Keywords

1 Introduction

1.1 The Layout of the Chapter

To cover the essential knowledge on this topic, the chapter is divided into several sections, including an outline of stroke as a life-threatening disease and the current medical strategies, the introduction of adipose-derived mesenchymal stem cells (ADSC) as a treatment tool for stroke and its therapeutic advantages, description of the general methodology of ADSC isolation, the mechanisms of action of ADSC against stroke, the current status of experimental and clinical studies as well as the perspectives of ADSC applications and the potential risks and limitations.

1.2 Stroke, Current Treatment Strategies, and Their Limitations

Stroke, which can be of ischemic or hemorrhagic origins due to a disruption of blood supply to the brain tissue, has been reported to be the second leading cause of death worldwide (Feigin et al. 2014). Not only is stroke a ruthless killer, but it also leaves behind devastating neurological deficits (i.e., those of motor, sensory, and cognitive) that account for their being the third leading cause of disability (Rosado-de-Castro et al. 2013). The cost for chronic care and rehabilitation as well as the remarkable impairment of the quality of life for the affected individuals impose an enormous socioeconomic burden on a society (Go et al. 2013).

The current mainstay of treatment for acute ischemic stroke, which accounts for over 80 % of all cases of stroke (Liu et al. 2013), includes the use of thrombolytic agents (e.g. recombinant tissue plasminogen activator, rtPA) that are supposed to be given within 4.5 h after the attack (Del Zoppo et al. 2009) with the hope of resuming patency of the supplying artery to restore the function of tissue surrounding the core region of infarction (i.e. ischemic penumbra) to minimize the ischemic insult rather than the irreversible infarction itself (Smith et al. 2011). However, the narrow therapeutic time window and the significant risk of symptomatic intracranial hemorrhage (i.e. up to 5.6 %) and death (Wardlaw et al. 2009; Seet and Rabinstein 2012) substantially hinder its use which is only suitable for only 2–4 % of patients with ischemic stroke (Molina 2011). The popularity of rtPA use is further hampered by its limited efficacy in disability prevention which is only six patients per 1000 ischemic strokes and its lack of beneficial impact on mortality rate (Hacke et al. 2004). As a result, various non-pharmaceutical strategies have been proposed including neuroprotective approaches such as hypothermia, ischemic/hypoxic conditioning, acupuncture, certain medical gases, and transcranial laser therapy as well as mechanical endovascular recanalization and recovery devices for treating the chronic phase of stroke (Chen et al. 2014b).

Since ischemic injury of the brain involves a cascade of events, a multi-faceted therapeutic approach is preferred (Chen et al. 2014b). The clinical possibility of cell therapy (i.e., “cell replacement therapy” or “cell transplant therapy”) for central nervous system disorders, including stroke, gained much attention in the year 2000 (Bjorklund and Lindvall 2000; Zivin 2000) when the first clinical trial on neuronal cellular transplantation in patients with stroke was reported (Kondziolka et al. 2000). Although it was not stem cell that was transplanted, it opened up the avenue for exploring the therapeutic potential of stem cell transplantation for stroke (Cairns and Finklestein 2003).

1.3 Stem Cells: Their Natures, Sources, and Therapeutic Potentials

The two distinctive properties that distinguish stems cells from other somatic cells are “self-renewal” and “potency” (Kuhl and Kuhl 2013). Self-renewal refers to the process in which a stem cell undergoes mitotic cell division to produce at least one daughter cell with equal developmental potential as the mother cell, in other words, another stem cell . On the other hand, potency is the ability of a stem cell to differentiate into different mature specialized cell types (i.e. multi-lineage differentiation). Regarding the use of stems cells in the treatment of stroke, experimental studies using embryonic stem cells (Chang et al. 2013; Drury-Stewart et al. 2013), mesenchymal stem cells (Ikegame et al. 2011), hematopoietic stem cells (Tsuji et al. 2014), neural stem cells (Andres et al. 2011), induced pluripotent stem cells (iPSC) (Oki et al. 2012), and also multipotent adult progenitor cells (Mora-Lee et al. 2012) in animal models of stroke have been reported with unanimous positive therapeutic results. Although mesenchymal stem cells were first identified four decades ago as adherent cells with fibroblastoid morphology being able to differentiate into cells of mesodermal origin such as osteocytes, chondrocytes, and adipocytes (Friedenstein et al. 1974), they were later found to be also capable of differentiating into ectodermal and endodermal elements (Lakshmipathy and Verfaillie 2005; Ikegame et al. 2011). The fact further highlights their observed therapeutic versatility against a variety of diseases of different pathological origins as reflected in their abilities of vascular endothelial (Li et al. 2013a), neuronal (Gao et al. 2013), and musculoskeletal (Gardner et al. 2013) repairs.

