Environmental cues to guide stem cell fate decision for tissue engineering applications

Transcription

1 Review 1. Introduction 2. Mesenchymal stem cell populations 3. Neural stem cell populations 4. Endothelial precursor cells 5. Expert opinion Cell- & Tissue-based Therapy Environmental cues to guide stem cell fate decision for tissue engineering applications Eben Alsberg, Horst A von Recum & Melissa J Mahoney Case Western Reserve University, Department of Biomedical Engineering, Euclid Avenue, Wickenden Building, Room 204, Cleveland, OH , USA The human body contains a variety of stem cells capable of both repeated self-renewal and production of specialised, differentiated progeny. Critical to the implementation of these cells in tissue engineering strategies is a thorough understanding of which external signals in the stem cell microenvironment provide cues to control their fate decision in terms of proliferation or differentiation into a desired, specific phenotype. These signals must then be incorporated into tissue regeneration approaches for regulated exposure to stem cells. The precise spatial and temporal presentation of factors directing stem cell behaviour is extremely important during embryogenesis, development and natural healing events, and it is possible that this level of control will be vital to the success of many regenerative therapies. This review covers existing tissue engineering approaches to guide the differentiation of three disparate stem cell populations: mesenchymal, neural and endothelial. These progenitor cells will be of central importance in many future connective, neural and vascular tissue regeneration technologies. Keywords: biomaterials, endothelial precursor cells, mesenchymal stem cells, neural stem cells, regenerative medicine, tissue engineering Expert Opin. Biol. Ther. (2006) 6(9): Introduction The human body contains a variety of stem cells capable of both repeated self-renewal and production of specialised, differentiated progeny [1]. The ability of these cells to become multiple different tissue-forming cell types has generated intensive research efforts over the last two decades to investigate their potential use as a promising cell source for tissue engineering and gene therapy strategies. Critical to the implementation of stem cells in such approaches is a thorough understanding of which external signals in the stem cell microenvironment provide cues to control their fate decision in terms of proliferation or differentiation into a desired, specific phenotype (Figure 1). These signals must then be incorporated into tissue regeneration approaches for regulated exposure to stem cells. The precise spatial and temporal presentation of factors directing stem cell behaviour is extremely important during embryogenesis, development and natural healing events, and it is possible that this level of control will be vital to the success of many regenerative therapies. This review covers existing tissue engineering approaches to guide the differentiation of three disparate stem cell populations: mesenchymal, neural and endothelial. Although these different cell populations do not respond in the same way to individual signals, their inclusion will highlight some of the differences and similarities in strategies to guide stem cell behaviour and stimulate new avenues of study in this exciting area of research / Informa UK Ltd ISSN

2 Environmental cues to guide stem cell fate decision for tissue engineering applications A B soluble factors - gene therapy - growth factors, cytokines, hormones and chemicals other cell populations insoluble factors - extracellular matrix molecules - immobilised adhesion ligands - biomaterial mechanical and structural properties STEM CELLS fate decision - self-renewal - differentiation - apoptosis - migration external mechanical loading - compressive stress - shear stress - hydrostatic pressure - cyclic stretch C D Figure 1. Multiple signals in the local microenvironment influence stem cell fate decision. 2. Mesenchymal stem cell populations The ability of bone marrow to form bone tissue when implanted under the renal capsule of mice provided the first evidence that osteogenic precursor cells are housed in this tissue [2]. Subsequent studies demonstrated that these stem cells with osteogenic potential are a distinct population of cells that are different from haematopoietic stem cells, which are also found in the bone marrow [3]. A heterogeneous population of marrow-derived stromal cells (MDSCs) were first isolated based on their preferential adherence to tissue culture polystyrene (TCP) [4]. A more homogenous stem cell population was later isolated by fractionating the bone marrow aspirates via density-gradient centrifugation to obtain mononuclear cells, plating the cells on TCP and then removing cells of the haematopoietic lineage that cannot adhere to TCP [5]. These cells have subsequently been identified as mesenchymal stem cells (MSCs), which can undergo extensive self-replication and produce progeny capable of forming bone, cartilage, marrow stroma, ligament, tendon, muscle and connective tissue [6,7]. So far, MSCs have been isolated not only from bone marrow, but also from numerous other adult tissues, including periosteum, muscle, fat and skin [8]. Although the vast majority of research efforts to control MSC fate decision have focused on culturing the cells with exogenous soluble growth factors and cytokines, it is rapidly becoming recognised that other biochemical and physical cues can play an important role in regulating MSC behaviour. In addition to soluble factors, the effects of extracellular matrix (ECM) molecules, structural and intrinsic biomaterial Figure 2. Presentation of multiple signals to MSCs can enhance bone tissue engineering. Photomicrographs of haematoxylin and eosin-stained sections demonstrate that when MSCs were transplanted subcutaneously on the backs of mice for 15 weeks in alginate scaffolds (A) without growth factor or with only a single growth factor [(B) TGF-β3 or (C) BMP-2], minimal bone tissue was formed. In contrast, significant bone tissue (pink) was generated in implants containing MSCs and both growth factors combined (D). Size bars represent 100 µm. BMP: Bone morphogenetic protein; MSC: Mesenchymal stem cell; TGF: Transforming growth factor. properties, external mechanical forces and other cell populations on MSCs are starting to be elucidated. Below, the authors present recent findings in this area and review how these signals have been incorporated into biomaterial systems to guide MSC differentiation and the formation of engineered musculoskeletal tissues. In this review, both MDSCs and MSCs will be referred to as MSCs for simplicity. 2.1 Response of MSCs to soluble factors Growth factors, cytokines, hormones and chemicals In vitro cell culture conditions have been developed in which a chemically defined, serum-free medium can support the proliferation of MSCs while preserving their ability to differentiate into cells that comprise musculoskeletal tissues [9]. MSCs can then be guided down multiple mesenchymal lineages when exposed to defined medium containing specific soluble supplements. These in vitro culture conditions have been thoroughly reviewed elsewhere [10,11]. However, researchers are now trying to incorporate these soluble signalling molecules into biomaterial scaffolds in order to guide MSC differentiation. For example, culture of MSCs with dexamethasone, β-glycerol phosphate, ascorbate and members of the 848 Expert Opin. Biol. Ther. (2006) 6(9)

