Canadian Journal of Cardiology 30 (2014) 1350e1360

Review

Regenerative Cell and Tissue-based Therapies for Pulmonary Arterial Hypertension William S. Foster, BPHE, Colin M. Suen, BMSc, and Duncan J. Stewart, MD Ottawa Hospital Research Institute, Sinclair Centre for Regenerative Medicine and Regenerative Medicine Program, and University of Ottawa, Faculty of Medicine, Department of Cellular and Molecular Medicine, Ottawa, Ontario, Canada

ABSTRACT

  RESUM E

Within the span of 2 decades, cell-based regenerative therapies for pulmonary arterial hypertension have progressed from bench-side hypotheses to clinical realities. Promising preclinical investigations that examined the therapeutic potential of endothelial progenitor cell and mesenchymal stem cell populations have demonstrated the safety and efficacy of these cell types and provided the foundation for first-in-man clinical trials. Moreover, these studies have improved our understanding of the therapeutic mechanisms by which stem/progenitor cells exert their regenerative functions. Ultimately, these discoveries have led to new applications for stem and progenitor cells including the autologous cell reseeding of decellularized or synthetic lung scaffolds. In this review, an overview of established and emerging cell and tissue regenerative therapies for pulmonary lung diseases are presented, along with discussion of recent advancements in the emerging field of repopulating decellularized or bioengineered lung scaffolds with stem/progenitor cells for allogeneic transplant.

cennies, les the rapies cellulaires re ge  ne ratrices de l’hyEn 2 de rielle pulmonaire sont passe es des hypothèses fondapertension arte alite s cliniques. Les recherches pre cliniques mentales aux re rapeutique des poprometteuses qui examinaient le potentiel the nitrices endothe liales et de cellules pulations de cellules proge senchymateuses souches ont de montre  l’innocuite  et l’efficacite  de me ces types de cellules et ont fourni la base sur laquelle reposent les tudes ont premiers essais cliniques sur l’homme. De plus, ces e liore  notre compre hension des me canismes the rapeutiques par ame nitrices exercent leurs fonctions lesquels les cellules souches et proge ge  ne ratrices. Finalement, ces de couvertes ont mene  vers de noure nitrices, y compris le velles applications des cellules souches et proge ensemencement des prothèses de cellularise es ou des prothèses de re tiques par des cellules autologues. Dans cette revue, poumons synthe sentons un survol des the rapies cellulaires et tissulaires nous pre tablies et e mergentes contre les maladies pulmonaires, et nous e es re centes dans le domaine e mergent de la discutons des avance cellularise es ou de prothèses de pourepopulation des prothèses de tiques à partir de cellules souches et proge nitrices en mons biosynthe vue d’une allogreffe.

In this review, an overview of established and emerging celland tissue-based regenerative therapies for pulmonary arterial hypertension (PAH) are discussed along with advancements in the emerging field of ex vivo lung bioengineering.

transport of deoxygenated blood from the right side of the heart to the lungs for reoxygenation. Pulmonary hypertension (PH) is a disease that occurs when the resistance to blood flow in the lung increases, resulting in progressive increases in pulmonary arterial pressure (PAP). Thus, PH is defined by an increase in mean PAP
25 mm Hg measured using direct catheterization.1 Although there are many reasons for increased PAP, for example, increased pulmonary pressures due to left heart disease, for the purpose of this review, we will focus on disorders of the precapillary arteriolar bed that result in PAH. Even though PAH is classified as a single PH subgroup (group 1; see Table 1), it is a heterogeneous disease with several clinical classifications based on pathology, hemodynamic characteristics, and/or treatment strategies.1 According to the most recent guidelines established at the fifth World Symposium on PH, PAH can be subdivided into the following classifications: (1) PAH of unknown aetiology or idiopathic (IPAH); (2) heritable PAH (HPAH); (3) drug- and toxin-induced PAH; and (4) PAH associated with connective

PAH The lung is unique of all organs of the body in that it must accommodate all of the cardiac output to perform its vital function of oxygenating the venous blood. In physiological conditions the pulmonary circulation functions as a lowpressure, high-throughput system that facilitates the Received for publication July 2, 2014. Accepted August 24, 2014. Corresponding author: Dr Duncan J. Stewart, Ottawa Hospital Research Institute, 501 Smyth Rd, Ottawa, Ontario K1H8L6, Canada. Tel.: þ1-613737-8899, x79017; fax: þ1-613-739-6294. E-mail: [email protected] See page 1358 for disclosure information.