2 Adipose-Derived Stem Cells: Therapeutic Advantages, Sources, and Isolation

2.1 Advantages of Therapeutic use of Adipose-Derived Mesenchymal Stem Cells Compared with Stem Cells of Other Origins

Compared with embryonic stem cells , autologous mesenchymal stem cells have the advantage of being self-derived without the concern of ethics and that of possible infection from unknown donors. The reported sources of mesenchymal stem cells include bone marrow (Skvortsova et al. 2008), adipose tissue (Gutierrez-Fernandez et al. 2013a), embryo (Liu et al. 2009), placenta (Kranz et al. 2010), dental pulp and periodontal ligament (Moshaverinia et al. 2014; Vasandan et al. 2014). Other sources, including palatine tonsil (Janjanin et al., 2008), dermis (Feisst et al. 2014), and skeletomuscular system (Aydin et al. 2014; Mason et al. 2014), have also been reported. In particular, the two most readily available sources of autologous ADSC, bone marrow and adipose tissue, have been widely investigated both experimentally and clinically regarding their therapeutic potentials in treating a myriad of diseases, especially ischemic and microvascular disorders (Calio et al. 2014; Liu et al. 2014).

Previous studies have shown that not only are adipose-derived stem cells (ADSC) (also known as “adipose-derived mesenchymal stem cells”, “adipose tissue-derived multipotent stromal cells” or “adipose-derived mesenchymal stromal cells”) relatively easy to obtain with less invasive procedures compared to bone marrow stem cells, but the former also exhibit better proliferative activity, differentiating capacity, immunomodulatory function, and trophic factor-releasing ability than the latter including greater production of vascular endothelial growth factor (VEGF), angiopoietin-1, and hepatocyte growth factor (HGF) (Ikegame et al., 2011) as well as interleukin 1 receptor (IL-1R), IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 1, nerve growth factor (Banas et al., 2008; Ikegame et al. 2011), and transforming growth factor (TGF)-β1 (Melief et al. 2013b). Furthermore, the lack of major histocompatibility complex class II (MHC-II) expression in adipose-derived stem cells also enables their storage and allogeneic administration to individuals with acute ischemic stroke (Gutierrez-Fernandez et al. 2012). On the other hand, although the discovery of iPSC seems to offer a solution to the problem of the limited availability of stem cells through reprogramming of autologous somatic cells (Takahashi and Yamanaka, 2006), the requirement for the activation of potentially tumorigenic genes (e.g. c-myc) for its induction (Araki et al. 2011) has raised much clinical concerns. ADSC, therefore, appear to be an applicable clinical tool for routine clinical practice.

2.2 Source- and Donor-Dependent Variability in ADSC Quality

Previous animal experimental studies have used adipose tissue from different regions for autologous ADSC isolation, including inguinal (Chen et al. 2012) and peri-epididymal (Leu et al. 2010; Jiang et al. 2014) fat depots. Besides, other studies using human ADSC for animal studies also showed positive therapeutic results (Kang et al. 2003b; Kim et al. 2007; Yang et al. 2012; Liu et al. 2014). Therefore, it appears that the source of ADSC has no remarkable bearing on the treatment outcomes. As a result, although subcutaneous adipose tissue especially that from the abdomen, thigh, and buttock removed through fat-extracting body-shaping procedures such as liposuction and lipectomy (Chia and Theodorou 2012) used to be regarded as medical waste products, it now serves as a readily available clinical source of adipose-derived stem cells. On the issue of the optimal source of ADSC, a previous clinically-oriented study comparing the properties of ADSC isolated from human adipose tissues of different depots, including abdominal subcutaneous fat, omentum, pericardial adipose tissue, and thymic remnants, has demonstrated that ADSC isolated from different sources exhibited varied proliferation and differentiation capacities that should be taken into account to serve a specific therapeutic purpose (Russo et al. 2014). For instance, ADSC isolated from intrathoracic depots exhibited a longer average doubling time and a higher proportion of CD34+ cells compared with those isolated from subcutaneous fat or the omentum. Moreover, subcutaneous and pericardial adipose tissue yielded ADSC with enhanced adipogenic differentiation potential, while ADSC from the omentum displayed high levels of osteogenic markers (Russo et al. 2014). Another study that underscores the importance of the origins of ADSC from a therapeutic point of view is its finding that adipose tissues of slightly different origins in close vicinity (i.e., epicardial fat, pericardial fat, and the right atrium) exhibited significantly different capacities of secreting trophic and inflammatory cytokines, different degrees of upregulation of inflammation- and fibrosis-related genes, as well as different therapeutic effects in a rat model of myocardial infarction to which mesenchymal stem cells from right atrium and epicardial fat were even found to be detrimental (Naftali-Shani et al. 2013). On the other hand, specific origins of ADSC may also be taken into account for special therapeutic needs. For instance, a clonogenic population of metabolically active stem cells has been reported to reside in adult human brown adipose tissue that may be activated to participate in energy homeostasis in vivo and, therefore, may be of therapeutic importance for obesity and related metabolic disorders (Silva et al. 2014).