3 Alsberg, von Recum & Mahoney transforming growth factor (TGF)-β superfamily is known to promote osteogenic differentiation. These factors are now being integrated directly into biomaterials, either by mixing [12] or surface functionalisation [13], so that MSCs in these scaffolds transplanted into bone defects will receive appropriate soluble signals to drive new bone tissue formation. In addition, it has also been found that delivery of multiple growth factors with MSCs can have a synergistic effect on new tissue formation (Figure 2) [14] Gene therapy Genetically modifying MSCs to produce these bioactive factors themselves is an attractive alternative to the direct delivery of growth factors to MSCs from biomaterials. MSCs may be engineered to express growth factors that can then act in an autocrine manner to regulate their own function and also in a paracrine manner to influence surrounding host tissue cells. Transfecting MSCs to serve as gene delivery vehicles has the distinct advantage of longer periods of protein expression compared with the direct, local delivery of a finite amount of protein from a biomaterial over a limited time span [15]. MSCs have been stably transfected in vitro with plasmid DNA or infected with adenovirus to express members of the TGF-β superfamily, such as bone morphogenetic protein (BMP)-2 and -4. Genetically altering the MSCs greatly enhanced the formation of bone and cartilage tissue when combined with biomaterial carriers and cultured in vitro or implanted in vivo [16-19]. A different approach is to develop biomaterials that incorporate cells, plasmid DNA encoding for a gene of interest and a growth factor. In an attempt to replicate natural bone tissue development, Huang and colleagues engineered a polymer system that could simultaneously deliver MSCs, plasmid DNA for BMP-4 and vascular endothelial growth factor (VEGF) [20]. Combined delivery of both a growth factor promoting vascularisation and plasmid DNA for a gene that promotes osteogenic differentiation resulted in significantly increased bone tissue formation compared with delivery of either soluble bioactive factor alone. Such combinatorial approaches will probably be of great value in MSC regenerative therapies. 2.2 Effects of insoluble factors on MSCs Natural and synthetic biomaterials A variety of biomaterials have been investigated as temporary three-dimensional (3D) scaffolds for MSC adhesion, proliferation, migration and differentiation. This scaffolding provides mechanical support while MSCs multiply and eventually differentiate into tissue-specific cell phenotypes. It is then desirable for the scaffold to degrade in concert with the production of new ECM by differentiated MSCs. Natural (e.g., silk fibroin, collagen type I, alginate, hyaluronan, coral calcium carbonate-derived apatite, bone-derived apatite [21-25]) and synthetic (e.g., β-tricalcium phosphate, hydroxyapatite, poly(ethylene glycol) [PEG], poly(lactic-co-glycolic acid) [PLGA], oligo(polyethylene glycol) fumarate [25-30]) biomaterials have been used to try to regenerate orthopaedic tissues using MSCs. In addition to the incorporation of soluble signals, as discussed in the preceding section, efforts are underway at present to tailor the biochemical and physical properties of these scaffolds in order to control MSC behaviour Immobilised adhesion molecules Cells embedded in the ECM within the tissues of the body employ adhesion interactions as one of their primary modes of physical communication with their surroundings. They recognise and attach to specific amino acid sequences in ECM protein molecules via integrins [31], which are a family of cell surface receptors comprised of interacting heterodimers of α and β subunits [32]. Cells can receive signals from the ECM through ligand receptor occupancy that can alter individual cell functions such as proliferation, differentiation [33], apoptosis [34] and migration [35]. In fact, it has recently been reported that ECM molecules, such as type I collagen and vitronectin, can regulate the osteogenic differentiation of MSCs through distinctly different mechanisms [36]. Although several natural biomaterials such as these have cell adhesion sites, they present numerous adhesion signals, thereby limiting specific cellular control. Therefore, many research groups are using materials that lack adhesion ligands and/or intrinsic protein binding capacity and immobilising adhesion molecules to these biomaterial surfaces via covalent chemical bonds in efforts to investigate the role of substrate adhesiveness on MSC behaviour. Covalently coupling the adhesion molecules to biomaterials is often accomplished with crosslinker molecules and/or photochemistries. Although whole proteins are frequently denatured or degraded during biomaterial modifications, short peptide sequences offer advantages of increased specificity, increased stability and incorporation at higher concentrations through basic chemical modification [37]. Specific peptide sequences, such as the ubiquitous arginine glycine aspartic acid (RGD) sequence present in ECM proteins (i.e., fibronectin [FN]), have been coupled to natural [38] and synthetic [39,40] biomaterials, and have been found to regulate MSC attachment, viability, growth and differentiation. In some studies, these effects are dependent on peptide type and density. It is exciting to note that some of these surface modifications can elicit desired responses, such as the differentiation of MSCs down the osteogenic lineage, without the addition of inductive soluble factors Biomaterial structural and intrinsic properties Recent work has provided evidence that not only are the biochemical properties of biomaterials important in influencing MSC function, but that the microarchitectural features of a biomaterial can also provide powerful signalling cues. A synthetic biodegradable polymer, poly-ε-caprolactone (PCL), was electrospun in order to form nanofibrous scaffolds with high porosity and high surface area:volume ratios. In the presence of chondrogenic media, these scaffolds Expert Opin. Biol. Ther. (2006) 6(9) 849

