Isolation Of Adipose-derived Stem Cells Research Articles

Soft Tissue Reconstruction.

Autologous fat transplantation has revolutionized soft tissue reconstruction, but conventional methods remain unpredictable as graft resorption rates are high due to lack of vascularization. The advent of adipose-derived stem cells (ASCs) has led to improvement of fat grafting outcomes, in part to their ability to undergo facile differentiation into adipose tissue, their angiogenic properties, and their ability to express and secrete multiple growth factors. This chapter discusses the isolation and characterization of human ASCs, its expansion in vitro, and relevant in vivo models for adipose tissue engineering.

A Novel Method for Human Adipose-Derived Stem Cell Isolation and Cryopreservation.

Adipose-derived stem cells (ADSCs) are isolated from abundant adipose tissue and have the capacity to differentiate into multiple cell lineages. ADSCs have raised big interest in therapeutic applications in regenerative medicine and demonstrated to fulfill the criteria for a successful cell therapy. There are several methods for isolation of ADSCs from adipose tissue and cryopreservation of ADSCs. Here, novel methods for the isolation and cryopreservation of ADSCs are presented and focused. Microscopic pieces of adipose tissue were placed on transwell inserts, and the ADSCs were induced to migrate to the lower wells for 1 week. We compared the properties of our ADSCs with those isolated by enzymatic digestion and enzyme-free method of culture plate, and our ADSCs were found to be more stable and healthier. In addition, we proposed a novel cryoprotectant solution (FNCP) containing pectin and L-alanine, which was compared with standard cryoprotectant solution. Overall, our methods proved more useful for ADSCs isolation than other methods and did not require consideration of "minimal manipulation" by the U.S. Food and Drug Administration (FDA). Furthermore, our FNCP did not contain dimethyl sulfoxide and fetal bovine serum, therefore stable storage is possible in xeno-free and animal-free cryopreservation solutions.

Isolation methods, proliferation, and adipogenic differentiation of adipose-derived stem cells from different fat depots in bovines.

The adipose-derived stem cells (ASCs) are a valuable resource for regenerative medicine and essential materials for research in fat deposition. However, the isolation procedure of ASCs has not been standardized and needs to be harmonized; differences in proliferation and adipogenic differentiation of ASCs obtained from different fat depots have not been well characterized. In the present study, we compared the efficiency of ASCs isolation by enzymatic treatment and explant culture methods and the proliferation ability and adipogenic differentiation potential of ASCs isolated from subcutaneous and visceral fat depots. The explant culture method was simple and with no need for expensive enzymes while the enzymatic treatment method was complex, time consuming and costly. By the explant culture method, a larger number of ASCs were isolated from subcutaneous and visceral fat depots. By contrast, fewer ASCs were obtained by the enzymatic treatment method, especially from visceral adipose. ASCs isolated by the explant culture method performed well in cell proliferation and adipogenic differentiation, though they were slightly lower than those by the enzymatic treatment method. ASCs isolated from visceral depot demonstrated higher proliferation ability and adipogenic differentiation potential. In total, the explant culture method is simpler, more efficient, and lower cost than the enzymatic treatment method for ASCs isolation; compared with visceral adipose, subcutaneous adipose is easier to isolate ASCs; however, the visceral ASCs are superior to subcutaneous ASCs in proliferation and adipogenic differentiation.

Single-cell transcriptomics reveals an angiogenic cell population for therapeutic angiogenesis in adipose tissue

