Inflammatory mediators orchestrate the host immune and metabolic response to acute bacterial infections and mediate the events leading to septic shock. Tumor necrosis factor (TNF) has long been identified as one of the proximal mediators of endotoxin action. Recent studies have implicated peroxisome proliferator-activated receptor alpha (PPARα) as a potential target to modulate regulation of the immune response. Since PPARα activators, which are hypolipidemic drugs, are being prescribed for a significant population of older patients, it is important to determine the impact of these drugs on the host response to acute inflammation. Therefore, we examined the role of PPARα activators on the regulation of TNF expression in a mouse model of endotoxemia. CD-1 mice treated with dietary fenofibrate or Wy-14,643 had fivefold-higher lipopolysaccharide (LPS)-induced TNF plasma levels than LPS-treated control-fed animals. Higher LPS-induced TNF levels in drug-fed animals were reflected physiologically in significantly lower glucose levels in plasma and a significantly lower 50% lethal dose than those in LPS-treated control-fed animals. Utilizing PPARα wild-type (WT) and knockout (KO) mice, we showed that the effect of fenofibrate on LPS-induced TNF expression was indeed mediated by PPARα. PPARα WT mice fed fenofibrate also had a fivefold increase in LPS-induced TNF levels in plasma compared to control-fed animals. However, LPS-induced TNF levels were significantly decreased and glucose levels in plasma were significantly increased in PPARα KO mice fed fenofibrate compared to those in control-fed animals. Data from peritoneal macrophage studies indicate that Wy-14,643 modestly decreased TNF expression in vitro. Similarly, overexpression of PPARα in 293T cells decreased activity of a human TNF promoter-luciferase construct. The results from these studies suggest that any anti-inflammatory activity of PPARα in vivo can be masked by other systemic effects of PPARα activators.
Biological aging alters the metabolism and volume of adipose tissue depots. Recent evidence suggests that circadian mechanisms play a role in promoting adipogenesis, obesity, and lipodystrophy. The current study compared cohorts of younger (5–9 months) and older (24–28 months) C57BL/6 mice as a function of biological age and circadian time. Advanced age significantly reduced the weight of the brown, epididymal, inguinal, and retroperitoneal adipose depots but not total body weight. The older mice reduced their physical activity by >50% and delayed their activity initiation after light offset. The expressed transcriptome in brown and white adipose depots and liver of both cohorts displayed evidence of circadian rhythmicity; however, the oscillating mRNAs differed significantly between age groups and across tissues. The amplitude of Cry1, a component of the negative arm of the circadian apparatus, and downstream regulators such as Rev-erbα were elevated in the older relative to the younger cohorts as a function of circadian time. Overall, transcript levels differed significantly for 557 (inguinal adipose), 1,016 (liver), and 1,021 (brown adipose) expressed sequences between the cohorts as a function of age. These included transcripts encoding proteins within the canonical and non-canonical Wnt pathways. Since the Wnt pathway regulates adipose stem cell differentiation and shares a critical enzyme, glycogen synthase kinase 3β, with the circadian mechanism, the intersection between these two fundamental regulatory mechanisms merits further investigation with respect to biological aging of adipose tissues.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-012-9389-7) contains supplementary material, which is available to authorized users.