4 Environmental cues to guide stem cell fate decision for tissue engineering applications significantly enhanced the differentiation of MSCs compared with the micromass cell pellet culture system that is employed extensively in MSC chondrogenesis studies [41]. Similarly, the unique cobweb-like structure of a collagen PLGA hybrid scaffold was recently shown to promote human MSC chondrogenesis. The large surface area of the PLGA mesh promoted cell adhesion and cell cell contact, and regularly spaced open pores facilitated the formation of cell aggregates analogous to those found in cell pellet cultures [42]. In another study, MSCs seeded onto 3D non-woven fabrics of polyethylene terephthalate exhibited increased adhesion and spreading on fabrics comprised of thicker fibres compared with those with thinner fibres. Altering the porosity and fibre diameter of the meshes was also shown to influence adherent MSC proliferation and osteogenic differentiation [43]. In a study where hydroxyapatite microparticles were used as an injectable carrier for MSCs, it was found that particle size and surface microporosity influenced the in vivo development of bone and bone marrow, respectively [44]. Particles with diameters of µm exhibited no bone formation, whereas particles with diameters of µm promoted extensive bone formation. In addition, bone marrow was only formed with particles in the smaller size range that were microporous. Freeform fabrication (FFF) technologies are being employed to create scaffolds with defined 3D architecture down to the micron scale to allow more precise investigation of the importance of structural properties such as porosity, pore size and connectivity on MSC function. For example, a PCL calcium phosphate scaffold with a honeycomb-like design was engineered using the FFF technique of fused deposition modelling and was shown to be conductive for osteogenic differentiation of MSCs [45]. In addition to the aforementioned variables, factors such as scaffold surface texture, or microtopography [21], and intrinsic mechanical properties [46] may also play a role in controlling MSC behaviour. It is important to note that all of these in vitro studies utilised differentiation medium and the in vivo studies occurred in locations rich in growth factors, cytokines and hormones. It has yet to be conclusively demonstrated whether these scaffold architectural features alone have the capacity to elicit MSC differentiation External mechanical forces Many tissues in the body are subject to various forms of mechanical stimulation. These forces applied at the tissue level can be transferred to adherent resident cells within these tissues. Cells are then able to convert these mechanical signals into biochemical responses through a mechanism termed mechanotransduction [47]. Investigators are studying how these mechanical signals, which are imparted to musculoskeletal tissues and resident MSCs, such as compressive stress, shear stress, hydrostatic pressure and cyclic stretch, may affect their gene expression. A variety of two-dimensional (2D) in vitro culture systems have been used to apply defined mechanical loads to MSCs. For example, markers specific to osteoblast differentiation were upregulated [48] and matrix mineralisation was increased [49] when MSCs were subject to cyclic equibiaxial strain. Other studies demonstrated that different types of 2D loading regimes can have significantly different effects. Cyclic uniaxial strain was shown to promote MSC differentiation into smooth muscle cells, whereas cyclic equibiaxial strain did not [50]. More recently, the effects of mechanical stimulation on MSCs in 3D in vitro culture systems that more closely mimic in vivo environments have been studied. MSCs seeded on partially demineralised bone scaffolds and subject to four-point bending loads exhibited increased expression of osteoblast-specific differentiation markers and increased formation of mineralised matrix. These effects were modulated by the concentration of dexamethasone in the culture media [51]. Similarly, mechanical stimulation resulting from shear stress in a perfusion bioreactor induced osteoblastic differentiation of MSCs seeded on titanium mesh scaffolds. Addition of dexamethasone to the perfusion bioreactor system enhanced the osteogenic effects in a synergistic manner [52], and scaffold mesh size was also shown to influence MSC differentiation under these flow conditions [53]. MSCs cultured on a gelatine β-tricalcium phosphate scaffold in a stirred bioreactor promoted increased cellular proliferation and osteogenic differentiation compared with static culture conditions [54]. It was hypothesised that the shear stress mechanical stimulation of the agitated culture system, in addition to increased exposure to oxygen and nutrients, was responsible for this finding. A complex, multiaxial loading bioreactor system has been designed to partially replicate the stresses ligaments experience in vivo. MSCs in collagen gels that were subjected to both axial and rotational strains in this system assumed a ligament-like cell morphology, produced a more organised ECM and expressed increased mrna of typical ligament cell markers compared with static controls [55]. Some of the factors that appear to regulate the effects of mechanical stimulation on MSCs are the type, frequency, magnitude and duration of loading, the presence of other bioactive factors and the type of scaffold used. Recent evidence indicates that mechanically induced changes in cytoskeletal tension and cell shape also play an important role in the MSC fate decision [56]. 2.3 Influence of other mature cell populations on MSC behaviour Other cell types can affect MSC differentiation through paracrine signalling of secreted growth factors, hormones and cytokines, and also via direct cell cell contact. Several groups have investigated the ability of more fully differentiated cells from the mesenchymal lineage to guide the MSC fate decision down the same pathway. For example, during osteogenesis through endochondral ossification, a cartilaginous anlage is always present prior to bone formation. It is, therefore, not surprising that chondrocytes have been found to secrete 850 Expert Opin. Biol. Ther. (2006) 6(9)

