Adipose-derived adult stem cells for cartilage tissue engineering

Tissue engineering is a promising therapeutic approach that uses combinations of implanted cells, biomaterial scaffolds, and biologically active molecules to repair or regenerate damaged or diseased tissues. Many diverse and increasingly complex approaches are being developed to repair articular cartilage, with the underlying premise that cells introduced exogenously play a necessary role in the success of engineered tissue replacements. A major consideration that remains in this field is the identification and characterization of appropriate sources of cells for tissue-engineered repair of cartilage. In particular, there has been significant emphasis on the use of undifferentiated progenitor cells, or "stem" cells that can be expanded in culture and differentiated into a variety of different cell types. Recent studies have identified the presence of an abundant source of stem cells in subcutaneous adipose tissue. These cells, termed adipose-derived adult stem (ADAS) cells, show characteristics of multipotent adult stem cells, similar to those of bone marrow derived mesenchymal stem cells (MSCs), and under appropriate culture conditions, synthesize cartilage-specific matrix proteins that are assembled in a cartilaginous extracellular matrix. The growth and chondrogenic differentiation of ADAS cells is strongly influenced by factors in the biochemical as well as biophysical environment of the cells. Furthermore, there is strong evidence that the interaction between the cells, the extracellular biomaterial substrate, and growth factors regulate ADAS cell differentiation and tissue growth. Overall, ADAS cells show significant promise for the development of functional tissue replacements for various tissues of the musculoskeletal system.     

Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo

In this study, we isolated CD31(-), CD34(-), CD106(-) (VCAM-1(-)), and fetal liver kinase(+) (Flk1(+)) cells from adipose tissue. These cells can be induced to differentiate into cells of osteogenic and adipogenic lineages in vitro and were termed adipose derived adult stem cells (ADAS cells). We also showed that they have characteristics of endothelial progenitor cells. In vitro, ADAS cells expressed endothelial markers when cultured with VEGF. In vivo, ADAS cells can differentiate in response to local cues into endothelial cells that contributed to neoangiogenesis in hindlimb ischemia models. PI3 kinase inhibitor LY294002 blocked the differentiation of ADAS cells into endothelial cells in vitro. Because ADAS cells can be expanded in culture without obvious senescence for more than 20 population doublings, they may be a potential source of endothelial cells for cellular pro-angiogenic therapies.     

Neurogenic differentiation of murine and human adipose-derived stromal cells

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1-3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.     

Extended passaging, but not aldehyde dehydrogenase activity, increases the chondrogenic potential of human adipose-derived adult stem cells

Adipose-derived adult stem (ADAS) cells represent an abundant population of multipotent mesodermal cells residing in various adipose tissue depots. ADAS cell preparations appear heterogeneous, yet at a clonal level, greater than 50% of these cells exhibit multilineage differentiation potential. To date, there have been few attempts to define prospectively a homogenous population of multipotent cells. In this study, we investigated whether aldehyde dehydrogenase (ALDH) can be used to enrich ADAS cells with increased chondrogenic potential. ALDH has been previously used to isolate primitive hematopoietic progenitors and has been implicated in early neurogenesis. Human ADAS cells were purified based on ALDH activity, and the cells were expanded and induced for chondrogenic differentiation using BMP-6 in a 3-D alginate culture. No significant differences in chondrogenic potential were observed in the ALDH-positive cells compared to unsorted controls. In contrast, significant differences were noted between cells assayed at passage 4 (P4) and cells assayed at passage 9 (P9). Following BMP-6 induction, AGC1 gene expression in P9 cells increased 290-fold over P4 cells. Similarly, COL2A1 expression in P9 cells increased fivefold compared to P4 cells, while COL10A1 levels remained unchanged. Immunohistochemical analysis over 28 days revealed consistent findings at the protein level for collagen II, collagen X, and aggrecan. No changes in telomerase activity were detected across passage, suggesting that ADAS cells retain some level of "stemness" in monolayer culture. These findings suggest that the chondrogenic potential of ADAS cells increases with passage number, although ALDH may not be a suitable marker for chondrogenesis.     

Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2

BACKGROUND: Adipose tissue has been demonstrated to contain a population of progenitor cells that can differentiate into bone and cartilage. Studies have suggested that adipose-derived adult stem (ADAS) cells can be induced to differentiate into chondrocytes by transforming growth factor-beta (TGF-beta). In this study, we examined whether bone morphogenetic protein-2 (BMP-2), as a member of the TGF-beta superfamily, could regulate ADAS cells to differentiate into a chondrolineage. METHODS: ADAS cells were isolated and induced by rhBMP-2. These cells were cultured in pellets for 2 weeks, and the chondrogenic phenotype was observed in vitro and in vivo. ADAS cells cultured without BMP-2 were used as controls. RESULTS: After 2 weeks of culture, the differentiated ADAS cells reacted positively to Alcian blue and collagen II, and the content of collagen II protein was obviously up-regulated at day 14. Glycosaminoglycan (GAG) content gradually increased from day 2 to day 14 (P < 0.05). However, H&E staining and collagen II expression were weak, and there was a little collagen II protein and GAG detected in the control group. Additionally, the pellets of ADAS cells induced by rhBMP-2 were transplanted into BALB/C nude mice and formed cartilage lacuna at week 8 in vivo. DISCUSSION: These data demonstrate that rhBMP-2 induce ADAS cells to differentiate into chondrocytes in vitro and in vivo. This is useful for basic and clinical studies aimed at repairing cartilage damage. But in a control group, ADAS cells tended towards differentiation into chondrocytes, which was affected by ITS. We will be exploring the mechanism further.     

In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult stem cells expressing BMP-7

BACKGROUND: Adipose-derived adult stem (ADAS) cells are multipotent cells capable of differentiating into osteoblasts, adipocytes and chondrocytes. The aim of this study was to determine whether BMP-7-expressing ADAS cells would elicit bone formation invitro and in vivo. METHODS: ADAS cells were harvested from Lewis rats and transduced with adenovirus carrying the recombinant human bone morphogenetic protein-7 (Ad-BMP-7) gene. Untransduced cells and cells transduced with adenovirus carrying the enhanced green fluorescence protein (Ad-EGFP) gene served as controls. BMP-7 expression was assessed by RT-PCR, immunofluorescence on day 1, and Western blot on days 4, 8 and 12. Alkaline phosphatase (ALP) activity was assayed on days 2, 4, 6, 8, 10 and 12. Osteocalcin production and bone nodule formation were detected by immunohistochemistry and von Kossa stain on day 12. A total of 1 x 10(6) cells mixed with type I collagen were implanted into the subcutaneous pocket in Lewis rat and subjected to histologic analysis 1, 2 and 4 weeks post-implantation. RESULTS: The Ad-BMP-7-transduced ADAS cells expressed BMP-7 at both mRNA and protein levels. ALP activity was detected in Ad-BMP-7-transduced cells from day 2 to day 12, peaking on day 8. Osteocalcin production and matrix mineralization further confirmed that these cells differentiated into osteoblasts and induced bone formation in vitro. Histologic examination revealed that implantation of BMP-7-expressing ADAS cells could induce new bone formation in vivo. DISCUSSION: ADAS cells would be a promising source of adult autologous stem cells for BMP gene therapy and tissue engineering.     

Epithelial differentiation of human adipose tissue-derived adult stem cells

Adult human stem cells are employed in novel treatments and bio-artificial devices. Recent studies have identified an abundant source of stem cells termed adipose-derived adult stem (ADAS)-cells in the subcutaneous adipose tissue. Under appropriate culture conditions ADAS-cells differentiate to various cell types, including chondrocytes, adipocytes, and smooth muscle cells. Aiming at epithelial differentiation this study investigated the effect of all-trans retinoic acid (ATRA) on human ADAS-cells. ATRA-induced cytokeratin 18 expression in ADAS-cells and nearly abolished vimentin expression as shown by Western blot. In immunofluorescence, the formation of keratin fibers in ATRA-treated ADAS-cells could be observed. The percentage of ADAS-cells being able to undergo epithelial differentiation as quantified by FACS-analysis was above 80%. Inhibition of cell growth by ATRA was shown using DAPI- and MTT-assays. ATRA can differentiate ADAS-cells toward the epithelial lineage. This finding, along with a previously described neural differentiation, shows that ADAS-cells have epithelial potential.     

Differentiation of human adipose-derived adult stem cells into neuronal tissue: Does it work?

Adipose tissue contains many cells and proteins that are of value not only for their potential therapeutic applications, but also for the low cost of their harvest and delivery. Mesenchymal stem cells (MSC) were originally isolated from the bone marrow, although similar populations have been isolated from adipose and other tissues. At one time, neural tissues were not regarded as regenerative populations of cells. Therefore, the identification of cell populations capable of neuronal differentiation has generated immense interest. Adipose tissue may represent an alternative source of cells that are capable of neuronal differentiation, potentially enhancing its use in the treatment of neurological disease. The aim of this review is to cover the current state of knowledge of the differentiation potential of human adipose-derived stem (ADAS) cells, specifically their ability to give rise to neuronal cells in vitro. This review presents and discusses different protocols used for inducing human ADAS cells to differentiate in vitro, and the neuronal markers utilized in each system.     

