Adipose-derived Stem Cells Population Research Articles

Distinct subpopulations of human subcutaneous adipose tissue precursor cells revealed by single-cell RNA sequencing.

Adipose-derived stem cells (ADSCs) play an important role in the differential capacity for excess energy storage between upper body abdominal (ABD) adipose tissue (AT) and lower body gluteofemoral (GF) AT. We cultured ADSCs from subcutaneous ABD AT and GF AT isolated from eight women with differential body fat distribution and performed single-cell RNA sequencing. Six populations of ADSCs were identified and segregated according to their anatomical origin. The three ADSC subpopulations in GF AT were characterized by strong cholesterol/fatty acid (FA) storage and proliferation signatures. The two ABD subpopulations, differentiated by higher expression of committed preadipocyte marker genes, were set apart by differential expression of extracellular matrix and ribosomal genes. The last population, identified in both depots, was similar to smooth muscle cells and when individually isolated and cultured in vitro they differentiated less than the other subpopulations. This work provides important insight into the use of ADSC as an in vitro model of adipogenesis and suggests that specific subpopulations of GF-ADSCs contribute to the more robust capacity for GF-AT to expand and grow compared with ABD-AT in women.NEW & NOTEWORTHY Identification of distinct subpopulations of adipose-derived stem cells (ADSCs) in upper body abdominal subcutaneous (ABD) and lower body gluteofemoral subcutaneous (GF) adipose tissue depots. In ABD-ADSCs, subpopulations are more committed to adipocyte lineage. GF-ADSC subpopulations are enriched for genes involved in lipids and cholesterol metabolism. Similar depot differences were found in stem cell population identified in freshly isolated stoma vascular fraction. The repertoire of ADSCs subpopulations was different in apple-shaped versus pear-shaped women.

Fisetin Attenuates Cellular Senescence Accumulation During Culture Expansion of Human Adipose-Derived Stem Cells.

Mesenchymal stem cells (MSCs) have long been viewed as a promising therapeutic for musculoskeletal repair. However, regulatory concerns including tumorgenicity, inconsistencies in preparation techniques, donor-to-donor variability, and the accumulation of senescence during culture expansion have hindered the clinical application of MSCs. Senescence is a driving mechanism for MSC dysfunction with advancing age. Often characterized by increased reactive oxygen species, senescence-associated heterochromatin foci, inflammatory cytokine secretion, and reduced proliferative capacity, senescence directly inhibits MSCs efficacy as a therapeutic for musculoskeletal regeneration. Furthermore, autologous delivery of senescent MSCs can further induce disease and aging progression through the secretion of the senescence-associated secretory phenotype (SASP) and mitigate the regenerative potential of MSCs. To alleviate these issues, the use of senolytic agents to selectively clear senescent cell populations has become popular. However, their benefits to attenuating senescence accumulation in human MSCs during the culture expansion process have not yet been elucidated. To address this, we analyzed markers of senescence during the expansion of human primary adipose-derived stem cells (ADSCs), a population of fat-resident MSCs commonly used in regenerative medicine applications. Next, we used the senolytic agent fisetin to determine if we can reduce these markers of senescence within our culture-expanded ADSC populations. Our results indicate that ADSCs acquire common markers of cellular senescence including increased reactive oxygen species, senescence-associated β-galactosidase, and senescence-associated heterochromatin foci. Furthermore, we found that the senolytic agent fisetin works in a dose-dependent manner and selectively attenuates these markers of senescence while maintaining the differentiation potential of the expanded ADSCs.