Abstract Background Therapeutic angiogenesis mediated by stem/progenitor cells is an attractive therapeutic option against cardiovascular disease (CVD). Adipose tissue (AT) can be safely obtained even in CVD patients with anti-platelet medications, and it is a readily available source of culture-expanded adipose-derived stem cells (ADSCs) for transplantation. Single-cell transcriptome enables us to screen all the surface markers at once, while conventional strategies have been limited for the number of target markers. Furthermore, gene profiling at single-cell resolution can be used for the quantification of each marker by how many favorable cells can be purified without mixing of detrimental cells. Purpose We aimed to identify and characterize a cell population with in vivo angiogenic potential by single-cell RNA sequencing (scRNA-seq) analysis and xenograft experiments. Methods We revisited scRNA-seq datasets of single cell fraction from AT, bone-marrow (BM), and umbilical-cord blood (UCB, n=6/organ) to find cell populations with pro-angiogenic potential. Next, we collected AT from CVD patients (n=23) and used multicolor flow cytometry to quantify and sort the specific populations. PBS, the specific marker-negative and unsorted ADSCs were used as controls. Xenograft models of PKH26 pre-labeled human ADSC transplantation in limb ischemia were used to evaluate the lectin capillary density, PKH+ engrafted ADSCs, and blood flow recovery. Results Clustering divided CD45–CD31–CD34+ progenitor fraction into 3 clusters. We identified pro-/anti-angiogenic clusters based on the expressions of well-known pro-/anti-angiogenic factors. All genes encoding cell-surface proteins were compared in this functional clustering, resulted in 17 markers screened (Fig. 1A, B). Taken together with enrichment analysis, CD271+ cells showed predominant and pro-angiogenic gene profile from the other top candidates including CD36 and CD54 (Fig. 1C, D). Next, we evaluated the number and gene profile of CD271+ cells in well-known stem cell sources including BM and UCB. Surprisingly, the number of CD271 expressing cells were significantly lower and did not show angiogenic gene profile in BM and UCB (Fig. 2A). In analysis of AT from 23 CVD patients, CD271+ cells were significantly decreased by donor insulin resistance (Fig. 2B). Cell therapy using CD271+ ADSCs demonstrated in vivo angiogenic capacity compared to those of CD271– ADSCs and PBS in limb ischemia model. Furthermore, CD271+ ADSC transplantation showed enhanced efficacy compared to unsorted ADSCs from the same donors (Fig. 2C–E). Conclusion In this study, we identified CD271+ cell population in AT as an angiogenic cell population through scRNA-seq analysis and cell therapy experiments. AT obtained from donors without insulin resistance would be the most suitable for CD271+ ADSC isolation. CD271+ ADSC transplantation with a promising angiogenic capacity could contribute better cell-based therapy tackling CVD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science (JSPS) KAKENHI (Tokyo, Japan)

Expression Profile of New Gene Markers Involved in Differentiation of Canine Adipose-Derived Stem Cells into Chondrocytes

The interest in stem cell research continuously increased over the last decades, becoming one of the most important trends in the 21st century medicine. Stem cell-based therapies have a potential to become a solution for a range of currently untreatable diseases, such as spinal cord injuries, type I diabetes, Parkinson’s disease, heart disease, stroke, and osteoarthritis. Hence, this study, based on canine material, aims to investigate the molecular basis of adipose-derived stem cell (ASC) differentiation into chondrocytes, to serve as a transcriptomic reference for further research aiming to introduce ASC into treatment of bone and cartilage related diseases, such as osteoarthritis in veterinary medicine. Adipose tissue samples were harvested from a canine specimen subjected to a routine ovariohysterecromy procedure at an associated veterinary clinic. The material was treated for ASC isolation and chondrogenic differentiation. RNA samples were isolated at day 1 of culture, day 30 of culture in unsupplemented culture media, and day 30 of culture in chondrogenic differentiation media. The resulting RNA was analyzed using RNAseq assays, with the results validated by RT-qPCR. Between differentiated chondrocytes, early and late cultures, most up- and down-regulated genes in each comparison were selected for further analysis., there are several genes (e.g., MMP12, MPEG1, CHI3L1, and CD36) that could be identified as new markers of chondrogenesis and the influence of long-term culture conditions on ASCs. The results of the study prove the usefulness of the in vitro culture model, providing further molecular insight into the processes associated with ASC culture and differentiation. Furthermore, the knowledge obtained could be used as a molecular reference for future in vivo and clinical studies.