Brown adipose; Circadian; Liver; Oscillation; Transcriptomics; White adipose
Adipose-derived adult stem cells (ASCs), bone marrow mesenchymal stem cells (bmMSCs), and human umbilical cord perivascular cells (HUCPVCs) tissue have been widely tested for regenerative applications, such as bone regeneration. Moreover, olfactory ensheathing cells (OECs) show promise in promoting spinal cord injury (SCI) regeneration. Our group recently proposed the use of a hybrid scaffold targeting both vertebral bone repair and SCI regeneration. According to this concept, both MSCs and OECs should be in close contact to be influenced by the factors that are involved in secretion. For this reason, here we studied the effects of the OEC secretome on the metabolic activity and proliferation of ASCs, bmMSCs, and HUCPVCs. The stem cells' secretome effects on metabolic activity and proliferation of the OECs were also considered. In co-cultures of OECs with ASCs, bmMSCs, or HUCPVCs, the metabolic activity/viability, proliferation, and total cell numbers were measured after 2 and 7 days of culture. The results demonstrated that the secretome of OECs has a positive effect on the metabolic activity and proliferation of MSCs from different origins, especially on ASCs. Furthermore, in general, the stem cells' secretome also had a positive effect on the OECs behavior, particularly when ASCs were in co-culture with OECs. These results suggest that the most suitable combination of cells to be used in our hybrid scaffold is the OECs with the ASCs. Finally, this work adds new knowledge to the cell therapy field, bringing new information about paracrine interactions between OECs and distinct mesenchymal stems.
Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population.
Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature.
In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction.
The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters.
adipose-derived stromal/stem cells; adipose tissue; characterization; function; phenotype; stromal vascular fraction
Fat grafting is used to restore breast defects after surgical resection of breast tumors. Supplementing fat grafts with adipose tissue-derived stromal/stem cells (ASCs) is proposed to improve the regenerative/restorative ability of the graft and retention. However, long term safety for ASC grafting in proximity of residual breast cancer cells is unknown. The objective of this study was to determine the impact of human ASCs derived from abdominal lipoaspirates of three donors, on a human breast cancer model that exhibits early metastasis.
Human MDA-MB-231 breast cancer cells represents “triple negative” breast cancer that exhibits early micrometastasis to multiple mouse organs . Human ASCs were derived from abdominal adipose tissue from three healthy female donors. Indirect co-culture of MDA-MB-231 cells with ASCs, as well as direct co-culture demonstrated that ASCs had no effect on MDA-MB-231 growth. Indirect co-culture, and ASC conditioned medium (CM) stimulated migration of MDA-MB-231 cells. ASC/RFP cells from two donors co-injected with MDA-MB-231/GFP cells exhibited a donor effect for stimulation of primary tumor xenografts. Both ASC donors stimulated metastasis. ASC/RFP cells were viable, and integrated with MDA-MB-231/GFP cells in the tumor. Tumors from the co-injection group of one ASC donor exhibited elevated vimentin, matrix metalloproteinase-9 (MMP-9), IL-8, VEGF and microvessel density. The co-injection group exhibited visible metastases to the lung/liver and enlarged spleen not evident in mice injected with MDA-MB-231/GFP alone. Quantitation of the total area of GFP fluorescence and human chromosome 17 DNA in mouse organs, H&E stained paraffin sections and fluorescent microscopy confirmed multi-focal metastases to lung/liver/spleen in the co-injection group without evidence of ASC/RFP cells.
Human ASCs derived from abdominal lipoaspirates of two donors stimulated metastasis of MDA-MB-231 breast tumor xenografts to multiple mouse organs. MDA-MB-231 tumors co-injected with ASCs from one donor exhibited partial EMT, expression of MMP-9, and increased angiogenesis.
Several studies have documented relationships between adipose tissue and bone mineral density (BMD); however, the degree to which there are racial differences in this relationship is not known. The purpose of this study was to examine the relationships between abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) and BMD among white and African American adults. The sample included 330 white women, 328 African American women, 307 white men, and 116 African American men 18–74 years of age. Dual-energy X-ray absorptiometry scans were used to measure BMD and computed tomography scans were used to measure abdominal VAT and SAT. Linear regression was used to assess the relationships between abdominal adiposity and BMD and to explore possible sex and race differences in the associations. In the total sample as well as in all sex-by-race groups, VAT and SAT were negatively related to BMD, after adjustment for lean body mass (LBM) and several covariates. The VAT model (including covariates) explained 33.3% of the variance in BMD and the SAT model (including covariates) explained 32.7% of the variance in BMD. Being African American, being male, and having high LBM were all associated with higher BMD. Race and sex interactions were not significant, indicating that the relationships were similar across race and sex groups. In conclusion, BMD was inversely related to abdominal VAT and SAT in white and African American adults after adjustment for LBM.