Effect of various freezing parameters on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells

The effect of four thermal parameters on post-thaw membrane integrity of adipose tissue derived adult stem (ADAS) cells after controlled-rate freezing was investigated with the help of a two-level four-parameter (2(4)) experimental design. The four thermal parameters studied were cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Several passages, including Passage-0 (P0), Passage-1 (P1), Passage-2 (P2), Passage-3 (P3), and Passage-4 (P4), obtained from the suspended culture of stromal vascular fraction (SVF) of the ADAS cells were used for this study. The two levels (low and high) of the four parameters [CR (1 and 40 degrees C/min); ET (-80 and -20 degrees C); HT (1 and 15 min); and TR (10 and 200 degrees C/min)] are chosen in such a way that they enclosed all parameter values possible using commercially available controlled-rate freezing equipment. Individual effect of each parameter on the immediate post-thaw membrane integrity was determined through the calculation of parameter effect values (E), and any synergy among the parameters on post-thaw membrane integrity was assessed through the calculation of two or more parameter interaction effect values (I). Nonlinearity in the experimental results was represented through the calculation of curvature value (CV). The results suggest that for 99% confidence level the parameters CR and ET have considerable effect on post-thaw membrane integrity of all passages of ADAS cells. A significant individual effect of TR was observed with P3 and P4 cells and a significant two-parameter interaction was observed between CR-ET for all passages. These observed results will be used as a basis to further develop freezing storage protocols of ADAS cells.     

Adult stem cells in neural repair: Current options,limitations and perspectives

Stem cells represent a promising step for the future of regenerative medicine. As they are able to differentiate into any cell type, tissue or organ, these cells are great candidates for treatments against the worst diseasesthat defy doctors and researchers around the world. Stem cells can be divided into three main groups:(1) embryonic stem cells;(2) fetal stem cells; and(3) adult stem cells. In terms of their capacity for proliferation, stem cells are also classified as totipotent, pluripotent or multipotent. Adult stem cells, also known as somatic cells, are found in various regions of the adult organism, such as bone marrow, skin, eyes, viscera and brain. They can differentiate into unipotent cells of the residing tissue, generally for the purpose of repair. These cells represent an excellent choice in regenerative medicine, every patient can be a donor of adult stem cells to provide a more customized and efficient therapy against various diseases, in other words, they allow the opportunity of autologous transplantation. But in order to start clinical trials and achieve great results, we need to understand how these cells interact with the host tissue, how they can manipulate or be manipulated by the microenvironment where they will be transplanted and for how long they can maintain their multipotent state to provide a full regeneration.     

成体干细胞的可塑性研究进展

人们传统观念认为成体干细胞局限于生成它们所在组织的分化细胞类型。但近年来的实验结果表明,从一个组织来的成体干细胞能被诱导分化成另外的一个组织的分化细胞,即成体干细胞具有可塑性。在此,我们对成体干细胞可塑性的证据、几种假设、调控机制和应用前景等方面做一综述。     

Planarian Immobilization,Partial Irradiation,and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology^1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts³. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal⁴, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a naïve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.Both tissue transplantation^5,6 and partial irradiation⁷ have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of naïve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals⁸. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.     

Adipose-derived stem cells: isolation, expansion and differentiation

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in adipose-derived stem cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.     

Stem cell: balancing aging and cancer

Stem cells are defined by their self-renewing capacity and the ability to differentiate into one or more cell types. Stem cells can be divided, depending on their origin, into embryonic or adult. Embryonic stem cells derive from early stage embryos and can give rise to cells from all three germ layers. Adult stem cells, first identified in hematopoietic tissue, reside in a variety of adult tissues. Under normal physiologic conditions, adult stem cells are capable of differentiating into the limited cell types that comprise the particular tissue or organ. Adult stem cells are responsible for tissue renewal and exhaustion of their replicative capacity may contribute to tissue aging. Loss of unlimited proliferative capacity in some of the adult stem cells and/or their progenitors may have involved the evolutionary trade-off: senescence prevents cancer but may promote aging. Embryonic stem cells exhibit unlimited self-renewal capacity due to the expression of telomerase. Although they possess some cancer cell characteristics, embryonic stem cells exhibit a remarkable resistance to genomic instability and malignant transformation. Understanding the tumor suppressive mechanisms employed by embryonic stem cells may contribute to the development of novel cancer treatments and safe cell-based therapies for age-related diseases.     