In Vitro Migration of Porcine Adipose-Derived Stem Cells in Platelet-Rich Plasma

The proliferation and migration of porcine adipose-derived stem cells (ASCs) have only begun to be studied in detail in vitro. The primary aim of these studies was to analyse in vitro ASC migration in varying concentrations of growth factors derived from the platelet-rich fraction of centrifuged plasma, the so-called platelet-rich plasma (PRP). ASCs were hypothesized to migrate at a faster velocity with increasing PRP concentrations. Whole blood was collected with a sodium citrate anticoagulant and subjected to two centrifugations. The first slower spin was to remove red blood cells to collect plasma, and the second faster spin was to collect the fraction with a high platelet concentration. Platelet concentration increased 3.5-fold compared to plasma, within the prescribed therapeutic range. For long-term culture of ASCs, DMEM supplemented with no more than 20% was necessary to maintain cell viability. ASC proliferation rate in PRP-supplemented media was comparable to that in fetal bovine serum (FBS), a standard cell culture media supplement. In addition, the velocity of ASC migration increased in cultures supplemented with 10%, 20% or 30% PRP. Generally, PRP was determined to be a media supplement with similar effects as FBS, potentially making it a suitable substitute for in vitro expansion of ASC populations. These results are likely partly explained by similarities in growth factor concentrations and their effects. Further characterization of PRP will be necessary to tease out the specific growth factor(s) responsible for the increase in swine ASC migration.

CD146 expression regulates osteochondrogenic differentiation of human adipose-derived stem cells.

Tissue engineering aims to develop innovative approaches to repair tissue defects. The use of adipose-derived stem cells (ASCs) in tissue regeneration was extensively investigated for osteochondrogenesis. Among the ASC population, ASCs expressing the CD146 were demonstrated to be multipotent and considered as perivascular stem cells, although the functional role of CD146 expression in these cells remains unclear. Herein, we investigated the influence of CD146 expression on osteochondrogenic differentiation of ASCs. Our results showed that, in two-dimensional culture systems, sorted CD146+ ASCs proliferated less and displayed higher adipogenic and chondrogenic potential than CD146- ASCs. The latter demonstrated a higher osteogenic capacity. Besides this, CD146+ ASCs in three-dimensional Matrigel/endothelial growth medium (EGM) cultures showed the highest angiogenic capability. When cultured in three-dimensional collagen scaffolds, CD146+ ASCs showed a spontaneous chondrogenic differentiation, further enhanced by the EGM medium's addition. Finally, CD146- ASCs seeded on hexafluoroisopropanol silk scaffolds displayed a greater spontaneous osteogenetic capacity. Altogether, these findings demonstrated a functional and relevant influence of CD146 expression in ASC properties and osteochondrogenic commitment. Exploiting the combination of specific differentiation properties of ASC subpopulations and appropriate culture systems could represent a promising strategy to improve the efficacy of new regenerative therapies.

Congenital muscular dystrophy-associated inflammatory chemokines provide axes for effective recruitment of therapeutic adult stem cell into muscles

BackgroundCongenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin α2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1−/− mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue.MethodsProteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations.ResultsComprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2, and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect.ConclusionsCollectively, identified molecules provided insight into the mechanisms governing directional migration and intramuscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment.

Extensive Characterization of Mesenchymal Stem Cell Marker Expression on Freshly Isolated and In Vitro Expanded Human Adipose-Derived Stem Cells from Breast Cancer Patients.

Variation in numbers and functions of cells in fat tissues may affect therapeutic outcomes and adverse events after autologous fat tissue grafting in postmastectomy breast cancer patients; however, the relevant information regarding cellular components is still incomplete. Phenotypic characterization of heterogeneous cell subsets in stromal vascular fraction (SVF) isolated from fat tissues by flow cytometry was also limited to a combination of few molecules. This study, therefore, developed a polychromatic staining panel for an in-depth characterization of freshly isolated SVF and expanded adipose-derived stem cells (ADSC) from the patients. ADSC were found predominant in SVF (~65% of CD45− cells) with a homogenous phenotype of CD13+CD31−CD34+CD45−CD73+CD90+CD105−CD146− (~94% of total ADSC). Endothelial progenitor cells (EPC) and pericytes were minor (~18% and ~11% of CD45− cells, respectively) with large heterogeneity. Downregulation of CD34 and upregulation of CD105 in ADSC were profound at passage 3, showing a phenotype similar to the classical mesenchymal stem cells from the bone marrow. Results from this study demonstrated that fat tissue collected from patients contains ADSC with a highly homogenous phenotype. The in vitro culture of these cells maintained their homogeneity with modified CD34 and CD105 expression, suggesting the expansion from a single population of ADSC.

