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There are known two types of AT: white adipose tissue and brown adipose tissue. The BAT is specialized in heat production thermogenesis and it can be found only in fetuses and newborn infants, being practically absent in adult humans. Its adipocytes are small and have many cytoplasmic lipid droplets, spherical and mildly eccentric nuclei and many mitochondria, which release heat via oxidation of fatty acids. Calorigenesis is guaranteed by uncoupling protein-1 (UCP-1 or thermogenin) which is located in the internal mitochondrial membrane and which acts as a proton channel, discharging the potential generated by the accumulation of protons in the intermembrane space during the Krebs Cycle, preventing synthesis of ATP and allowing it to be dissipated as heat.
While WAT's participation in thermogenesis is insignificant, its functional capacity is of much wider scope. WAT is distributed through the entire body and since it is an excellent thermal insulator it has an important role in conservation of body temperature. Due to its capacity to store energy (~200,000-300,000 Kcal in normal adults) and provide it when necessary, it is the most important buffer system for energy balance.
Adipocytes, which vary enormously in size, are embedded in a connective matrix and are the only cells specialized and perfectly adapted to store lipids without compromising their functional integrity. They possess the full enzymatic equipment necessary to synthesize fatty acids (lipogenesis), to store TAG during periods of abundant energy supply and to release them via lipolysis when there is a calorie deficit. The regulation of these two processes is controlled by the central nervous system.
Once discovered the adipocyte's capacity to secrete hormones (adipokines), the concept of AT biological function has completely changed, consolidating the idea that it is not just an energy supplier, but also a dynamic organ, which plays a central role in energy metabolism regulation
In a dynamic view, WAT, particularly adipocytes, originate a wide range of signal-molecules such as growth factors, proteins of the alternative complement pathway, proteins related to the immune system, adipokines involved in the regulation of pressure, of blood coagulation, of glycemic homeostasis and of angiogenesis.
WAT also secretes important regulators of lipoprotein metabolism and other key regulator molecules for many physiological functions of the body, such as insulin-like growth factor I (IGF-I), glucocorticoids, sex steroids, acylation-stimulating protein (ASP), adipophilin, adipoQ, adipsin, monobutyrin, agouti protein, and factors related to pro-inflammatory and immune processes.
All these features show that white adipose tissue lies at the heart of a network of autocrine, paracrine, and endocrine signals.
Adipsin, a serine protease secreted by fat cells, was proved to have identical sequence with complement D, the initial and rate-limiting enzyme in the alternative complement pathway. It was shown that fat cells synthesize all of the proteins of the alternative complement pathway, namely factors C3, D (adipsin), and B.
ASP (Acylation Stimulation Protein)
Acylation-stimulating protein (ASP) is a 14-kDa-serum protein resulting from the cleavage of the terminal arginine residue from C3a factor by plasma carboxypeptidases. Because C3a is the end product of the alternative complement pathway, of which factor D (adipsin) is the main component, it was designated the "adipsin-ASP pathway".
ASP increases after a fat-containing meal and stimulates triglyceride synthesis via diacylglycerol acyltransferase (DGAT) in adipocytes and fibroblasts. Although ASP is expressed by both preadipocytes and fibroblasts, its formation is a feature of mature and fully differentiated adipocytes.
By activation of the diacylglycerol-protein kinase C (DAG-PKC) pathway it also stimulates translocation of glucose transporters to the cell surface.
ASP stimulates greater triglyceride production than insulin and displays an additive effect with that of insulin. This feature supports ASP's role in determining the rate at which fatty acids are stored in adipose tissue.
aP2 (Adipose Fatty Acid-Binding Protein)
Fatty acid-binding proteins are abundant low-molecular- weight cytoplasmic proteins that are thought to be involved in the intracellular transport and metabolism of fatty acids. Members of this family are expressed in a tissue-specific manner. The adipose-specific fatty acid-binding protein (aP2), is expressed during adipocyte differentiation and comprises up to 6% of cytosolic proteins in the mature fat cell.
aP2 has been shown to be involved in intracellular trafficking and targeting of fatty acids as it shuttles fatty acids within the aqueous cytosol toward the membranes of the relevant intracellular organelles that are involved in triglyceride synthesis or fatty acid oxidation.
Acrp30 (Adipocyte Complement-Related Protein of 30 kDa)
Acrp30, also known as adipoQ, ApM-1 or adiponectin, is a serum protein (~0.01% of the total plasmatic proteins) which shows similarity to complement factor C1q.
Like adipsin, its secretion is modulated by insulin, revealing the possibility that its expression to be regulated by the nutritional state. It has been shown that adiponectin suppresses the attachment of monocytes to endothelial cells, an early event in atherosclerotic vascular change, thus suggesting a protective role against vascular damage. Furthermore, decreased plasma adiponectin concentrations may be an indicator of macroangiopathy in type 2 diabetic patients. In addition, its expression was found to be markedly reduced in adipose tissue of obese mice and humans.
The agouti protein is expressed only in the skin of mice and is a secreted product that regulates the coat color. Dominant mutations in the agouti locus cause its expression in all tissues, which subsequently produces a syndrome consisting of yellow fur, obesity, hyperinsulinemia, and insulin resistance. In contrast to mice, the human agouti gene is normally expressed in adipose tissue and testis, suggesting a possible role in regulating adipose tissue function. The participation of the agouti protein in the development of insulin resistance has been associated with increasing intracellular free calcium concentrations.