成体干细胞的研究及潜在应用

成体干细胞(adultstemcells)存在于人和哺乳动物的多种成体中,具有自我更新和一定的分化潜能.现已从骨髓、软骨、血液、神经、肌肉、脂肪、皮肤、角膜缘、肝脏、胰腺等许多组织中获得干细胞,并在部分成体干细胞的体外分离培养、扩增及诱导分化等研究中取得突破性进展,发现部分成体干细胞具有预想不到的分化潜能.成体干细胞不仅是发育生物学研究的理想模型,而且是细胞移植治疗、人工组织或器官构建的种子细胞和基因治疗的理想载体细胞,因此,在揭示生命的本质和规律及再生医学中有十分广阔的应用前景.     

Adipose tissue: a subtle and complex cell system

White adipose tissue (WAT) represents a large amount of all adult tissues. For a long time, it was considered as a poorly active, overgrown and undesirable tissue. It was mainly studied for its involvement in energy metabolism and disorders, as well as for its endocrine functions. WAT is composed of two main populations, matures adipocytes and stroma vascular fraction (SVF) that can be separated easily. The SVF contains two compartments, stromal and hematopoietic that have been recently characterized. The stromal population (or ADAS for Adipose Derived Stromal Cells) presents functional features of, as well as lineage relationship with, macrophages. These stromal cells, that are able to differentiate into adipocytes, also display endothelial potential, and could be considered as vascular progenitors. Differentiation of various adipose-derived cell subsets towards functional cardiomyocytes, osteoblasts, chondrocytes, muscle, hematopoietic and neural cells was also obtained in vitro or in vivo. Adipose tissue thus appears as a complex tissue composed of different cell subsets that could vary according to the nature and the location of fat pads, or to the physiological or pathological status. WAT appears as a very plastic and heterogeneous tissue that is very easy to sample. This represents a great advantage when considering adipose tissue as a potential and suitable source of stem cell for cell therapy. Further investigations in this way have to lead to the emergence of new insights fundamental to progress in our knowledge of adipose tissue biology.     

Adult neural stem cells: plasticity and developmental potential.

Stem cells play an essential role during the processes of embryonic tissue formation and development and in the maintenance of tissue integrity and renewal throughout adulthood. The differentiation potential of stem cells in adult tissues has been thought to be limited to cell lineages present in the organ from which they derive, but there is evidence that somatic stem cells may display a broader differentiation repertoire. This has been documented for bone marrow stem cells (which can give rise to muscle, hepatic and brain cells) and for muscle precursors, which can turn into blood cells. The adult central nervous system (CNS) has long been considered incapable of cell renewal and structural remodeling. Recent findings indicate that, even in postnatal and adult mammals, neurogenesis does occur in different brain regions and that these regions actually contain adult stem cells. These cells can be expanded both in vivo and ex vivo by exposure to different combinations of growth factors and subsequently give rise to a differentiated progeny comprising the major cell types of the CNS. Almost paradoxically, adult neural stem cells display a multipotency much broader than expected, since they can differentiate into non-CNS mesodermal-derivatives, such as blood cells and skeletal muscle cells. We review the recent findings documenting this unforeseen plasticity and unexpected developmental potential of somatic stem cells in general and of neural stem cells in particular. To better introduce these concepts, some basic notions on the functional properties of adult neural stem cells will also be discussed, particularly focusing on the emerging role of the microenvironment in determining and maintaining their peculiar characteristics.     

Stem cells from adipose tissue

This is a review of the growing scientific interest in the developmental plasticity and therapeutic potential of stromal cells isolated from adipose tissue. Adipose-derived stem/stromal cells (ASCs) are multipotent somatic stem cells that are abundant in fat tissue. It has been shown that ASCs can differentiate into several lineages, including adipose cells, chondrocytes, osteoblasts, neuronal cells, endothelial cells, and cardiomyocytes. At the same time, adipose tissue can be harvested by a minimally invasive procedure, which makes it a promising source of adult stem cells. Therefore, it is believed that ASCs may become an alternative to the currently available adult stem cells (e.g. bone marrow stromal cells) for potential use in regenerative medicine. In this review, we present the basic information about the field of adipose-derived stem cells and their potential use in various applications.     

Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius)

Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well.