Multipotent stem cells with neural crest stem cells characteristics exist in bovine adipose tissue

Neural crest stem cells (NCSCs) often referred to as the fourth germ layer, comprise a migratory, stem and progenitor cell population and are synonymous with vertebrate evolution and development. The cells follow specific paths to migrate to different locations of the body where they generate a diverse array of cell types and tissues. There are NCSCs which are maintained in an undifferentiated state throughout the life in the animal tissues. Based on some cells migratory property, we successfully developed a separation strategy to isolate and identify a population of adipose-derived stem cells with neural crest stem cell features in adult bovine adipose tissues within minimally-invasive surgical procedures. The cells have a high degree of multi-potency and self-renewal capabilities, can be cultured and maintained in feeder-free adhesion conditions as monolayer cells, and also be able to grow in the suspension condition in the form of neurosphere. For the purpose of simple description, we name this type cell as bovine adipose-derived neural crest stem cell (baNCSC). Taken together our study describes a readily accessible source of multipotent baNCSC for autologous tissue engineer and cell-based therapeutic researches.

P3495Long-term engraftment of human CD271-positive adipose-derived stem cells with pericytic and less-aged gene profile in a mouse model of hindlimb ischemia

Abstract Background Therapeutic angiogenesis using adipose-derived stem cells (ADSCs) is an attractive strategy for ischemic cardiovascular diseases. We previously reported that human CD271+ population of adipose-derived stem cells (ADSCs) promoted neovascularization with enhanced engraftment in a mouse model of hindlimb ischemia. However, whether and how CD271+ ADSCs promote the long-term engraftment is still uncertain. Purpose We aimed to examine whether the angiogenic effect and cell engraftment capacity of CD271+ ADSCs would be sustained in long-term period. Then, comparative gene profiling between CD271+ and CD271- ADSCs were analyzed. Finally, cell proliferation and endothelial differentiation assays were conducted. Methods ADSCs were isolated from subcutaneous adipose tissue of 5 patients received cardiovascular surgery. CD271+ and CD271- ADSCs were sorted from CD45-CD31-CD34+ ADSCs fraction by FACS sorting (Fig. A). Cultured CD271+ and CD271- ADSCs at passage 6 were labeled by PKH26 cell linker dye and used for xenograft experiments. Briefly, athymic nude mice were subjected to hindlimb ischemia and one million of human ADSCs were injected into the ischemic muscles. In control group, PBS was solely injected. At 2 and 5 weeks, neovascularization was evaluated by immunohistochemistry (capillary density using lectin perfusion). Cell engraftment was assessed by counting PKH26-positive cells. Furthermore, we compared gene profiling between CD271+ and CD271- ADSCs by microarray. Proliferative capacity was evaluated by colony-forming unit (CFU) assay with Giemsa staining. In endothelial differentiation assay, CD271+ and CD271- ADSCs were cultured in differentiation induction medium containing vascular endothelial growth factor for 2 weeks and stained with anti-human CD31 antibody. Results Cell therapy using CD271+ ADSCs demonstrated approximately 3-fold more enhanced neovascularization than those using CD271- ADSCs or PBS in histological analysis of capillary density at 2 weeks from cell therapy (Fig. B and C). At 5 weeks, mice treated with CD271+ ADSCs were significantly rescued from limb ischemia and this was accompanied by sustained engraftment of ADSCs (Fig. D). In microarray analysis, the differentially expressed 2167 genes were extracted to classify CD271+ and CD271- ADSCs. Pathway analysis demonstrated CD271 expression on ADSCs was associated with the pathways related to stemness and cell differentiation. Indeed, we found that genes related to cell proliferation (PI3K, Cyclin D, and Cyclin D2) were up-regulated in CD271+ ADSCs. Additionally, we found the pericytic marker nestin which was significantly up-regulated in CD271+ ADSCs. Consistent with these findings, CD271+ ADSCs were more proliferative and capable for endothelial differentiation while CD271- ADSCs were not. FACS and cell therapy experiments Conclusion These results suggest that CD271+ ADSCs possess long-term engraftment and angiogenic capacity due to their less-aged and more pericytic gene profile. Acknowledgement/Funding Japan Society for the Promotion of Science (JSPS) KAKENHI (Tokyo, Japan) Grant Number JP16H06828

Adipose-Derived Stem Cells from Fat Tissue of Breast Cancer Microenvironment Present Altered Adipogenic Differentiation Capabilities.