http://dx.doi.org/10.1016/j.cjca.2014.08.022 0828-282X/Ó 2014 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Foster et al. Regenerative Cell and Tissue-based Therapies for PAH Table 1. Group 1 pulmonary hypertension classification 1. PAH 1.1 Idiopathic PAH 1.2 Heritable PAH 1.2.1 BMPR2 1.2.2 ALK-1, ENG, SMAD9, CAV1, KCNK3 1.2.3 Unknown 1.3 Drug- and toxin-induced 1.4 Associated with: 1.4.1 Connective tissue disease 1.4.2 Portal hypertension 1.4.3 Congenital heart disease 1.4.4 Schistosomiasis ALK-1, activin-like receptor kinase-1; BMPR2, bone morphogenetic protein receptor type 2; CAV1, caveolin-1; ENG, endoglin; KCNK3, potassium channel subfamily K member 3; PAH, pulmonary arterial hypertension; SMAD9, mothers against decapentaplegic homolog 9. Modified from Simonneau et al.1 with permission from Elsevier Inc.

tissue disease, HIV infection, portal hypertension, congenital heart diseases, or schistosomiasis (Table 1).1 In patients with PAH, blood flow through the pulmonary arteries is restricted by a number of pathological processes leading to vasoconstriction, vascular remodelling, and obliteration,2 which contribute to increased pulmonary vascular resistance, right ventricle hypertrophy, and in many cases, death due to right heart failure.2,3 Although the exact mechanisms leading to structural remodelling of the pulmonary vasculature remain yet to be fully elucidated, endothelial dysfunction is widely considered to be an initiating event in the development of PAH.4,5 Insults to the endothelium inflicted by environmental toxins, viruses, reactive oxygen species, increased levels of serotonin, anorexigens, or shear stress have been implicated in the disruption of endothelial cell (EC) homeostasis and function (Fig. 1).4 Most notably, damage to the endothelium can reduce the production of vasodilators such as prostacyclin,6 nitric oxide,7 and increase the production of vasoconstrictors such as thromboxane A28 and endothelin-1.9 PAH is further characterized by several histological and functional abnormalities such as intimal hyperplasia and fibrosis, medial smooth muscle cell hypertrophy, increased deposition of extracellular matrix (ECM) inflammatory cell infiltrates, and/or pulmonary EC injury and dysfunction at the level of the intra-alveolar (precapillary) arteriole.2 Unlike the proximal pulmonary arteries, in which there are well developed medial and adventitial layers composed of smooth muscle cells and connective tissue that support the structure of the vessel, the distal lung arteriole is composed of a single endothelial layer with scant matrix support and only an occasional mural pericyte. Thus, the lung microvasculature might be particularly vulnerable to injury and EC apoptosis which might result directly in the loss of the fragile precapillary lung arterioles and lead to the loss of functional microcirculation (ie, the degenerative hypothesis) (Fig. 1).10 However, in the advanced stages of the disease, PAH is often characterized by complex and highly proliferative vascular intimal lesions.11 It has been hypothesized EC apoptosis is also a trigger for the emergence of apoptosis-resistant and growth dysregulated vascular cells (ie, the proliferative hypothesis), and that these abnormal cells are associated with the formation of obliterative structures called plexiform lesions (Fig. 1).5,10 At present, the relative contribution of these degenerative and proliferative mechanisms to

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the increase in pulmonary vascular resistance in PAH is not known for certain, and might indeed vary in individual cases. Rationale for Cell-Based Therapies for PAH Traditional pharmacological therapies for PAH have focused primarily on restoring pulmonary endothelial function by restoring imbalances to vasoactive factors. Among the most common “vasodilator” therapies for PAH are the administration of prostanoids, endothelin receptor antagonists, calcium channel blockers, and phosphodiesterase type 5 inhibitors.2 Although these therapies have been shown to modestly improve functional capacity, and possibly survival in some cases,12-14 they fall far short of providing a cure for PAH and generally do not address the widespread loss of functional microcirculation: the root cause of the hemodynamic abnormalities in this disease. In healthy individuals, the lung is normally endowed with an abundance of lung microcirculation that can be recruited to accommodate even maximal increases in cardiac output with exercise. Thus, clinical symptoms and signs of PH might only develop when a certain threshold has been reached, usually associated with the loss of most of the lung vascular bed (Fig. 2). This means that in patients with severe PAH even trivial loss of the remaining microvascular cross-sectional area can result in large changes in hemodynamics and functional status. The reverse is also true and even small improvements in microvascular area could produce large functional benefits. Therefore, regenerative therapeutic approaches, with the goal of repairing endothelial damage and/or regenerating lung microvasculature, represent a potential paradigm shift from current approaches for treating this devastating disease. The use of cell-based therapies in PAH is further facilitated by the anatomic location of the vascular injury and pathology in this disease at the intra-alveolar (precapillary) arteriole. In addition to its vital gas exchange function, the lung also functions as a filter to remove small particles (ie, thrombi) from the circulation. Thus, noncirculating cells injected intravenously are transported through the venous circulation to the lungs toward the distal pulmonary arterioles. This natural targeting process has greatly facilitated the use of cell or cell-based gene therapies in PAH. Stem and Progenitor Cell Types Used in Regenerative Therapies for PAH There is a wide range of cell types that could be used to repair and regenerate the lung microvasculature in PAH. Although autologous adult stem and progenitor cells present the least risk, they likely offer the least potential for direct tissue repair and regeneration. In contrast, pluripotent stem cells have the greatest capacity for transdifferentiation and regeneration, but their use in clinical translational research programs will be delayed until methods are established to maintain their intended differentiation state and eliminate the risk of neoplasia.15 Endothelial progenitor cells Origins and functions. In 1997, Takayuki Asahara and colleagues published a seminal report describing endothelial progenitor cells (EPCs) as population culture-modified