Resistin (12.5 KDa) is a hormone and it belongs to a family of proteins generically known as resistin-like molecules (RELM), which are characterized by the presence of a cysteine rich segment at the C-terminal end. Resistin was first described in 2001 when a relationship between resistin and insulin resistance induced by obesity was demonstrated. Apparently, its secretion is stimulated by insulin and down-regulated by the inflammatory processes, glucocorticoids, lipopolysaccharides (LPS), tumor necrosis factor (TNF-Î±), Î²-adrenergic stimulation and peroxisome proliferator activated receptor-gamma (PPARÎ³). Studies of human adipose tissue (AT) have shown that resistin is predominantly expressed in preadipocytes.
The adipocytes also secrete vascular function-related proteins such as angiotensinogen and plasminogen activator inhibitor type 1 (PAI-1). White adipose tissue (WAT) contains all the main components of the renin-angiotensin system such as angiotensinogen, angiotensin converting enzyme, angiotensin II, and angiotensin receptors.
Angiotensinogen is primarily synthesized by the liver, although angiotensinogen mRNA is present in several tissues, including adipose tissue. Angiotensinogen is the substrate of renin in the renin-angiotensin system and is converted into angiotensin I, the precursor of angiotensin II. It has been suggested that the interaction between angiotensin II and angiotensin receptors induce the prostacyclin synthesis in fat cells, which subsequently influence adipocyte differentiation. Furthermore, angiotensinogen expression is increased in obesity and its expression is thought to be regulated by the nutritional status.
PAI-1 (Plasminogen Activator Inhibitor-1)
PAI-1 is a member of the family of serine protease inhibitors or serpins. Increased concentrations of PAI-1 favor the development of thromboembolic complications. Among the multiple mechanisms that may explain the relationship between obesity and cardiovascular disease, disorders of the fibrinolytic system seem to play a central role. Insulin appears to be the main inducer of PAI-1 synthesis in adipose tissue. In addition, TNF-Î±, as well as IL-1Î², also has a stimulatory effect on PAI-1 protein secretion and may contribute to the elevated PAI-1 concentrations reported in obesity and insulin resistance.
GH (Growth Hormone)
Growth hormone (GH) is an important regulator of body mass throughout life. GH deficiency in both children and adults is characterized by abnormal body composition, with increased fat mass and decreased muscle mass.
Adipocytes have specific GH receptors, as the hormone exerts a variety of direct metabolic effects such as inhibition of glucose uptake and stimulation of lipolysis (2).
The presence of estrogens in the plasma of postmenopausal women led to the discovery that adipose tissue is an active extraglandular producer of certain steroid hormones. Two enzymes of relevance to sex steroid metabolism were found in adipose tissue: 17b-hydroxysteroid oxidoreductase and cytochrome-P-450-dependent aromatase.
A net release of testosterone, estradiol, and estrone from abdominal subcutaneous adipose tissue in women, but not in men, has been demonstrated, showing a sexual dimorphism in relation to the influence of sex steroids on adipose tissue function.
IL-6 is a multifunctional cytokine produced by many different cell types, including immune cells, fibroblasts, stromal-vascular cells, endothelial cells, myocytes, and a variety of endocrine cells (172).
Production of IL-6, as well as systemic concentrations, has been shown to be positively correlated with body mass index (175). Furthermore, a third of total circulating concentration of IL-6 has been estimated to originate from adipose tissue (110, 184). In this view, IL-6 may be both an autocrine and a paracrine regulator of adipocyte function. IL-6 increases hepatic triglyceride secretion (116) and may contribute to the visceral obesity associated hypertriglyceridemia. Glucocorticoids and catecholamines were shown to be important modulators of IL-6 expression in different fat depots (40, 119). Dexamethasone suppresses IL-6 production, whereas insulin has no effect, suggesting that cortisol may act physiologically in the modulation of IL-6 production.
IL-6 decreases adipose tissue liporprotein lipase (LPL) activity and has been involved in the fat depletion taking place during cancer cachexia and other wasting disorders (51, 158). IL-6 is an inflammatory mediator as well as a stress-induced cytokine (189).
Tumor Necrosis Factor (TNF-Î±)
TNF-Î± is a cytokine, first identified in macrophages involved in the metabolic disturbances of chronic inflammation and malignancy. The biological actions of TNF-Î± include induction of insulin resistance, anorexia, and weight loss.
Subcutaneous fat depots exhibit higher TNF-Î± mRNA expression than omental fat depots (68). The amount of TNF-Î± mRNA is positively correlated with body adiposity and decreases in obese subjects after weight loss (112). TNF-Î± mRNA expression is also closely correlated with hyperinsulinemia, showing positive associations with fasting insulin and triglyceride concentrations (62). Expression of TNF-Î± takes place even in preadipocytes, although the amount of specific mRNA increases moderately in a differentiation dependent manner (69, 70).
