain reaction (RT-PCR), immunofluorescent staining, and flow cytometry were used to examine the expression of NO-synthesizing enzymes. The proliferative capacity of PDLSCs was determined by EdU assays. The osteogenic potential of PDLSCs was tested using alkaline phosphatase (ALP) staining, Alizarin Red staining, and calcium concentration detection. Oil Red O staining was used to analyze the adipogenic ability. Western blot, RT-PCR, and staining were used to examine the signaling pathway.ResultsHuman PDLSCs expressed both inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) and produced NO. Blocking the generation of NO with the NOS inhibitor l-NG-monomethyl arginine (l-NMMA) had no influence on PDLSC proliferation and apoptosis but significantly attenuated the osteogenic differentiation capacity and stimulated the adipogenic differentiation capacity of PDLSCs. Increasing the physiological level of NO with NO donor sodium nitroprusside (SNP) significantly promoted the osteogenic differentiation capacity but reduced the adipogenic differentiation capacity of PDLSCs. NO balances the osteoblast and adipocyte lineage differentiation in periodontal ligament stem cells via the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling pathway.ConclusionsNO is essential for maintaining the balance between osteoblasts and adipocytes in PDLSCs via the JNK/MAPK signaling pathway.Graphical NO balances osteoblast and adipocyte lineage differentiation via JNK/MAPK signaling pathway

Effect of TGF\u03b21, TGF\u03b23 and keratinocyte conditioned media on functional characteristics of dermal fibroblasts derived from reparative (Balb/c) and regenerative (Foxn1 deficient; nude) mouse models

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin’s pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor β family (TGFβ) expression showed significantly higher levels of Tgfβ3 transcript between nude and Balb/c mice but no differences were detected for Tgfβ1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFβ3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFβ3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.

Menstrual blood-derived mesenchymal stem cells as a potential tool for cartilage regeneration

Purpose: Human mesenchymal stem cells (MSCs) are multipotent cells found in almost all human tissues. These cells are of great interest in regenerative medicine due to their high potential to differentiate into several directions including osteocytes, adipocytes and chondrocytes. Menstrual blood has been proposed as one of the perspective MSCs sources (MenSCs), due to their ease of access, as compared to the classical bone marrow MSCs (BMMSCs). As MSCs exhibit high chondrogenic differentiation potential, they ideally suit for therapeutic use in cartilage regeneration. Transforming growth factor β (TGF-β) has been considered as one of the major factors for induction of chondrogenic response in BMMSCs, however a member of the TGF-β superfamily Activin A also seems to contribute in the early stages of MSC chondrogenesis. Activin A seems necessary for the maintenance of self-renewal and pluripotency of MSCs. Furthermore there is an increasing body of evidence that Ca2+-dependent regulatory mechanisms can influence differentiation potential of chondrogenic progenitor cells, whereas Activin A was shown to increase free calcium concentrations in cells. Since Activin A also regulates menstrual cycle, we hypothesized that it may influence chondrogenic differentiation of MenSCs. The aim of this study was to characterize MenSCs and their chondrogenic differentiation capacity using two different growth factors - TGF-β3 and Activin A. The data was compared to the classical BMMSCs. Methods: BMMSCs and MenSCs were isolated from different healthy donors. Different cell surface markers (CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD271, CD106, CD34, CD10, Stro1, Notch1) were analysed by flow cytometry. Cell proliferation capacity was determined by CCK-8 proliferation kit. Intracellular calcium was measured using fluorescent dye Cal-520. Adipogenic differentiation capacity was determined by Oil-Red staining and osteogenic differentiation by Alizarin Red staining. Chondrogenic differentiation (Activin A and TGF-b3 stimulation) was investigated by immuno- and histochemical staining with collagen II antibodies and safranin O, respectively. Results: In contrast to BMMSCs, MenSCs exhibited higher expression of CD10 and Notch1, and higher proliferative capacity. Furthermore, MenSCs exhibited higher level of intracellular calcium concentration as compared to BMMSCs. Adipogenic differentiation of MenSCs was weaker, whereas osteogenic differentiation capacity was higher compared to BMMSCs. Chondrogenic differentiation in BMMSCs was stronger with TGF-β3 than with Activin A, while MenSCs chondrogenic differentiation was remarkably more pronounced with Activin A than with TGF-β3. Conclusions: Taken together, our results demonstrate that MenSCs exhibit similar stem cell properties to BMMSCs, better proliferation potential and different regulatory mechanisms in chondrogenesis in vitro. Menstrual blood seems a convenient stem cells source for isolation of MSCs with chondrogenic differentiation potential, as does not require any invasive surgical interventions, and thus provides an option of repeated sample collection in the same donor, which make MenSCs a perspective tool in cartilage regenerative medicine.