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Figure 1. Pathophysiological mechanisms of pulmonary arterial hypertension (PAH). Exposure to environmental insults such as increased serotonin levels, anorexigens, viruses, increased levels of inflammatory cytokines such as interleukin 6 (IL-6), or sheer stress can contribute to endothelial cell (EC) damage and injury. In healthy individuals, physiological repair processes restore normal lung function via proliferation of nearby ECs and/or the recruitment of circulating endothelial progenitor cells (EPCs). Alternatively, in individuals with PAH, pulmonary vascular cell damage contributes to the degeneration of microvasculature and/or arteriolar remodelling. In patients with hereditary PAH underlying genetic mutations to the genes encoding for bone morphogenetic protein receptor type 2 (BMPR2), activin-like receptor kinase-1 (ALK-1), and endoglin are associated with increased susceptibility to EC damage and injury. Traditional pharmacotherapies aimed at restoring imbalances in vasoactive factors are presented alongside emerging therapies aimed at regenerating the microvasculature. iPSCs, induced pluripotent stem cells; MSCs, mesenchymal stromal/ stem cells; PDE-5, phosphodiesterase type 5; ROS, reactive oxygen species; SMC, smooth muscle cell.

mononuclear cells (MNCs) with EC-like characteristics and vasculogenic properties in vivo.16 Before this report, it was widely believed that the process of de novo blood vessel formation was restricted to embryogenesis and carried out largely by EC precursors such as angioblasts.17 Thus, the identification of putative human EPCs represented a paradigm shift in our understanding of postnatal vasculogenesis and the discovery of a cell type with considerable therapeutic potential for PAH patients.

Characterization. Asahara and colleagues first isolated an “EPC” population by selecting CD34 or fetal liver kinase 1positive circulating MNCs.16 Since then, other groups have demonstrated the ability to derive putative EPCs from bone marrow18 and umbilical cord blood MNCs19 using a variety of protocols. Often referred to using the same term, these different EPC populations vary considerably with regard to cell surface marker expression and morphology.20 The most common markers used for EPC identification include mature

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Figure 2. Conceptual relationship between lung microvascular loss and clinical pulmonary arterial hypertension (PAH).

EC markers such as VEGFR2 (fetal liver kinase 1; kinase insert domain receptor), CD31 (platelet-EC adhesion molecule-1), CD34, Tie-2, von Willebrand factor, and classic hematopoietic stem cell markers such as CD34, CD133 (AC133; prominin 1), and CD117 (c-kit; stem cell growth factor receptor).21 In addition, functional assays used to assess mature EC phenotypes include the ability to bind Ulex eurpaeus agglutin-1, uptake acetylated low-density lipoprotein, adhere to ECM analogues such as fibronectin, and form tubelike structures in vitro or in vivo.20 Although at present there is no consensus concerning the criteria to define “true” EPCs,22 it has recently been suggested that precursors of endothelial colony-forming cells (ECFCs), also referred to as late-outgrowth EPCs, might be reliably identified within populations of circulating MNCs using a panel of surface markers (CD34þ, CD133, CD146þ).23,24 However, the downside of using such an approach is in the fact that only a small fraction (approximately 0.01%) of the original MNC population meet these strict criteria. Such a limited amount of starting material presents a difficult challenge when scaling up to the number of cells required to treat a human patient. An alternative approach is to culture unselected circulating MNCs on fibronectin or collagen I matrices. Two subsets of EPCs derived from circulating MNCs have been described: early-outgrowth EPCs and late-outgrowth EPCs. Earlyoutgrowth EPCs, often referred to as circulating angiogenic cells or bone marrow angiogenic cells,25 appear as rod-shaped adherent cells after 3-7 days of culture.26 This early outgrowth population expresses variable endothelial (CD31 and kinase insert domain receptor), but strong leukocyte (CD45), and monocyte (CD11c and CD14) markers.27 Circulating angiogenic cells also lack a defining characteristic of true progenitor cells because of their lack of proliferation capacity in vitro. Lateoutgrowth EPCs, or ECFCs, appear as adherent colonies after approximately 2 weeks in culture, display a “cobble-stone” morphology, and express all mature EC markers.28 Moreover, these cells have been shown to form blood vessels de novo and participate directly in angiogenesis.29 When grown from single cells, late-outgrowth EPCs/ECFCs demonstrate clonal growth and the ability to form functional blood vessels when transplanted into immunodeficient mice.30,31 Presently, it is still unclear whether late-outgrowth EPCs differentiate from