The correlation between donor-dependent variability and the quality of ADSC has also been widely investigated. For instance, one study demonstrated a negative association of donor age with the proliferation and differentiation potential of ADSC (Choudhery et al. 2014). Donors of advanced age has also been reported to reduce the yield of ADSC expressing low-affinity nerve growth factor receptor (CD271) (Cuevas-Diaz Duran et al. 2013) and also produce ADSC of impaired angiogenic capacities (De Barros et al. 2013). On the other hand, infant-derived cells have been shown to be morphologically more elongated with long telomeres, and exhibit augmented angiogenic and osteogenic abilities compared with older cells (Wu et al. 2013). Nevertheless, other studies using adipose tissue from human adults showed that donor age, body-mass index, and harvest site do not influence cell yield and proliferation rate (Buschmann et al. 2013). Moreover, doubling time, telomere length, the osteogenic and chondrogenic differentiation capacity, as well as osteogenic paracrine activity were also found to be similar among ADSC from adult donors of different ages (Ding et al. 2013; Wu et al. 2013). Consistently, another experimental study investigating the impact of donor age on the function of adipose-derived stem cells also demonstrated that aged ADSC from rats still retained potential to support axon regeneration (Mantovani et al. 2012). One interesting finding is that ADSC from older donors were found to exhibit compromised adipogenic potential that actually favors their application in regeneration therapy (Ding et al. 2013). The overall promising proliferation and differentiation capabilities of ADSC regardless of the donor’s age, therefore, open up an avenue to their clinical application, taken into account that the elderly will be the greatest beneficiaries of autologous stem cell treatment. On the other hand, the morphology, proliferation rate, and doubling time of ADSC have also been shown to vary with the nature of the coatings on which they were cultured (Marycz et al. 2013).

2.3 Isolation, Culture, and Identification of Adipose-Derived Mesenchymal Stem Cells

The isolation, culturing, and identification of ADSC are straightforward, including the procedures of mincing, digestion, filtering, centrifugation, culturing, and flow cytometric identification as described previously (Leu et al. 2010). Briefly, the harvested adipose tissue is minced into < 1 mm3 size pieces using a pair of sharp, sterile surgical scissors to maximize the surface areas for enzyme digestion (Fig. 9.1). Then 200–300 μL of sterile saline is added to every 0.5 g of tissue to prevent dehydration. Sterile saline (37 °C) is added to the homogenized adipose tissue in a ratio of 3:1 (saline: adipose tissue), followed by the addition of stock collagenase solution to a final concentration of 0.5 Units/mL. The tubes with the contents are placed and secured on a Thermaline shaker and incubated with constant agitation for 60 ± 15 min at 37 °C. After 40 min of incubation, the content is triturated with a 25 mL pipette for 2–3 min. The cells obtained are placed back to the rocker for incubation. The contents of the flask were transferred to 50 mL tubes after digestion, followed by centrifugation at 600 g, for 5 min at room temperature. The fat layer and saline supernatant from the tube are poured out gently in one smooth motion or removed using vacuum suction. The cell pellet thus obtained is resuspended in 40 mL saline and then centrifuged again at 600 g for 5 min at room temperature. After being resuspended again in 5 mL saline, the cell suspension is filtered through a 100 μm filter into a 50 mL conical tube to which 2 mL of saline is added to rinse the remaining cells through the filter. The flow-through is pipetted to a 40 μm filter into a new 50 mL conical tube. The tubes are centrifuged for a third time at 600 g for 5 min at room temperature. The cells, which are a mixture of lymphocytes, macrophages, fibroblasts , endothelial cells, and other cell populations, are resuspended in saline. An aliquot of cell suspension can then be removed for cell culturing in DMEM-low glucose medium contain 10 % FBS for two weeks. Flow cytometric analysis is subsequently used for the identification of cellular characteristics after cell labeling with appropriate antibodies. The flow cytometric characteristics and typical morphology of ADSC are also shown in Fig. 9.1. A previous study comparing the phenotypes of different mesenchymal stem cells isolated from human term placental chorionic villi, umbilical cord , adult bone marrow and adipose tissue demonstrated that, although the phenotypes were mostly similar among stem cells of different origins, vascular cell adhesion molecule 1 (VCAM-1) (i.e. CD106) was highly expressed on chorionic villi-derived mesenchymal stem cells, whereas it was moderately expressed on bone marrow-derived mesenchymal stem cells and absent on ADSC (Yang et al. 2013). Another study also showed consistent results (Zhu et al. 2012).