Development of hydroxyapatite-coated nonwovens for efficient isolation of somatic stem cells from adipose tissues.

Adipose-derived stem cells (ASCs) are an attractive cell source for cell therapy. Despite the increasing number of clinical applications, the methodology for ASC isolation is not optimized for every individual. In this study, we developed an effective material to stabilize explant cultures from small-fragment adipose tissues.MethodsPolypropylene/polyethylene nonwoven sheets were coated with hydroxyapatite (HA) particles. Adipose fragments were then placed on these sheets, and their ability to trap tissue was monitored during explant culture. The yield and properties of the cells were compared to those of cells isolated by conventional collagenase digestion.ResultsHydroxyapatite-coated nonwovens immediately trapped adipose fragments when placed on the sheets. The adhesion was stable even in culture media, leading to cell migration and proliferation from the tissue along with the nonwoven fibers. A higher fiber density further enhanced cell growth. Although cells on nonwoven explants could not be fully collected with cell dissociation enzymes, the cell yield was significantly higher than that of conventional monolayer culture without impacting stem cell properties.ConclusionsHydroxyapatite-coated nonwovens are useful for the effective primary explant culture of connective tissues without enzymatic cell dissociation.

Transplantation of Autologous Adipose-Derived Stem Cells for Chronic Nonhealing Wounds

<p>細胞治療是國際上廣受醫學界矚目的一種新興療法，可促進慢性困難傷口癒合。幹細胞因其獨特的生物特性，具有豐富的醫療應用潛力，其中脂肪幹細胞(ASCs)為間質幹細胞之來源之一，可透過微創手術（如：抽脂手術）從脂肪組織中取得，因其可用於促進組織再生及傷口癒合而受到關注。脂肪幹細胞再生能力的潛在機制涉及細胞分化和多種旁分泌作用，可增加膠原沉積、血管新生及減少發炎。2018年9月，衛生福利部頒布《特定醫療技術檢查檢驗醫療儀器施行或使用管理辦法》，有條件地允許6項自體細胞治療技術在臺 灣進行臨床應用，包含脂肪幹細胞應用在慢性困難傷口癒合。執行此項新興醫療技術前必須接受審查，以判定其在臨床場所及人體細胞組織優良操作規範實驗室中的安全性及執行效率，且須在脂肪組織的採集與運輸、脂肪幹細胞的分離與培養，以及細胞的運輸與移植實施標準化方案。藉由臺灣現行法規對製造自體脂肪幹細胞的嚴格品質管制，可讓脂肪幹細胞療法成為慢性困難癒合傷口病人的治療選擇之一。</p> <p> </p><p>Cell therapy is an emerging practice in the international medical community used to facilitate the healing of chronic wounds. Stem cells have exhibited potential for therapeutic applications because of their unique biological properties. Adipose-derived stem cells (ASCs) can be obtained from fat tissue through minimally invasive procedures such as liposuction. Thus, ASCs have gained attention as a source of mesenchymal stem cells, which can facilitate regeneration and wound healing. The mechanism underlying the regenerative properties of ASCs involves cell differentiation and several paracrine effects, which increases collagen deposition, causes angiogenesis, and prevents inflammatory states. In September 2018, the Ministry of Health and Welfare issued the Regulations Governing the Application or Use of Specific Medical Techniques or Examinations, which conditionally allow for the clinical application of six autologous cell therapy technologies in Taiwan. The clinical use of ASCs for chronic nonhealing wounds is included. This novel medical technology must be investigated to determine its safety and efficiency in both clinical facilities and good tissue practice labs. Standardized protocols, including those for fat tissue harvest and transportation, ASC isolation and expansion, and cell transportation and transplantation, should be implemented throughout the process. With stringent quality control for autologous ASC generation in compliance with regulations in Taiwan, ASC-based therapy can be a promising treatment option for chronic nonhealing wounds.</p> <p> </p>

Application of Free Skin Flap Transplantation in Skin Malignant Tumor Resection.