5 Alsberg, von Recum & Mahoney soluble morphogenetic factors that induce osteogenic differentiation of MSCs [57]. Bone formation during embryogenesis, growth and fracture healing also involves the close association of evolving vascular endothelium and osteogenic cells. In fact, endothelial cells have been shown to direct osteogenic differentiation of MSCs in vitro, but only when the two cell types are in direct contact [58]. In addition, cotransplantation of both groups of cells led to increased bone formation in a subcutaneous in vivo mouse model. Platelets, which are abundant in fibrin clots that form following fractures, have also been found to promote new bone formation by transplanted MSCs [59]. Curiously, osteoblasts themselves have not generally been found to promote MSC osteogenic differentiation [57,60]. MSCs can also be stimulated to differentiate into other non-osteogenic mesenchymal cell phenotypes using coculture systems. Synovial cells induce chondrogenesis [61], and periodontal ligament cells [62], nucleus pulposus cells [63] and skeletal myoblasts [64] increase MSC expression of genes specific for those respective cell types. These heterotypic interactions may be exploited when engineering tissues. For example, human umbilical vein endothelial cells seeded together with 10T1/2 mesenchymal precursor cells in a FN collagen type I scaffold resulted in the differentiation of the precursor cells into mural cells and the formation of stable, functional blood vessels [65]. The effects of coculture systems on MSCs are not, however, limited to differentiation down the mesenchymal lineage. Coculture systems with non-mesenchymal cells have resulted in MSC differentiation into neurons [66], hepatocytes [67] and small airway epithelial cells [68]. Critical to incorporating coculture systems into tissue regeneration strategies is the determination of whether direct cell cell contact is required for the observed effects, or whether cell-secreted soluble factors alone are sufficient. It will also be important to establish if the observed MSC plasticity effects elicited from cell cell contact in some of these cocultures are a result of actual differentiation or cell fusion, as has been reported by some groups [68]. 2.4 The future of MSCs in tissue engineering Extensive progress has been made in identifying the microenvironmental signals that can affect the MSC fate decision process. As MSCs, as well as all other cells in the body, are exposed to numerous different signals in precise temporal sequence and spatial orientation during tissue morphogenesis, repair and homeostasis, future work in this area of research will focus on determining how combinatorial presentation of these factors influences MSCs. Once the results of delivering bioactive factors at various time points are more fully understood in 3D MSC systems [69,70], novel biomaterials can be developed to release multiple bioactive factors with temporal and sequential control [71]. In addition, MSC-derived chondrogenic and osteogenic cells have recently been used to form osteochondral grafts [72-74], and developing complex tissues comprised of multiple cell types is an exciting new direction for the field, with many possibilities. 3. Neural stem cell populations Neural stem cells (NSCs) have been identified in developing tissue and, more recently, in two isolated regions of the adult brain, including the subventricular zone and the dentate gyrus of the hippocampus [75-78]. NSCs can also be formed from mouse and human embryonic stem cells (ESCs) [79,80]. This is a particularly attractive possibility, as ESCs offer the possibility of an unlimited supply of cells for transplantation therapies. NSCs derived from each of these sources undergo limited self-renewal and can differentiate to form neurons or glial cells, the two major cell types found in the CNS. Multiple cues are involved in the choice between these fates, including ECM molecules, growth factors and cell cell interactions. 3D cell culture systems based on natural and synthetic polymer materials that mimic a subset of these cues are being developed to control NSC fate. Below, the authors provide a summary of approaches that have been developed for this purpose in traditional monolayer culture systems, and describe how this information is being built into 3D culture systems that may one day be transplantable. The authors describe the extent to which these approaches have achieved therapeutic benefits in animal models of degenerative disease and injury, and the limitations that will need to be addressed in future work. 3.1 Response of NSCs to soluble factors Expansion of NSCs with soluble factors Mitogens are used to expand populations of NSCs. In general, mitogenic factors are more effective when applied to NSCs grown above a non-adhesive surface as spherical tissues, or neurospheres. For example, the mitogen fibroblast growth factor (FGF)-2 has been used to successfully expand NSCs derived from developing [81] and adult rodent tissues [82], from developing human tissues [83] and, more recently, from rodent [84] and human ESCs [80]. Although FGF-2 promoted NSC proliferation, the fate of newly generated cells shifted towards the neuronal phenotype [81]. Mitogens that maintain NSCs in a proliferative state without impacting the lineage potential of the cells have not been clearly identified. However, the results from a recent study suggest that maintenance of NSC self-renewal may involve simultaneous exposure to multiple cues, including factors derived from endothelial cells [85] Differentiation of NSCs with soluble factors Soluble factors have been applied to cultures of NSCs to encourage differentiation towards a specific phenotype. When exposed to leukaemia inhibitory factor [86,87] and ciliary neurotrophic factor [88], NSCs tended to differentiate into glial cells. Exposure to triiodothyronin [89] and insulin growth factors I and II [90] favoured the development of Expert Opin. Biol. Ther. (2006) 6(9) 851

6 Environmental cues to guide stem cell fate decision for tissue engineering applications oligodendrocytes, a glial cell subtype that is of interest in the development of cell-based therapies for the treatment of spinal cord injuries. Neuronal differentiation is initiated in NSC cultures following withdrawal of mitogen. The number of newly generated neurons is increased when NSC cultures are exposed to members of the neurotrophin family of molecules, including nerve growth factor, brain-derived neurotrophic factor (BDNF), and neurotrophin-3, -4 and -5 [91-94]. These molecules may exert their effect by shifting the differentiation potential of the NSCs and/or by improving the survival of newly committed neurons, as there is a large body of evidence documenting a survival effect on maturing postmitotic neurons. To be functional, newly generated neurons must develop the capacity to synthesise neurotransmitters, generate action potentials and form synaptic contacts with other cell types. This maturation process is improved when postmitotic neurons are exposed to neurotrophins. For example, addition of BDNF to NSC cultures increases synapse formation and synaptic transmission [95]. Neurotrophins also increase the length and branching pattern of neurons [96-97]. This may be one mechanism by which neurotrophins increase synapse number, as increasing the complexity of neuronal architecture increases the surface area of axons and dendrites, and increases the number of potential contact sites for synapse formation [99]. A related molecule, glial-derived neurotrophic factor (GDNF) has proven to be most effective at increasing the yield of dopaminergic neurons from NSC cultures. For example, treatment of NSC cultures derived from the mesencephalic region