Fig. 9.1
figure 1

Simplified procedures of harvesting, processing, culturing, and characterization of adipose-derived stem cells. After being removed from the human body through procedures such as liposuction and lipectomy, the adipose tissue is minced to small pieces of size less than 1 mm3 to maximize the efficiency of enzymatic digestion for freeing the cells from connective tissue. After cell-harvesting through filtration and centrifugation, the cells are purified with the number of cells expanded through culturing. The cultured cells subsequently undergo flow cytometric analysis for the identification of surface markers characteristic of mesenchymal stem cells. (Note the typical spindle-shaped morphology of mesenchymal stem cells at right lower corner)

Full size image

2.4 Automated Devices for Adipose-Derived Stem Cell Isolation

Compared with other tissues from which stem cells are isolated, adipose tissue has been shown to have at least two log greater concentrations of available stem and progenitor cells. This knowledge enables the direct utilization of these useful cellular elements without prior ex vivo expansion (Hicok and Hedrick 2011). Indeed, the Celution system, which is a closed, commercially available automated platform for adipose tissue processing for the isolation of adipose-derived stem and progenitor cells, has been described in 2011 (Hicok and Hedrick 2011). The system has been reported to take only 2.5 h for processing and successfully applied clinically (Marino et al. 2013). The “stromal vascular fraction” (SVF) thus obtained comprises both live and dead cells. Therefore, one noteworthy concern is that the cell debris may contribute to subsequent inflammatory responses that would potentially alter cell differentiation (Ye and Gimble 2011). Accordingly, several approaches have been proposed for retrieving the viable cells from SVF, including fluorescence-activated cell sorting (FACS), magnetic activated cell sorting (MACS), and dielectrophoresis (Wu and Morrow 2012). The former two involve the use of antibodies, while the latter retrieves life cells based on the presence of charge on their surface.

3 ADSC as a Therapeutic Option Against Stroke: Principles and Mechanisms

3.1 Therapeutic Actions of ADSC Implicated in Pathophysiological Changes of Stroke

The therapeutic role of ADSC against stroke could best be understood by reviewing the essential pathological changes and the physiological recovery mechanisms involved. Ischemia-induced inflammatory responses in stroke involve not just the neurons, but also other components of the neurovascular unit (del Zoppo 2009). This finding underscores the importance of immunomodulation in the management of stroke instead of merely restoring tissue perfusion (Iadecola and Anrather 2011). In addition, investigation of brain recovery from ischemic stroke has revealed the plasticity of the repairing process that involves axonal outgrowth and myelination (Ueno et al. 2012). Moreover, beside necrosis, apoptosis initiated after the stroke attack also results in irreversible loss of cellular elements in the central nervous system (Ouyang and Giffard 2013). Furthermore, although resuming patency of the obstructed vessel through fibrinolysis or angioplasty theoretically salvages the region at risk of ischemic infarction, the resulting ischemia-reperfusion injury actually triggers a cascade of inflammatory events (Iadecola and Anrather 2011; Liu et al. 2014). The major contributor to injuries following reperfusion is the reactive oxygen species (ROS) generated both from inflammatory cells and damaged mitochondria (Manzanero et al. 2013).

Pathologically, similar to the microscopic changes observed in animal models of stroke, evidence of reactive gliosis has been reported in human subjects after ischemic stroke including increased numbers of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes and ED1-positive activated microglia as well as enhanced expression of chondroitin sulphate proteoglycans (CSPG) in the cortical penumbra regions (Huang et al. 2014). Hence, stroke involves a series of pathological changes that require a number of corresponding measures for the subsequent repairing. This is reflected in the results of a previous study that demonstrated the activation of hundreds of genes responsible for not only tissue repair, but also nervous system development and cell proliferation both in the penumbra and core of infarct as early as 24 h after ischemic stroke in rats (Ramos-Cejudo et al. 2012), highlighting the complexity of the repairing process. Since it is proposed that stem cells , which are known to participate in physiological tissue repair in various organs, may have a significant role to play in the recovery process after stroke (Gutierrez-Fernandez et al. 2012), numerous previous studies have been conducted to investigate the therapeutic potential of ADSC using the known recovery mechanisms of stroke as referring parameters.

