**3. Basic science background**

### **3.1. The outline and the anatomy of adipose tissue**

Adipose tissue is a complex and multi-depot organ, constituted for one third by mature adipocytes and for the other two thirds by a combination of a large variety of other cells. [1] Among represented cell lines are included small blood vessels, nervous cells, fibroblasts and, importantly, adipocyteprogenitor cells, also known aspreadipocytes orAdiposeDerivedStem Cells (ADSCs). Evolution has preserved in mammals two histologically different qualities of adiposetissue:whiteadiposetissue(WAT)andbrownadiposetissue(BAT),whicharecomposed by different types of mature adipocytes [Table 1]. In particular, white adipocytes are spherical, having a diameter ranging between 30 and 70 μm according to the amount of lipid depots, and lipids within the cells are organized in a single large "uni-locular" droplet, the size of which can exceed50μm.Thus,the lipiddroplet occupies the vastmajority ofthe whole intracellular space, pushingtheremainingcytoplasmandnucleusintoathinmarginalrim.Ontheotherhand,brown adipocytes are polygonal with a centrally placed nucleus and their cellular size ranges from 20 to 40μm.They accumulate lipids in smaller"multi-locular"droplets andthey are rich of specific mitochondria, containing the protein UCP-1 which is responsible for uncoupling of oxidative phosporylation and production of heat. WAT and BAT are both innervated by noradrenergic fibers of the sympathetic nervous system. As for the vascularization of adipose tissue, white adipocytes are organized in collections of fat lobules, each supplied by a selective arteriole and surrounded by septae of connective tissue. An individual adipocyte is supplied by an adjacent capillary and it is associated to a glycoprotein layer, reticular fibrils, fibroblasts, mastocytes and macrophages. Compared to WAT, BAT provides a more extensive vascular tree, characterized by dense multiple capillaries. The relevant vascularization of the latter in combination with the presence of a significantly high number of mitochondria, account for the typical "brown" color. WAT and BAT have also different roles in energy metabolism. Primary function of white adipocytes is to store excess energy as lipid, which is then mobilized in response to metabolic needs. Brown adipocytes, on the other hand, use accumulated lipids primarily as a source of energy released in the form of heat. WAT can be found in several anatomically distinct and separate collections, or "depots." There are two major anatomic subdivisions of these depots, eachshowinguniqueanatomic,metabolic,endocrine,paracrine,andautocrineproperties:intraabdominal or visceral adipose tissue and subcutaneous adipose tissue. In addition, WAT can alsobefoundinsmallamountsoffattylayerssurroundingotherorgans,suchastheheart,kidney and genitalia. Intra-peritoneal fat, composed of omental and mesenteric adipose tissue, comprises the vast majority of visceral fat. Importantly, subcutaneous adipose tissue shows different structural features in different anatomical districts. [2] In fact, fat depots in the abdominal area are characterized by the presence of large adipocytes, densely packed togeth‐ erandsurroundedbyapoor stromal(collagen)network.Instead,inmore localizeddepots (such as throcanteric areas, the sovra-pubic area, arm pits, medial regions of the knees, tights, arms, pectoralandmammaryareas)adipocytespresentasmallerdiameter,amorerepresentedstromal componentandamore extensivevascularnetwork.BATinnewborns andchildrencanbe found in several body areas. However, while in other small mammals these depots persist during growth,inhumans brownadipocytesundergo amorphologic transformation,rapidly accumu‐ lating lipids, becoming uni-locular and losing their typical ultrastructural and molecular properties, including mitochondria [Figure 1.]. As a consequence, there are no discrete collec‐ tions of BAT that can be found in human adults.