mononuclear cells in culture or are derived from a rare specialized cell within the circulating MNC population. EPC preclinical studies. As mentioned, pulmonary EC apoptosis is believed to be a key trigger in the development of PAH followed by subsequent microvascular pruning and/or the emergence of growth dysregulated vascular cells.10 Thus, therapies with the ability to promote endothelial repair and microvascular regeneration via the angiogenic or neoangiogenic mechanisms could be highly beneficial in preventing or reversing established PAH. Since the mid-2000s, several groups have demonstrated the efficacy of putative EPC populations in rat and dog models of monocrotaline (MCT)-induced PAH.32 This evidence indicates that EPCs might exert their therapeutic benefits via 2 potential mechanisms: direct engraftment into the injured endothelium to repair or replace damaged cells,16,33 or, the release of proangiogenic cytokines and growth factors that support the regeneration of the lung microcirculation (Fig. 3).29 Indeed, it has been shown that fluorescently labelled EPCs have the ability to integrate into the pulmonary endothelium at the distal arteries and limit the progression of MCT-induced PAH.33 However, the number of transplanted EPCs that show long-term persistence in the lung is very low, and it is unlikely that engraftment and direct transdifferentiation into news ECs can represent an important mechanism accounting for their reported efficacy in PAH.34 There is growing appreciation that early-outgrowth EPCs might exert their therapeutic effects largely though indirect paracrine mechanisms.35-37 Compared with mature ECs, myeloid-derived EPCs release significantly higher levels of VEGF, stromal cell-derived factor-1 (SDF-1), and insulin growth factor-1 (IGF-1).35 Human EPCs injected intramyocardially into mice promote a sustained upregulation of proangiogenic and anti-apoptotic factors such as VEGF-A, FGF-2, angiopoietin 1, angiopoietin 2, placental growth factor, hepatocyte growth factor, IGF-1, platelet-derived growth factor subunit B, and SDF1.36 Cell-free conditioned media (CdM) induced a migratory response in mature ECs and could be inhibited by VEGF and SDF-1 antibodies.35 Furthermore, daily intraperitoneal injections of CdM from human cord blood-derived ECFCs have been shown to preserve alveolar, lung vascular growth, and attenuate PH in neonatal rats exposed to hyperoxia.38

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Figure 3. Origins, lineages, and functions of endothelial progenitor cells (EPCs), mesenchymal stromal/stem cells (MSCs), and induced pluripotent stem cells (iPSCs) in the context of pulmonary arterial hypertension (PAH). Blue arrows indicate stem/progenitor cell origins and differentiation pathways from peripheral blood, bone marrow, and/or adult somatic cells; represented by skin fibroblasts. Solid red arrows and hashed red arrows highlight the known and hypothetical therapeutic mechanisms by which selected cell populations act on areas of pulmonary precapillary arteriole injury/dysfunction, respectively. CACs, circulating angiogenic cells; EC, endothelial cell; ECFCs, endothelial colony forming cells; iPSCs, induced pluripotent stem cells; MNCs, mononuclear cells.

EPC clinical trials. The first clinical trial to access the safety, efficacy, and feasibility of autologous EPC transplantation for adult IPAH patients was published in 2007.39 The authors reported on their prospective, randomized trial that compared with conventional pharmacotherapy alone, a single intravenous infusion of culture-derived EPCs in patients taking background PAH therapy resulted in modest, but significant, improvements in exercise capacity and hemodynamic measurements at 12 weeks.39 In a subsequent clinical trial assessing the efficacy of EPC transplantation in a pediatric IPAH population, EPC cell infusion was associated with a significant reduction in mean PAP and pulmonary vascular resistance, improvements in cardiac output, and a greater exercise capacity determined using the 6-minute walk test.40 However, the main limitations of these pilot studies were the lack of a true placebo control group, no double-blinding, and a follow-up period of 12 weeks that would be insufficient to monitor long-term efficacy and safety. Moreover, the small sample sizes reflect the low prevalence of IPAHdan unfortunate hurdle in designing large clinical trials for PAH. Although these are the first to demonstrate proof-of-concept in a patient population, subsequent trials need to be performed to validate these pilot studies. Larger and more rigourously designed multicentre double-blind placebocontrolled clinical trials would provide greater confidence in the efficacy, safety, and generalizability of EPC therapy for PAH.