One of the most important surgical approaches for restoring cutaneous abnormalities caused by tumor resection is the insertion of a skin flap. Flap transplantation has been used to cover and fill different deficiencies after tumor removal. However, distal necrosis is among the most prevalent postsurgical consequences for skin flaps, particularly when treating large regions. Blood circulation failure causes flap necrosis, which may lead to serious problems, particularly in patients undergoing body cavity reconstruction following tumor excision. ADSCs (adipose-derived stem cells) are a kind of stem cell separated from the adipose tissue that has multilineage differentiation, simple availability, high proliferation capability, and self-renewal capabilities. This paper uses human adipose-derived stem cell (ADSC) therapy for skin flap transplantation in skin malignant tumor resection. ADSC-based therapy is an applicable technique for assisting flap transplantation. The isolation of ADSC is performed using different trypsin concentrations, and then, the population doubling time is determined. The isolated ADSCs are differentiated and then employed for the skin flap model. The performance of the suggested method is analyzed using various assays. The usage of adipose-derived stem cells to boost the vitality of the skin flaps proved successful.

Update on the Basic Science Concepts and Applications of Adipose-Derived Stem Cells in Hand and Craniofacial Surgery.

Adipose-derived stem cell therapy offers plastic surgeons a novel treatment alternative for conditions with few therapeutic options. Adipose-derived stem cells are a promising treatment because of their broad differentiation potential, capacity for self-renewal, and ease of isolation. Over the past decade, plastic surgeons have attempted to harness adipose-derived stem cells' unique cellular characteristics to improve the survival of traditional fat grafting procedures, a process known as cell-assisted lipotransfer. However, the full implications of cell-assisted lipotransfer in clinical practice remain incompletely understood, stressing the urgent need to assess the scientific evidence supporting adipose-derived stem cell-based interventions. Furthermore, with the strict regulatory climate surrounding tissue explantation therapies, reviewing the safety and efficacy of these treatments will clarify their regulatory viability moving forward. In this report, the authors provide a comprehensive, up-to-date appraisal of best evidence-based practices supporting adipose-derived stem cell-derived therapies, highlighting the known mechanisms behind current clinical applications in tissue engineering and regenerative medicine specific to plastic and reconstructive surgery. The authors outline best practices for the harvest and isolation of adipose-derived stem cells and discuss why procedure standardization will elucidate the scientific bases for their broad use. Finally, the authors discuss challenges posed by U.S. Food and Drug Administration oversight of these cell-based therapies and examine the role of adipose-derived stem cell-based applications in the future of plastic surgery.

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells.

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4-9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14-21 days of differentiation using a cocktail of 0.5 μg/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 μM indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

Osteogenic Capacity and Mettl14 and Notch1 Expression of Adipose-Derived Stem Cells from Osteoporotic Rats

To investigate the differences in the osteogenic capacity of osteoporotic adipose-derived stem cells (OP-ASCs) and normal control adipose-derived stem cells (Ctrl-ASCs), and to examine the expression levels of RNA methyltransferase like 14 (Mettl14) and the Notch signaling molecule 1 (Notch1). The osteoporosis (OP) model of SD rats was established with ovariectomy (OVX). Micro-CT, HE staining and Masson staining were performed to identify the successful establishment of the OP model, OP-ASCs and Ctrl-ASCs were isolated and cultured adherently. Then, the three-way differentiation capacity of the adipose-derived stem cells (ASCs) was determined through alizarin red staining, alcian blue staining and oil red O staining and flow cytometry was conducted to examine the surface antigens CD29, CD44, CD90, CD31, CD34, and CD45. Alizarin red staining and comparison of the mRNA and protein expression of Run-related transcription factor 2 (Runx2) were done to explore the differences in osteogenic potential of OP-ASCs and Ctrl-ASCs. Real-time PCR and Western blot were performed to explore the expression differences of Mettl14 and Notch1 at mRNA and protein levels between OP-ASCs and Ctrl-ASCs. Micro-CT, HE and Masson staining results showed that the number of trabecular bone decreased and the spacing increased in the tibias of the osteoporosis group (OP group) compared with those of the control group (Ctrl group), indicating that the OP model was established successfully. Three-way differentiation and flow cytometry results confirmed the successful isolation and culture of ASCs. After osteogenic induction, alizarin red staining showed that OP-ASCs had fewer number and more scattered distribution of mineralized nodules than Ctrl-ASCs did. The expression of Runx2 in OP-ASCs was lower than that in Ctrl-ASCs ( P<0.05). Mettl14 as well as Notch1 showed lower expression in OP-ASCs than they did in Ctrl-ASCs ( P<0.05). The osteogenic capacity of OP-ASCs was lower compared with that of Ctrl-ASCs, Mettl14 expression of OP-ASCs was decreased compared with that of Ctrl-ASCs, and the Notch signaling pathway was inhibited in OP-ASCs. The study helps build the foundation for further investigation in the specific mechanisms of Mettl14 and Notch1 during osteogenic differentiation of OP-ASCs.