**Table 1.** Main differences between White and Brown adipocytes.

stem cell therapies for the repair and regeneration of various tissues and organs in a large number of different diseases. Thus, over the past years, this field has become a very active and attractive area of clinical and experimental research, providing significant outcomes and reaching important milestones. Today adipose tissue embodies an hot spot of regenerative

Giving the increasing amount of experimental and clinical data regarding adipose tissue and ADSCs, in this chapter we are going to briefly review the concepts and the insights behind the role of adipose tissue in regenerative medicine and tissue engineering. In particular we are going to focus the attention on current cutting edge translational research from bench to bedside, including the investigation of biological properties of ADSCs, the state of art of their manipulation, the latest progresses in their clinical adoption, the development of bio-engi‐

Adipose tissue is a complex and multi-depot organ, constituted for one third by mature adipocytes and for the other two thirds by a combination of a large variety of other cells. [1] Among represented cell lines are included small blood vessels, nervous cells, fibroblasts and, importantly, adipocyteprogenitor cells, also known aspreadipocytes orAdiposeDerivedStem Cells (ADSCs). Evolution has preserved in mammals two histologically different qualities of adiposetissue:whiteadiposetissue(WAT)andbrownadiposetissue(BAT),whicharecomposed by different types of mature adipocytes [Table 1]. In particular, white adipocytes are spherical, having a diameter ranging between 30 and 70 μm according to the amount of lipid depots, and lipids within the cells are organized in a single large "uni-locular" droplet, the size of which can exceed50μm.Thus,the lipiddroplet occupies the vastmajority ofthe whole intracellular space, pushingtheremainingcytoplasmandnucleusintoathinmarginalrim.Ontheotherhand,brown adipocytes are polygonal with a centrally placed nucleus and their cellular size ranges from 20 to 40μm.They accumulate lipids in smaller"multi-locular"droplets andthey are rich of specific mitochondria, containing the protein UCP-1 which is responsible for uncoupling of oxidative phosporylation and production of heat. WAT and BAT are both innervated by noradrenergic fibers of the sympathetic nervous system. As for the vascularization of adipose tissue, white adipocytes are organized in collections of fat lobules, each supplied by a selective arteriole and surrounded by septae of connective tissue. An individual adipocyte is supplied by an adjacent capillary and it is associated to a glycoprotein layer, reticular fibrils, fibroblasts, mastocytes and macrophages. Compared to WAT, BAT provides a more extensive vascular tree, characterized by dense multiple capillaries. The relevant vascularization of the latter in combination with the

medicine that may give rise to a new era of active stem cell therapy.

neered products and the actual therapeutic prospective opportunities.