Adipogenic inducers, such as the nonselective phosphodiesterase inhibitor IBMX and the thiazolidinediones, are inhibitors of TNF-Î± expression. The adipogenic effect of these compounds may be at least partially mediated by a suppression of endogenous TNF-Î± production. Triglycerides and free fatty acids play an important role as physiological inducers of TNF-Î± expression.
TNF-Î± has a catabolic effects in adipose tissue (69) as it inhibits the expression of the two master regulators of adipose differentiation: the transcription factor CCAAT/enhancer binding protein-a (CEBPÎ±) and the nuclear receptor peroxisome proliferator-activated receptor-Î³2 (PPARÎ³2) (156, 184). This suppression may result in the subsequent downregulation of many adipocyte-specific proteins, such as LPL, aP2, fatty acid synthetase, acetyl-CoA carboxylase, glycerol-3-phosphate dehydrogenase (GPDH), and GLUT-4 among others.
Furthermore, mature adipocytes are stimulated to mobilize lipids upon TNF-Î± exposure, possibly via hormone sensitive lipase activation (56) and chronic treatment of fat cells with TNF-Î± has been shown to reverse the adipocyte phenotype back to a fibroblast-like morphology (122). TNF-Î± can modulate adipose tissue cellularity by controlling the programmed cell death. Thus TNF-Î± could decrease adipose tissue mass by reducing not only fat cell volume but also adipocyte number.
The discovery of leptin in 1994 (196) brought a major development in energy balance regulation knowledge. Leptin consists of four antiparallel Î±-helices, connected by two long crossover links and one short loop, arranged in a left-hand twisted helical bundle. Its main role is to inform the brain about the abundance of body fat, thereby regulating feeding behavior, metabolism, and endocrine physiology in accordance with the nutritional state of the organism (1, 44).
Leptin is secreted by fat cells in a direct proportion to the body fat stores, thus having the potential to play a key regulatory role in fuel homeostasis (166). Fasting induces a fall in its mRNA levels, which is rapidly reversed on refeeding (44, 166). Glucose and lipid increase leptin expression in adipose tissue and induce its synthesis, raising the possibility that leptin acts as a sensor of nutrient flux.
In addition, leptin has been shown to repress acetyl-CoA carboxylase gene expression, fatty acid synthesis, and lipid synthesis (145, 180, 181). Thus leptin is involved in the direct regulation of adipose tissue metabolism by both inhibiting lipogenesis and stimulating lipolysis (44). Insulin stimulates the expression of leptin's encoding gene, as do estrogens and glucocorticoids, while androgens and GH down-regulate it (1, 6, 44). In addition, sexual dimorphism is evident in leptin concentrations, with almost twofold higher leptin concentrations in women (44). Adipocyte size is an important determinant of leptin synthesis, because larger fat cells contain more leptin than smaller adipocytes from the same individual (1, 23).
Leptin has many effects in addition to appetite and body weight regulation. It has been shown to be involved in quite diverse physiological functions, such as reproduction (17), hematopoiesis (20), angiogenesis (147), immune responsiveness (98), blood pressure control (42), and bone formation (30). In addition, leptin appears to be able to enhance the production of cytokines in macrophages and to increase the attachment and subsequent receptor-mediated process of phagocytosis (46). Leptin has been demonstrated to have a direct proliferative effect on T cells, showing an adaptive response of this hormone to enhance the immune competence of the organism against the immunosuppression associated with starvation (98). Leptin is also an angiogenic factor secreted by adipose tissue, as it cause cultured endothelial cells to aggregate, form tubes, and display a reticular array reminiscent of tissue vasculature (147). It has also been observed that leptin accelerates wound healing, a process depending on blood vessel growth.
In conclusion, adipocytes are continuously turning over under a tight hormonal control. Apart from its central role in triglyceride metabolism, adipose tissue possesses a major role in regulating metabolic functions. Fat cells modulate their own metabolism, and hence their size, via autocrine and paracrine mechanisms. The production of the cytokines IL-6, TNF-Î±, and leptin plays decisive role in the development of obesity and insulin resistance. The local production of angiotensinogen may be the etiological cause of obesity-related hypertension development. Furthermore, synthesis of estrogens by adipose tissue may mediate effects of obesity on the risk for osteoporosis and cancer.
Taken together, these features prove that the adipocytes behave as endocrine as well as paracrine/autocrine cells. Along with its active role in regulating energy balance, WAT has the potential to play a dynamic role in a variety of other physiological processes, including the regulation of its own growth and development.
2. Adipose tissue, a rich source of MSCs for ATE
While embryonic stem cells, fetal stem cells or induced pluripotent stem cells exhibit nearly unlimited potential to differentiate in vitro and in vivo, the applications of these cells are limited by legal and ethical concerns, as well as by scientific and clinical issues of safety and efficacy. Tissue-specific stem cells derived from adults offer alternative approaches that circumvent many of these concerns.
Because mature blood cells are mainly short-lived, stem cells must be produced throughout life to replenish the multilineage progenitors. In adults, the hematopoietic stem cells (HSCs) reside in the bone marrow and are arranged in a hierarchy of progenitors that become progressively restricted to several or single lineages. As with all other stem cells, HSCs have the ability to self-renew and differentiate, specifically to all blood cell lineages. Furthermore, some reports claim that these cells have the capacity to transdifferentiate into e.g. hepatocytes, which gives them broader potential in regenerative medicine than expected.
