Cell-Based Therapy for Neonatal Lung Diseases

CHAPTER 21 Cell-Based Therapy for Neonatal Lung Diseases Karen C. Young, Bernard Thébaud, and Won Soon Park

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• The postnatal lung contains populations of stem cells with the capacity to reconstitute the lung following injury. • Prematurity and its antecedent factors may alter lung stem cell programming leading to dysfunctional repair. • Preterm infants in whom bronchopulmonary dysplasia develops exhibit decreased and/or dysfunctional stem cells. • Preclinical studies provide robust evidence that stem cells reduce lung injury and improve survival in experimental models of bronchopulmonary dysplasia via paracrine-mediated mechanisms. • Mesenchymal stem cells are an attractive population for lung repair as they are immunoprivileged, easily isolated, and have pleiotropic therapeutic effects. • Early clinical evidence demonstrates the safety and feasibility of mesenchymal stem cell therapy for bronchopulmonary dysplasia. However, barriers for implementation, including optimal dosing, patient, timing, and culture techniques, need to be overcome for successful translation. The lungs are remarkably complex organs with more than 40 different cell types uniquely organized to facilitate gas transport and exchange. This intrinsic complexity along with low cell turnover has led to challenges in understanding lung stem cell biology.1,2 Despite this, real progress has been made in the past decade and recent reports indicate that following injury, the damaged lung epithelium has the capacity to repair itself by a population of resident lung stem cells, with possibly minor contribution of bone marrow (BM)-derived stem cells. More data are also accumulating on the complex niches within which stem cells reside and the molecular mechanisms that regulate stem cell self-renewal and differentiation, and their impact on disease pathogenesis.3,4 Evidence is also mounting that environmental perturbations alter stem cell function and fate, leading to dysfunctional repair and remodeling. Aberrant stem cell reprogramming in preterm infants and their antecedent diseases now support the notion that early alterations in stem cell function contribute to bronchopulmonary dysplasia (BPD) pathogenesis. New exciting preclinical data provide evidence that stem cell–based therapies reduce lung injury in BPD models. Among the cells being tested, mesenchymal stem cells have shown tremendous promise owing to their ability to replenish the endogenous stem niche, their immunomodulatory properties, and their availability. Clinical trials in several countries using mesenchymal stem cell–based therapies are now underway in preterm infants. This chapter discusses recent advances in lung stem cell biology, our current understanding of the impact of BPD on endogenous stem cells, and the application as well as challenges of implementing stem cell–based therapies for BPD. 347

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B

Asymmetric division

Symmetric division

Fig. 21.1  Stem cell division. Stem cells may divide asymmetrically or symmetrically. During symmetric division, the stem cells divide and produce two daughter stem cells or two differentiated daughter progeny. Alternatively, during asymmetric division, the stem cell produces one differentiated daughter progeny and one stem cell.

Stem Cells In the early 1900s the Russian histologist Alexander Maximow discovered that all blood cells have a common ancestral origin.5 Since then our knowledge of stem cell biology has grown exponentially, and stem cells are now known to be essential for organogenesis and tissue homeostasis. By definition, stem cells must have the ability to both replenish themselves through self-renewal and to differentiate into mature progeny.6–8 They may divide symmetrically or asymmetrically, giving rise to a stem cell and a more committed progenitor cell (Fig. 21.1).9,10 They may also be classified as totipotent, pluripotent, or multipotent (Fig. 21.2).10 Totipotent stem cells are capable of differentiating into all adult and embryonic tissues, including extraembryonic tissues such as trophectoderm.11,12 In mammals, only the zygote and the first cleavage blastomeres are totipotent.12 Pluripotent stem cells are capable of differentiating into derivatives of all three germ layers (ectoderm, mesoderm, and endoderm) and are typically derived from embryos at different embryonic stages of development.13 Multipotent stem cells are able to differentiate into multiple cell types of one lineage.14 The most prominent example remains hematopoietic stem cells, which are capable of differentiating into all cell types of the hematopoietic system.15 Stem cells may also be categorized as embryonic or adult stem cells. Embryonic stem cells are derived from blastocysts in the developing embryo and are pluripotent.16 Adult stem cells are found in tissues in specialized microenvironments.17,18 These cells are typically multipotent and following an asymmetric cell division, they produce a population of transit-amplifying progenitor cells.19 They may act as intermediates between dedicated stem cells and mature differentiated cells. Within tissues, there may also be facultative stem cells or progenitors, which are normally quiescent differentiated cells, but following injury, they may self-renew and give rise to other differentiated progeny.2,20 