of the brain, which contains dopaminergic precursor cells, with GDNF increased the total yield of dopaminergic neurons [100]. GDNF also impacted the final architecture of neurons by increasing the total length and branching pattern of neurites [101,102] Gene therapy for NSC differentiation Although soluble factors are clearly potent regulators of NSC fate, transcription factors intrinsic to cells also impact fate choice. One emerging approach to control NSC fate is to overexpress genes that encode for these transcription factors in NSCs. For example, Nurr 1 is a transcription factor critical to the proper development of midbrain dopaminergic neurons [103]. When overexpressed, Nurr-1 results in increased expression of dopaminergic features in mouse ESCs and NSCs isolated from developing tissue [104,105]. In one study, depolarisation-induced dopamine release was demonstrated from engineered cells [100]. These studies suggest the potential of genetic manipulation for producing functional dopaminergic neurons. However, these genetically modified cells have not been tested in animal models of degenerative disease. Even more intriguing is the possibility of using a gene therapy approach to recruit endogenous NSCs to repopulate injured or diseased tissue structures in the CNS. In a recent study, the adult rat ventricular lining was transfected with an adenoviral vector encoding the gene for BDNF [106]. This resulted in an increase in the number of newly generated neurons in the olfactory bulb, a region of the brain that undergoes neurogenesis during adulthood, and in the striatum. These findings suggest that it may one day be possible to initiate self-repair strategies for treating degenerative diseases such as Huntington s disease, which is characterised by a loss of striatal neurons. 3.2 Influence of ECM on NSC fate During development of the CNS, when NSCs undergo fate choice naturally, immunocytochemistry and in situ hybridisation studies indicate that there is abundant ECM in the CNS. The ECM components include FN, laminin (LN), vitronectin, collagens, proteoglycans, tenascin and thrombospondin [107,108]. Interactions between these ECM molecules and cell surface receptors modulate NSC fate during morphogenesis. Differentiation protocols that are used in cell culture mimic some of these interactions. For example, LN is one ECM molecule that is often used to support the differentiation of NSCs in culture. When grown on LN-coated surfaces, the number of newly generated neurons increases [109]. As with neurotrophins, this effect may be the result of an increase in the phenotypic specification of NSCs, or could be the result of an increase in the survival of postmitotic neurons [110]. LN also impacts the maturation of committed postmitotic neurons. It is a potent stimulator of neurite outgrowth for several cell types [ ] and stimulates synapse formation [116]. 3.3 Effects of other cells on NSC proliferation and differentiation Indeed, ECM and soluble factors are not the only important cues that impact NSC expansion and differentiation. Signals derived from other surrounding cells impact NSC fate. These signals can be membrane-derived and require cell contact or are secreted by local cell populations. For example, in culture, it has been shown that soluble factors released from microglia enhance the differentiation of both adult and embryonic NSCs towards the neuronal phenotype [117]. Microglia release a number of molecules that may underlie this effect, including BDNF, cytokines and chemokines. Vascular cells also impact NSC fate. When cultured in the presence of endothelial cells, NSCs continued to proliferate without differentiating [85]. This is very interesting, as FGF-2 alone does not maintain NSC self-renewal, but endothelial factors acting in conjunction with FGF-2 achieve this outcome. Astrocytes, a type of glial cell in the CNS, also produce soluble factors and membrane-associated factors that affect the fate of NSCs. For example, a coculture of adult NSCs with astrocytes derived from adult hippocampal tissue (a neurogenic region of the brain) resulted in an increase in neuronal fate commitment [118]. Astrocyte-derived signals also increased the maturation of committed neurons. NSCs grown on a layer of astrocytes extend longer, more branched neurites [118]. Astrocytes derived from a non-neurogenic 852 Expert Opin. Biol. Ther. (2006) 6(9)

7 Alsberg, von Recum & Mahoney region of the brain, the spinal cord, did not induce the same effect, suggesting that there is some regional specificity. 3.4 Biomaterials for NSCs 3D cell culture platforms are being developed to control the growth and differentiation of NSCs. Ultimately, when placed into tissue, the 3D material may reduce glial scar formation and guide the development of new tissue. This is particularly important within the CNS, which contains oriented tracts of fibres that transmit information to targeted locations in the brain. Several types of matrices are being developed for these purposes. For example, experimental conditions have been developed to seed NSCs throughout macroporous (pore size µm in diameter) PLGA polymer scaffolds [119]. NSCs adhered to the surface of these matrices, and their attachment to the synthetic material impacted differentiation. When cultured on PLGA matrices, immature markers were downregulated, whereas differentiation markers were upregulated [120]. Injectable matrices based on natural or synthetic materials that gel in response to ionic strength or temperature are being developed for neural cell delivery and may be particularly useful for the treatment of deep tissue structures. Materials that are being explored for this purpose include alginate [121], fibrin [122], agarose [121, ], collagen, methylcellulose [126] and peptide-based hydrogels [127]. In gel-based materials, cells are assembled throughout a network of crosslinked polymer chains. Several features of the gel material impact the function of neural cells within the hydrogel. For example, neurite growth through gel-like materials depends on the mechanical properties of the material. Using agarose gel matrices, neurite outgrowth from DRG neurons was shown to be inhibited at higher gel concentrations [123]. Neurite growth is also impacted by charge in the 3D material [125]. Negatively charged surfaces tend to inhibit neurite growth, whereas positively charged surfaces promote neurite growth. Neurite extension in gels can be supported by incorporating cell-adhesive peptide sequences and neurite extension sequences in the matrix. The specific peptide sequences RGD and tyrosine isoleucine glycine serine arginine (YIGSR) have been found to promote neurite attachment [128]. The sequence isoleucine lysine valine alanine valine (IKVAV) induces neurite extension [129]. Culture of neurites on peptide-modified alginate surfaces [121] and in 3D environments promotes neurite extension [122,124]. These