3.2 Observed Therapeutic Effects of ADSC Against Stroke

To date, most results of the therapeutic use of ADSC against stroke came from animal studies for which middle cerebral artery occlusion (MCAO) is the commonly used model. The parameters for assessment were based on the established pathological changes after stroke at molecular, cellular, and functional levels. For instance, the findings of increased levels of chemokine receptor type 4 (CXCR4), stromal cell-derived factor 1 (SDF-1), IL-8/Gro, Doublecortin (DCX) (i.e., marker of migrating neuroblasts), von Willebran factor (vWF), and endothelial cell markers as well as enhanced microvessel proliferation after ADSC treatment in a rat ischemic stroke model in one study (Leu et al. 2010), together with consistent observation of augmented expressions of basic fibroblast growth factor (bFGF) and VEGF with enhanced angiogenesis in the brain in another animal investigation (Wang et al. 2008), highlight the roles of ADSC in nerve repair and revascularization in the ischemic brain. Reinforcing evidence was provided by another study that demonstrated elevated levels of VEGF, synaptophysin (SYP), oligodendrocyte (Olig-2) and neurofilament (NF) in rats after ADSC treatment compared to those in untreated animals 14 days after MCAO (Gutierrez-Fernandez et al. 2013b). The reduction in expression of GFAP in the previous studies also signifies an amelioration of reactive gliosis after ADSC treatment (Leu et al. 2010; Gutierrez-Fernandez et al. 2013b; Jiang et al. 2014). Besides, the suppressed mRNA expressions of Bax and caspase 3 as well as the increased expression of Bcl-2 in animals with stroke after ADSC treatment compared to those in the untreated group suggest an anti-apoptotic function of ADSC (Leu et al. 2010; Jiang et al. 2014). On the other hand, intravenous infusion of human ADSC has also been reported to attenuate neurological deficits (Kim et al. 2007; Yang et al. 2012), brain edema, atrophy, glial proliferation , inflammation , and apoptosis (Kim et al. 2007) in a rat model of hemorrhagic stroke.

However, the effect of ADSC treatment on infarct volume after experimental ischemic stroke is equivocal. Although one study demonstrated a significant reduction (Leu et al. 2010), other studies demonstrated no notable change in infarct volume (Gutierrez-Fernandez et al. 2013b; Jiang et al. 2014) despite the same number of cells being administered each time (2 × 106) and the unanimous findings of significantly improved neurological function, reduced cell death, and enhanced cellular proliferation in all studies (Leu et al. 2010; Gutierrez-Fernandez et al. 2013b; Jiang et al. 2014). The discrepancies in infarct volume among the studies may partly be explained by the differences in the choice of ligation procedure (i.e. permanent (Gutierrez-Fernandez et al. 2013b) vs. transient (Leu et al. 2010; Jiang et al. 2014)), the timing and frequency of ADSC administration (once at 30 min after stroke (Gutierrez-Fernandez et al. 2013b) vs. 3 times at 0, 12 and 24 h after stroke (Leu et al. 2010) vs. once at 3 days after stroke induction (Jiang et al. 2014)), the time of sacrificing animals for histological analysis after induction (14 days (Gutierrez-Fernandez et al. 2013b) vs. 21 days (Leu et al. 2010) vs. 28 days (Jiang et al. 2014)), and the route of ADSC administration (systemic intravenous (Leu et al. 2010; Gutierrez-Fernandez et al., 2013b) vs. intra-carotid arterial (Jiang et al. 2014)). Therefore, although the timing of sacrificing animals and the route of cell injection do not seem to be the significant causes of discrepancies in the size of infarct, it appears that an early timing and increased frequency of ADSC administration (Leu et al. 2010) offered significant benefit in the reduction of infarct volume in a rodent experimental setting of ischemic stroke. Accordingly, a meta-analysis demonstrated that the efficacy in structural restoration drops by 1.5 % for each day delay in treatment and that a significant dose-response relationship exists between the number of stem cells administered and the improvement of structural outcome after ischemic stroke (Lees et al. 2012). Another interesting comparison based on animal experimentation between the therapeutic effects of autologous and allogeneic cells on structural and functional outcomes after ischemic stroke revealed that the former is more effective in preserving structural integrity, while the latter is more beneficial for functional outcome (Lees et al. 2012).

3.3 Mechanisms Underlying Therapeutic Actions of ADSC from Experimental Studies

Taken together, experimental investigations, both in vitro and in vivo, have provided significant insight into some of the mechanisms involved in repair of the nervous system after stroke. The mechanisms underlying the above-mentioned therapeutic benefits of ADSC against stroke can be summarized into (i) paracrine effects, (ii) transdifferentiation, and (iii) immunomodulation (Fig. 9.2) .