In 1968, Friedenstein and co-workers reported that a small number of adherent cells from rat whole bone marrow that were heterogeneous in appearance and after a transient dormant phase began to multiply rapidly. After several passages in culture, these adherent cells became uniformly spindle-shaped and demonstrated ability to differentiate into colonies that resembled small deposits of bone or cartilage. Friedenstein's initial observations of the potential of these marrow stromal cells were further investigated in the 1980s, particularly by Piersma and co-workers and by Owen and co-workers. In the early 1990s, Caplan popularized the term mesenchymal stem cell (MSC), but some investigators still preferred not to refer to these cells as stem cells when publishing pre-clinical or clinical studies of MSC.
Since the stem cell label has scientific implications that may or may not be strictly correct, the International Society for Cellular Therapy (ISCT) proposed a uniform nomenclature for these important cells in two position statement papers. They proposed that the plastic-adherent cells currently described as mesenchymal stem cells should be termed multipotent mesenchymal stromal cells, while the term mesenchymal stem cell should be reserved for a subset of these cells that demonstrate stem cell activity by clearly stated criteria. The criteria state that the acronym MSC may be used for both populations, as long as the acronym is defined in the presentation of the work. To define MSCs, some minimal criteria were suggested by the ISCT for in vitro demonstrations of long-term survival with self-renewal capacity and tissue repopulation with multilineage differentiation. MSCs must be surface-adherent in standard culture conditions, they must express CD105, CD73 and CD90 surface markers, lack the expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface molecules and they also must differentiate into osteoblasts, adipocytes and chondroblasts in vitro.
MSCs are able to differentiate into a large variety of specialized mesenchymal tissues including bone, cartilage, muscle, marrow stroma, tendon, ligament, fat and a range of other connective tissues.
Furthermore, MSCs reside in various locations throughout the body, e.g. in bone marrow, around blood vessels (as pericytes), in fat, skin, muscle and other locations.
In addition to their potential to differentiate into cells of different lineages, MSCs have also been shown to possess some level of plasticity. Until recently, it was believed that tissue-specific stem cells only differentiated into mature phenotypes within their restricted lineages. This novel notion of stem cell plasticity is perhaps not surprising, since within the mesenchymal cell lineages, plasticity of mature cells was proposed several decades ago by showing that chondrocytes could transdifferentiate into osteoblasts, and that adipocytes could switch their phenotype to that of osteoblasts.
In summary, the performance of MSCs have an impact on the overall health status of individuals by controlling the body's capacity to naturally remodel, repair, and upon demand, rejuvenate various tissues.
Although the bone marrow (BM) MSCs (BM-MSCs) continue to be a viable option for a stem cell population for tissue engineering applications, there are drawbacks in using this source. A bone marrow harvest is a painful procedure with possible donor site morbidity as a result. Although BM-MSCs grow well under standard culture conditions, ex vivo expansion is necessary due to relatively low numbers MSCs in the harvested marrow. In this perspective, adipose tissue has become an attractive option and it is an attractive alternative source of stem cells. Subcutaneous adipose depots are accessible and abundant. Adipose tissue is comprised of adipocytes and a heterogeneous set of cell populations that surround and support them, which upon isolation are termed the stromal vascular fraction (SVF). The adipose derived stem cells found in SVF have the ability to differentiate into cells of several lineages such as adipocytes osteoblasts, chondrocytes, myocytes, endothelial cells, hematopoietic cells, hepatocytes and neuronal cells. Furthermore, the SVF contains cells from the microvasculature, such as vascular endothelial cells and their progenitors, vascular smooth muscle cells and also cells with hematopoietic progenitor activity.
Despite the fact that the SVF is a heterogeneous cell population, subsequent expansion results in a relatively homogeneous cell population, enriching with cells expressing a stromal immunophenotype.
In 1964, Martin Rodbell was the first to present a method for in vitro isolation of mature adipocytes and progenitors from rat fat tissue. In his protocol, the tissue was minced into small fragments, digested at 370C with type I collagenase, and the cellular components were separated by centrifugation. Following centrifugation, the supernatant contained the mature adipocytes, which floated due to their high lipid content, and the pellet contained the SVF components, including the presumptive adipocyte progenitor cells in addition to cells of the hematopoietic lineages. The protocol to isolate human adipocyte progenitors was later modified by Van, Roncari, Deslex, Hauner and others, who found that when the SVF components are cultured in the presence of inductive factors, the cells accumulated lipid vacuoles and expressed the adipogenic enzymes. Katz, Zuk and co-workers were the first to show that the SVF isolated from human lipoaspirates contained cells with multilineage potential. They termed these cells processed lipoaspirate (PLA) cells. Since then, several groups working independently have developed and refined procedures of isolating and characterizing adipose stem cells.
As in many rapidly developing fields, a range of names have been used to describe the plastic adherent cell population isolated from adipose tissue, e.g. lipoblast, pericyte, preadipocyte, processed lipoaspirate (PLA) cells, adipose derived stem/stromal cells (ASCs), adipose-derived adult stem (ADAS) cells, adipose-derived adult stromal cells, adipose-derived stromal cells (ADSCs), adipose stromal cells (ASCs) and adipose mesenchymal stem cells (AdMSCs). To address the problem, the International Fat Applied Technology Society (IFATS) proposed a standardized nomenclature by adopting the term adipose-derived stem cells (ADSCs) to identify the isolated, plastic-adherent, multipotent cell population.
