ISSN: 1539-6509; eISSN: 1944-7930
Neural Potential of Adipose Stem Cells BArBArA ZAvAN, vINceNZo vINdIgNI, chIArA gArdIN, doMeNIco d’AvellA, AleSSANdro dellA PuPPA, gIovANNI ABATANgelo, ANd roBerTA corTIvo Abstract: In the last few years, adipose tissue, which has been largely ignored by anatomists and physicians for centuries, has found new brightness thanks to the stem cells contained within. These adipose derived stem cells (ADSC) have the same characteristics of the mesenchymal stem cells (MSC) residing in bone marrow. They have the same cell surface markers and are capable of differentiating into the same cell types, including osteoblasts, chondrocytes, myoblasts, adipocytes, and neuron-like cells. Adipose tissue is ubiquitous and uniquely expandable. Most patients possess excess fat that can be harvested, making adipose tissue the ideal largescale source for research on clinical applications. In this review focused on the neural potential of adipose-derived stem cells. Current strategies for their isolation, differentiation, and in vitro characterization, as well as their latest in vivo applications for neurological disorders or injury repair, were discussed. [Discovery Medicine 10(50):37-43, July 2010]
Barbara Zavan, Ph.D., Chiara Gardin, Ph.D., Giovanni Abatangelo, B.S., and Roberta Cortivo, Ph.D., are at the Department of Histology, Microbiology, and Medical Biotechnology, University of Padova, Padova, Italy. Vincenzo Vindigni, M.D., Ph.D., is at the Unit of Plastic and Reconstructive Surgery, University of Padova, Padova, Italy. Domenico D’Avella, M.D., and Alessandro Della Puppa, M.D., are at the Unit of Neurosurgery, University of Padova, Padova, Italy. Corresponding author: Barbara Zavan, Ph.D. ([email protected]
). © Discovery Medicine. All rights reserved.
Introduction Stem cell therapy is undoubtedly the most promising therapeutic approach for neurological disorders owing to their characteristics such as the ability to self-renew, long term viability, and multilineage potential. By definition, neural tissue has long been regarded as incapable of regeneration, and the identification of cell populations capable of neuronal differentiation has generated intense interest in the last decade (Kokai et al., 2005). In recent years, neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells, neuronal stem cells, and mesenchymal stem cells. Although embryonic stem cells are theoretically highly beneficial due to their ability of undergoing expansion and neuronal differentiation in vitro and in vivo, there are various limitations to their use imposed by cell regulations, ethical considerations, and genetic manipulation. These obstacles have encouraged researchers to find alternative cell sources capable of neuronal differentiation. Adult stem cells, on the other hand, are more easily available, with neither ethical nor immunoreactive considerations, as long as they are of autologous tissue origin (Galli et al., 2003). In this context the first approach applied to the discovery of adult stem cells for neuronal differentiation has been carried out at the natural site of neurogenesis. It is known that adult neurogenesis occurs within the unique local environment called “niche,” which provides both supportive and instructive signals for the development of adult neural progenitors. Recent studies have identified some of the cellular architectures of the neurogenic niches, including blood vessels, ependymal cells, endothelial cells, and astrocytes. Neuronal stem cells (NSC) are multipotent stem cells with properties of indefinite growth and division and pluripotency to dif-
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ferentiate into three major central nervous system (CNS) cell types: neurons, astrocytes, and oligodendrocytes. NSC within the developing brain are found in the germinal zones, called the ventricular zones, but also found within specialized stem cell niches in the adult brain, including the subependymal zones. Other proliferative cells located within the subgranular zone of the dentate gyrus are defined as progenitors, because separate such cell populations give rise to neurons or glial cells and they show only limited self-renewal. NSC have been tested in experimental models of neurodegenerative diseases which make it possible to test potentially powerful new therapeutic strategies for a board spectrum of human neurological disorders. The large amount of information derived from studies regarding the in vitro commitment of stem cells to a neuronal phenotype has led the researchers to focus their attention also on other sites of easier accessibility (Gritti et al., 2002). Neural progenitors, indeed, have been isolated from non-neurogenic regions, including spinal cord, white matter, striatum of adult mammals, skin, bone marrow, and adipose tissue. These neural progenitors are multipotent, capable of giving rise to neurons, astrocytes, and oligodendrocytes both in culture and after transplanting back to neurogenic regions of the adult. These results support the hypothesis that multipotent progenitors exist in non-neurogenic regions and their developmental potentials are limited by the local environment. Injury and diseases appear to induce changes in the environment to allow neurogenesis to occur in these regions (Miller et al., 2010; Orlacchio