Obesity; Osteoporosis; Race/ethnicity; Gender; Adiposity
Until recently, the complexity of adipose tissue and its physiological role was not well appreciated. This changed with the discovery of adipokines such as leptin. The cellular composition of adipose tissue is heterogeneous and changes as a function of diabetes and disease states such as diabetes. Tissue engineers view adipose tissue as a rich source of adult stromal/stem cells isolated by collagenase digestion. In vitro and in vivo studies have documented that adipose stromal/stem cells are multipotent, with the ability to differentiate along the adipocyte, chondrocyte, osteoblast and other lineage pathways. The adipose stromal/stem cells secrete a wide range of cytokines and growth factors with potential paracrine actions. Furthermore, adipose stromal/stem cells exert immunomodulatory functions when added to mixed lymphocyte reactions, suggesting that they can be transplanted allogeneically. This review article focuses on these mechanisms of adipose stromal/stem cell action and their potential utility as cellular therapeutics.
Adipokine, Adipose Stromal/stem Cells; Experimental Autoimmune Encephalitis; Hematopoietic Stem and Progenitor Cell; Mesenchymal Stem Cell or Multipotent Stromal Cell; Progressive Osseous Heteroplasia
Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources—bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP+ cells 48 hr post-transfection, 58±7.1% and 54±3.8%, respectively, versus 33±4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP+ cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.
Boura and colleagues compare liposome-mediated gene transfer efficiencies to human mesenchymal stem cells (MSC) derived from bone marrow (BM MSC), adipose tissue (ASC), or umbilical cord matrix (UCM MSC). MSC isolated from BM and UCM were more amenable to genetic modification when compared to ASC. Modified cells from all sources maintained transgene expression for 10 days and retained their characteristic immunophenotypic profile and multilineage differentiation capacity.
Multiple sclerosis (MS), characterized by chronic inflammation, demyelination, and axonal damage, is a complicated neurological disease of the human central nervous system. Recent interest in adipose stromal/stem cell (ASCs) for the treatment of CNS diseases has promoted further investigation in order to identify the most suitable ASCs. To investigate whether MS affects the biologic properties of ASCs and whether autologous ASCs from MS-affected sources could serve as an effective source for stem cell therapy, cells were isolated from subcutaneous inguinal fat pads of mice with established experimental autoimmune encephalomyelitis (EAE), a murine model of MS. ASCs from EAE mice and their syngeneic wild-type mice were cultured, expanded, and characterized for their cell morphology, surface antigen expression, osteogenic and adipogenic differentiation, colony forming units, and inflammatory cytokine and chemokine levels in vitro. Furthermore, the therapeutic efficacy of the cells was assessed in vivo by transplantation into EAE mice. The results indicated that the ASCs from EAE mice displayed a normal phenotype, typical MSC surface antigen expression, and in vitro osteogenic and adipogenic differentiation capacity, while their osteogenic differentiation capacity was reduced in comparison with their unafflicted control mice. The ASCs from EAE mice also demonstrated increased expression of pro-inflammatory cytokines and chemokines, specifically an elevation in the expression of monocyte chemoattractant protein-1 and keratin chemoattractant. In vivo, infusion of wild type ASCs significantly ameliorate the disease course, autoimmune mediated demyelination and cell infiltration through the regulation of the inflammatory responses, however, mice treated with autologous ASCs showed no therapeutic improvement on the disease progression.
While administration of ex vivo culture-expanded stem cells has been used to study immunosuppressive mechanisms in multiple models of autoimmune diseases, less is known about the uncultured, nonexpanded stromal vascular fraction (SVF)-based therapy. The SVF is composed of a heterogeneous population of cells and has been used clinically to treat acute and chronic diseases, alleviating symptoms in a range of tissues and organs.