2.1. Isolation and proliferation of adipose stem cells
The first method of cell isolation from adipose tissue was reported by the pioneer Rodbell who isolated cells from the adipose tissue of rats by enzymatic digestion with collagenase. However, Zuk and collaborators were the first to report on the existence of stem cells in the SVF of fat tissue. They isolated the adipose stem cells from liposuction aspirates using the collagenase digestion method, and subsequently allowing the ADSCs to adhere to the plastic surface of tissue culture flasks, which is still the basis of most methods used to date. An alternative method developed by Sengenes and co-workers to isolate mesenchymal stem cells from SVF is to use immunomagnetic beads to separate CD34+/CD31- cells.
A further aspect to take into account when isolating ADSCs is the method used to harvest the adipose tissue. In humans, ADSCs can be isolated from fat tissue wastes resulting from plastic surgery, i.e. liposuction aspirates and from reconstructive surgeries, through resection of a large tissue fragment. When the starting material is obtained from liposuction procedures, the isolation method is simplified, as the procedure generates finely minced tissue fragments that are more homogeneous, allowing a more efficient enzymatic digestion. When working with whole tissue pieces as starting material, the tissue is minced manually, requiring more time and effort for thorough enzymatic digestion.
Furthermore, to speed up the isolation procedure, companies are developing commercial bench top closed systems for isolating unexpanded ASCs directly for cell therapy, such as Cytori's Celutionâ„¢ system and Tissue Genesis' TGI 1000â„¢ platform (Tissue Genesis Cell Isolation System). The Cytori's Celutionâ„¢ system is a CE Marked medical technology and has also been approved by the U.S. FDA as a medical device (FDA, 2009), while the Tissue Genesis' TGI 1000â„¢ platform has not yet received approval from the FDA for any application.
Also, the effects on yield and cell proliferation using different harvesting techniques and harvesting sites have also been investigated, and contradictory reports have been published. In a report by Fraser and co-workers the results showed that neither the site of harvest nor the harvesting technique (liposuction, syringe-based and pump-assisted) affected the number of ADSCs obtained. Nevertheless, the number of clonogenic cells varied with the harvesting site. Oedayrajsingh-Varma and co-workers studied three harvesting techniques (resection, tumescent liposuction and ultrasound-assisted liposuction) and the results suggested that the harvesting technique affected the recovery of ADSCs, with ultrasound-assisted liposuction yielding the lowest number of proliferative ADSCs. Later, the same group also concluded that the site of harvest also affected the yield of ADSCs. Furthermore, von Heimburg and co-workers reported that resection yielded lower numbers of viable progenitors as compared to liposuction aspirates. However, due to the small number of reports published and the variations in the protocols used, it is difficult to conclude the optimal harvesting technique, site of harvest and optimal isolation procedure.
2.2. Characterization of adipose stem cells
Adipose stem cells are commonly characterized by their immunophenotype in the undifferentiated state and by their differentiation potential towards the adipogenic, osteogenic, and chondrogenic lineages in the presence of lineage-specific induction factors.
2.2.1. hADSCs' immunophenotype
Unlike embryonic stem cells, undifferentiated ADSCs cannot be identified by a single surface marker but rather by a panel of markers that are used for identification of the population. Still, a number of reports have been published suggesting markers for identifying the mesenchymal stem cell population, such as STRO-1, CD271, STRO-3 and MSCA-1+CD56+.
BM-MSCs and ADSCs show very similar surface marker expression patterns. Furthermore, both cell sources express the surface markers characteristic for MSCs, meeting the criteria set by ISCT. However, minor differences exist as BM-MSCs lack the expression of CD49d, which is strongly expressed on ADSCs, while ADSCs lack expression of CD106, which is expressed on BM-MSCs. This reciprocal expression pattern is interesting because CD106 is the cognate receptor of CD49d and both molecules are involved in hematopoietic stem and progenitor cell homing to and mobilization from the bone marrow.
Markers CD13, CD29, CD73, CD90, CD133, MHC I and MHC II have been detected with highly consistent patterns of expression on the surface of ADSCs. Markers that are uniformly reported to have strong positive expression are CD13, CD29, CD44, CD73, CD90, CD105, CD166 and MHC I, while markers of the hematopoietic and angiogenic lineages, such as CD31, CD45 and CD133, have been reported to show low or lack of expression on ADSCs. Moreover, MHC II has also been found to be absent on ADSCs. Moderate expression has been reported for markers CD9, CD34, CD49d, CD106, CD146 and STRO-1, i.e. surface marker expression levels of lower than 50%.
The presence or absence of STRO-1 is particularly controversial because while Gronthos and co-workers reported absence of this marker in ADSC cultures Zuk and co-workers reported its presence.
Similar controversies are seen for CD34 and CD106, where Gronthos and co-workers reported detection of these markers in ADSCs, while Zuk and co-workers and Katz and co-workers reported their absence or expression on a small population of cells.