Perivascular Adipose Tissue as a Relevant Fat Depot for Cardiovascular Risk in Obesity.

Obesity is associated with increased risk of premature death, morbidity, and mortality from several cardiovascular diseases (CVDs), including stroke, coronary heart disease (CHD), myocardial infarction, and congestive heart failure. However, this is not a straightforward relationship. Although several studies have substantiated that obesity confers an independent and additive risk of all-cause and cardiovascular death, there is significant variability in these associations, with some lean individuals developing diseases and others remaining healthy despite severe obesity, the so-called metabolically healthy obese. Part of this variability has been attributed to the heterogeneity in both the distribution of body fat and the intrinsic properties of adipose tissue depots, including developmental origin, adipogenic and proliferative capacity, glucose and lipid metabolism, hormonal control, thermogenic ability, and vascularization. In obesity, these depot-specific differences translate into specific fat distribution patterns, which are closely associated with differential cardiometabolic risks. The adventitial fat layer, also known as perivascular adipose tissue (PVAT), is of major importance. Similar to the visceral adipose tissue, PVAT has a pathophysiological role in CVDs. PVAT influences vascular homeostasis by releasing numerous vasoactive factors, cytokines, and adipokines, which can readily target the underlying smooth muscle cell layers, regulating the vascular tone, distribution of blood flow, as well as angiogenesis, inflammatory processes, and redox status. In this review, we summarize the current knowledge and discuss the role of PVAT within the scope of adipose tissue as a major contributing factor to obesity-associated cardiovascular risk. Relevant clinical studies documenting the relationship between PVAT dysfunction and CVD with a focus on potential mechanisms by which PVAT contributes to obesity-related CVDs are pointed out.

De novo adipocyte differentiation from Pdgfr\u03b2+ preadipocytes protects against pathologic visceral adipose expansion in obesity

Pathologic expansion of white adipose tissue (WAT) in obesity is characterized by adipocyte hypertrophy, inflammation, and fibrosis; however, factors triggering this maladaptive remodeling are largely unknown. Here, we test the hypothesis that the potential to recruit new adipocytes from Pdgfrβ+ preadipocytes determines visceral WAT health in obesity. We manipulate levels of Pparg, the master regulator of adipogenesis, in Pdgfrβ+ precursors of adult mice. Increasing the adipogenic capacity of Pdgfrβ+ precursors through Pparg overexpression results in healthy visceral WAT expansion in obesity and adiponectin-dependent improvements in glucose homeostasis. Loss of mural cell Pparg triggers pathologic visceral WAT expansion upon high-fat diet feeding. Moreover, the ability of the TZD class of anti-diabetic drugs to promote healthy visceral WAT remodeling is dependent on mural cell Pparg. These data highlight the protective effects of de novo visceral adipocyte differentiation in these settings, and suggest Pdgfrβ+ adipocyte precursors as targets for therapeutic intervention in diabetes.

Human Umbilical Cord Mesenchymal Stem Cells: Subpopulations and Their Difference in Cell Biology and Effects on Retinal Degeneration in RCS Rats.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are potential candidates for treating retinal degeneration (RD). To further study the biology and therapeutic effects of the hUC-MSCs on retinal degeneration. Two hUC-MSC subpopulations, termed hUC-MSC1 and hUC-MSC2, were isolated by single-cell cloning method and their therapeutic functions were compared in RCS rat, a RD model. Although both subsets satisfied the basic requirements for hUC-MSCs, they were significantly different in morphology, proliferation rate, differentiation capacity, phenotype and gene expression. Furthermore, only the smaller, fibroblast-like, faster growing subset hUC-MSC1 displayed stronger colony forming potential as well as adipogenic and osteogenic differentiation capacities. When the two subsets were respectively transplanted into the subretinal spaces of RCS rats, both subsets survived, but only hUC-MSC1 expressed RPE cell markers Bestrophin and RPE65. More importantly, hUC-MSC1 showed stronger rescue effect on the retinal function as indicated by the higher b-wave amplitude on ERG examination, thicker retinal nuclear layer, and decreased apoptotic photoreceptors. When both subsets were treated with interleukin-6, mimicking the inflammatory environment when the cells were transplanted into the eyes with degenerated retina, hUC-MSC1 expressed much higher levels of trophic factors in comparison with hUC-MSC2. The data here, in addition to prove the heterogeneity of hUC-MSCs, confirmed that the stronger therapeutic effects of hUC-MSC1 were attributed to its stronger anti-apoptotic effect, paracrine of trophic factors and potential RPE cell differentiation capacity. Thus, the subset hUC-MSC1, not the other subset or the ungrouped hUC-MSCs should be used for effective treatment of RD.