In this study, the ability of human SVF cells was compared with culture-expanded adipose stem cells (ASCs) and bone-derived marrow stromal cells (BMSCs) as a treatment of myelin oligodendrocyte glycoprotein (35–55)-induced experimental autoimmune encephalitis in C57Bl/6J mice, a well-studied multiple sclerosis model (MS). A total of 1 × 106 BMSCs, ASCs, or SVF cells were administered intraperitoneally concomitantly with the induction of disease. Mice were monitored daily for clinical signs of disease by three independent, blinded investigators and rated on a scale of 0 to 5. Spinal cords were obtained after euthanasia at day 30 and processed for histological staining using luxol fast blue, toluidine blue, and hematoxylin and eosin to measure myelin and infiltrating immune cells. Blood was collected from mice at day 30 and analyzed by enzyme-linked immunosorbent assay to measure serum levels of inflammatory cytokines.
The data indicate that intraperitoneal administration of all cell types significantly ameliorates the severity of disease. Furthermore, the data also demonstrate, for the first time, that the SVF was as effective as the more commonly cultured BMSCs and ASCs in an MS model. All cell therapies also demonstrated a similar reduction in tissue damage, inflammatory infiltrates, and sera levels of IFNγ and IL-12. While IFNγ levels were reduced to comparable levels between treatment groups, levels of IL-12 were significantly lower in SVF-treated than BMSC-treated or ASC-treated mice.
Based on these data, it is evident that SVF cells have relevant therapeutic potential in an animal model of chronic MS and might represent a valuable tool for stem cell-based therapy in chronic inflammatory disease of the central nervous system. SVF offers advantages of direct and rapid isolation procedure in a xenobiotic-free environment.
Bone marrow-derived mesenchymal stromal cells (BMSCs) are a cell population of intense exploration for therapeutic use in inflammatory diseases. Secreted factors released by BMSCs are responsible for the resolution of inflammation in several pre-clinical models. New studies have uncovered that adipose tissue also serves as a reservoir of multipotent, non-hematopoietic stem cells, termed adipose-derived stromal/stem cells (ASCs), with many common characteristics to BMSCs. We hypothesized that ASC and BMSC secreted factors would lead to a comparable benefit in the context of generalized inflammation.
Proteomic profiling of conditioned media revealed that BMSCs express significantly higher levels of sVEGFR1 and sTNFR1, two soluble cytokine receptors with known therapeutic activity in sepsis. In a prophylactic study of endotoxin-induced inflammation in mice, we observed that BMSC secreted factors provided a greater survival benefit and tissue protection of endotoxemic mice compared to ASCs. Neutralization of sVEGFR1 and sTNFR1 did not significantly affect the survival benefit experienced by mice treated with BMSC secreted factors.
Our findings suggest that BMSCs may be more effective as a cell therapeutic for use in endotoxic shock and that ASCs may be positioned for continued exploration in immunomodulatory diseases. Soluble cytokine receptors can distinguish stromal cells from different tissue origins, though they may not be the sole contributors to the therapeutic benefit of BMSCs. Furthermore, other secreted factors not discussed in this study may also differentiate these stromal cell populations from one another.