studies indicate that two factors, the mesh size and the chemistry of the surrounding polymer network, are both important influences on process growth through gel-based materials. Fewer studies have examined the fate of NSCs within 3D environments. Neural progenitor cells (NPCs) proliferate, extend neurites and reconstruct neural tube-like structures that are composed of postmitotic neurons and glial cells when placed in a collagen gel matrix [130,131]. Using patch-clamp recording it was demonstrated that CNS stem and progenitor cells form excitatory and inhibitory synaptic connections that Figure 3. NPCs encapsulated in three-dimensional degradable PEG-based hydrogels generate new neurons and glial cells. Neural precursor cells proliferated to form large multicellular aggregates of cells when cultured for 2 weeks in synthetic degradable PEG hydrogels. Cells in the degradable hydrogels were fluorescently labelled with calcein-am (green, live) or ethidium bromide (red, dead). As the hydrogel degraded, the porosity increased and neurites emerged from aggregates to penetrate and grow throughout the hydrogel. Size bar represents 20 µm. NPC: Neural progenitor cell; PEG: Poly(ethylene glycol). generate action potentials spontaneously [132]. In another study, incorporation of peptide sequences increased neuronal differentiation of NPCs in 3D culture [127]. Recently, it has been found that NPCs generate new neurons and glial cells following encapsulation in 3D degradable PEG-based hydrogels. Interestingly, the synthetic environment promoted neurosphere formation during the initial stages of degradation and promoted neurite elongation at late stages of degradation. This finding was probably due to temporal changes in porosity that occur during degradation of the PEG hydrogel polymer network (Figure 3) [133]. 3.5 Stem cell therapies in neural regeneration In general, neurons of the mature CNS are postmitotic, and degenerative disease can result in the permanent loss of a large number of cells. NSC therapies are being developed for degenerative diseases of this kind, including retinal degeneration, Parkinson s disease (PD), spinal cord injury, amyotropic lateral sclerosis and muscular dystrophy. For example, cell replacement therapies are being developed to treat PD, a disease characterised by the progressive loss of dopaminergic neurons in the brain. Intrastriatal transplants of fetal nigral neurons reinnervate the striatum, form synaptic Expert Opin. Biol. Ther. (2006) 6(9) 853

8 Environmental cues to guide stem cell fate decision for tissue engineering applications connections, release dopamine and improve motor deficits in animal models of PD [ ]. Recently, ESC-derived dopaminergic neurons were shown to extend axons into the host striatum, form functional synaptic connections, and modulate spontaneous and pharmacologically induced behaviours [84]. These data support the notion that ESC-derived neurons may one day serve as a plentiful source of cells for transplantation therapies. In humans, application of fetal transplants has been met with variable but significant improvements in behavioural function [ ]. When examined histologically, surviving neurons are present within grafts at 18 months post-transplant [141]. At 10 years post-transplant, basal and drug-induced dopamine release were found to occur at normal levels, indicating that, despite the ongoing disease process, grafted neurons remain phenotypically stable and functional over clinically relevant timescales in the adult human CNS [142]. Several recent studies indicate that NSC transplants hold promise in improving axonal regeneration following spinal cord injury (for a review see [144,145]). When transplanted into the site of injury, NSCs survive and differentiate into neurons, astrocytes and oligodendrocytes; studies indicate that the size of the injury is reduced and that animal motor function is improved [146,147]. The mechanism by which stem cells promote functional recovery following spinal cord injury has not yet been determined. Stem cells may impact recovery on multiple levels. Transplanted stem cells may create a permissive healing environment for host axon regeneration by secreting growth promoting factors. They may alter scar formation that typically occurs near the site of injury as resident glial cells are activated. They may also affect recovery indirectly, by secreting factors that signal the recruitment of endogenous cell populations to aid in repair. 3.6 The future of NSCs in tissue engineering Although transplantation therapy has significant potential as a treatment for degenerative diseases and injury in the CNS, application of this approach is limited by several factors. First, NSCs do not endure the transplantation procedure and the acute tissue environment following surgery well. As few as 10% of the transplanted cell population survives 1 week following grafting [ ]. Second, simply replacing lost neurons is not likely to be sufficient. Robust, reproducible transplantation therapy will require methods to specifically reconstruct neuroanatomical circuitry within the CNS. Biomaterials targeted to improve cell survival and direct morphology offer a significant opportunity to improve long-term outcomes and expand clinical applications of NSCs as therapeutics in the CNS. Biomaterials laden with NSCs have already demonstrated some promising improvements in neural cell function following implantation in animal models of disease. For example, in one recent study, a PLGA polymer scaffold seeded with NSCs was implanted into the cerebral hemisphere of an animal that had been subjected to a hypoxic ischaemic injury that results in extensive tissue loss. A meshwork of highly arborised donor and host neurites was observed in the implant and in surrounding tissue regions [151]. In a different study, PLGA polymer scaffolds seeded with NSCs were found to improve the functional degree of recovery in an animal model of spinal cord injury [152]. Although there is some evidence in the literature that suggests that PLGA polymers result in some degree of inflammation when implanted in the CNS, when seeded with NSCs and implanted into the CNS, a lower degree of inflammation and glial scarring was observed relative to controls. As a result, the scaffolding may result in a more physiologically relevant regenerated tissue structure by both impeding glial scar formation (which can inhibit tissue regeneration) and by directing the growth of new processes from donor and host cells. Direct imaging studies indicate that transplants of NSCs remain localised to the transplant epicenter when grafted into adult CNS tissue [153,154]. Long-distance migration away from the transplant epicenter can occur if grafts are placed in developing CNS tissue. The dependence of the migratory potential of grafted NSCs on host environment age is probably related to differences in the chemical composition