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell types, including adipocytes, osteoblasts, and chondrocytes. The role of adipose-derived stem cells (ADSCs) in cancers is significantly relevant. They seem to be involved in the promotion of tumour development and progression and relapse processes. For this reason, investigating the effects of breast cancer microenvironment on ADSCs is of high importance in order to understand the relationship between tumour cells and the surrounding stromal cells. With the current study, we aimed to investigate the specific characteristics of human ADSCs isolated from the adipose tissue of breast tumour patients. We compared ADSCs obtained from periumbilical fat (PF) of controls with ADSCs obtained from adipose tissue of breast cancer- (BC-) bearing patients. We analysed the surface antigens and the adipogenic differentiation ability of both ADSC populations. C/EBPδ expression was increased in PF and BC ADSCs induced to differentiate compared to the control while PPARγ and FABP4 expressions were enhanced only in PF ADSCs. Conversely, adiponectin expression was reduced in PF-differentiated ADSCs while it was slightly increased in differentiated BC ADSCs. By means of Oil Red O staining, we further observed an impaired differentiation capability of BC ADSCs. To investigate this aspect more in depth, we evaluated the effect of selective PPARγ activation and nutritional supplementation on the differentiation efficiency of BC ADSCs, noting that it was only with a strong differentiation stimuli that the process took place. Furthermore, we observed no response in BC ADSCs to the PPARγ inhibitor T0070907, showing an impaired activation of this receptor in adipose cells surrounding the breast cancer microenvironment. In conclusion, our study shows an impaired adipogenic differentiation capability in BC ADSCs. This suggests that the tumour microenvironment plays a key role in the modulation of the adipose microenvironment located in the surrounding tissue.

Single-Cell Profiles and Clinically Useful Properties of Human Mesenchymal Stem Cells of Adipose and Bone Marrow Origin

Background: Mesenchymal stem cells (MSCs) can be isolated from various tissues and can present themselves as a promising cell source for cell-based therapies. Although adipose- and bone marrow–derived mesenchymal stem cells have already been used in a considerable number of clinical trials for osteoarthritis treatment, systematic analyses from single- to bulk-cell resolution as well as clinical outcomes of these 2 MSCs are still insufficient. Purpose: To explore the characteristics and differences of adipose-derived stem cells (ADSCs) and bone marrow MSCs (BMSCs) at single- and bulk-cell levels, to study the clinical outcomes of these 2 cells on the treatment of osteoarthritis, and to provide potential guidance on the more precise clinical application of these MSCs. Study Design: Controlled laboratory study and meta-analysis. Methods: Same donor–derived ADSCs and BMSCs were isolated and cultured. Single- and bulk-cell assays were used to identify the characteristics of these 2 cells. Meta-analysis of clinical trials was done to compare the clinical therapeutic effects in osteoarthritis treatment with ADSCs and BMSCs. Results: Single-cell RNA sequencing analysis showed that the population of ADSCs showed lower transcriptomic heterogeneity when compared with BMSCs. Additionally, as compared with BMSCs, ADSCs were less dependent on mitochondrial respiration for energy production. Furthermore, ADSCs had a lower expression level of human leukocyte antigen class I antigen and higher immunosuppression capacity when compared with the BMSC population. Meta-analysis of current clinical trials of osteoarthritis treatment with MSCs consistently showed that ADSCs are more stable than BMSCs in their therapeutic effect. Conclusion: These results provide basic biological insights into human ADSCs and BMSCs at the single-cell resolution. Findings indicated that ADSCs may be a more controllable stem cell source, may be more adaptable to surviving in the hypoxic articular cavity niche, and may exhibit superiority in regulating inflammation. Based on the meta-analysis results of the different characteristics of ADSCs and BMSCs, ADSCs were implicated as being a better cell source for osteoarthritis treatment. Clinical Relevance: These results guide a more precise clinical application of adipose and bone marrow mesenchymal stem cells.