et al., 2010). The existence of active adult neurogenesis in neurogenic regions of normal brain and in non-neurogenic regions after injury demonstrates the striking plasticity and regenerative capacity of the adult CNS. It is the ability of the mature CNS to functionally incorporate new neurons into its existing circuitry that offers the promise of successful cell-replacement therapies for degenerative neurological diseases (Benn et al., 2004). The current goals are to gain a better understanding of environmental factors and signaling mechanisms that regulate adult neurogenesis and to translate such knowledge into successful therapeutics targeted to neurodegenerative diseases. To this end, the research attention has been directed at alternative sources of neuronal stem cells, such as the mesenchymal lineages (Anghileri et al., 2008). Up to now, the large amount of evidence accumulated on mes-
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enchymal stem cell behavior demonstrated that, under appropriate conditions, mesenchymal stem cells (MSC) selectively differentiate not only into mesenchymal lineages but also into endodermal and ectodermal cell lineages in vitro (Venkataramana et al., 2009). The adult tissue in which MSC are resident is bone marrow and adipose tissue (Bonfield and Caplan, 2010). Bone marrow procurement is excessively painful for patients and yields low numbers of harvested cells; on the contrary adipose tissue shows several attractive characteristics -- the accessibility and the low cost of MSC harvest and delivery, to name a few (Romanov et al., 2005). When compared with bone marrow-derived mesenchymal stem cells, adipose-derived stem cells are equally capable of differentiating into cells and tissues of mesodermal origin (Baglioni et al., 2009). Since human adipose tissue is ubiquitous and easily attainable in large quantities under local anesthesia with little patient discomfort, it may provide an important alternative source of stem cells for mesenchymal tissue regeneration and engineering (Dhar et al., 2007). Indeed most patients possess excess fat that can be harvested without creating contour deformities, making adipose tissue the ideal large-scale source for research on the clinical application of neural progenitor cells. MSC derived from adipose tissue have previously been referred with several acronyms starting from processed lipoaspirate (PLA), first used by Zuk et al. (2002) on their pioneer studies, passing on to adipose tissue derived stromal cells (ADAS), adipose tissue derived stem cells (ASC), and adipose derived stem cells (ADSC). In this review the term adipose derived stem cells (ADSC) will be used. Among the various research methodologies described in research articles related to the neuronal potential of ADSC, the authors focused their attention on the following protocol for obtaining cells with a well defined neuronal phenotype (Ning et al., 2006). The protocol includes the following steps: a) Digestion of the adipose tissue b) In vitro expansion of ADSC c) In vitro differentiation d) In vitro characterization e) In vivo application
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Harvesting of Adipose Tissue and Pre-adipocyte Isolation The first step obligatory for achieving a successful isolation of ADSC is the processing of adipose tissue. Adipose tissue can be obtained from both lipoaspiration (liposuction) and abdominoplasty (”tummy tuck”) operations. Aspiration of adipose tissue is performed through manually applying negative pressure using a 10-ml syringe with a blunt tip cannula and no addition of any solutions (Coleman, 2006). Liposuction samples are weighed after washing in PBS, centrifugation, and elimination of supernatant. During abdominoplasty operations, waste material is obtained en bloc and subsequently bisected: fibrous structures and visible blood vessels are removed and tissue is minced with scissors into pieces with a diameter of 2-6 mm (Torio-Padron et al., 2010; von Heimburg et al., 2004). At this point the digestion process is the same for both types of tissues. Adipose tissue is digested with 0.075% collagenase (type 1A; Sigma-Aldrich) in Hank’s balanced salt’s solution (HBSS) followed by 0.25% trypsin. Floating adipocytes are discarded, and cells from the stromal-vascular fraction were pelleted, rinsed with media, and centrifuged. Red blood cells are lysed in NH4Cl. The viable cells obtained are counted using the trypan blue exclusion assay. In Vitro Expansion of Adipose Derived Stem Cells This step is strongly emphasized with an aim to gather as much neuroprogeny as possible through the least number of cellular passages (Yamamoto et al. 2007). The following are key methodologies. 1) Strictly follow the formation of neurosphere colonies that contain neural stem cells. 2) Adopt strategies directed at enriching these structures. One example of a correct approach is avoiding the use of a coating condition or attachment factor such as poly-lysine. In our laboratory, we recently performed a study on improving the yield of neurospheres. ADSC have been colored in monolayer conditions, with and without coating condition. After 7 days, most cells adhered to the tissue culture plastic, assuming a fibroblast-like phenotype -- large,