of each host environment. Interestingly, endogenous NSCs can migrate across long distances in the adult brain to repopulate damaged areas of the CNS [155,156]. As the mechanisms that underlie the recruitment of endogenous NSCs in adult tissue become clear, it may one day be possible to design self-repair strategies, a potentially powerful approach to repair and cure injuries and diseases of the CNS. 4. Endothelial precursor cells 4.1 Endothelial precursor cell populations The growth and differentiation of endothelial cells from precursor cells has been examined through many models, from embryonic development to disease states, such as ischaemia or cancer, to tissue engineering. There is a substantial body of research on how to identify, amplify and purify such endothelial precursors both of embryonic and adult origin. Adult endothelial precursors can be derived from circulating blood, bone marrow sources or tissue. They differ from fully differentiated endothelium in that they express certain markers not present on mature cells. The importance of reliable sources of proliferating endothelial cells cannot be overstated. While tissue engineers desire such cells for use in endothelialisation of vascular grafts and prevascularisation of engineered tissue beds (such as for bone and liver regeneration), these cells are also in high demand for cellular therapy. Endothelial cells or endothelial precursors are sought for therapy in any poorly perfused tissue from ischaemic heart to brain to limbs. These cells directly, or indirectly through secreted factors, show much promise in their capacity to rebuild vascular networks and to restore oxygenation and nutrient transport to those tissues. Although fully differentiated adult endothelial cells can be isolated, this isolation often requires a large mass of tissue due to the 854 Expert Opin. Biol. Ther. (2006) 6(9)

9 Alsberg, von Recum & Mahoney somewhat limited proliferation capacity of the cells. In addition, the tissue-specific phenotypic expression of differentiated endothelial cells in one tissue type may interfere with their cellular function in another tissue type. Therefore, the advantages of endothelial precursors, namely high proliferation capacity and lack of tissue specific markers, make them quite desirable. This section describes the isolation, expansion and differentiation of such adult precursors. In addition, the authors examine the growing body of knowledge on the selection and differentiation of endothelial precursors from ESCs. Finally, this section speculates on what the future holds for adult and embryonic endothelial precursors Circulating endothelial precursors The presence of endothelial cells in the circulatory system, particularly in patients with cancer or sickle cell anaemia, has long been described [ ]. These cells were originally presumed to be fully differentiated endothelial cells that had been mobilised or sloughed off in response to injury and disease. It was not until 1997 that this population was discovered to contain endothelial precursors expressing primitive markers, maintaining a high proliferation capacity and contributing to vasculogenesis in vivo [161]. Since then, many other groups have confirmed these findings [ ]. In the literature these circulating precursors are often referred to as endothelial precursor cells (EPCs); however, they may be more accurately referred to as circulating endothelial precursors (CEPs) to distinguish them from tissue- or bone marrow-resident EPCs and from fully differentiated circulating endothelial cells (CECs) [167]. The exact origin of these cells is unclear, but it is presumed to be the bone marrow. Indeed, the association of the endothelial lineage and the haematopoietic lineage to a common precursor, the haemangioblast, has been observed for > 80 years [168]. The fate of these two cell lineages has led to research on common precursors of both embryonic and adult sources; studies are examining endothelial precursors from the ESC-derived putative haemangioblast, as well as the angiogenic potential of endothelial precursors found in adult bone marrow Haematopoietically derived endothelial precursors The ability of bone marrow cells to contribute to the population of CEPs was examined clinically in humans through fluorescent in situ hybridisation (FISH) [164]. The peripheral blood of patients who had received gender-mismatched bone marrow was examined for CECs. Using FISH for the Y chromosome, it was possible to determine whether the cells were from the recipient or donor. More than 95% of the CECs were recipient in origin, but these cells had a limited proliferation capacity and were deemed to be mature CECs. The remaining 5% were of donor origin, presumably from the bone marrow transplantation, and had a high proliferation capacity, similar to that previously observed in CEPs. Using a single green fluorescent protein (GFP)-positive haematopoietic stem cell, Grant and colleagues was able to reconstitute the haematopoietic system of lethally irradiated non-gfp mice [169]. These mice showed GFP-positive neovascularisation in the eye following laser-induced ischaemia. This conclusively demonstrated that the cell responsible for reconstituting the haematopoietic system also contributed to the CEP population. Similar experiments were demonstrated in the brain with stroke models [170]. Subsequently, bone marrow-derived cells have been implicated in angiogenesis in limb ischaemia [171,172], myocardial infarction [ ], atherosclerosis [176], as well as neonatal and tumour growth [ ]. When a mixed population of bone marrow cells is used [182], however, it is difficult to determine if the angiogenic effect seen is wholly due to EPCs or if it is due to soluble signals secreted by other cells in the bone marrow, such as platelets, erythrocytes and monocytes [ ] Tissue-resident endothelial precursors In addition to CEPs from adult and umbilical cord blood [186] and bone marrow sources of EPCs, a further location for endothelial precursors is resident within tissue. It has been demonstrated that an endothelial progenitor could be isolated from a side population of skeletal muscle cells based on efflux of Hoechst dye [187]. In addition, Hirschi and colleagues and Reyes and colleagues independently demonstrated that an endothelial progenitor exists within the non-haematopoietic MSCs found in bone marrow [180,188]. There is also some evidence of vascular precursor cells resident in the walls of larger blood vessels that can contribute both to endothelium and to the smooth muscle cell populations [189]. 4.2 Response of EPCs to soluble factors Expansion of EPCs with soluble factors It is estimated that there are on the order of 2 3 CEPs per ml of peripheral blood [164], which makes up 0.01% of the mononuclear cells. However, following vascular trauma, the number of these CEPs increases by orders of magnitude with an observed 12% of mononuclear cells 24 h after injury [167]. This rapid mobilisation is believed to be primarily due to elevated levels of circulating VEGF-A observed in blood vessels after injury [190,191]. This observation has been used therapeutically, where patients with lower limb ischaemia receiving VEGF gene transfer observed a > 200% increase in circulating EPCs [192]. This could theoretically be used to increase yield prior to the harvest of cells for tissue engineering purposes, similar to what is done to improve cell precursor yield prior to bone marrow transplantation [193]. The amount of circulating VEGF can be increased indirectly by administering matrix metalloproteases (MMP), such as MMP-9, which is presumed to free matrix-bound VEGF [194]. Release of VEGF can itself have indirect effects, such as inducing secretion of haematopoietic growth factors (i.e., Expert Opin. Biol. Ther. (2006) 6(9) 855