In Vitro Study of Deuterium Effect on Biological Properties of Human Cultured Adipose-Derived Stem Cells.

In current in vitro study we have shown the impact of deuterium content in growth medium on proliferation rate of human cultured adipose-derived stem cells (ADSC). ADSCs have also demonstrated morphological changes when cultured in deuterated growth medium: the cell cultures did not reach confluence but acquired polygonal morphology with pronounced stress fibers. At high deuterium concentrations the ADSCs population doubling time increased which indicated the cell cycle retardation and decrease of cell proliferation rate. The deuterated and deuterium-depleted growth media demonstrated acute and chronic cytotoxicity, respectively. The minimal migration ability was observed in deuterated medium whereas the highest migration activity was observed in the medium with the deuterium content close to natural. The cells in deuterated growth medium demonstrated decrease in metabolic activity after three days in culture. In contrast, in deuterium-depleted medium there was an increase in ADSC metabolic activity.

Phenotypic characteristics of feline adipose-derived stem cells affected by cell passage number.

In this study we evaluated the influence of passage number on the phenotypic characteristics of feline adipose-derived stem cells (ASCs) in order to develop a broader understanding of their dynamics. Feline ASCs were individually isolated from five domestic cats and subjected to proliferative culture at passage (P) 2, 6 and 10. The cells at each sub-passage were examined in regard to their phenotypic properties associated with multipotent mesenchymal stem cells (MSCs), such as morphology, proliferation kinetics, self-renewal, and expression of MSCs-specific surface markers. The differentiation capacity into adipocytes and osteoblasts was also identified. Feline ASCs appeared with a fibroblast-like morphology with minimal alteration through P10. The rate of cell proliferation gradually decreased, while cell doubling time gradually increased with each passage. A significant decrease in CFU-F efficiency was observed with increasing cell passage number. The ASC population uniformly expressed their characteristic markers CD44 and CD90, but did not express the hematopoietic marker CD45. However, MSC markers gradually decreased in the later passage stages. Feline ASCs were capable of undergoing both adipogenesis and osteogenesis at P2. These findings suggested that the phenotypic characteristics of feline ASCs could be affected by long-term passages, which is potentially very important in regard to their therapeutic application.

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

Regulation of ADSC Osteoinductive Potential Using Notch Pathway Inhibition and Gene Rescue: A Potential On/Off Switch for Clinical Applications in Bone Formation and Reconstructive Efforts.

Although there has been tremendous research in the ability of mesenchymal-derived adipose derived stem cells (ADSCs) to form bone, less is known regarding the molecular mechanisms that regulate the osteogenic potential of ADSCs. Notch, which consists of a key family of regulatory ligands involved in bone formation, is expressed in the bone marrow-derived mesenchymal stem cell niche and is critical for proliferation, migration, and ultimately osseous differentiation. The authors investigate how Notch impacts ADSC proliferation and osteogenic differentiation to determine a translatable application of these cells in bone regeneration. Enriched ADSC populations were isolated from tissue and examined for their ability to respond to Notch pathway signaling events. Proliferation, viability, extracellular matrix deposition, and osteoinduction were assessed following Notch activation and inhibition. Notch pathway rescue was conducted using a lentiviral vector encoding a downstream Notch-1 intracellular domain (NICD). Proliferation, osteogenic induction, and the ability to form bone elements were reduced following Notch inhibition (p < 0.05). However, ADSCs, while in the presence of the Notch inhibition, were able to be rescued following lentiviral transduction with NICD, restoring osteogenic potential at both the molecular and cellular functional levels (p < 0.05). These data suggest a potential translatable "on/off switch," using endogenous Notch signaling to regulate the proliferation, differentiation, and osteogenic potential of ADSCs. Although Notch inhibition reduced ADSC proliferation and down-regulated osteoinduction, targeted gene therapy and the delivery of the downstream NICD peptide restored bone formation, suggesting pragmatic clinical utility of ADSCs for bone regeneration.

Adipose-Derived Stem Cells as a Tool in Cell-Based Therapies.