The biological changes of umbilical cord mesenchymal stem cells in inflammatory environment induced by different cytokines.

Mesenchymal stem cells (MSCs) are used as therapeutic tool for the treatment of immune diseases. The inflammatory environment also influences the characteristics of MSCs after transplantation. The aim of the study was to investigate the effects of pro-inflammatory cytokines on the characteristics of umbilical cord mesenchymal stem cells (UCMSCs). UCMSCs were exposed to pro-inflammatory cytokines in vitro for 3 and 7 days, and the biological properties were analyzed. The results showed that the proliferation ability was suppressed by interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The adipogenic capacity was inhibited in all conditioned medium, while the chondrogenic and osteogenic capacity was enhanced by TNF-α and IL-1β in vitro. Prostaglandin E2 (PGE2) was increased by IL-1β on the third day, and angiopoietin-1 (Ang-1) was inhibited appreciably by TNF-α on the seventh day. Interleukin-6 (IL-6) was increased by TNF-α and IL-1β, and hepatocyte growth factor (HGF) was inhibited by all inflammatory cytokines. IFN-γ secretion level from human peripheral mononuclear cells (hPBMCs) was lowered by UCMSCs which had been stimulated by TNF-α or IL-1β for 3 days. Moreover, IFN-γ and TNF-α secretion level was only inhibited by UCMSCs which had been by stimulated IFN-γ for 3 days but not 7 days. Our data demonstrated that different inflammatory cytokines and the duration of treatment had different effects on the properties of UCMSCs, which might be instructive for clinical pretreatment in cellular therapeutics.

Reduced satellite cell density and myogenesis in Wagyu compared with Angus cattle as a possible explanation of its high marbling

Mechanisms responsible for excellent marbling in Japanese black cattle, Wagyu, remain to be established. Because both muscle cells and intramuscular adipocytes are developed from mesenchymal progenitor cells during early muscle development, we hypothesized that intramuscular progenitor cells in Wagyu cattle have attenuated myogenic capacity in favor of adipogenesis, leading to high marbling but reduced muscle growth. Biceps femoris muscle biopsy samples were obtained from both Angus (n=3) and Wagyu (n=3) cattle at 12 months of age. Compared with Angus, the density of satellite cells was much lower in Wagyu muscle (by 45.8±10%, P<0.05). Consistently, the formation of myotubes from muscle-derived progenitor cells was also lower (by 64.2±12.9%, P<0.05), but adipogenic capacity was greater in Wagyu. The average muscle fiber diameter was larger in Wagyu (by 23.9±6.8%, P=0.089) despite less muscle mass, suggesting less muscle fiber formation in Wagyu compared with Angus cattle. Because satellite cells are derived from fetal myogenic cells, the reduction in satellite cell density together with lower muscle fiber formation suggests that myogenesis was attenuated during early muscle development in Wagyu cattle. Given the shared pool of mesenchymal progenitor cells, the attenuated myogenesis likely shifts progenitor cells to adipogenesis during early development, which may contribute to high intramuscular adipocyte formation in Wagyu cattle.

Stemness Characteristics of Periodontal Ligament Stem Cells from Donors and Multiple Sclerosis Patients: A Comparative Study.

Multiple sclerosis (MS) is the most prevalent and progressive autoimmune disease that affects the central nervous system, and currently, no drug is available for the treatment. Stem cell therapy has received substantial attention in MS treatment. Recently, we demonstrated the immunosuppressive effects of mesenchymal stem cells derived from neural crest-originated human periodontal ligament tissue (hPDLSCs) in an in vivo model of MS. In the present study, we comparatively investigated the stemness properties of hPDLSCs derived from healthy donors and relapsing-remitting MS patients. Stem cell marker expression, cell proliferation, and differentiation capacity were studied. We found that both donor- and MS patient-derived hPDLSCs at early passage 2 showed similar expression of surface antigen markers and cell proliferation rate. Significant level of osteogenic, adipogenic, chondrogenic, and neurogenic differentiation capacities was observed in both donor- and MS patient-derived hPDLSCs. Interestingly, these cells maintained the stemness properties even at late passage 15. Senescence markers p16 and p21 expression was considerably enhanced in passage 15. Our results propose that hPDLSCs may serve as simple and potential autologous stem cell niche, which may help in personalized stem cell therapy for MS patients.