In Vitro Cultures of Adipose-Derived Stem Cells: An Overview of Methods, Molecular Analyses, and Clinical Applications.

Adipose-derived stem cells (ASCs) exhibiting mesenchymal stem cell (MSC) characteristics, have been extensively studied in recent years. Because they have been shown to differentiate into lineages such as osteogenic, chondrogenic, neurogenic or myogenic, the focus of most of the current research concerns either their potential to replace bone marrow as a readily available and abundant source of MSCs, or to employ them in regenerative and reconstructive medicine. There is close to consensus regarding the methodology used for ASC isolation and culture, whereas a number of molecular analyses implicates them in potential therapies of a number of pathologies. When it comes to clinical application, there is a range of examples of animal trials and clinical studies employing ASCs, further emphasizing the advancement of studies leading to their more widespread use. Nevertheless, in vitro studies will most likely continue to play a significant role in ASC studies, both providing the molecular knowledge of their ex vivo properties and possibly serving as an important step in purification and application of those cells in a clinical setting. Therefore, it is important to consider current methods of ASC isolation, culture, and processing. Furthermore, molecular analyses and cell surface properties of ASCs are essential for animal studies, clinical studies, and therapeutic applications of the MSC properties.

Xenogen-free isolation and culture of human adipose mesenchymal stem cells.

Adipose-derived Stem Cells (ASCs) present great potential for reconstructive procedures. Currently, isolation by enzyme digestion and culturing using xenogenic substances remain the gold standard, impairing clinical use. Abdominal lipo-aspirate and blood samples were obtained from healthy patients. A novel mechanical isolation method for ASCs was compared to (the standard) collagenase digestion. ASCs are examined by flowcytometry and multilineage differentiation assays. Cell cultures were performed without xenogenic or toxic substances, using autologous plasma extracted from peripheral blood. After eGFP-transfection, an in vivo differentiation assay was performed. Mechanical isolation is more successful in isolating CD34+/CD31-/CD45-/CD13+/CD73+/CD146- ASCs from lipo-aspirate than isolation via collagenase digestion (p <0 .05). ASCs display multilineage differentiation potential in vitro. Autologous plasma is a valid additive for ASCs culturing. eGFP-ASCs, retrieved after 3 months in vivo, differentiated in adipocytes and endothelial cells. A practical method for human ASC isolation and culturing from abdominal lipo-aspirate, without the addition of xenogenic substances, is described. The mechanical protocol is more successful than the current gold standard protocol of enzyme digestion. These results are important in the translation of laboratory-based cell cultures to clinical reconstructive and aesthetic applications.

Isolation and Characterisation of Human Adipose-Derived Stem Cells.

Recently, adipose-derived stem cells (ASCs), obtained from fresh human lipoaspirate, have shown promise as immunomodulatory agents having demonstrated immunosuppressive functionality both in vitro and in vivo. A number of researchers have described the isolation of ASCs through the enzymatic digestion of fat samples, followed by a number of purification steps, involving centrifugation and filtration. Here, we utilize a standard isolation technique, with the added purification of putative ASCs by fluorescence activated cell sorting (FACS). ASCs are an extremely valuable resource in clinical applications, including reconstruction, regeneration, and investigations into immune activity. This method could be used to isolate and purify ASCs for such downstream applications.