flat, and spindle shaped cells, with a small population of expanded cells organized into spheres growing in suspension. At day 7, neurospheres present in the medium were collected and their number analyzed. Total cells forming neurospheres were determined from a standard curve (μg DNA vs. cell number). As reported above, in the presence of a coating condition ADSC are not able to generate neurospheres. The presence of neurospheres is detectable only in absence of any attachment factor (Vindigni et al., 2009). 3) Incubate ADSC with a proliferative medium for at least 2-3 weeks. In this proliferative phase, serum also contributed to neurosphere production: the highest number of neurospheres was produced from cells cultured in medium supplemented with serum. Similarly, the combined presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) ensured the highest number of neurospheres. Although EGF alone and bFGF alone stimulated neurosphere production, results were greatly improved when the combination of the two was used in the medium (Zavan et al., 2010; Vindigni et al., 2009). In Vitro Differentiation The phase following ADSC proliferation represents the one in which more variants are documented in literature. Neurospheres previously obtained as described in Kang et al. (2006) could be expanded with several protocols directly to obtain different final phenotypes. Undifferentiated adipose stem cells cultured at high densities will spontaneously form spherical clumps of cells that can be isolated in 0.25% trypsin/2.21mM EDTA. Free-floating neurospheres released from the cell culture plate surface into the culture medium can also be collected. The spheres of cells are transferred to an ultra low cluster culture dish and cultured in neurobasal medium supplemented with B27 (1:50), 20 ng/ml bFGF, and 20 ng/ml EGF for 4-7 days. The culture density of the spheroid bodies should be maintained at 10-20 spheroids/cm2 to prevent self aggregation. Neuronal differentiation medium • Neurobasal medium containing 1-5% fetal bovine serum (FBS) plus 50 ng/ml each of nerve growth factor
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(NGF) and brain-derived neurotrophic factor (BDNF), and 10 ng/ml NT-3 (Toma et al., 2005) • DMEM-F12 3:1, 1% FBS, 2% B27 serum free supplement, 10 μg/ml NGF, and antibiotics (Gingras et al., 2007) • Neurobasal medium containing 1-5% FBS plus BDNF and retinoic acid (Anghileri et al., 2008) Schwann cell differentiation medium • Neurobasal medium containing 1% FBS (Toma et al., 2005) • DMEM-F12 3:1, containing 1% FBS plus 1% N2 supplement, 4 μM forskolin, and 10 ng/ml heregulin β (Biernaskie et al., 2006) In Vitro Characterization Cell population obtained by the cultivation in neurodifferentiative medium is often a mix of cells expressing one or more neurospecific markers (Fujimura et al., 2005). The most cited proteins whose expression is studied by both immunohistological and molecular analyses include the following. Intermediate filament: Neurofilaments specifically present in neurons. Myelin basic protein: Protein with a role in both the formation and stabilization of myelin. It is present in oligodentrocytes. Nestin: Protein expressed in the developing central nervous system. βIII tubulin. Protein abundant in the central and peripheral nervous systems. Glial fibrillar acidic protein (GFAP): Protein specifically found in mature glial cells (astrocytes). S100: Protein belonging to the family of calcium binding proteins. It is located in the cytoplasm and nuclei of astrocytes, Schwann cells, ependymoma tumor cells, and astroglioma tumor cells. The above obtained cells often express more than one marker, for example: newly born neurons: nestin + βIII tubulin, or nestin + GABA receptor + tyroxine hydroxilase
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astrocytes: GFAP + / CNPase oligodendrocytes: GFAP - / CNPase + Schwann cells: GFAP / CNPase / S100 Another test used to confirm neuronal phenotype is based on patch clamp strategy. With this approach Anghileri et al. (2008) obtained differentiated cells with a negative membrane potential (-60 mV), delayed rectifier potassium currents, and tetrodotoxin-sensitive sodium currents. In Vivo Application Human neurological disorders and disease pathology are largely caused by a loss of neurons and glial cells in the brain or spinal cord. Cell based therapies are emerging as innovative approaches for the treatment of such defects for which there are no effective therapies. Critical to the success of cell therapies is the selection and mode of delivery of therapeutic cells. Despite intense efforts, the ability to promote functional recovery after contusion injuries, ischemic insults, or the onset of neurodegenerative diseases in the brain and spinal cord remains very limited, while the need for such therapies is increasing with an aging population. Recent studies suggest that cellular therapies may provide a functional benefit in a wide range of neurological insults. MSC derived from a variety of tissue sources have been therapeutically evaluated in animal models of stroke, spinal cord injury, and multiple sclerosis. In each situation, treatment with MSC results in substantial functional benefit and these pre-clinical studies have led to the initiation of a number of clinical trials worldwide in neural repair. Here we report the in vivo application of MSC derived from adipose tissue in several neurological disorders as described in literature. Spinal cord injury Chi et al. (2010) engrafted Schwann cells derived from nestin-expressing ADSC to spinal cord injury lesions. Prior to engraftment, these cells were induced and differentiated in vitro and tested for the expression of several neuronal markers such as A2B5, GFAP, O4, p75, S100, Sox10, Krox-20, and L1. After the engraftment, the induced Schwann cells have been able to form a peripheral nervous system (PNS)-type myelin sheath on central nervous system (CNS) axons. In addition to direct participation in repairing tissue as myelin sheath-