Autophagy controls mesenchymal stem cell properties and senescence during bone aging.

SummaryBone marrow‐derived mesenchymal stem cells (BMMSCs) exhibit degenerative changes, including imbalanced differentiation and reduced proliferation during aging, that contribute to age‐related bone loss. We demonstrate here that autophagy is significantly reduced in aged BMMSCs compared with young BMMSCs. The autophagy inhibitor 3‐methyladenine (3‐MA) could turn young BMMSCs into a relatively aged state by reducing their osteogenic differentiation and proliferation capacity and enhancing their adipogenic differentiation capacity. Accordingly, the autophagy activator rapamycin could restore the biological properties of aged BMMSCs by increasing osteogenic differentiation and proliferation capacity and decreasing adipogenic differentiation capacity. Possible underlying mechanisms were explored, and the analysis revealed that autophagy could affect reactive oxygen species and p53 levels, thus regulating biological properties of BMMSCs. In an in vivo study, we found that activation of autophagy restored bone loss in aged mice. In conclusion, our results suggest that autophagy plays a pivotal role in the aging of BMMSCs, and activation of autophagy could partially reverse this aging and may represent a potential therapeutic avenue to clinically treat age‐related bone loss.

Minced Pulp as Source of Pulpal Mesenchymal Stem Cells with Odontogenic Differentiation Capacity

IntroductionPulp tissue regeneration is becoming a reality after discovery of mesenchymal stem cells (MSCs) residing in the pulp tissues through various clinical innovations, although MSC transplantation into the pulp space has met with challenges of in vitro cell expansion and cultures. As a way to circumvent the regulatory and technical complexities of in vitro MSC culture, we investigated the use of minced pulp tissues as a source of pulpal MSCs for tissue regeneration. MethodsWe characterized the phenotype of cells explanted from minced pulp (MP), namely MP-derived MSCs (MP-MSCs), compared with dental pulp stem cells (DPSCs) established from pulp tissues by enzyme digestion. Phenotypic characterization included replication kinetics, immunophenotyping, and multilineage differentiation. Using the tooth slice model, we assessed odonto/osteogenic differentiation of DPSCs, MP-MSCs, and minced pulp tissues in situ. ResultsIn vitro replication of MP-MSCs occurred more rapidly during the initial phase of subcultures compared with DPSCs; however, MP-MSCs arrived at senescence at population doubling 47, whereas DPSCs replicated until population doubling 64, indicating shorter replicative lifespan. MP-MSCs also demonstrated stronger odonto/osteogenic differentiation than DPSCs by alkaline phosphatase activity and the protein expression. Both MP-MSCs and DPSCs demonstrated odonto/osteogenic and adipogenic differentiation capacities. Both cell types also showed mineralized tissue formation in the tooth slice model. Seeding minced pulp tissue on poly-L-lactic acid scaffold allowed for migration of MP-MSCs from the tissues and odontogenic differentiation with dentin sialophosphoprotein expression in the tooth slice model. ConclusionsThese data indicated that MP may be an alternative source of pulpal MSCs that may allow de novo pulp-dentin regeneration without the need for in vitro culture and expansion.

Aldehyde dehydrogenase activity helps identify a subpopulation of murine adipose-derived stem cells with enhanced adipogenic and osteogenic differentiation potential.

AIMTo identify and characterize functionally distinct subpopulation of adipose-derived stem cells (ADSCs).METHODSADSCs cultured from mouse subcutaneous adipose tissue were sorted fluorescence-activated cell sorter based on aldehyde dehydrogenase (ALDH) activity, a widely used stem cell marker. Differentiation potentials were analyzed by utilizing immunocytofluorescece and its quantitative analysis.RESULTSApproximately 15% of bulk ADSCs showed high ALDH activity in flow cytometric analysis. Although significant difference was not seen in proliferation capacity, the adipogenic and osteogenic differentiation capacity was higher in ALDHHi subpopulations than in ALDHLo. Gene set enrichment analysis revealed that ribosome-related gene sets were enriched in the ALDHHi subpopulation.CONCLUSIONHigh ALDH activity is a useful marker for identifying functionally different subpopulations in murine ADSCs. Additionally, we suggested the importance of ribosome for differentiation of ADSCs by gene set enrichment analysis.