Methods of Isolation, Characterization and Expansion of Human Adipose-Derived Stem Cells (ASCs): An Overview.

Considering the increasing interest in adipose-derived stem cells (ASCs) in regenerative medicine, optimization of methods aimed at isolation, characterization, expansion and evaluation of differentiation potential is critical to ensure (a) the quality of stem cells also in terms of genetic stability; (b) the reproducibility of beneficial effects; and (c) the safety of their use. Numerous studies have been conducted to understand the mechanisms that regulate ASC proliferation, growth and differentiation, however standard protocols about harvesting and processing techniques are not yet defined. It is also important to note that some steps in the procedures of harvesting and/or processing have been reported to affect recovery and/or the physiology of ASCs. Even considering the great opportunity that the ASCs provide for the identification of novel molecular targets for new or old drugs, the definition of homogeneous preparation methods that ensure adequate quality assurance and control, in accordance with current GMPs (good manufacturing practices), is required. Here, we summarize the literature reports to provide a detailed overview of the methodological issues underlying human ASCs isolation, processing, characterization, expansion, differentiation techniques, recalling at the same time their basilar principles, advantages and limits, in particular focusing on how these procedures could affect the ASC quality, functionality and plasticity.

Soft Tissue Reconstruction.

Autologous fat transplantation has revolutionized soft tissue reconstruction, but conventional methods remain unpredictable as graft resorption rates are high due to lack of vascularization. The advent of adipose-derived stem cells (ASCs) has led to improvement of fat grafting outcomes, in part to their ability to undergo facile differentiation into adipose tissue, their angiogenic properties, and their ability to express and secrete multiple growth factors. This chapter discusses the isolation and characterization of human ASCs, its expansion in vitro, and relevant in vivo models for adipose tissue engineering.

The Use of Vibrational Energy to Isolate Adipose-Derived Stem Cells.

Background:Adipose-derived stem cell (ADSC)–based treatments have the potential to treat numerous soft-tissue pathologies. It would be beneficial to develop an efficient and reliable intraoperative, nonenzymatic method of isolating ADSCs for clinical use. This study aims to determine the (1) viability and proliferative capacity of ADSCs after exposure to vibrational energies and (2) efficacy of vibrational energy as a method of ADSC isolation from surgically harvested infrapatellar fat pad (IFP).Methods:Cultured ADSCs were exposed to 15 minutes of vibration (60 Hz) with displacements ranging from 0 to 2.5 mm to assess cell viability and proliferation. Then, arthroscopically harvested adipose tissue (IFP; n = 5 patients) was filtered and centrifuged to separate the stromal vascular fraction, which was exposed to 15 minutes of vibration (60 Hz; 1.3 mm or 2.5 mm displacement). A viability analysis was then performed along with proliferation and apoptosis assays.Results:Vibration treatment at all displacements had no effect on the viability or proliferation of the cultured ADSCs compared with controls. There was an increased apoptosis rate between the 2.5 mm displacement group (7.53%) and controls (5.17%; P < 0.05) at day 1, but no difference at days 2, 3, and 14. ADSCs were not isolated from the IFP tissue after vibration treatment.Conclusions:ADSCs maintained viability and proliferative capacity after 15 minutes of vibration at 60 Hz and 2.5 mm displacement. ADSCs were not isolated harvested IFP tissue after the application of vibrational energy.

Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat.