EPC enhancement strategies. Although preclinical studies have demonstrated the therapeutic potential of unmodified EPCs to prevent PH in experimental models, EPCs alone have proven to been less effective in restoring the lung microcirculation in preclinical models in the context of hemodynamic abnormalities (ie, treatment model).33,41 We have previously shown that cell-based gene therapy can be effective in preventing and reversing established PH in the MCT model33 even when using somatic cells (smooth muscle cells or fibroblasts) as vectors to deliver the transgenes to the distal arterial bed.42,43 Because of the efficacy of cell-based gene transfer with nonregenerative “carrier” cells, it was logical to assume that the genetic enhancement of EPCs could result in synergistic benefits from the reparative cell activity and the therapeutic transgene. Thus, a number of different therapeutic transgenes have been assessed, including angiopoietin-1,44 VEGF,42,43 adrenomedullin,45 calcitonin gene-related peptide,46 and nitric oxide synthase (endothelial nitric oxide synthase [eNOS]).33,43,47,48 Of particular note, our group showed that the administration of eNOS transfected EPCs could effectively reverse established MCT-induced rodent PAH, evidenced by normalized right ventricular pressures, improved microvascular architecture, and alveolar perfusion compared with naive EPCs.33 Based on these promising results in preclinical prevention and treatment models of PH, the first EPC-based eNOS gene therapy clinical trial (Pulmonary Hypertension: Assessment of Cell Therapy; PHACeT; Clinicaltrials.gov

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NCT00469027) was initiated in Toronto and Montreal in 2007. Mesenchymal stem cells Origins and functions. Mesenchymal stromal/stem cells (MSCs) are one of the most widely studied regenerative cell types. To date, there are already hundreds of clinical trials for a variety of diseases including, but not limited to, myocardial infarction, stroke, sepsis, graft vs host disease, and Crohn’s disease.49 So far, human clinical data suggest that MSCs can be safely administered without adverse events for a variety of conditions.49 Characterization. MSCs are most often defined according to the minimal criteria outlined by the International Society for Cellular Therapy in 2006.50 These characteristics include plastic adherence, positive expression of mesenchymal (CD73, CD95, and CD105) but not hematopoietic (human leukocyte antigen-DR [HLA-DR], CD11b, CD14, CD34, CD45, CD79a, and CD19) cell surface antigens, and differentiation potential for osteogenic, chondrogenic, and adipogenic lineages.51 Based on this set of characteristics, MSCs have been isolated from many different sources in the body, including most commonly bone marrow, umbilical cord blood, and Wharton’s jelly, adipose tissue, and almost every organ including the lung.52-54 The existence of MSC-like populations in almost every organ bed has led to the concept that there are multiple sources of tissue-resident MSCs.54 The in vivo function and origin of MSCs bears much in common with pericytes because of their perivascular localization and phenotypic similarity.52 However, the relative differences in cellular function and regenerative potential in different tissues between MSCs derived from different origins are largely unknown. Therefore, one must closely examine the origin and methodology of isolation and cell culture before extrapolating findings about MSCs. The purported effects of MSCs range from wound healing and tissue repair to angiogenesis and immune modulationdprocesses that are dysregulated in many diseases and are desirable targets for therapy.55 When administered exogenously, it has been shown that MSCs can home to sites of injury, differentiate, and repair the tissue microenvironment.32 Remarkably, in most preclinical studies using MSCs, their effects appear not to be mediated by the “classical” stem cell paradigm of regeneration by direct differentiation because of their low rates of long-term persistence and engraftment in the target organ.56 Rather, MSCs release many trophic and immunomodulatory paracrine factors such as interleukin (IL)10, PGE2, IL-8, VEGF, and transforming growth factor beta that facilitate tissue repair.55 Therefore, the paracrine mechanism of MSCs might allow them to produce dramatic effects in vivo despite low levels of cell persistence.57 It is increasingly recognized that MSCs interact with a wide variety of immune cells to promote the resolution of inflammation, tissue healing, and repair. When exposed to inflammatory cytokines, MSCs can inhibit T-cell proliferation and alter cytokine production of other immune cells such as natural killer cells, dendritic cells, and macrophages by mechanisms involving IL10, indoleamine 2,3-dioxygenase, nitric oxide, transforming growth factor beta, and PGE2.58,59 Because of the role of