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forming cells, the induced Schwann cells also expressed several neurotrophic factors, as native Schwann cells did, which may suggest an additional role for induced Schwann cells in stimulation of endogenous healing responses. Thus, spheroid-forming cells from subcutaneous fat tissue demonstrated rapid and efficient induction into Schwann cells, and such cells showed therapeutic promise for repairing of damage to the CNS and PNS. Kang et al. (2006) reported that intravenous infusion of oligodendrocyte precursor cells (OPCs) derived from rat ADSC autograft cell sources improves motor function in rat models of spinal cord injury. After 4-5 weeks, transplanted oligodendrocytes survived and migrated into the injured region of spinal cord very efficiently (30-35%) and migrated cells were partially differentiated into neurons and oligodendrocytes. The authors found that some of the engrafted cells migrated and integrated in the kidney, brain, lung, and liver through the intravenous system. Behavioral analyses revealed that the locomotor functions of OPC-autografted rats with spinal cord injury were significantly restored. Efficient migration of intravenously engrafted rat adipose tissue-derived stromal cells (rATSC)-OPCs cells into spinal cord lesions suggests that spinal cord injury-induced chemotactic factors facilitate the migration of rATSC-OPCs. Here, the authors demonstrated that engrafted rATSCs and spinal cord injury-induced chemotactic factors indeed play an important role in proliferation, migration, and differentiation of endogenous spinal cord-derived neural progenitor cells in the injured region. In transplantation paradigms, the interaction between engrafted oligodendrocyte precursor cells derived from ADSC and endogenous spinal cordderived neuronal progenitor cells will be important in promoting healing through cell fate decisions, resulting in coordinated induction of cell migration and differentiation. Brain injury Kulikov et al. (2008) evaluated possible therapeutic effect of ADSC derived cells of neuronal phenotype induced with retinoic acid on Wistar rats that were subjected to toxic effect of 3-nitropropionic acid. Results showed decreased neurological symptoms, normalized exploratory activity (open field test), and long-term memory (Morris test), which correlated with normalization of pathomorphological manifestations in the brain. Destructive changes in the caudate nucleus caused by treatment with 3-nitropropionic acid
(reduced size of neurons, changes in their shape, and cell edema) tended to decrease under the effect of ADSC: the number of neurons increased 2-fold, the cells acquired typical round shape, and cell edema decreased. Stroke Kim et al. (2007) investigated the neuroprotective effect of ADSC in an intracerebral hemorrhage model. Intracerebral hemorrhage was induced via the stereotactic infusion of collagenase. Human ADSC were intravenously administered at 24 hours after the induction of intracerebral hemorrhage. Acute brain inflammation was monitored with several biochemical parameters and by measuring hemispheric atrophy and perihematomal glial thickness at 6 weeks post induction of intracerebral hemorrhage, and the modified limb placing behavioral scores were determined weekly over 5 weeks post induction of intracerebral hemorrhage. The results showed that ADSC transplantation attenuated neurological deficits from 4 to 5 weeks post induction of intracerebral hemorrhage, and reduced both the brain atrophy and the glial proliferation at 6 weeks. Transplanted ADSC were found to densely populate perihematomal areas at 6 weeks, and to express endothelial markers but not neuronal or glial markers. In summary, the authors showed that ADSC transplantation in the intracerebral hemorrhage model reduced both acute cerebral inflammation and chronic brain degeneration, and promoted long-term functional recovery. Neural protection Wei et al. (2009) provided evidence that one of the mechanisms with which ADSC induce repair is through paracrine mechanisms effected by secreted pro-survival and repair-inducing trophic factors and not through ADSC’s differentiation. In their studies, the authors have found that adipose stromal cell-conditioned medium potently protected cerebellar granule neurons from apoptosis induced by serum and potassium deprivation. Neural cell protection was mostly attributable to activated caspase-3 and Akt-mediated neuroprotective pathway signaling. Specific neutralization of neurotrophic factor activity demonstrated that serum and potassium deprivation-induced Akt-mediated neuroprotection and caspase-3-dependent apoptosis were mainly modulated by insulin growth factor 1 (IGF-1). These data suggest that among the many neuroprotective factors secreted by adipose stromal cells, IGF-1 is the major factor that mediates protection Discovery Medicine, volume 10, Number 50, July 2010