Ultrasound-assisted liposuction provides a source for functional adipose-derived stromal cells

Background aimsRegenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. MethodsAfter lipoaspirate harvest and processing, we sorted for ASCs using fluorescent-assisted cell sorting based on an established surface marker profile (CD34+CD31–CD45–). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. ResultsBoth UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. ConclusionsTaken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications.

The effects of flavonoid compound from Agrimonia pilosa Ledeb on promotting 3T3-L1 preadipocytes differentiation by activating PPARγ partially

This study aimed to investigate the mechanism of intervening type II diabetes by flavonoid compound from Agrimonia pilosa Ledeb in vitro. We found flavonoid compound exhibited moderate peroxisome proliferator-activated receptor gamma agonist activity which was lower than that of rosiglitazone by luciferase reporter assay. 3T3-L1 cell differentiation was induced typically in the presence of various concentrations of flavonoid compound or pioglitazone for assaying insulin sensitization. We found that flavonoid compound effectively promoted 3T3-L1 preadipocyte differentiation and lipid deposition, but its intensity was lower than that of pioglitazone. Quantitative polymerase chain reaction assays showed that flavonoid compound promoted the expression of peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein-α, and sterol regulatory element-binding protein 1 in a dose-dependent manner. As a result, the expressions of Glut4 and adiponectin were significantly enhanced. Additionally, the expressions of AP2 and lipoprotein lipase induced by flavonoid compound were lower than those by pioglitazone. These findings indicate that flavonoid compound from Agrimonia pilosa Ledeb exhibits the good property of partial peroxisome proliferator-activated receptor gamma agonism, and shares similar insulin-sensitizing effects with pioglitazone, but has lower adipogenic capacity. It is implied that flavonoid compound has a promising prospect of fighting insulin resistance as a partial agonist of peroxisome proliferator-activated receptor gamma.

Botulinum toxin A improves adipose tissue engraftment by promoting cell proliferation, adipogenesis and angiogenesis.

Adipose tissue engraftment has become a well-established therapy in plastic and reconstructive surgery used to restore age-related or injury-related soft tissue loss. However, the unpredictable absorption rates limit its further application. Some clinicians have noted that more optimal aesthetic results are achieved when botulinum toxin A (BoNTA) is applied prior to adipose tissue grafting. In the present study, we transplanted allogeneic adipose tissue treated with or without BoNTA in SD rats in vivo. We subsequently evaluated the survival rate (weight, volume, apoptosis and cellular integrity) and revascularization of the adipose tissue. The results revealed that BoNTA improved the long-term weight and volume retention of the graft, and preserved cellular integrity. BoNTA significantly increased the expression levels of CD31 and vascular endothelial growth factor (VEGF), suggesting enhanced vasodilation and endothelial cell proliferation. In vitro, adipose-derived stem cells (ASCs) were isolated, identified and induced to proliferate and differentiate with or without BoNTA. Furthermore, to evaluate the proliferative, adipogenic and angiogenic ability of the ASCs, CCK-8 assay and Oil Red O staining were conducted. Gene and protein expression levels were analyzed by RT-qPCR and western blot analysis. The results revealed that 8×10−2 U/ml BoNTA as the optimal dose increased ASC proliferation and adipogenic differentiation capacity, as well as the expression level of the key cytokine of angiogenesis. On the whole, our findings indicate that BoNTA improves adipose tissue engraftment and promotes ASC regeneration, which could benefit future clinical applications.

A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield.