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inflammation and angiogenesis in PAH, MSCs have been an attractive cell type for PAH therapy. MSC preclinical studies. Because of their multilineage potential, low immunogenicity, ease of cell culture, and scalability for clinical trials, MSCs are an ideal cell type for developing stem cell therapies. A hallmark of MSCs is low or lack of expression of major histocompatibility complex and costimulatory molecules, which are key components in antigen presentation and T-cell stimulation.32 These properties contribute to the potential of MSCs as immunoprivileged cells, which allow them to evade the host immune system and its natural inflammatory response to foreign bodies.60 Therefore, an advantage of MSC therapy is its potential use as an allogeneic, off-the-shelf product. Because of their potential benefits, a number of regenerative approaches using MSCs have been studied in the context of PAH. Indeed, syngeneic transfer of bone marrow-derived MSCs has been shown to effectively reduce pulmonary pressures, right ventricular hypertrophy, and pulmonary vascular remodelling in the MCT rat model of PAH.56,61-63 In a study that examined cell engraftment, intratracheally administered MSCs integrated mostly in the alveolar epithelium rather than the pulmonary vasculature, yet still provided benefit in the therapy of PAH.56 These observations would support the paracrine hypothesis, in which secreted soluble factors support tissue regeneration and repair (Fig. 3). Along these lines, Lee et al. recently demonstrated that enrichment of MSC-derived exosomes ameliorated PAH in the mouse hypoxia model of PAH.64 Exosomes are 40-100 nm membrane-bound vesicles, which have the ability to facilitate extracellular transport of protein, mRNA, and microRNA.65 Cell surface signalling proteins such as integrins and cell surface receptors allow exosomes to home and target specific tissues.66 Therefore, it is possible that the therapeutic benefits of cell therapies might be recapitulated using cell-free CdM. Using CdM would provide many regulatory and practical advantages over live cells such as long-term storage. MSC enhancement strategies. The engineering of MSCs to overexpress transgenes, or MSC-based gene therapy, has been shown to enhance the regenerative activity of MSCs in vivo. Target genes such as eNOS and prostacyclin synthase, pathways that have been traditionally targeted in PAH, and heme oxygenase-1 have all demonstrated efficacy in ameliorating PAH in preclinical animal models.61,67,68 Overexpression of transgenes after ex vivo transfection in controlled conditions is one of the most direct strategies to study specific molecular pathways in MSCs and other stem cells. However, genetic manipulation is rather costly, difficult to scale-up, and introduces potential toxicity of the nonviral or viral transfection vectors.32 However, these studies might form the basis for novel, less disruptive strategies, such as pharmacological or physicochemical preconditioning. Using small molecules, it might be possible to achieve the same or similar benefit by targeting the common molecular pathways. An example of such a strategy could involve priming MSCs to respond to the inflammatory milieu found in disease states. Poly(I:C) has been used to stimulate toll-like receptor 3, an important pathway in pathogen recognition and innate immunity.69 MSCs preconditioned with poly(I:C), a toll-

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like receptor 3 ligand, have increased immunosuppressive effects by potentiating key anti-inflammatory mediators, indoleamine 2,3-dioxygenase and PGE2.70 Currently, this preconditioning strategy has shown favourable outcomes as a treatment for diseases of severe inflammation such as experimental sepsis,70,71 and might have some utility in modulating chronic inflammation in PAH. As well, preconditioning methods such as hypoxia might also prime MSCs toward favourable therapeutic phenotypes. In contrast to routine cell culture (21% O2), hypoxic (1%-3% O2) conditions more closely resemble the microenvironment in which MSCs are found. Indeed, hypoxia promotes survival, proliferation, migratory capacity, and enhances revascularization in hind limb ischemia.72 As we gain a better understanding of the regulation of this fascinating cell type, we will be able to develop effective and minimally invasive methods of MSC enhancement. Induced pluripotent stem cells Origins and functions. Originally described by Takahashi and Yamanaka, induced pluripotent stem cells (iPSCs) are generated by reprogramming adult somatic cells to a more pluripotent state comparable with that of an embryonic stem cell (ESC).73 Reprogramming was initially achieved by transducing skin fibroblasts with a cocktail of pluripotency factors (Octamer-binding transcription factor 4, SRY (sex determining region g)-box 2, Krüppel-like factor 4, and c-myc) using a genome-integrative retrovirus, but later modified to include other nonintegrative methods.73,74 After approximately 4 weeks in culture, iPSCs display similar morphological and proliferative characteristics nearly identical to ESCs. Furthermore, iPSCs share a molecular signature highly similar to ESCs in terms of gene expression and DNA methylation patterns.74 Recently it has been shown that reprogrammed fibroblast iPSCs can be differentiated into endothelial-like cells (ie, iPSC-ECs) with a similar gene expression profile to human umbilical vein ECs.75 Differentiation is achieved by culturing iPSCs in the presence of activin A, bone morphogenetic protein, and VEGF.75 Emerging along the edge of embryoid bodies, iPSC-ECs can be subsequently harvested, expanded, and sorted using fluorescent activated cell sorting (FACS) by selecting for CD31- and/or CD144 (vascular endothelial cadherin)-expressing cells.75 iPSC preclinical studies. Using iPSCs or iPSC-ECs in a therapeutic capacity represents an exciting new avenue for PAH research. Compared with fibroblasts, iPSC-ECs injected intramuscularly have been shown to promote neovascularization and improve blood flow in hind-limb ischemia models.76,77 iPSC-ECs also displayed enhanced engraftment ability over fibroblasts evidenced by human CD31 antibody staining.77 When grown in hypoxic conditions to mimic the microenvironment of ischemic tissues, iPSC-ECs significantly upregulate the expression of major proangiogenic factors epidermal growth factor, HGF, VEGF, placental growth factor, and SDF-1 compared with normoxic conditions.76 Interestingly, the magnitude of cytokine upregulation in hypoxic conditions was noted to be more dramatic in iPSC-ECs than bone marrow-derived endothelial-like cells.76 Taken together, these results suggest that iPSC-ECs might share some