Moreover, the expressions of some surface markers change during cell culturing and passaging. For instance, the expression level of CD29, CD44, CD73, CD90 and CD166 increase from the SVF to passage 2, whereupon they stabilize at a high expression level. On the contrary, hematopoietic cell markers, such as CD11, CD14, CD34 and CD45, expressed on cells in the SVF decrease or are lost with increasing passage number, suggesting that adherence to plastic and subsequent expansion will select for a relatively homogeneous cell population compared with the SVF.
To note, these inconsistencies in the results reported by different teams is by no means unique for ADSCs; similar differences in expression profiles have been detected for BM-MSCs. They may be explained by the differences in marker antibodies sources and sensitivity differences between detection methods used in the referred studies, the proliferative stage of the cells in culture or donor heterogeneity. As a consequence, it may prove impossible to unify the protocols of surface marker characterization due to the reasons mentioned above; however, some minimal criteria for characterization of ADSCs by surface markers may prove useful.
2.2.2. hADSCs' differentiation potential
In order to use adipose stem cells for clinical tissue engineering applications, the multipotentiality of ADSCs must be established. In the following sections, some in vitro characterization methods will be described. The differentiation protocols for BM-MSCs/ADSCs from different species may vary, however in this review, only protocols related to the determination of human ADSC differentiation potential are discussed.
The cells of the adipose lineage differentiate from a multipotent stem cell population residing in the vascular stroma of adipose tissue and undergo a multi-step process by an initial commitment step, in which cells become restricted to the adipocyte lineage, but do not yet express markers of terminal differentiation. Subsequent differentiation occurs by activation of several transcription factors resulting in the adipocyte phenotype factors secreted by cells within the stromal vascular population and/or adipocytes undergoing hypertrophy. Factors that lead to the commitment of mesenchymal stem cells to the adipose lineage ex vivo have been identified, but the molecular mechanisms by which these pathways are regulated have not been determined.
The main promoters of adipogenic differentiation, peroxisome proliferator-activated receptor Î³ (PPARÎ³) and CCAAT/enhancer binding protein, alpha (C/EBPÎ±) act synergistically to activate transcription of genes producing the adipocyte phenotype, although hormones are required for terminal differentiation. The expression of PPARÎ³ rapidly increases after hormonal induction of differentiation, preceded by an increase in the expression of C/EBP-Î² and C/EBP-Î´. A decrease in C/EBP-Î² and C/EBP-Î´ in early to intermediary stages of differentiation is associated with the induction of C/EBP-Î± mRNA and occurs slightly before the expression of adipocyte-specific genes. The C/EBPÎ± encoded protein has been shown to modulate the expression of the gene encoding leptin. Adipocyte enhancer binding protein 1 (AEBP) acts as a transcriptional repressor on C/EBP proteins, which bind to adipocyte enhancer 1 (AE-1), in the promoter region of the adipose P2 (aP2)/FABP4 gene; this gene encodes adipocyte fatty-acid binding protein. Detailed adipogenic differentiation program will be further discussed in section 4.
When applying the appropriate induction factors in vitro, human ADSCs are capable of differentiating into their original differentiation pathway, adipogenesis. The first adipogenic induction media reported was a chemically defined serum-free (SF) media containing insulin or IGF-1, triiodothyronine and transferrin, with serum only used briefly for cell attachment. Further refinements to the induction media supplement composition have been made with the addition of isobutylmethylxanthine (IBMX; a phosphodiesterase inhibitor resulting in elevated cyclic AMP levels), hydrocortisone or dexamethasone (glucocorticoid receptor agonist), indomethacin or thiazolidinedione (PPARÎ³ ligand), pantothenate, biotin and serum.
After a week of induction, neutral lipid containing vacuoles accumulate in ADSCs and the production of adipogenic mRNAs, such as lipoprotein lipase, PPARÎ³, C/EBPÎ±/Î²/Î´, followed by FABP4/aP2 and leptin, can be detected. The expression of LPL has often been cited as an early sign of adipocyte differentiation; however, its expression occurs spontaneously at confluence and is independent of the adipogenic factors addition.
hADSCs osteogenic differentiation is still not elucidated but the process has been studied more extensively in BM-MSCs. It is commonly believed that osteogenic cells arise from multipotential mesenchymal cells found in bone marrow or adipose tissue that have the capacity to undergo a number of restriction steps to give rise to progenitor cells with more and more limited capacities. These commitment steps are mediated by many kinds of inducers such as glucocorticoids and molecules of TGF-Î² superfamily, including bone morphogenic proteins (BMPs).
As the stem cells or progenitor cells differentiate, expression of osteoblast-associated genes, e.g. type I collagen (COLL I), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) and receptor (PTH1R) are active and/or lost, as the bone matrix matures and mineralizes. ALP is thought to increase until the mineralization, while OPN is strongly expressed during both the proliferation and differentiation stages, being upregulated prior to other matrix proteins, including BSP and OCN. OCN appears to be upregulated in parallel with mineralization.
In the presence of ascorbate, Î²-glycerophosphate, dexamethasone and/or vitamin D3, ADSCs differentiate into osteoblast-like cells in vitro and express ALP, RUNX2, BMP-2, BMP-4, BMP receptors I and II, and PTH receptor genes characteristic of osteoblast-like cells.