**2. Purpose**

**2.1. Meeting the adipose tissue**

180 Regenerative Medicine and Tissue Engineering

**3. Basic science background**

**3.1. The outline and the anatomy of adipose tissue**

**Figure 1.** Monolayered culture of adipocytes in vitro with adipogenic medium.

#### **3.2. The living image of adipose derived stem cells**

The understanding of biochemical characteristics, molecular/cellular biology, immunebiological characteristics and phenotype of adipose tissue has significantly advanced in the last years. Adipose tissue has shown to consist mostly of cells of mesenchymal origin with few others endothelial cells, smooth muscle cells and pericytes, all showing low levels of cell senescence. Adipose tissue derives from the mesodermal layer of the embryo and develops both during pre-natal and post-natal growth. The microscopic location of the adipogenic progenitor cells in the adult is still controversial. [3] It remains to be proven whether the origin of the cells correlates with endothelial, pericytic or stromal compartments. A large number of surface antigens are in common with endothelial cells, suggesting a common origin. According to some researchers, adipogenic progenitor cells could be released directly by the bone marrow and distributed systemically by blood flow: experimental evidences of bone marrow derivedcells capable of differentiating into adipocytes in vivo have already been described but the contribution of these circulating cells to the overall growth and development of adipose tissue is still under investigation. Mesenchymal stem cells (MSC) were first described as immature cells in the bone marrow, capable to give rise to mesenchymal lineages such as osteoblasts, chondrocytes and adipocytes. [4] MSCs represent a small fraction of nucleated cells of human bone marrow (0.01%-0,0001%). MSCs are defined by three minimal criteria, as established by the International Society for Cellular Therapy in 2005: adherence to plastic dishes, specific surface antigen (CD73+, CD90+, CD105+, CD45-, CD34-, CD14 or CD11b-, CD79- or CD19-, HLA-DR) and in vitro capability to give rise to adipocytes, osteoblasts and chondrocytes. A similar protocol has been used for a long time to isolate adipose tissue progenitors: the resulting immature adherent cells were thus called pre-adipocytes. To obtain these cells fat pads are minced and digested with collagenase, separating an upper layer of floating mature adipocytes from a lower layer of pelleted stromal vascular fraction (SVF). [5] The SVF is an heterogeneous cell population of circulating blood cells, fibroblasts, pericytes, endothelial cells and preadipocytes. Pre-adipocytes may be isolated from the SVF by plating and washing. This cell population, adopting appropriate differentiating agents, can give rise to mature adipocytes, demonstrating their nature of adipose progenitors. Cell cultures have provided evidence of regenerative capacities in both the heterogeneous stromal vascular fraction (SVF) and in the more homogeneous adipose-derived stem cells (ADSCs). In 2002 pre-adipocytes were better characterized and they were demonstrated to show clear multi-potency potential: thus, they were named Adipose Derived Stem Cells (ADSCs). [6] In particular, ADSCs represent a mesodermal stem cell population with clonal mesodermal, ectodermal, and endodermal potentials capabilities that express multiple CD marker antigens similar to those of other mesenchymal stem cells as those residing in bone marrow. Several investigations have reported a differentiation into adipogenic, osteogenic, chondrogenic and myogenic lineages in vitro by means of specific culture media. In particular, the potential to differentiate into nonmesodermal lineages is exciting. The differentiation into neural precursors, which are of an ectodermal origin, has been described. In addition, evidence of differentiation into hepato‐ cytes, pancreatic islet cells, endothelial cells and other epithelial cells has been provided in different reports. By definition, a stem cell is characterized by the ability to self-renew and to differentiate along multiple lineage pathways. Since the self-renewal of ADSCs has not been fully established yet, it is accepted that some investigators may use the same acronym to mean "adipose-derived stromal cells", in agreement with the statement of the International Society for Cellular Therapy. Indeed, ADSCs present several differences from MSCs at genomic, proteomic and functional levels. For instance, during the earliest rounds of proliferation, ADSCs express the CD34 antigen: the frequency of these cells is much higher (100 to 500 folds higher) than that of MSCs in the bone marrow. In addition, MSCs are probably more committed towards osteoblastic and chondrogenic lineages than ADSCs. Thus, although numerous author use the same term "MSCs" both for cells derived from bone marrow and for those derived from adipose tissue, MSCs and ADSCs are probably two distinct cell populations. A more precise definition of ADSCs, based on their immune-phenotype and/or differentiation capabilities, has not been yet provided. Some authors believe that ADSCs are a heterogeneous group of progenitor cells with differences in their stem cell potential. Thus, ADSCs and SVFs cells represent an autologous alternative to pluri-potent embryonic stem cells with a multilineage differentiation potential, a significant therapeutic impact and a critical role in the rapidly expanding fields of tissue engineering and regenerative medicine. Significantly, further investigations are needed to better clarify these aspects. Importantly, the most important characteristics of ADSCs, with a possible interest for clinical applications, comprise their multi-potency, secretory functions and immune-modulatory capabilities.