Endogenous Lung Stem Cells The postnatal lung contains stem cells capable of reconstituting the lung following injury21 (Table 21.1) and these are predominantly facultative stem cells. These endogenous lung stem cells are limited in their differentiation potential, have potency restricted to lung cell lineages and topographically, are localized to specific anatomic regions and tissue micro-environments.22 Despite attempts to identify a dedicated lung stem cell, there is no clear evidence of such a cell. Instead, it is more likely that there are several niches of multipotent cells with the

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Blastocyst

Zygote Inner cell mass

Totipotent stem cell

Pluripotent stem cell

Multipotent stem cell

Embryo and placenta

Endoderm Mesoderm Ectoderm

Multiple cell types from one germ layer

21

Fig. 21.2  Stem cell potency. Totipotent stem cells (zygote or first cleavage blastomeres) can give rise to all cells in the embryo and placenta. Pluripotent stem cells (e.g., embryonic stem cells) can give rise to the entire embryo and all cell lineages (endoderm, mesoderm, and ectoderm). Multipotent stem cells typically give rise to cells in one cell lineage.

Table 21.1 SUMMARY OF LUNG EPITHELIAL PROGENITOR CELL POPULATIONS Cell Type

Marker Genes

Location

Differentiation Potential

Basal cells

Trp63, Krt5, Krt14, NGFR, Pdpn Scgb1a1, Cyp2f2

Trachea

Self, ciliated, club Unknown

23–26

Bronchiole

Self, ciliated

28,29

Club cells

Variant club cells Scgb1a1, Cyp2f2low BASCs SP-C, Scgb1a1

Near neuroepithelial bodies

Integrin α6β4 alveolar progenitors

Itgα6, Itgβ4

Conducting airways, BADJ, alveolar wall

Distal airway stem cells

Trp63, Krt5, Krt14, NGFR, Pdpn

Distal airways

AEC2

SP-C

Alveoli

Role in BPD

Unknown, killed by naphthalene Self, ciliated, club Unknown but survive naphthalene injury Self, club, ciliated, Unchanged in rodent AEC2 hyperoxia-models of BPD but gives rise to AEC2 cells following bleomycin injury and increases after intratracheal MSC therapy in rodent hyperoxia BPD models Self, club, Unknown but may ciliated, AEC2, contribute to alveoAEC1 lar repair following bleomycin-induced lung injury Self, club, Unknown but migrate ciliated, AEC2, and give rise to AEC1 alveolar cells following severe influenza Self, AEC1 Proliferates in response to hyperoxia and gives rise to AEC1 cells

References

31,32 46–50

44,45

40–42

33–36,37,121

AECA, Alveolar type 1 epithelial cells; AEC2, alveolar type 2 epithelial cells; BADJ, bronchoalveolar duct junction; BASC, bronchoalveolar stem cell; BPD, bronchopulmonary dysplasia, MSC, mesenchymal stem cells; NGFR, nerve growth factor receptor; SP-C, surfactant protein C.

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potential to reenter the cell cycle during injury. It is also conceivable that prematurity and its associated exposures dysregulate signaling pathways that modulate stem cell programming, leading to perturbation of these endogenous niches and dysfunctional repair.

Airway Candidate Stem Cells

B

Within the proximal gas-conducting system (the trachea and main bronchi) is a distinct population of undifferentiated basal cells expressing cytokeratins 5 and 14, transcription factor 63, and nerve growth factor that functions as bronchiolar stem cells.23–26 These basal cells self-renew and give rise to ciliated and club cells (formerly known as Clara cells)27 under steady-state conditions and following sulfur dioxide inhalation injury. Within the distal airways, lineage tracing experiments show that club cells self-renew and differentiate into ciliated cells.28,29 Evidence also points to different club cell populations with variable proliferative capacity and lineage potential.30,31 One club cell subpopulation is resistant to the toxin, naphthalene, which poisons the cytochrome P450 enzyme.31 These cells have been termed “variant club cells” and are located around the neuroendocrine bodies and at the bronchoalveolar duct junction. Variant club cells are quiescent in the steady state; however following naphthalene-induced injury they proliferate quite rapidly.32 The role of these cells in BPD is unknown. 