Sir: We have read with great interest the article entitled “A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield.”1 In the article, Tevlin et al. developed a new method that allows a higher extraction rate of adipose-derived stem cells with similar differentiation capacities compared with the conventional method.2 We would like to thank the authors for the study because it is increasingly important to develop new techniques that can facilitate the extraction of adipose-derived stem cells for use in cell therapy. Nevertheless, some points raised questions and need some discussion. First, the percentage of adipose-derived stem cells present in the stromal vascular fraction obtained with the “conventional” method seems very low (1.95 percent). Indeed, in our experience and in the literature, the average proportion of adipose-derived stem cells present in the stromal vascular fraction is approximately 30 to 40 percent.3 Moreover, stromal vascular fraction is a heterogeneous mix of cells, and the percentage of immune cells modified the adipogenic differentiation capacities of stromal vascular fraction. Therefore, it would have been interesting to compare the percentage of erythrocytes, endothelial cells, and immune cells to determine whether this new method modified the extraction rate of other cells that could be responsible for the modification of the adipogenic differentiation capacities of the adipose-derived stem cells. Moreover, the authors said that they studied the function of the adipose-derived stem cells isolated with their new methods; however, it seems that they characterized the functions of the adipose-derived stem cells but also the function of all the cells that are present in the stromal vascular fraction. The authors demonstrated that the adipose-derived stem cells extracted with the new method expressed more bone morphogenetic protein (BMP) receptor 1a and 1b. It would be interesting to determine also the expression of BMP receptor 2, receptors of BMP4 and BMP7, inducers of osteogenic differentiation of adipose-derived stem cells. Moreover, it would be interesting to determine whether this new technique isolates a different population of adipose-derived stem cells based on this BMP receptor expression. A minor remark is that the study was realized with a human sample; therefore, it should be important to indicate the number of samples analyzed for this study knowing the interpatient variability. The authors claimed that their new method of stromal vascular fraction extraction is different with regard to two aspects compared with the conventional method. Foremost, this method uses a stronger and quicker shaking during digestion, and the modification of the digestion media. Indeed, the media are enriched with different products and thus it is really difficult to determine which product is responsible for the higher extraction rate. Moreover, the higher percentage of extracted adipose-derived stem cells could be simply linked to the concentration of collagenase, which is three times more important in the new digestion media compared with the conventional one. The development of rapid, simple, and enzyme-free procedures to isolate stromal vascular fraction is the new goal of cell therapy. Therefore, the use of digestion media containing more of the chemical products, even if they seem to not affect cell viability, could be a real issue for clinical use and from the regulatory framework point of view.4,5 DISCLOSURE None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this communication. The authors received no funding support for this work. Jérôme Laloze, M.D.Department of Plastic and Reconstructive SurgeryRangueil University HospitalToulouse, FranceDepartment of Maxillofacial and Reconstructive Surgeryand StomatologyCHU DupuytrenLimoges, France Nicolas Bertheuil, M.D.Department of Plastic, Reconstructive and Aesthetic SurgeryHospital SudUniversity of Rennes 1Rennes, France Julie Usseglio-Grosso, M.D.Department of Maxillofacial and Reconstructive Surgeryand StomatologyCHU DupuytrenLimoges, France Audrey Varin, Ph.D.STROMAlabUMR5273 CNRS/UPS/EFS - INSERM U1031Rangueil University HospitalToulouse, France Benoit Chaput, M.D.Department of Plastic and Reconstructive SurgeryRangueil University HospitalToulouse, France

Diet-induced adipose tissue expansion is mitigated in mice with a targeted inactivation of mesoderm specific transcript (Mest).

Interindividual variation of white adipose tissue (WAT) expression of mesoderm specific transcript (Mest), a paternally-expressed imprinted gene belonging to the α/β-hydrolase fold protein family, becomes apparent among genetically inbred mice fed high fat diet (HFD) and is positively associated with adipose tissue expansion (ATE). To elucidate a role for MEST in ATE, mice were developed with global and adipose tissue inactivation of Mest. Mice with homozygous (MestgKO) and paternal allelic (MestpKO) inactivation of Mest were born at expected Mendelian frequencies, showed no behavioral or physical abnormalities, and did not perturb expression of the Mest locus-derived microRNA miR-335. MestpKO mice fed HFD showed reduced ATE and adipocyte hypertrophy, improved glucose tolerance, and reduced WAT expression of genes associated with hypoxia and inflammation compared to littermate controls. Remarkably, caloric intake and energy expenditure were unchanged between genotypes. Mice with adipose tissue inactivation of Mest were phenotypically similar to MestpKO, supporting a role for WAT MEST in ATE. Global profiling of WAT gene expression of HFD-fed control and MestpKO mice detected few differences between genotypes; nevertheless, genes with reduced expression in MestpKO mice were associated with immune processes and consistent with improved glucose homeostasis. Ear-derived mesenchymal stem cells (EMSC) from MestgKO mice showed no differences in adipogenic differentiation compared to control cells unless challenged by shRNA knockdown of Gpat4, an enzyme that mediates lipid accumulation in adipocytes. Reduced adipogenic capacity of EMSC from MestgKO after Gpat4 knockdown suggests that MEST facilitates lipid accumulation in adipocytes. Our data suggests that reduced diet-induced ATE in MEST-deficient mice diminishes hypoxia and inflammation in WAT leading to improved glucose tolerance and insulin sensitivity. Since inactivation of Mest in mice has minimal additional effects aside from reduction of ATE, an intervention that mitigates MEST function in adipocytes is a plausible strategy to obviate obesity and type-2-diabetes.