Soluble receptors; Mesenchymal stem cells; Adipose stem cells; Endotoxic shock; Tissue necrosis factor alpha (TNF-α); Vascular endothelial growth factor (VEGF)
Adipose-derived stromal/stem cells (ASCs) are a promising cell source for vascular-based approaches to clinical therapeutics, as they have been shown to give rise to both endothelial and perivascular cells. While it is well known that ASCs can present a heterogeneous phenotypic profile, spontaneous interactions among these subpopulations that result in the formation of complex tissue structures have not been rigorously demonstrated. Our study reports the novel finding that ASCs grown in monolayers in the presence of angiogenic cues are capable of self-assembling into complex, three-dimensional vascular structures. This phenomenon is only apparent when the ASCs are seeded at a high density (20,000 cells/cm2) and occur through orchestrated interactions among three distinct subpopulations: CD31-positive cells (CD31+), α-smooth muscle actin-positive cells (αSMA+), and cells that are unstained for both these markers (CD31−/αSMA−). Investigations into the kinetics of the process revealed that endothelial vessel-like structures initially arose from individual CD31+ cells through proliferation and their interactions with CD31−/αSMA− cells. During this period, αSMA+ cells proliferated and appeared to migrate toward the vessel structures, eventually engaging in cell-cell contact with them after 1 week. By 2 weeks, the lumen-containing CD31+ vessels grew greater than a millimeter in length, were lined with vascular basement membrane proteins, and were encased within a dense, three-dimensional cluster of αSMA+ and CD31−/αSMA− cells. The recruitment of αSMA+ cells was largely due to platelet-derived growth factor (PDGF) signaling, as the inhibition of PDGF receptors substantially reduced αSMA+ cell growth and vessel coverage. Additionally, we found that while hypoxia increased endothelial gene expression and vessel width, it also inhibited the growth of the αSMA+ population. Together, these findings underscore the potential use of ASCs in forming mature vessels in vitro as well as the need for a further understanding of the heterotypic interactions among ASC subpopulations.
Obesity has been associated with increased incidence and mortality of breast cancer. While the precise correlation between obesity and breast cancer remains to be determined, recent studies suggest that adipose tissue and adipose stem cells (ASCs) influence breast cancer tumorigenesis and tumor progression.
Breast cancer cells lines were co-cultured with ASCs (n = 24), categorized based on tissue site of origin and body mass index (BMI), and assessed for enhanced proliferation, alterations in gene expression profile with PCR arrays, and enhanced tumorigenesis in immunocompromised mice. The gene expression profile of ASCs was assess with PCR arrays and qRT-PCR and confirmed with Western blot analysis. Inhibitory studies were conducted by delivering estrogen antagonist ICI182,780, leptin neutralizing antibody, or aromatase inhibitor letrozole and assessing breast cancer cell proliferation. To assess the role of leptin in human breast cancers, Oncomine and Kaplan Meier plot analyses were conducted.
ASCs derived from the abdominal subcutaneous adipose tissue of obese subjects (BMI > 30) enhanced breast cancer cell proliferation in vitro and tumorigenicity in vivo. These findings were correlated with changes in the gene expression profile of breast cancer cells after co-culturing with ASCs, particularly in estrogen receptor-alpha (ESR1) and progesterone receptor (PGR) expression. Analysis of the gene expression profile of the four groups of ASCs revealed obesity induced alterations in several key genes, including leptin (LEP). Blocking estrogen signaling with ICI182,780, leptin neutralizing antibody, or letrozole diminished the impact of ASCs derived from obese subjects. Women diagnosed with estrogen receptor/progesterone receptor positive (ER+/PR+) breast cancers that also expressed high levels of leptin had poorer prognosis than women with low leptin expression.
ASCs isolated from the abdomen of obese subjects demonstrated increased expression of leptin, through estrogen stimulation, which increased breast cancer cell proliferation. The results from this study demonstrate that abdominal obesity induces significant changes in the biological properties of ASCs and that these alterations enhance ER+/PR+ breast cancer tumorigenesis through estrogen dependent pathways.