Fig. 9.2
figure 2

Summary of reported mechanisms underlying adipose-derived stem cell treatment for stroke. The three major mechanisms by which adipose-derived stem cells exert therapeutic functions include paracrine effects , transdifferentiation, and immunomodulation . The paracrine effects stem from the release of a variety of trophic factors from stem cells that elicit a number of biological responses such as angiogenesis, neurogenesis, and abatement of apoptosis. Transdifferentiation of stem cells involves the transformation of implanted stem cells into specific cellular elements with distinct functions and cell markers (e.g., neuron-like, endothelium-like, or glial-like cells). Immunomodulation includes stem cell-mediated modification of the immunological system, such as inhibition of T cell polarization for alleviating immune responses, suppression of transformation of monocytes to antigen-presenting immunogenic cells (e.g., dendritic cells) for inducing tolerance, and the release of various immuomodulatory cytokines for suppressing inflammatory reactions. APC: Antigen-presenting cell; VEGF: Vascular endothelial growth factor; FGF2: Fibroblast growth factor 2; HGF: Hepatocyte growth factor; IGF-1: Insulin-like growth factor-1; BMP2: Bone morphogenetic protein 2; BDNF: Brain-derived neurotrophic factor; NGF: Nerve growth factor; GFAP: Glial fibrillary acidic protein; MAP2: Microtubule-associated protein 2; NeuN: Neuronal nuclei; vWF: von Willebran factor

Full size image

3.3.1 Paracrine Effects

Although several previous experimental studies have demonstrated the presence of ADSC in the brain up to several weeks after being administered , the scarcity of stem cells in brain tissue could not account for the observed therapeutic outcomes (Leu et al. 2010; Jiang et al. 2014). The finding of stem cells not yet embedded into the brain tissue (Gutierrez-Fernandez et al. 2011; Ikegame et al. 2011) also precludes the possibility of their direct participation as fully functional neurons, implying their help through other mechanisms in the recovery process (Gutierrez-Fernandez et al. 2011). In concert with that finding, ADSC has been reported to produce a number of trophic factors including VEGF, angiopoietin-1, and HGF (Ikegame et al. 2011), insulin-like growth factor-1 (IGF-1) (Wei et al. 2009), TGF-β1 (Melief et al. 2013b), bone morphogenetic protein 2 (BMP2) and fibroblast growth factor 2 (FGF2) (Moriyama et al. 2012) as well as nervous system-related molecules including nerve growth factor (Banas et al. 2008; Ikegame et al. 2011), brain-derived neurotrophic factor (BDNF) (Iadecola and Anrather 2011; Liu et al. 2014), GFAP, nestin, and microtubule-associated protein 2 (MAP2) (Yang et al. 2011). Therefore, based on the actions of these trophic factors, it is rational to attribute the observed enhancement of angiogenesis and neurogenesis as well as the abatement of apoptosis to the paracrine effects of the administered ADSC (Leu et al. 2010; Gutierrez-Fernandez et al. 2012) .

Consistently, another intriguing finding is the discovery of therapeutic effects against stroke using cell-free ADSC culture medium (Cho et al. 2012; Egashira et al. 2012). One study applying human adipose-derived stem cell-conditioned medium to the lateral ventricle of a rat model of ischemic stroke 8 h after MCAO continuously for 7 days demonstrated not only a reduction of infarction volume and preservation of motor function, but also enhanced endothelial cell proliferation , reduced neural cell apoptosis, and suppressed astrogliosis in the penumbra regions (Cho et al. 2012). Another similar study using intracerebroventricular administration of concentrated murine adipose-derived stem cell-conditioned medium in a murine model of MCAO-induced ischemic stroke shed some light on the importance of the timing of treatment (Egashira et al. 2012). The result of that study showed that, while administration of conditioned medium prior to MACO exhibited a dose-dependent reduction in infarction volume of the brain and administration 5 min after MACO was still effective, the therapeutic effect vanished if conditioned medium was administered 2 h after MCAO (Egashira et al. 2012). By contrast, the former study reported effectiveness up to 8 h after MCAO before starting conditioned medium treatment (Cho et al. 2012). Other than the possible variations arising from the differences in the source of conditioned medium and the animal model used, the discrepancy in therapeutic effects between the two studies appears to be due to the way of conditioned medium administration. While the former adopted the approach of continuous intracerebroventricular infusion (Cho et al. 2012), the latter used single intracerebroventricular injection (Egashira et al. 2012) . Again, consistent with the results of previous experimental studies using ADSC transplantation for ischemic stroke (Leu et al. 2010; Gutierrez-Fernandez et al. 2012), it appears that early timing and repeated (if not continuous) treatment are of therapeutic advantage for both ADSC transplantation and conditioned medium therapy. In vitro, murine ADSC-derived conditioned medium has also been demonstrated to reduce glutamate-induced excitotoxicity in human neuroblastoma cells (Egashira et al. 2012).