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therapeutic characteristics with other stem/progenitor cell types and might theoretically be used to treat PAH (Fig. 3). iPSC future studies. In addition to therapeutic applications, it has been hypothesized that iPSC-derived ECs might serve as a useful platform for studying functional abnormalities related to gene variants in IPAH and HPAH patients and that ECs derived from pulmonary artery EC-iPSCs might reveal DNA methylation changes associated with disease pathology.78,79 In support of this rationale, our group recently demonstrated that ECFCs, also called blood outgrowth ECs,25 could be used to interrogate the molecular underpinnings of abnormalities in EC gene expression in patients with HPAH and known bone morphogenetic protein receptor type 2 mutations. Using a proteomic approach, we reported dysregulated expression of a number of proteins in blood outgrowth ECs from HPAH patients, among these translationally controlled tumour protein was identified as a possible link between EC apoptosis and the emergence of growth dysregulated, hyperproliferative vascular cells.80,81 Ex Vivo Lung Tissue Engineering for Lung Transplantation in PAH Lung transplantation is the ultimate lung “regenerative” strategy and the only treatment option for patients with latestage lung diseases such as PAH, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and cystic fibrosis. Despite current efforts, recent estimates indicate that the demand for lung transplants greatly exceeds the availability of suitable donor lungs.82 Moreover, patients who are fortunate enough to receive donor organs are faced with complications such as organ rejection and increased risk of death associated with chronic immunosuppressive therapies.83 Decellularized lung scaffolds might provide a potential solution for these limitations. These lung scaffolds could be prepared from donor lungs deemed unsuitable for transplantation, or from nonhuman species such as farmed pigs. After removal of resident lung cells by various detergents, the lung scaffold could then be recellularized using somatic or autologous stem/ progenitor cells. iPSC technology provides the possibility of personalized lung bioengineering with the use of autologous cells to repopulate a decellularized scaffold, derived from differentiation of patient-specific iPSC cells into the main pulmonary cell types.77 Although this approach has shown some promise in experimental studies, even when coupled with fairly crude recellularization strategies using populations of somatic or transformed endothelial and epithelial cell types,84,85 there are many significant hurdles that need to be overcome before it will be possible to contemplate the clinical use of recellularized lung scaffolds. However, a similar approach has been successfully used clinically for a simpler structure. In 2008, Paolo Macchiarini and colleges published a seminal report describing the first successful human tissue-engineered trachea transplantation for a female patient with end-stage left-main bronchus malacia.86 Before implantation, the donor trachea was decellularized and subsequently repopulated with a combination of the recipient’s autologous epithelial cells and chondrocytes in a specially designed bioreactor for a period of 92 hours.86 In a recently published follow-up report, the