Alveolar Candidate Stem Cells Within the alveoli, substantial in vivo and in vitro evidence suggest that alveolar type 2 epithelial cells (AEC2) are the progenitors of alveolar type 1 epithelial cells (AEC1).33–36 AEC2 cells play a crucial role in replenishing the AEC1 population under both steady-state and injury conditions, and lineage tracing experiments reveal that AEC2 cells not only self-renew but also give rise to AEC1 progeny.37 In three-dimensional (3D) culture, AEC2 cells form alveolospheres containing cells that express both AEC2 and AEC1 cell markers.37 Emerging data also show different subpopulations of AEC2 cells with variable regenerative potential. In whole lung and primary cultures of adult rat AEC2 cells, there is a hyperoxia-resistant subpopulation of telomerase-positive AEC2 that expands in response to injury.38 AEC2 cells expressing high surface levels of telomerase but low epithelial (E)-cadherin are more proliferative and less likely to undergo hyperoxic damage compared with AEC2 cells that express high levels of E-cadherin.39 More recently, rare populations of undifferentiated basally located distal airway stem cells expressing cytokeratin 5, transcription factor 63, and cytokeratin 14 have also been identified as potential alveolar progenitors.40–42 These cells originate from SOX2+ airway progenitors43 and are inactive under steady-state conditions, but they proliferate and migrate into the alveoli following severe influenza-induced lung injury, differentiating into both airway and alveolar lineages.40–42 Additional populations of multipotent epithelial stem cells that express the laminin receptor α6β4 have also been described.44,45 These cells are localized within the epithelium of the conducting airways and alveoli and have the capacity to give rise to AEC2 cells, suggesting the possibility that these multipotent distal airway stem cells may contribute to alveolar repair. Other candidate alveolar progenitors include rare populations of epithelial cells located at the bronchoalveolar duct junction, so-called bronchoalveolar stem cells (BASCs).46 These cells express both surfactant protein C (SP-C) as well as secretoglobin (Scgb1a1), are resistant to naphthalene, and proliferate rapidly after naphthalene- and bleomycin-induced lung injury47 Although BASCs are unchanged in hyperoxia-induced lung injury48 and their role in lung development is unclear, BASCs are able to give rise to AEC2 cells following bleomycin-induced lung injury, albeit to a minor degree.49,50 In specialized culture media, BASCs give rise to cells that express pro-SP-C, aquaporin 5, as well as Scgb1a1, suggesting that these cells could give rise to cells in the conducting as well as gas exchange portion of the lung. However, more research is needed to elucidate the regenerative capacity of these alveolar progenitor cells in BPD, their interactions with cells in the lung vasculature

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and mesenchymal compartment, and the molecular mechanisms that drive these alveolar progenitors toward quiescence or activation. 