Recent development in stem cell isolation methods and expansion under laboratory conditions create an opportunity to use those aforementioned cells in tissue engineering and regenerative medicine. Particular attention is drawn towards mesenchymal stem cells (MSCs) being multipotent progenitors exhibiting several unique characteristics, including high proliferation potential, self-renewal abilities and multilineage differentiation into cells of mesodermal and non-mesodermal origin. High abundance of MSCs found in adipose tissue makes it a very attractive source of adult stem cells for further use in regenerative medicine applications. Despite immunomodulating properties of adipose-derived stem cells (ASCs) and a secretion of a wide variety of paracrine factors that facilitate tissue regeneration, effectiveness of stem cell therapy was not supported by the results of clinical trials. Lack of a single, universal stem cell marker, patient-to-patient variability, heterogeneity of ASC population combined with multiple widely different protocols of cell isolation and expansion hinder the ability to precisely identify and analyze biological properties of stem cells. The above issues contribute to conflicting data reported in literature. We will review the comprehensive information concerning characteristic features of ASCs. We will also review the regenerative potential and clinical application based on various clinical trials.

Obesity-driven alterations in adipose-derived stem cells are partially restored by weight loss.

The therapeutic potential of adipose-derived stem cells (ASCs) is reduced by various stress-inducing conditions that affect tissue homeostasis such as diabetes, aging, and obesity. Previous works have provided evidence of negative effects of obesity on ASC populations, but it is unclear whether this persists after a weight loss. This study evaluated whether weight loss can restore the attenuated properties found in ASCs derived from populations with obesity (oASCs). In vitro functional analyses were performed to investigate the possible recovery properties in mouse oASCs. Using ASCs isolated from subcutaneous tissue from formerly obese mice (dASCs) and control mice (cASCs), cell proliferation, viability, and some regenerative properties in these cells were analyzed compared with oASCs to evaluate the functional cell state. Cell proliferation, viability, and some regenerative properties are strengthened in dASCs and cASCs compared with oASCs. Nevertheless, metabolic analysis reveals a mitochondrial load misbalance and function leading to impaired respiration in dASCs. This study demonstrates that an initial obese environment triggers a detrimental state in ASCs that is not completely recovered after weight loss.

Standardization and Optimization for the Use of Human Adipose-Derived Stem Cells on an Extracellular Matrix

PURPOSE: Extracellular matrix (ECM) use in breast reconstruction surgery has gained popularity with utilization in 56% of tissue-expander based and/or implant-based surgical procedures.1 Combining adipose-derived stem cells (ASC) with an ECM has demonstrated improved tissue incorporation and increased strength of repair in hernia repair.2 Obtaining sufficient ASC number and improving ASC attachment to ECM are two key limiting factors for clinical treatments utilizing ASC on an ECM. With this study, we hope to establish (1)a safe and efficacious population doubling (PD) range under which in vitro expansion doesn’t affect ASC population doubling time (PDT) and/or differentiation capacity (DC); and determine if (2)human platelet rich plasma (PRP) can be used to promote the attachment and proliferation of ASC on an ECM. METHODS: Freshly isolated ASC from 3 individuals were cultured at 4 different seeding densities (n=4) ranging from 500 cells/cm2 to 5000 cells/cm2. Cell numbers were determined at 90% confluence to calculate PDT and PD. Osteogenic, chondrogenic, and adipogenic DC were measured by Alizarin red, Alcian blue, and Oil red O staining, respectively. Freshly isolated PRP from 3 individuals (n=3) or phosphate-buffered saline (PBS) (n=3) was pre-incubated on ECM before seeding with ASC. MTT assays were utilized to assess cell attachment at day 1 and cell proliferation at day 7. RESULTS: ASC were cultured for 9 to 31 days prior to 90% confluence and no significant differences in growth rates were observed among individual specimens and overall for PD 1.6 to 6.2 (n=4, p=0.184)(Fig. 1). The DC over PDs 1.6 to 6.2 also showed no significant changes (osteogenic, n=3, p=0.472; chondrogenic, n=3, p=0.878; adipogenic, n=3, p=0.256). PRP treatment significantly increased cell attachment on ECM (P<0.001) and also significantly increased proliferation compared to the PBS controls across all samples (P<0.001)(Fig. 2).Figure 1: Cell Growth Rate: Population doubling time (PDT) observed among seeding densities 500 cells/cm2 to 5000 cells/cm2 (n=4, p=0.184).Figure 2: Cell Attachment and Proliferation: Significantly increased MTT readings across all groups (PBS:PBS, PRP:PRP, All:All) (n=3, P<0.001). Significantly increased cell presence demonstrated after 7 days (n=3, P<0.001).CONCLUSION: Our results demonstrate that in vitro expansion of ASC within the PD range 1.6 to 6.2 causes no significant change in cell growth rate or regenerative capacity. This suggests in vitro expansion prior to clinical application in this PD range is both safe and effective. Also, PRP treatment was shown to significantly improve ADSC attachment and proliferation on an ECM. We hope that this information will benefit ASC application to ECM in future in vivo studies and clinical applications.