The thermoresponsive behavior of a Methylcellulose (MC) polymer was systematically investigated to determine its usability in constructing MC based hydrogel systems in cell sheet engineering applications. Solution-gel analyses were made to study the effects of polymer concentration, molecular weight and dissolved salts on the gelation of three commercially available MCs using differential scanning calorimeter and rheology. For investigation of the hydrogel stability and fluid uptake capacity, swelling and degradation experiments were performed with the hydrogel system exposed to cell culture solutions at incubation temperature for several days. From these experiments, the optimal composition of MC-water-salt that was able to produce stable hydrogels at or above 32 °C, was found to be 12% to 16% of MC (Mol. wt. of 15,000) in water with 0.5× PBS (~150mOsm). This stable hydrogel system was then evaluated for a week for its efficacy to support the adhesion and growth of specific cells in culture; in our case the stromal/stem cells derived from human adipose tissue derived stem cells (ASCs). The results indicated that the addition (evenly spread) of ~200 µL of 2 mg/mL bovine collagen type -I (pH adjusted to 7.5) over the MC hydrogel surface at 37 °C is required to improve the ASC adhesion and proliferation. Upon conﬂuence, a continuous monolayer ASC sheet was formed on the surface of the hydrogel system and an intact cell sheet with preserved cell–cell and cell–extracellular matrix was spontaneously and gradually detached when the grown cell sheet was removed from the incubator and exposed to room temperature (~30 °C) within minutes.
cell sheet engineering; temperature-responsive polymers; adult stem cells; scaffolds
Obesity is associated with a higher risk of developing cancer and co-morbidities that are part of the metabolic syndrome. Adipose tissue is recognized as an endocrine organ, as it affects a number of physiological functions, and contains adipose tissue-derived stem cells (ASCs). ASCs can differentiate into cells of multiple lineages, and as such are applicable to tissue engineering and regenerative medicine. Yet the question of whether ASC functionality is affected by the donor’s body mass index (BMI) still exists.
ASCs were isolated from patients having different BMIs (BMI-ASCs), within the ranges of 18.5-32.8. It was hypothesized that overweight BMI-ASCs would be more compromised in early adipogenic and osteogenic potential, and ability to form colonies in vitro. BMI was inversely correlated with ASC proliferation and colony forming potential as assessed by CyQUANT proliferation assay (fluorescence- based measurement of cellular DNA content), and colony forming assays. BMI was positively correlated with early time point (day 7) but not later time point (day 15) intracytoplasmic lipid accumulation as assessed by Oil-Red-O staining. Alizarin red staining and RT-PCR for alkaline phosphatase demonstrated that elevated BMI resulted in compromised ASC mineralization of extracellular matrix and decreased alkaline phosphatase mRNA expression.
These data demonstrate that elevated BMI resulted in reduced ASC proliferation, and potentially compromised osteogenic capacity in vitro; thus BMI is an important criterion to consider in selecting ASC donors for clinical applications.
Adipose stromal/stem cells (ASCs); Body mass index (BMI); Osteogenesis; Proliferation; Colony formation; Cell size
The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic agents for a wide range of diseases. However, the highly demanding cell doses used in MSC clinical trials (up to millions of cells/kg patient) currently require labor intensive methods and incur high reagent costs. Moreover, the use of xenogenic (xeno) serum-containing media represents a risk of contamination and raises safety concerns. Bioreactor systems in combination with novel xeno-free medium formulations represent a viable alternative to reproducibly achieve a safe and reliable MSC doses relevant for cell therapy. The main goal of the present study was to develop a complete xeno-free microcarrier-based culture system for the efficient expansion of human MSC from two different sources, human bone marrow (BM), and adipose tissue. After 14 days of culture in spinner flasks, BM MSC reached a maximum cell density of (2.0±0.2)×105 cells·mL−1 (18±1-fold increase), whereas adipose tissue-derived stem cells expanded to (1.4±0.5)×105 cells·mL−1 (14±7-fold increase). After the expansion, MSC expressed the characteristic markers CD73, CD90, and CD105, whereas negative for CD80 and human leukocyte antigen (HLA)-DR. Expanded cells maintained the ability to differentiate robustly into osteoblast, adipocyte, and chondroblast lineages upon directed differentiation. These results demonstrated the feasibility of expanding human MSC in a scalable microcarrier-based stirred culture system under xeno-free conditions and represent an important step forward for the implementation of a Good Manufacturing Practices–compliant large-scale production system of MSC for cellular therapy.
Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and demonstrated ability to support bone formation, in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASC), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol - HFIP), pore sizes (250–500μm vs. 500–1000μm) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes with decellularized trabecular bone (DCB) included as a “gold standard”. The scaffolds were seeded with hASC and cultured for 7 weeks in osteogenic media. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400–600μm porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2, and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.
Bone; Tissue engineering; Silk; Scaffold; Adipose stem cells
Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis.
Adipogenesis; Adipose-derived Stem Cells; Botanical Extracts; Lipogenesis; Lipolysis; Subcutaneous; Visceral
Krabbe disease, also known as globoid cell leukodystrophy, is an autosomal recessive neurodegenerative disease caused by the genetic deficiency of galactocerebrosidase (GALC), a lysosomal enzyme responsible for the degradation of several glycosphingolipids like psychosine and galactosylceramide. In order to investigate whether GALC deficiency in Krabbe disease affects adipose-derived stromal/stem cell (ASC) properties and if the ASCs could be used as a source of autologous stem cell therapy for patients with Krabbe disease, ASCs isolated from subcutaneous adipose tissue of Twitcher mice (a murine model of Krabbe disease) and their normal wild type littermates were cultured, expanded, and characterized for their cell morphology, surface antigen expression, osteogenic and adipogenic differentiation, colony forming units, growth kinetics, and immune regulatory capacities in vitro.
ASCs from Twitcher mice (TwiASCs), when compared to ASCs from normal mice (WtASCs), have a reduced osteogenic differentiation potential, have less self-replicating and proliferative capacity, although they have the same fibroblast morphologies and cell sizes. However, surprisingly, the TwiASCs demonstrated similar immune-suppressive capacities as their counterparts WtASCs did when they were transwell co-cultured with macrophages in vitro.
This study reveals that Twitcher ASCs exhibit differences in the biologic potential when compared to their counterparts from normal mice. The changes in Twitcher ASCs may be influenced by the GALC deficiency in Twitcher mice. Nevertheless, none of the changes preclude the use of the TwiASCs for autologous applications.
Adipose stem cells; ASCs; Krabbe disease; Twitcher mice; Autologous transplantation
We report engineering of half centimeter size bone constructs created in vitro using human Adipose-derived Stem Cells (hASC), decellularized bone scaffolds and perfusion bioreactors. The hASCs are easily accessible, can be used in an autologous fashion, are rapidly expanded in culture, and are capable of osteogenic differentiation. hASCs from four donors were characterized for their osteogenic capacity, and one representative cell population was used for tissue engineering experiments. Culture-expanded hASCs were seeded on fully decellularized native bone scaffolds (4 mm Ø × 4 mm) providing the necessary structural and mechanical environment for osteogenic differentiation, and cultured in bioreactors with medium perfusion. The interstitial flow velocity was set to a level necessary to maintain cell viability and function throughout the construct volume (400 μs−1), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-β-glycerophosphate, ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, bone osteopontin). Medium perfusion markedly improved the distribution of cells and bone matrix in engineered constructs. In summary, a combination of hASCs, decellularized bone scaffold, perfusion culture and osteogenic supplements resulted in the formation of compact and viable bone tissue constructs.
Adipose-derived human stem cells; perfusion; bioreactor; scaffold; osteogenesis; tissue engineering
The objective of this study was to determine the tissue density, in vitro expansion and differentiation of canine adipose tissue-derived (ASC) and bone marrow-derived (BMSC) stromal cells. Primary (P0) and cell passages 1–6 (P1–6) cell doubling numbers (CD) and doubling times (DT) were determined in fresh cells. The P0, P3, and P6 adipogenic (CFU-Ad), osteogenic (CFU-Ob), and fibroblastic (CFU-F) colony forming unit frequencies, lineage specific mRNA levels in differentiated P3 cells and composition of P3 and P6 chondrogenic pellets were assessed in cryogenically preserved cells.