The success of stem cell application in regenerative medicine, usually require a stable source of stem or progenitor cells. Fat tissue represents a good source of stem cells because it is rich in stem cells and there are fewer ethical issues related to the use of such stem cells, unlike embryonic stem cells. Therefore, there has been increased interest in adipose-derived stem cells (ADSCs) for tissue engineering applications. Here, we aim to provide an easy processing method for isolating adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery. We used a homogenizer to mince fat and compared the results with those obtained from the traditional cut method involving a sterile scalpel and forceps. Our results showed that our method provides another stable and quality source of stem cells that could be used in cases with a large quantity of fat. Furthermore, we found that pregnancy adipose-derived stem cells (P-ADSCs) could be maintained in vitro for extended periods with a stable population doubling and low senescence levels. P-ADSCs could also differentiate in vitro into adipogenic, osteogenic, chondrogenic, and insulin-producing cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirates, adipose tissues obtained from pregnant women contain multipotent cells with better proliferation and showed great promise for use in both stem cell banking studies as well as in stem cell therapy.

Function of microRNAs in the Osteogenic Differentiation and Therapeutic Application of Adipose-Derived Stem Cells (ASCs).

Traumatic wounds with segmental bone defects represent substantial reconstructive challenges. Autologous bone grafting is considered the gold standard for surgical treatment in many cases, but donor site morbidity and associated post-operative complications remain a concern. Advances in regenerative techniques utilizing mesenchymal stem cell populations from bone and adipose tissue have opened the door to improving bone repair in the limbs, spine, and craniofacial skeleton. The widespread availability, ease of extraction, and lack of immunogenicity have made adipose-derived stem cells (ASCs) particularly attractive as a stem cell source for regenerative strategies. Recently it has been shown that small, non-coding miRNAs are involved in the osteogenic differentiation of ASCs. Specifically, microRNAs such as miR-17, miR-23a, and miR-31 are expressed during the osteogenic differentiation of ASCs, and appear to play a role in inhibiting various steps in bone morphogenetic protein-2 (BMP2) mediated osteogenesis. Importantly, a number of microRNAs including miR-17 and miR-31 that act to attenuate the osteogenic differentiation of ASCs are themselves stimulated by transforming growth factor β-1 (TGFβ-1). In addition, transforming growth factor β-1 is also known to suppress the expression of microRNAs involved in myogenic differentiation. These data suggest that preconditioning strategies to reduce TGFβ-1 activity in ASCs may improve the therapeutic potential of ASCs for musculoskeletal application. Moreover, these findings support the isolation of ASCs from subcutaneous fat depots that tend to have low endogenous levels of TGFβ-1 expression.

Zinc silicate mineral-coated scaffold improved in vitro osteogenic differentiation of equine adipose-derived mesenchymal stem cells

In current study we aimed to coat the PLLA scaffold with zinc (Zn) silicate mineral nanoparticles. Then, using equine adipose-derived stem cells (ASCs) we intended to compare the osteogenic induction potency of Zn silicate mineral-coated PLLA scaffold with uncoated PLLA scaffold and tissue culture plastic (TCPS). Adipose tissues were collected from 3 horses, and isolation of ASCs was achieved by enzymatic digestion. PLLA scaffold was successfully prepared using a phase separation method and coated with Zn silicate mineral nanoparticles. The coating efficiency was then characterized by scanning electron microscopy and further evaluated with the application of fourier transform infrared microscopic imaging. Viability and growth characteristics of ASCs on TCPS, uncoated and coated PLAA scaffolds were investigated by MTT assay. Alizarin Red staining was performed for determination of calcium deposition following the osteogenic induction. Furthermore, other common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium content, as well as osteogenic (Runx2, ALP, osteonectin, and collagen I) marker genes were also evaluated. Our data showed that Zn silicate mineral nanoparticles was coated successfully on PLLA scaffold and such scaffold had no detrimental effect on cell growth rate as indicated by MTT assay. Moreover, ASCs that differentiated on Zn silicate mineral-coated PLLA scaffold indicated higher ALP activity, more calcium content, and higher expression of bone-related genes than that on uncoated PLLA scaffold and TCPS. Adequate proliferation rate and higher expression of osteogenic markers of stem cells, provides this scaffold as a suitable substrate to support proliferation and differentiation of ASCs in equine.