#### *3.2.1. Differentiation potential of ADSCs*

**Figure 1.** Monolayered culture of adipocytes in vitro with adipogenic medium.

The understanding of biochemical characteristics, molecular/cellular biology, immunebiological characteristics and phenotype of adipose tissue has significantly advanced in the last years. Adipose tissue has shown to consist mostly of cells of mesenchymal origin with few others endothelial cells, smooth muscle cells and pericytes, all showing low levels of cell senescence. Adipose tissue derives from the mesodermal layer of the embryo and develops both during pre-natal and post-natal growth. The microscopic location of the adipogenic progenitor cells in the adult is still controversial. [3] It remains to be proven whether the origin of the cells correlates with endothelial, pericytic or stromal compartments. A large number of surface antigens are in common with endothelial cells, suggesting a common origin. According to some researchers, adipogenic progenitor cells could be released directly by the bone marrow and distributed systemically by blood flow: experimental evidences of bone marrow derivedcells capable of differentiating into adipocytes in vivo have already been described but the contribution of these circulating cells to the overall growth and development of adipose tissue is still under investigation. Mesenchymal stem cells (MSC) were first described as immature cells in the bone marrow, capable to give rise to mesenchymal lineages such as osteoblasts, chondrocytes and adipocytes. [4] MSCs represent a small fraction of nucleated cells of human bone marrow (0.01%-0,0001%). MSCs are defined by three minimal criteria, as established by the International Society for Cellular Therapy in 2005: adherence to plastic dishes, specific surface antigen (CD73+, CD90+, CD105+, CD45-, CD34-, CD14 or CD11b-, CD79- or CD19-, HLA-DR) and in vitro capability to give rise to adipocytes, osteoblasts and chondrocytes. A similar protocol has been used for a long time to isolate adipose tissue progenitors: the resulting immature adherent cells were thus called pre-adipocytes. To obtain these cells fat pads are minced and digested with collagenase, separating an upper layer of floating mature adipocytes

**3.2. The living image of adipose derived stem cells**

182 Regenerative Medicine and Tissue Engineering

ADSCs, like MSCs, have the ability to differentiate into mesodermal cells, such as adipocytes, fibroblasts, myocytes, osteocytes and chondrocytes, in a process called lineage-specific differentiation. The increasing evidence for the ability of ADSCs to differentiate into cells of non-mesodermal origin such as neurons, endocrine pancreatic cells, hepatocytes, endothelial

cells and cardiac myocytes, is surprising. This process is called "cross-differentiation". Lineage-specific differentiation can be tracked at a molecular level by the expression of key transcription factors of mature tissues. The earlier stages of differentiation, named "allocation" or "commitment", that drive the ADSCs into the specialized lineage are not completely known yet. In vitro, the differentiation of multi-potent cells into a desirable cell phenotype can be obtained by appropriate culture conditions and stimulation with a cocktail of known differ‐ entiating agents [Table 2].


**Table 2.** Experimental growth factors used for differentiation of ADSCs in different cell lineages.

#### **•** Adipogenic differentiation

ADSCs have an exceptional potential for differentiation into mature adipocytes, which is very promising in developing techniques for repairing soft-tissue defects. [7] Differentiation can be induced by a large variety of substances, including insulin, dexamethasone, rosiglitazone and indomethacin. During differentiation ADSCs, initially showing a fibroblast-like spindle or stellate shape, undergo morphologic changes with the appearance of one or more lipid vacuoles and they begin to express several genes and proteins characterizing the mature adipocyte, including leptin, peroxisome-proliferating activated receptor γ (PPARγ), glucose transporter type 4 (GLUT4) and glycerol-3-phosphate dehydrogenase (GPDH).

**•** Osteogenic differentiation

Osteogenic differentiation can be induced in vitro by supplementing the culture medium with dexamethasone, β-glycerophosphate and vitamin D3. The acquisition of the osteoblast phenotype is accompanied by expression of specific genes and proteins, including alkaline phosphatase, type I collagen, osteopontin, osteonectin, and Runx2. Osteogenic differentiation may also be obtained by transfection of osteogenic lineage-determining genes (BMP2 and Runx2): this approach has proved to be effective both in vitro and in vivo in a large number of reports. These experimental findings hold great promise for the use of ADSCs in bone regeneration.

**•** Chondrogenic differentiation

cells and cardiac myocytes, is surprising. This process is called "cross-differentiation". Lineage-specific differentiation can be tracked at a molecular level by the expression of key transcription factors of mature tissues. The earlier stages of differentiation, named "allocation" or "commitment", that drive the ADSCs into the specialized lineage are not completely known yet. In vitro, the differentiation of multi-potent cells into a desirable cell phenotype can be obtained by appropriate culture conditions and stimulation with a cocktail of known differ‐

Adipogenic Insulin; isobutylmethylxanthine (IBMX) ; dexamethasone; rosiglitazone;