3.3.2 Transdifferentiation

The role of direct cell participation regarding the use of ADSC for the treatment of ischemic stroke remains controversial. Previous studies using bone marrow-derived mesenchymal stem cells demonstrated that physical presence of the infused stem cells depends on the route of administration. Implantation of stem cells in the injured brain was evident when the cells were given through the carotid artery (Gutierrez-Fernandez et al. 2011; Jiang et al. 2014) but not through the intravenous route (Gutierrez-Fernandez et al. 2011) . Neurological deficits, however, were improved regardless of the presence of implanted stem cells in the brain (Gutierrez-Fernandez et al. 2011; Jiang et al. 2014), raising the question regarding the therapeutic significance of stem cell implantation in stroke. Indeed, it has been shown that only a small fraction (around 0.02 %) of intravenously administered bone marrow-derived hematopoietic stem cells migrate to the ischemic brain, and most of the transplanted cells express microglial but not neural protein markers (Schwarting et al. 2008). For ADSC, while a study failed to identify evidence of migration or implantation of cells into the damaged brain after their intravenous injection in an animal model of stroke despite significant functional recovery (Gutierrez-Fernandez et al. 2013b), other experimental studies (Kim et al. 2007; Leu et al. 2010; Yang et al. 2012) have demonstrated presence of the transplanted ADSC several weeks after intravenous administration with the expression of von Willebran factor, a marker of endothelial cell (Kim et al. 2007; Leu et al. 2010) . Another study using ADSC to treat a rat model of hemorrhagic stroke through right lateral cerebral ventricular injection demonstrated the differentiation of the infused ADSC into neuron-like (NeuN+) and glial-like cells (GFAP+) in region surrounding the hematoma (Chen et al. 2012). Despite the relatively small number of ADSC to explain the overall functional recovery in the reported studies, their presence signifies “transdifferentitation” as a possible mechanism underlying the positive therapeutic impact (Gutierrez-Fernandez et al. 2013a). Indeed, the capacity of neural differentiation for ADSC has been extensively investigated (Cardozo et al. 2010; Kompisch et al. 2010; Liao et al. 2010; Qian et al. 2010; Abdanipour et al. 2011; Yu et al. 2011; Ahmadi et al. 2012). It has also been reported that, compared with bone marrow-derived mesenchymal stem cells , ADSC have superior neurogenic potential (Kang et al. 2004) . Consistently, previous studies using ADSC after induced neural differentiation for treating experimental ischemic stroke were also found to be effective in improving functional recovery (Kang et al. 2003b; Yang et al. 2011). On the other hand, another finding of interest is the requirement for direct physical contact between human ADSC and murine neural stem cells in vitro for induction of neuronal differentiation of the latter, further emphasizing the existence of a mechanism that involves cell-cell interaction other than that of transdifferentiation and paracrine effects in promoting neurogenesis (Kang et al. 2003a) .

3.3.3 Immunomodulation

Taking into account the immunological nature of stroke-elicited damage and the subsequent repairing process (Iadecola and Anrather 2011) , it is not surprising to find that ADSC exert their therapeutic actions at least partly through immunomodulation. Indeed, ADSC have been reported to produce a variety of immunomodulatory cytokines, including IL-1R, IL-6, IL-8, IL-18, toll-like receptor (TLR)-4, TGF-β1, plasminogen activator inhibitor-1 (PAI-1), G-CSF, GM-CSF, and monocyte chemotactic protein 1 (Banas et al. 2008; Leu et al. 2010; Ikegame et al. 2011; Melief et al. 2013b). Moreover, ADSC have been shown to suppress the differentiation of monocytes towards antigen-presenting immunogenic cells and promote differentiation towards an anti-inflammatory IL-10-producing cell type through the production of IL-6 (Melief et al. 2013a). Consistently, coculturing ADSC with allogeneic dendritic cells revealed that ADSC could negatively modulate immunity and induce immune tolerance through downregulating costimulatory molecules (i.e., CD80, CD83, CD86, and secretion of IL-12 and tumor necrosis factor (TNF)-alpha), while induce dendritic cell tolerance through upregulating indoleamine-2,3-dioxygenase (IDO). Cocultured dendritic cells were also found to inhibit CD4+ T cell activation and naive T cells toward Th1 helper cell polarization (Peng et al. 2012). Again, another credit given to ADSC as compared with bone marrow-derived mesenchymal stem cells in the aspect of immunomodulation in stroke treatment is the finding of a higher immunomodulatory capacity in the former than that in the latter (Melief et al. 2013b) .