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authors indicated that the 5-cm long graph appeared to be well vascularized and displayed normal physiological function.87 Since this pioneering study, a number of groups have made marked advancements in whole-lung tissue engineering using several strategies outlined in the following sections. Lung decellularization strategies Unlike the comparatively simplistic tubular structure of the trachea, accurately recapitulating the intricate hierarchical structures of the pulmonary airway and vasculature within the mammalian lung represents a daunting challenge. The major advantage of decellularizing whole donor lungs is the ability to produce a complete and hypoallergenic “scaffold” to be used as a platform for cell growth. After decellularization, stem/ progenitor cells may be used to reseed the remaining airways, vasculature, and lymphatic structures formed by the remaining ECM proteins.84 A number of protocols and methodologies have been developed to decellularize explanted lungs, the most common use vascular and/or airway perfusion of mild detergents such as Triton X-100, sodium deoxycholate, sodium dodecyl sulphate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, or ethylenediaminetetraacetic acid in addition with hypertonic sodium chloride solution to lyse cells, DNA-ase, RNA-ase, and/or antibiotics.84,88 Ideally, the decellularization technique should be thorough enough to remove all traces of native donor cells without severely damaging the supportive ECM features and molecules. Composed of mostly collagen, elastin, laminin, fibronectin, entactin, proteoglycans, and tenascin, the ECM plays a key role in facilitating cellular proliferation and differentiation and therefore must be preserved.89 Furthermore, the remaining cellular components with immunogenic antigens might elicit an unwanted immunological response when recipient cells are used to inoculate the scaffold. Repopulating acellular lung scaffolds The mammalian lung is composed of several distinct cell populations such as airway epithelial cells, vascular ECs, and smooth muscle cells, and a myriad of endogenous and exogenous immune cells. Thus, selecting the most appropriate cellular candidates for scaffold repopulation is a critical component of ex vivo lung tissue engineering. To date, efforts have focused primarily on reseeding acellular lung scaffolds with either mature somatic or stem/progenitor cell populations.85,90-94 Petersen et al.92 and Ott et al.85 showed that acellular rat lung scaffolds maintained in bioreactors could be partially recellularized using combinations of neonatal lung epithelial cells, fetal lung cells, ECs, and A549 carcinomatous human alveolar basal epithelial cells. After orthotopic left lung transplantation, Ott et al. reported that scaffolds seeded with fetal lung cells exhibited successful gas exchange for up to 6 hours, although evidence of pulmonary edema was observed during gross examination of the explanted constructs.85 A subsequent study by Song et al. showed that rat lung scaffolds recellularized with a combination of fetal pneumocytes and human umbilical cord ECs were able to support oxygenation for up to 7 days at levels comparable with cadaveric lung transplant controls.94 In a separate study, bone marrow-derived MSCs delivered via intratracheal injection (2  106 cells per lung) were able to differentiate in

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parenchymal regions of decellularized mouse lung matrices.90 However, despite displaying several morphologies, mRNA analysis revealed that the reseeded MSCs expressed genes indicative of mesenchymal and osteoblast lineages, but failed to express genes associated with airway or alveolar cell types.90 The report also indicated that MSCs were able to preferentially home to and engraft in regions of rat lung ECM enriched with collagen type I and V, laminin, and fibronectin, demonstrating the importance of preserving ECM integrity during decellularization.90 Interestingly, despite narrowed vasculature compared with control lungs, decellularized lung scaffolds from rats with MCT-induced PAH maintained the ability to support cell reseeding.93 In addition to MSCs, Ghaedi et al. showed that alveolar epithelial type II and type I cells derived from human iPSCs could be used to inoculate decellularized rat and human lungs.91 The authors reported that after adhesion to the ECM and diffuse repopulation of alveolar lung structures, the iPSCs-derived alveolar cells displayed pulmonary epithelial cell markers possibly because of correct matrix interactions.91 Although these preclinical studies provide evidence to suggest that decellularized lung scaffolds can be partially repopulated with various cell populations, transiently supporting gas exchange when transplanted in vivo, much work remains to be done before whole lung tissue engineering using autologous stem/progenitor cells becomes a therapeutic reality. Major challenges include not only accurately recapitulating the complex structure and function of the mature human lung, but also scaling-up the manufacturing processes for decellularization and recellularization for whole human or xenogeneic (porcine) lungs for clinical application.95 Moreover, as in the past, ensuring that all potential transplants are biocompatible and hypoallergenic remains a top priority. Although still very promising, it is likely that the time needed to successfully overcome these challenges will be measured in decades rather than years. Conclusions Recent advancements in our understanding of therapeutic stem/progenitor cell populations have contributed to a rapid development of novel cell- and tissue-based regenerative therapies for PAH. Moving forward, it will be critical to place an emphasis on understanding the underlying cellular and molecular mechanisms responsible for the observed benefits of select cell types while continuing to explore safe enhancement strategies. Although preliminary studies in lung bioengineering have demonstrated promising results, a considerable amount of work still remains before clinical translation becomes a reality. Acknowledgements The authors thank Jose Mansilla-Miranda for his artistic contributions. Funding Sources W.S.F., C.M.S., and D.J.S. are supported by the Canadian Institutes of Health Research. W.S.F. and C.M.S. are Graduate Fellows in the Canadian Institutes of Health Research Training Program in Regenerative Medicine.

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Canadian Journal of Cardiology Volume 30 2014

Disclosures D.J.S. is a co-founder and share-holder of Northern Therapeutic Inc. and has received research funding from United Therapeutics.

18. Shi Q, Rafii S, Wu MH, et al. Evidence for circulating bone marrowderived endothelial cells. Blood 1998;92:362-7.

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