Osteogenic Dexamethasone; β-glycerophosphate; vitamin D3; bone morphogenetic

Chondrogenic insulin growth factor (IGF); BMPs; transforming growth factor-β (TGF-β)

Neurogenic Valproic acid; epidermal growth factor (EGF); fibroblast growth factor

Tendinous FGF; platelet derived growth factor (PDGF-BB); EGF; TGF-β; IGF-1; BMPs

ADSCs have an exceptional potential for differentiation into mature adipocytes, which is very promising in developing techniques for repairing soft-tissue defects. [7] Differentiation can be induced by a large variety of substances, including insulin, dexamethasone, rosiglitazone and indomethacin. During differentiation ADSCs, initially showing a fibroblast-like spindle or stellate shape, undergo morphologic changes with the appearance of one or more lipid vacuoles and they begin to express several genes and proteins characterizing the mature adipocyte, including leptin, peroxisome-proliferating activated receptor γ (PPARγ), glucose

Osteogenic differentiation can be induced in vitro by supplementing the culture medium with dexamethasone, β-glycerophosphate and vitamin D3. The acquisition of the osteoblast phenotype is accompanied by expression of specific genes and proteins, including alkaline phosphatase, type I collagen, osteopontin, osteonectin, and Runx2. Osteogenic differentiation may also be obtained by transfection of osteogenic lineage-determining genes (BMP2 and Runx2): this approach has proved to be effective both in vitro and in vivo in a large number

(FGF); nerve growth factor (NGF) and brain-derived neurotrophic factor

indomethacin.

protein (BMP-2)

Myogenic/cardiomyogenic Dexamethasone; hydrocortisone; IL-3; IL-6

(BDNF)

**Table 2.** Experimental growth factors used for differentiation of ADSCs in different cell lineages.

transporter type 4 (GLUT4) and glycerol-3-phosphate dehydrogenase (GPDH).

entiating agents [Table 2].

184 Regenerative Medicine and Tissue Engineering

**•** Adipogenic differentiation

**•** Osteogenic differentiation

**Type of differentiation Stimulating factors**

Vascular/endothelial Specific environment

Insulin growth factor (IGF), bone morphogenetic proteins (BMPs), and transforming growth factor-β (TGF-β) have shown to induce chondrogenic differentiation of ADSCs when added to the culture medium. Chondrogenic differentiation occurs also by seeding ADSCs into polyglycolic acid (PGA) scaffolds, as it was largely demonstrated in several other in vitro models and in vivo in nude mice.

**•** Differentiation into other lineages

Terminally differentiated myoblasts can be obtained in vitro, showing the ability to form multinucleated myotubules and to shrink/diastole under the influence of atropine. This property of ADSCs is of particular interest for the treatment of genetic muscular dystrophies: preclinical in vivo studies on animal models are currently ongoing. In addition, other studies have focused on the capability of ADSCs to differentiate into cardiomyocytes with a possible application in heart regeneration or repair after an ischemic injury. Furthermore, endothelial regeneration is another important field of research: ADSCs have shown to be able to differen‐ tiate into endothelial cells and to secrete several pro-angiogenic factors, like vascular endo‐ thelial growth factor (VEGF) and platelet-derived growth factor (PDGF). Differentiation into neuron-like cells has also been reported by different authors: ADSCs may acquire a neurallike morphology and they may express several proteins specific for the neuronal phenotype (Neuron Specific Enolase; Neuron Specific Nuclear Protein). Finally, some studies have explored the chance for ADSCs to differentiate into pancreatic islet cells, hepatocytes and epithelial cells with the purpose to find an alternative cellular therapy for diseases such as diabetes mellitus and liver disfunction: data and outcomes are however still preliminary and lacking of strong evidence.