When induced for 2 to 4 weeks in vitro, in the appropriate induction conditions, ADSCs start to produce calcium phosphate mineral within their extracellular matrix and begin to express osteogenic genes and proteins.
Similarly to adipocytes and osteoblasts, chondrocytes likely develop from multipotent mesenchymal cells that give rise to progeny with more limited capacities. Nevertheless, there is little evidence distinguishing whether bone and cartilage forming cells arise from a common bipotential progenitor, or whether the two cell types arise from two separate monopotential precursors.
Furthermore, a common set of genes has been elucidated to be necessary for early adipogenic, osteogenic and chondrogenic differentiation in both BM-MSCs and ADSCs, although osteogenesis and adipogenesis appear to be linked in a differentiation branch separate from chondrogenesis. Yet, there is a particularly intriguing connection between the osteogenic and chondrogenic lineages, not only due to the possibility of a common bipotential progenitor but also due to the fact that hypertrophic chondrocytes can transdifferentiate into osteoblast-like cells.
For chondrogenic differentiation, ADSCs are routinely cultured in micro mass culture or pellet culture systems. The micro mass or pellet culture model mimics pre-cartilage condensation during embryonic development, which increases the cell-to-cell interaction and leads to the production of a cartilage-like matrix. The suspension of cells in hydrogel scaffolds has been done in the attempt to mimic the composition of native cartilage.
Chondrogenic differentiation requires the use of a defined medium supplemented with certain bioactive factors, including ascorbate- 2-phosphate, dexamethasone, L-proline and TGF-Î²1. Other factors of the BMP family have also been studied for chondrogenic induction of ADSCs. For example, while BMP-6 promotes chondrogenic differentiation, BMP-7 induces chondrogenic differentiation only when present in high doses. With the addition of chondrogenic induction factors and when maintained in an appropriate 3D environment in vitro, ADSCs will start to secrete the extracellular matrix proteins of cartilage, including COLL II, COLL VI and aggrecan.
Differentiation potential towards other lineages
In vitro, expanded ADSCs contain progenitor cells that have the ability to differentiate into mature endothelial cells and participate in blood vessel formation although the capacity may be limited. ADSC induced vessel formation and growth may be related to the secretion of proangiogenic factors or through perivascular functions of the ASCs (Madonna et al., 2009) or perhaps both.
Together with the angiogenic inductive features, human ADSCs also have potential to differentiate along the cardiomyocyte pathway. These preliminary reports point towards ADSCs having potential in regenerating cardiac tissue damaged through infarctions or ischemic injury.
ADSCs cells also demonstrate in vitro evidence for differentiation along the skeletal myocyte pathway. Under appropriate induction conditions, ADSCs express myoD and myogenin, transcription factors regulating skeletal muscle differentiation. The cells fuse, form multi-nucleated myotubes, and express protein markers of the skeletal myocyte lineage, such as myosin light chain kinase. This suggests that ADSCs may have applicability in the repair of damaged skeletal muscle in tissue engineering applications.
There is some evidence to suggest that human ADSCs can differentiate into cells of ectodermal origin, such as hepatocytes and neurons. When hepatogenically induced, ADSCs differentiate into hepatocyte-like cells, although the mechanisms are not yet clear. The cells expressed albumin and Î±-fetoprotein and showed LDL uptake and production of urea. Additionally, when transplanted into a murine model, with a carbon tetrachloride induced hepatic injury, the transplanted cells were able to express albumin in vivo.
Furthermore, when proper induction cues were applied in vitro, ADSCs displayed neuronal and/or oligodendrocytic markers. ADSCs take on a bipolar morphology, similar to that of neuronal cells, while expressing neuronal associated proteins such as nestin, intermediate filament M, Neu N, as well as glial fibrillary acidic protein (GFAP), a protein associated with oligodendrocyte differentiation.
WAT expansion takes place rapidly after birth as a result of increased fat cell size as well as an increase in fat cell number. Even at the adult stage, the potential to generate new fat cells persists. It has been demonstrated that fat cell number can increase when rats are fed a high-carbohydrate or high-fat diet (67, 68, 176). Increase in fat cell number is also observed in severe human obesity. However, the relative contribution of fat cell size and fat cell number to human adipose tissue growth on nutritional stimulation remains to be clarified. Moreover, fat cell precursors isolated from adult WAT of various species, including humans, can be differentiated in vitro into mature adipocytes (21, 58, 98, 104, 160, 213, 264). The potential to acquire new fat cells from fat cell precursors throughout the life span is now undisputed. The committed preadipocyte maintains the capacity for growth but has to with draw from the cell cycle before adipose conversion.
During adipocyte differentiation, acquisition of the adipocyte phenotype is characterized by chronological changes in the expression of numerous genes. This is reflected by the appearance of early, intermediate, and late mRNA/protein markers and triglyceride accumulation. These changes take place primarily at the transcriptional level, although posttranscriptional regulation occurs for some adipocyte genes (180, 295).