Does the liposuction method influence the phenotypic characteristic of human adipose-derived stem cells?

Adipose-derived stem cells (ASCs) possess a high differentiation and proliferation potential. However, the phenotypic characterization of ASCs is still difficult. Until now, there is no extensive analysis of ASCs markers depending on different liposuction methods. Therefore, the aim of the present study was to analyse 242 surface markers and determine the differences in the phenotypic pattern between ASCs obtained during mechanical and ultrasound-assisted liposuction. ASCs were isolated from healthy donors, due to mechanical and ultrasound-assisted liposuction and cultured in standard medium to the second passage. Differentiation potential and markers expression was evaluated to confirm the mesenchymal nature of cells. Then, the BD LyoplateTM Human Cell Surface Marker Screening Panel was used. Results shown that both population of ASCs are characterized by high expression of markers specific for ASCs: cluster of differentiation (CD)9, CD10, CD34, CD44, CD49d, CD54, CD55, CD59, CD71 and low expression of CD11a, CD11c and CD144. Moreover, we have noticed significant differences in antigen expression in 58 markers from the 242 studied. Presented study shows for the first time that different liposuction methods are not a significant factor which can influence the expression of human ASCs surface markers.

Development of a Human Adipose-Derived Stem Cell Model for Characterization of Chemical Modulation of Adipogenesis

Abstract Environmental influence is a significant factor governing the relationship over an individual's genetic predisposition toward obesity. A largely unexplored avenue is the notion of obesogens: environmental endocrine disrupting chemicals (EDCs) that may alter the propensity toward obesity, particularly during prenatal and early-life exposure. In an effort to establish a human-based platform for concentration–response assessments of novel candidate EDCs, human adipose-derived stem cells (ASCs) were selected as a biologically relevant primary cell model. A quantitative fluorescence-based readout of neutral lipid accumulation was used as a phenotypic endpoint to define a priming cocktail that could initiate lineage progression, but maintain an appreciable assay window (Z′-factor=0.85) with consistency across multiple human donor ASC populations. Concentration–response analysis of the uncoupled cocktail components demonstrates the feasibility of testing compounds with suspected modes of action for the ...

Keratinocyte progenitor cells reside in human subcutaneous adipose tissue.

The differentiation of adipose-derived stem cells (ASCs) towards epithelial lineages has yet to be demonstrated using a standardized method. This study investigated whether keratinocyte progenitor cells are present in the ASC population. ASCs isolated from subcutaneous adipose tissue were cultured and examined for the expression of the keratinocyte progenitor markers p63 and desmoglein 3 (DSG3) by immunofluorescence microscopy and flow cytometry. In addition, p63 and DSG3 expression levels were assessed before and after differentiation of ASCs into adipocytes by real-time PCR and western blot analysis, as well as in subcutaneous adipose tissue by real-time reverse transcriptase polymerase chain reaction. Both markers were expressed in ASCs, but were downregulated after the differentiation of ASCs into adipocytes; p63-positive cells were also detected in subcutaneous adipose tissue. ASCs co-cultured with human fibroblasts and incubated with all-trans retinoic acid and bone morphologic protein 4 showed an upregulation in DSG3 level, which was also increased in the presence of type IV collagen. They also showed an upregulation in cytokeratin-5 level only in the presence of type IV collagen. These results provide the demonstration that keratinocyte progenitor cells reside in subcutaneous adipose tissue.