10 Environmental cues to guide stem cell fate decision for tissue engineering applications granulocyte-macrophage colony-stimulating factor) [195]. In addition to VEGF, other factors have been examined and found to increase EPC mobilisation, such as angiopoietin-1 [196], statins [ ], stromal cell-derived growth factor (SDF)-1 [200] and placental growth factor [201,202]. Exogenous delivery of biological factors is one approach to increase the number of EPCs that can potentially be isolated. Another option is through ex vivo proliferation in tissue culture. CEPs have been observed to proliferate for > 1000 passages in culture, allowing them to compete with the fully mature CECs that are limited to passages [164] Gene therapy to control EPC behaviour Although gene therapy is being investigated for many therapeutic applications, its use with endothelial progenitors is still limited. One area in which there has been some success is the use of gene therapy in reducing cell senescence. Several investigators have examined ex vivo transfection and selection of endothelial precursors with the human telomerase reverse transcriptase (htert) to increase cell proliferation beyond its usual limits [203,204]. This could increase the potential for single endothelial precursor clones to be used therapeutically. Another approach has been to transfect cells with a VEGF gene and thereby take advantage of the ability of VEGF to promote recruitment, attachment and differentiation [205]. 4.3 Response of EPCs to insoluble factors EPC homing to insoluble factors The ECM, in addition to providing attachment signals for endothelial cells, may also provide homing cues for endothelial precursors. VEGF, which is present in endothelial ECM, has been observed to influence homing (as has SDF-1, which binds to the CXCR-4 receptor found highly expressed on EPCs) [206,207]. Exogenous delivery of factors such as simvastin has been investigated to elucidate their role in upregulating integrin subunits specifically found on EPCs and thereby enhancing their attachment to ECM [199]. Several groups have attempted to exploit the idea of using EPC homing to improve endothelialisation and wound healing. Anti-CD34 antibody has been bound to a metal stent to improve stent endothelialisation [208]. Although CD34 is present on haematopoietic precursors such as EPCs, it is not present on fully differentiated endothelium. A similar strategy was employed using the same anti-cd34 on poly(tetrafluoroethylene) (PTFE) grafts [209]. As expected, recruitment of EPCs increased; however, inflammation also increased, which may have resulted from recruitment of other unwanted haematopoietic cells to the area (e.g., monocytes) [210]. Monocytes can have different responses to wound healing, either pro-angiogenic or pro-inflammatory, depending on the local signalling environment [211]. The pro-angiogenic capacity of monocytes is demonstrated both in the secretion of factors such as VEGF and also in their ability to differentiate into endothelial cells by administration of exogenous factors [212,213]. However, the inflammatory response of these cells, often observed on biomaterial implants, is inherently antiangiogenic and, therefore, undesirable Influence of ECM signalling and mechanical forces on EPCs Although research into recruitment and expansion of endothelial precursors has been fairly extensive, research on differentiation has not been so thorough. Typically, EPCs isolated from blood are allowed to attach to tissue culture dishes where over time they lose endothelial progenitor markers such as CD133 [163,165]. Alternatively, cells are cultured on matrix molecules, such as FN [ ], and collagen type I [217,218] and IV [219,220], in an effort to mimic the native endothelial cell ECM environment. Although it is known that integrins play an important role in endothelial differentiation, their exact role is not well understood [221,222]. Attachment of endothelial cells is critical for any tissue-engineered small-diameter graft, and this adhesion is dependent on precise biochemical and physical cues. For example, fluid flow and interfacial shear influence the attachment and orientation of differentiated endothelial cells [223]. The same has been observed for endothelial precursors [214,224]. 4.4 Mixed populations of endothelial precursors As discussed previously, one of the complications of EPC transplantation is that it is difficult to obtain a clonally pure population. During the isolation of CEPs from peripheral blood, one of the primary contaminants is other mononuclear blood cells, such as monocytes, which have both positive and negative effects toward endothelialisation, as mentioned earlier [183,210]. Isolation of EPCs from bone marrow, umbilical cord blood or other tissues would be expected to have similar complications. Use of ESCs as endothelial precursors has two major problems regarding other cell types. The first is the presence of cells that have undergone differentiation into an unwanted phenotype. The second problem is the presence of feeder cells or other stromal cells. However, there has been substantial progress in identifying the factors, such as leukaemia inhibitory factor, nanog and BMPs, involved in maintaining these cells in an undifferentiated state, and this has made feeder-free culture of mouse ESCs a possible option [ ]. 4.5 Biomaterials for vascular regeneration with EPCs Tissue-engineered blood vessels made using EPCs have been prepared with the same materials used in designs incorporating fully differentiated endothelial cells. These materials include non-degradable polymers [216,228], degradable polymers [214,229,230] and natural materials [215,216,231]. The non-degradable polymers, such as PTFE and polyesters (e.g., Dacron ), are the same as those that are used routinely to replace large-diameter vessels, for example, for an aortic aneurysm. Although these materials are effective in these high-shear environments, they typically fail 856 Expert Opin. Biol. Ther. (2006) 6(9)