3.1. Growth Arrest
In preadipose cell lines as well as in primary preadipocytes, growth arrest and not cell confluence or cell-cell contact appears to be required for adipocyte differentiation. Although confluence leads to growth arrest, cell-cell contact is not a prerequisite for adipocyte conversion. Primary rat preadipocytes plated at low density in serum free medium can also differentiate in the absence of cell-cell contact (277).
C/EBP-Î± and PPAR-Î³ transcription factors have been shown to transactivate adipocyte specific genes. Both C/EBP-Î± and PPAR-Î³ also appear to be involved in the growth arrest that is required for adipocyte differentiation. McKnight and coworkers (276) have demonstrated the antimitotic activity of C/EBP-Î± through the use of a C/EBP-Î±-estrogen receptor fusion protein. Activation by estrogen treatment results in cessation of cell growth as assessed by cell number and DNA synthesis (276). In PPAR-Î³-expressing cells, cell cycle withdrawal is accompanied by a decrease in the DNA binding. Therefore, C/EBP-Î± and PPAR-Î³ may act cooperatively to bring growth arrest (6). Although C/EBP-Î± and PPAR-Î³ expression increases dramatically during adipocyte differentiation, the low level of these factors expressed in preadipocytes may be sufficient to mediate growth arrest that precedes differentiation.
3.2. Clonal Expansion
After growth arrest at confluence, preadipocytes must receive an appropriate combination of mitogenic and adipogenic signals to continue through subsequent differentiation steps. Studies on preadipose cell lines have shown that growth arrested cells undergo at least one round of DNA replication and cell doubling. This has been proposed to lead to the clonal amplification of committed cells (196). However, primary preadipocytes derived from human adipose tissue do not require cell division to enter the differentiation process (63). In these cells, inhibition of mitosis with cytosine arabinoside does not impair adipocyte development, indicating that clonal amplification of committed cells is not a critical step. These cells may have already undergone potential critical cell divisions in vivo and may therefore correspond to a later stage of adipocyte development.
Similarly, another group of growth arrest-specific (gas) genes shows a distinct expression pattern during clonal expansion. Gas6 appears to be preferentially expressed during clonal expansion of post confluent preadipocytes, whereas gas1and gas3 are expressed in serum-starved preadipocytes (244). Combined, these observations suggest differential regulation of the cell cycle in preconfluent proliferation versus postconfluent hormonally stimulated clonal expansion.
3.3. Early Changes in Gene Expression
Growth arrest and clonal expansion are accompanied by complex changes in the pattern of gene expression that can differ with the cell culture models and the specific differentiation protocols employed. Expression of lipoprotein lipase (LPL) mRNA has often been cited as an early sign of adipocyte differentiation (3, 47, 98, 123, 166). LPL is secreted by mature adipocytes and plays a central role in controlling lipid accumulation (48, 86). However, LPL expression occurs spontaneously at confluence and is independent of the addition of agents required for adipocyte differentiation (8, 9, 277). This suggests that LPL expression may reflect the growth-arrest stage rather than being an early differentiation step. It is also synthesized and secreted by other mesenchymal cell types including cardiac muscle cells and macrophages (49, 267). Because LPL expression is not adipocyte specific and it is independent of the additional agents required for adipocyte differentiation, classification of LPL as an early marker of adipocyte differentiation remains somewhat questionable.
The early expression of C/EBP and PPAR is logical given their subsequent involvement in terminal differentiation by transactivation of adipocyte-specific genes. PPAR-Î³ is largely adipocyte specific and is expressed at low but detectable levels in preadipocytes. Its expression rapidly increases after hormonal induction of differentiation. A transient increase in the expression of C/EBP-Î² and C/EBP-Î´ isoforms precedes the increase in PPAR-Î³ expression (27, 167, 299). The subsequent decrease of C/EBP-Î² and C/EBP-Î´ in early to intermediary stages of differentiation is concomitant with the induction of C/EBP-Î± mRNA. This increase in C/EBP-Î± expression occurs slightly before the expression of adipocyte-specific genes (27, 153, 167).
During adipocyte differentiation, cells convert from a fibroblastic to a spherical shape, and dramatic changes occur in cell morphology, cytoskeletal components, and the level and type of extracellular matrix (ECM) components. It is likely that these changes could influence the expression and action of PPARs and/or C/EBPs during adipocyte differentiation. Decrease in actin and tubulin expression is an early event in adipocyte differentiation that precedes overt changes in morphology and the expression of adipocyte-specific genes (255). These changes in cell shape reflect a distinct process in differentiation and are not the result of accumulated lipid stores.
A switch in collagen gene expression is also an early event of adipocyte differentiation. The relative concentrations of fibroblast-expressed type I and type III procollagen mRNA decline by 80-90%, while secretion of type IV collagen and entactin/nidogen increases (13, 290).
The amount of pericellular fibronectin, as well as cellular synthesis of fibronectin, decreases by four- to five folds during differentiation (10).
Preadipocyte factor-1 (pref-1), a preadipocyte protein with epidermal growth factor (EGF)-like repeats, has been hypothesized to be involved in maintaining the preadipose phenotype (246-248). A dramatic decrease in pref-1 expression accompanies adipocyte differentiation; it is abundant in preadipocytes and is not detectable in mature fat cells. It is the only known gene whose expression is completely downregulated during adipocyte differentiation