OP-32 - 4-Hydroxynonenal as a biomarker of redox regulation in pathophysiology of metabolic stress

While pathophysiology of metabolic stress has yet to be revealed, it seems that obesity-associated ectopic fat deposition and increased risk of insulin resistance can result from impaired fat accumulation associated with oxidative stress in the adipose tissue. Therefore, our recent studies analyzed potential roles of 4-hydroxynonenal (HNE) on adipogenesis in respect to effects of metformin and insulin. The presence of HNE-protein adducts was assessed in subcutaneous (SC) and omental (OM) adipose tissues obtained from obese individuals along with its endogenous accumulation in proliferating and differentiating preadipocytes derived from these tissues, while the impact of HNE on preadipocyte redox homeostasis, proliferation, adipogenic capacity and signaling pathways were determined in vitro. Majority of HNE-protein adducts accumulated in adipocytes and blood vessels, while exposure of preadipocytes to HNE inhibited their growth increasig H2O2, CAT, PRX2, TRX1, SOD1 and SOD2. The HNE also up-regulated anti-adipogenic FABP4 and down-regulated pro-adipogenic genes SREBF1 and FASN, causing dephosphorylation of IRS-1 and p70S6K, while activating GSK3α/β and BAD. Therefore, HNE could play important roles in proliferation and differentiation of preadipocytes and in metabolic dysfunction of adipose tissue, while it seems that insulin and metformin might reverse the HNE impaired adipogenesis.

Gestational Diabetes Affects the Growth and Functions of Perivascular Stem Cells.

Gestational diabetes mellitus (GDM), one of the common metabolic disorders of pregnancy, leads to functional alterations in various cells including stem cells as well as some abnormalities in fetal development. Perivascular stem cells (PVCs) have gained more attention in recent years, for the treatment of various diseases. However, the effect of GDM on PVC function has not been investigated. In our study, we isolated PVCs from umbilical cord of normal pregnant women and GDM patients and compared their phenotypes and function. There is no significant difference in phenotypic expression, response to bFGF exposure and adipogenic differentiation capacity between normal (N)-PVCs and GDM-PVCs. However, when compared with N-PVCs, early passage GDM-PVCs displayed decreased initial rates of cell yield and proliferation as well as a reduced ability to promote wound closure. These results suggest that maternal metabolic dysregulation during gestation can alter the function of endogenous multipotent stem cells, which may impact their therapeutic effectiveness.

Microcarriers with Synthetic Hydrogel Surfaces for Stem Cell Expansion.

Microcarriers are scalable support surfaces for cell growth that enable high levels of expansion, and are particularly relevant for expansion of human mesenchymal stem cells (hMSCs). The goal of this study is to develop a poly(ethylene glycol) (PEG)-based microcarrier coating for hMSC expansion. Commercially available microcarriers do not offer customizability of microcarrier surface properties, including elastic modulus and surface cell adhesion ligands. The lab has previously demonstrated that tuning these material properties on PEG-based hydrogels can modulate important cellular growth characteristics, such as cell attachment and expansion, which are important in microcarrier-based culture. Eosin-Y is adsorbed to polystyrene microcarriers and used as a photoinitiator for thiol-ene polymerization under visible light. Resultant PEG coatings are over 100 µm thick and localized to microcarrier surfaces. This thickness is relevant for cells to react to mechanical properties of the hydrogel coating, and coated microcarriers support hMSC attachment and expansion. hMSC expansion is highly favorable on coated microcarriers in serum-free media, with doubling times under 25 h in the growth phase, and retained osteogenic and adipogenic differentiation capacity after culture on microcarriers. These microcarriers with defined, synthetic coatings enable tailorable surfaces for cell expansion that may be suitable for a variety of biomanufacturing applications.