Cell yields from bone marrow were significantly higher than adipose tissue. Overall ASC and BMSC CDs and DTs and P3 and P6 CFU-F, CFU-Ad, and CFU-Ob were comparable. The P0 BMSC CFU-Ob was significantly higher than ASC. Lineage specific mRNA levels were higher in differentiated versus control cells, but similar between cell types. Protein was significantly greater in P3 versus P6 ASC chondrogenic pellets. Based on these findings, fresh and revitalized canine ASCs are viable alternatives to BMSCs for stromal cell applications.
Canine; Multipotent stromal cells; Bone; Adipose tissue; Cryopreservation
Adipose-derived stem cells (ASCs) have emerged as important regulators of inflammatory/immune responses in vitro and in vivo and represent attractive candidates for cell-based therapies for diseases that involve excessive inflammation. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive due to lack of effective therapies. In this study, the therapeutic effects of ASC-based therapy were assessed in vivo by comparison of the anti-inflammatory properties of both human and murine ASCs in a mouse model of lipopolysaccharide (LPS)-induced ALI.
Human ASCs (hASCs) or mouse ASCs (mASCs) were delivered to C57Bl/6 mice (7.5 × 105 total cells/mouse) by oropharyngeal aspiration (OA) four hours after the animals were challenged with lipopolysaccharide (15 mg/kg). Mice were sacrificed 24 and 72 hours after LPS exposure, and lung histology examined for evaluation of inflammation and injury. Bronchoalveolar lavage fluid (BALF) was analyzed to determine total and differential cell counts, total protein and albumin concentrations, and myeloperoxidase (MPO) activity. Cytokine expression in the injured lungs was measured at the steady-state mRNA levels and protein levels for assessment of the degree of lung inflammation.
Both human and mouse ASC treatments provided protective anti-inflammatory responses. There were decreased levels of leukocyte (for example neutrophil) migration into the alveoli, total protein and albumin concentrations in BALF, and MPO activity after the induction of ALI following both therapies. Additionally, cell therapy with both cell types effectively suppressed the expression of proinflammatory cytokines and increased the anti-inflammatory cytokine interleukin 10 (IL-10). Overall, the syngeneic mASC therapy had a more potent therapeutic effect than the xenogeneic hASC therapy in this model.
Treatment with hASCs or mASCs significantly attenuated LPS-induced acute lung injury in mice. These results suggest a potential benefit for using an ASC-based therapy to treat clinical ALI and may possibly prevent the development of acute respiratory distress syndrome (ARDS).
Stem Cell Antigen-1 (Sca-1) is a member of the lymphocyte-activated protein 6 family and has served as a marker for the identification of stem cells in various tissues, including fat depots. In vitro and in vivo studies suggest the possible involvement of Sca-1 in adipogenic differentiation and link Sca-1 antigenicity with adipocyte progenitors. Previously, we showed that Sca-1-enriched populations of ear mesenchymal stem cells possess enhanced capacity to differentiate into adipocytes. Additionally, we determined the natural frequency and localization of Sca-1-positive progenitor/stem cells in brown and white fat in situ. The present study addressed the question whether Sca-1 deficiency alters the white adipose tissue response to a high-saturated-fat diet. Our results show that Sca-1 null mice (Sca-1–/–) fed high-fat diet developed obesity equally well as wild-type mice, suggesting either an indirect in vivo effect of Sca-1 or a compensatory response to Sca-1 deficiency. However, contrary to wild-type mice, high fat diet-fed Sca-1–/– mice showed no alterations in serum adipocytokines. The data lead to the conclusion that Sca-1 is either redundant or a nonessential marker of adipose progenitor/stem cells. Nevertheless, since Sca-1-deficient mice displayed elevated blood glucose at fasting and exhibited glucose intolerance and insulin resistance, Sca-1 has subtle effects on adipose function. Thus, the Sca-1-deficient mice may provide a useful model for metabolic studies.