#### *3.2.2. ADSCs as a secretome*

Importance of ADSCs does not only reside in their potential to differentiate in mature lineages. Similarly to the original adipose tissue from which they can be isolated, ADSCs have shown to act as a "secretome", accurately regulating proteins and growth factors secreted into the extracellular milieu and having a relevant impact on different organs and systems within the human body [Table 3.]. [8] Trophic effects of ADSCs include stimulation of angiogenesis, hematopoietic support, gene transfer and suppression of inflammation. Indeed ADSCs represent a source of several cytokine/soluble factors regulating the survival and differentia‐ tion of various endogenous cells/tissues. A large number of these molecules have been related to the regenerative attitude of ADSCs: among these, we may include hepatocyte growth factor (HGF), granulocyte and macrophage colony stimulating factors, interleukins (ILs) 6, 7, 8 and 11, tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF), brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), adipokines and others. Full characterization of the secretory profile of ADSCs, either by immune-enzymatic techniques (ELISA) or by mass spectrometry, is still object of investigation. Several adipokines such as adiponectin, angiotensin, cathepsin D, penetraxin, pregnancy zone protein and retinol binding protein, as well as stromal cell-derived growth factor (CXCL12) have been found in the conditioned media of ADSCs differentiating towards the adipocyte lineage. ADSCs secrete also oher different well characterized cytokines (GM-CSF, TGF-β, PGE2, IGF-1) and their release can be modulated by exposure to different agents, such as b-FGF and EGF or inflam‐ matory stimula, like lipopolysaccharide (LPS). The role of these and other factors has been investigated by multiple studies regarding one or more possible applications of ADSCs in the field of regenerative medicine. Brain Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Glial Derived Neurotrophic Factor (GDNF) are thought to be important molecules secreted by ADSCs mediating neurotrophic effects and modulating in animal models of Parkinson Disease the recovery after hypoxic-ischemic injuries. Hepatocyte Growth Factor (HGF) and Vascular Endothelial Growth factor (VEGF) are the most important factors capable of inducing angiogenesis in areas that have undergone ischemic episodes and their importance is particularly relevant in wound healing. In cardiac regeneration, IGF-1 and VEGF mediate respectively an anti-apoptotic and angiogenic action, to which is attributed the capacity of ADSCs to have beneficial effects when transplanted/injected in different animal models of myocardial infarction/failure. In conclusion, most of ADSCs secreted factors act through mechanisms that mediate protection against cell death or, alternatively, induce cell migration and proliferation. Alternatively, they can indirectly act on the targeted cell popula‐ tions: by promoting vascularization they can be indirectly linked to an increase of oxygen and nutrients in the affected areas, which may in turn promote local regenerative processes. Indeed, up to now most reports have focused on a limited set of known factors but it is expected that other molecules are responsible for the regenerative effects of ADSCs.

#### *3.2.3. Immunomodulatory properties of ADSCs*

The regenerative potential of ADSCs has been related also to their immune-modulatory abilities. ADSCs have been shown to be an immune-privileged site, preventing severe graftversus-host response after transplantation procedures in vitro and in vivo. A concern of fundamental importance is the interplay between ADSCs and the host tissue, with particular focus on the immune system. Several studies have shown that ADSCs can be used either for autologous or allogenic cell transplants: this feature would be a major advantage for the employment of adipose tissue as a source for cell-based therapies. Furthermore ADSCs seem to act also as modulators of the immune system. The allogenic potential of these cells could be explained by the property of ADSCs to decrease the expression of hematopoietic markers and HLA-DR after subsequent passages. In addition, it has been observed that ADSCs only express HLA class I, but not HLA class II molecules: the latter can only be induced in ADSCs after incubation with IFN-γ. Furthermore, several experiments have proved that ADSCs do not stimulate lymphocyte proliferation and they do not elicit a response by Mixed Lymphocyte Reaction (MLR): in addition, they can also inhibit phyohemagglutinin (PHA)-stimulated lymphocyte proliferation. These immune-suppressive effects are likely mediated by soluble factors, among which PGE-2 seems to be the most important. Notably, the secretion of cytokines by ADSCs can be modulated not only by the inflammatory stimulus but also by the surface upon which they are seeded: thus the bio-scaffold/environment provided could be another mechanism to control the immune-modulatory properties of ADSCs.


**Table 3.** Synopsis of properties of ADSCs.