4 Clinical Use of ADSC Against Stroke: Present Status, Perspectives, and Limitations

4.1 Clinical Application of ADSC: Probabilities and Possibilities

Given the promising experimental outcomes of applying stem cells to the treatment of stroke and the in-depth understanding of the underlying mechanisms, a number of clinical trials are either reported or still on-going in recent years despite the majority of them are small, nonrandomized, and uncontrolled. The cells administered included bone marrow mononuclear cells (Correa et al. 2005; Li et al. 2013b), bone marrow-derived mesenchymal stem cells, (Bang et al. 2005; Suarez-Monteagudo et al. 2009; Lee et al. 2010; Bringas et al. 2011; Honmou et al. 2011), human teratocarcinoma-derived neurons (Kondziolka et al. 2000), peripheral blood hematopoietic progenitor/stem cells (Chen et al. 2014a), umbilical cord-derived mesenchymal stem cells (Han et al. 2011; Jiang et al. 2013), as well as human (Rabinovich et al. 2005) and porcine fetal cells (Savitz et al. 2005). Except for premature termination of the study adopting porcine fetal cells because of overt complications (Savitz et al. 2005), the results of other published trials support the safety and effectiveness of stem cell/progenitor cells as a therapeutic tool in the clinical setting of ischemic and hemorrhagic stroke as reflected in the overall significantly improved neurological functions of the treated patients up to 5 years of follow-up (Lee et al. 2010). On the other hand, results on the use of ADSC in clinical trial have not been reported. To date, there is only one study still recruiting patients to explore the safety and effectiveness of applying autologous ADSC in patients after stroke on the National Institutes of Health clinical trial registry database (www.clinicaltrials.gov). Therefore, albeit optimistic, the exact therapeutic impact of ADSC on disease progression and functional recovery in the clinical setting of stroke remains to be elucidated for the years to come.

4.2 ADSC Against Stroke: Concerns and Speculations

Despite the promising outcomes of applying ADSC to the treatment of stroke in experimental settings, there have been serious concerns about possible tumorigenesis in the clinical scenario because of the multilineage differentiation potential of ADSC (Lee et al. 2012). A study investigating the fate of human ADSC from different human donors after being subcutaneously injected into immunodeficient SCID mice showed that the cells survived for at least 17 months with subsequent differentiation into fibroblasts of the subdermic connective tissue and into mature adipocytes of fat tissue, exclusively at the site of injection without evidence of migration or fusion with host cells (Lopez-Iglesias et al. 2011), underscoring the safety of ADSC transplantation. Moreover, the use of terminally differentiated ADSC may be a possible option for minimizing the risk especially when the protocols for in vitro transdifferentiation of ADSC into neuronal lineage have been well-documented (Cardozo et al. 2010; Kompisch et al. 2010; Liao et al. 2010; Qian et al. 2010; Abdanipour et al. 2011; Yu et al. 2011; Ahmadi et al. 2012). Indeed, the use of induced ADSC has been endorsed as a promising therapeutic option in stroke treatment (Yang et al. 2011; Shen et al. 2013). Furthermore, it has been shown that iPSC can be generated from human ADSC without transducing c-myc so that the proliferative and differentiation capacity of ADSC can be enhanced without increasing the risk of oncogenesis (Aoki et al. 2010).

On the other hand, taken into account the therapeutic advantage of early stem cell administration at the acute stage of stroke, the use of automated devices for adipose-derived stem cell isolation for direct injection without ex vivo expansion and purification may be a feasible option for daily clinical practice because of the high concentration of useful stem and progenitor cells from adipose tissue compared with other sources (Hicok and Hedrick 2011). At the other end of the spectrum, the use of gene-transfer techniques for producing stem cells over-expressing different neurotrophic factors, such as BDNF, glial derived neurotrophic factor (GDNF), or neurotrophin-3 (NT-3), has been reported to be effective options in the treatment of ischemic stroke in animal models (Chen et al. 2013). Finally, considering the wide therapeutic applicability and easy harvesting of ADSC, the establishment of autologous or allogeneic cell banks for ADSC storage to facilitate urgent or scheduled use is no longer a far-fetched idea (West et al. 2014).