against serum and potassium deprivation-induced apoptosis of cerebellar granule neurons. This study establishes a mechanistic basis supporting the therapeutic application of ADSC for neurological disorders, specifically through paracrine support provided by secreted trophic factors. Conclusions During the past decade a tremendous amount of research has focused on the elucidation of the molecular basis of neurodegenerative diseases, and significant progress has been made. With the recent advances in stem cell biology and the further investigation into the molecular mechanism of adult neurogenesis, the researchers’ attention has been drawn to stem cell based therapies which have potential to cure many of these neurodegenerative diseases (Benn et al., 2004; Benn and Woolf, 2005). Cellular-based transplantation is indeed a viable option as a therapy for neurodegenerative diseases by providing tropic support for diseased neurons or replacing the neurons themselves to correct the underlying disorder (Bunnell et al., 2008). This option has several benefits over the use of an endogenous approach. Endogenous neural progenitors have a limited proliferative capacity, while transplantation allows for the production and delivery of an unlimited number of well-defined and differentiated cell types. Furthermore, repair of the disease process with endogenous neurons does not correct the underlying cause of the initial degeneration; therefore, the newly generated neurons will also be prone to the same degeneration over time. Hopefully with transplantation of cells from an exogenous source one could correct the underlying cause or genetically protect the new neurons from the disease process. In this view MSC are emerging as an effective therapeutic approach to a wide range of neural insults. These applications are possible owing to their characteristics such as plasticity, immunoregulatory actions, immunosuppressive properties, neuromodulatory, and homing to areas of insults where they can release a wide range of tropic signals that influence surrounding tissues (Franco Lambert et al., 2009). Bone marrow and adipose tissue are the principal adult tissues in which MSC reside. Adipose-derived stem cells are now being examined as an alternative to bone marrow stromal cells and have been demonstrated, using flow cytometry and immunohistochemistry, to have a protein expression phenotype that is comparable Discovery Medicine, volume 10, Number 50, July 2010
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to that of bone marrow stromal cells (Case et al., 2009). Other similarities of adipose derived stem cells to bone marrow MSC are that both stem cell types are capable of differentiating into chondrocytes, osteoblasts, myocytes, and neural and glial cells (Mizuno, 2009). In this regard, the research related to the neuronal induction is at its first stages; new methods to induce transdifferentiation could replace the existing differentiation protocols. Neuron-like morphology or expression of selective neuronal or glial markers does not result from aberrantly induced gene expression. Instead, genuine neuronal differentiation of adult stem cells requires full cell functionality, which may be demonstrated through electrophysiology and expression of the complete profile of neuronal genes (Fraser et al., 2006). In summary, we conclude that ADSC provide unique opportunities for investigating novel treatments for a vast array of inherited and acquired diseases. In this review, a series of protocols for the isolation, expansion, neuronal differentiation characterization, and in vivo applications of ADSC have been described. These protocols can readily be adapted to adjust for differences in the size of the adipose tissue sample. The future aim is to apply ADSC to replacement therapies for neurodegenerative diseases such as Parkinson’s disease and inflammatory disorders such as multiple sclerosis (Taupin, 2009). References Anghileri E, Marconi S, Pignatelli A, Cifelli P, Galié M, Sbarbati A, Krampera M, Belluzzi O, Bonetti B. Neuronal differentiation potential of human adipose-derived mesenchymal stem cells. Stem Cells Dev 17(5):909-16, 2008. Baglioni S, Francalanci M, Squecco R, Lombardi A, Cantini G, Angeli R, Gelmini S, Guasti D, Benvenuti S, Annunziato F, Bani D, Liotta F, Francini F, Perigli G, Serio M, Luconi M. Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J 23(10):3494-505, 2009. Benn SC, Woolf CJ. Adult neuron survival strategies -- slamming on the brakes. Nat Rev Neurosci 5(9):686-700, 2004. Benn SC, CJ Woolf. How do adult neurons survive? Discov Med 5(27):309-18, 2005. Biernaskie JA, McKenzie IA, Toma JG, Miller FD. Isolation of skinderived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny. Nat Protoc 1(6):2803-12, 2006. Bonfield TL, Caplan AI. Adult mesenchymal stem cells: an innovative therapeutic for lung diseases. Discov Med 9(47):337-45, 2010. Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C. Adiposederived stem cells: isolation, expansion and differentiation. Methods
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