Lung Mesenchymal Stem Cells Originally characterized in 1968 by Friedenstein and colleagues, mesenchymal stem cells (MSCs) were first described as a population of BM stromal cells that were adherent, fibroblastic in appearance, and clonogenic.51 Because there are no specific cell markers for MSCs, the International Society for Cellular Therapy established minimal criteria for defining MSCs: (1) adherence to plastic under standard tissue culture conditions; (2) expression of cell surface markers, CD105, CD90, and CD73, and no expression for HLA-DR, CD79α, CD45, CD34, CD14, CD19, and CD11b; and (3) the capacity to differentiate into osteoblasts, chondroblasts, and adipocytes under appropriate in vitro conditions.52 Although these criteria were mainly used to describe BM-derived MSC, emerging data now suggest that most organs have endogenous MSC populations, albeit with varying functionality, differentiation potential, and phenotype, depending on micro-environmental cues.53,54 MSCs have been isolated from fetal and adult lungs based on their adherence to plastic,55 expression of adenosine-binding cassette G (ABCG2),56 Hoechst 33342 dye efflux,57 and their osteogenic, chondrogenic, and adipogenic differentiation potential.56,58 Compared with BM-MSCs, lung-derived MSCs seem to be constitutively more prone to epithelial differentiation59 and have a distinct pattern of Hox gene expression implicated in lung development.60 Although lung and BM-MSCs reduce elastase injury to the same extent,61 lung MSCs express more intercellular adhesion molecule 1 (ICAM-1), platelet-derived growth factor receptor alpha (PDGFRα), and integrin α2 than BM-MSCs,61 conferring potential differences in MSC adherence, migration, and invasion. Moreover, lung MSCs are now believed to be a part of a diverse population of mesenchymal progenitors62 directly involved in regulating the growth and function of epithelial progenitor cells.63 The role of lung MSCs in BPD is still being clarified. Lung MSCs are decreased in the lungs of newborn rodents exposed to hyperoxia,64 yet increased MSCs in the tracheal aspirate of preterm infants predicts an increased risk for BPD by more than 25-fold.65 Moreover, MSCs isolated from the lungs of preterm infants differentiate into myofibroblasts under the influence of the pro-fibrotic factor, transforming growth factor beta.66 Similarly, MSCs isolated from human lung allografts undergo pro-fibrotic differentiation in response to cytokines associated with bronchiolitis obliterans,67 but a significant decrease in lung MSCs is evident in the lungs of rodents with bleomycin-induced pulmonary fibrosis and replacement of lung MSCs attenuates fibrosis.68 One potential explanation for these apparent contradictory findings is the presence of different subpopulations of lung MSCs with variable epithelial-­ supportive capacity and myofibroblastic differentiation potential.69 Conceivably, depletion of lung MSCs may interfere with the endogenous regenerative response; conversely, lung microenvironmental cues may influence MSC behavior and function, potentially driving them toward a more dysfunctional fibrotic phenotype. 

Lung Endothelial Progenitors Endothelial progenitors (EPCs) were first described by Asahara et al. in 1997.70 This group described a population of peripheral blood mononuclear cells that could differentiate into endothelial cells. These cells expressed the hematopoietic stem marker, CD34, as well as the endothelial cell marker, vascular endothelial growth factor receptor 2 (VEGFR2), and were shown to contribute to physiologic and pathologic neovascularization.70 Since then various different markers have been used to identify this population, and although still marred with controversy, current data suggest that there is a hierarchy of EPCs based on their proliferative potential.71 One subset appears early in culture, displays endothelial markers, but does not form vessels in vivo.72 These cells exert mainly paracrine effects on endothelial cells. Another subset, termed endothelial colony-forming cells (ECFCs),71 gives rise to colonies 7 to 21 days after plating, has robust proliferative potential, and forms vessels when

21

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transplanted in vivo.71 These cells can not only be isolated from umbilical cord and aorta vessel walls,73 but recent evidence also points to similar cells within the pulmonary microvasculature.74 This is intriguing given the importance of angiogenesis in lung development and repair. Indeed, prior studies have linked circulating EPC quantity with adult lung disease outcomes. Patients with end-stage chronic lung disease have reduced circulating EPCs75 and patients with idiopathic pulmonary fibrosis showed a marked EPC depletion, particularly when pulmonary hypertension is evident.76 Interestingly, ECFCs isolated from neonatal rats with hyperoxia-induced BPD-like lung injury proliferate less and form fewer capillary-like networks, suggesting that functional deficiency in ECFCs may contribute to BPD pathogenesis.77 This is further supported by evidence of decreased and dysfunctional cord ECFCs in preterm infants in whom BPD subsequently develops.78,79 

B

Cell Therapies for Bronchopulmonary Dysplasia Evidence from Preclinical Studies The recent insights into stem cell biology have unraveled the therapeutic potential of stem cells. Specifically, MSCs have attracted much attention because of their ease of isolation, culture, and expansion; allogeneic use; and pleiotropic therapeutic effects. The latter is of particular interest for a multifactorial disease such as BPD, in which multiple pathophysiologic mechanisms contribute to impaired alveolar and lung vascular development. Early proof-of-concept studies in neonatal rodents exposed to hyperoxia showed that rodent BM-MSCs administered intravenously (IV) or intratracheally (IT) prevent the arrest in lung vascular and alveolar growth in this model (Fig. 21.3).64,80 An interesting source of MSCs, especially when treating neonatal diseases, is represented by perinatal tissue. The placenta and umbilical cord are usually discarded after birth, yet they contain a large number of various cells, including MSCs, with therapeutic potential and can be harvested without harm to the mother or the newborn. After the above-mentioned proof-of-concept studies, MSCs derived from the umbilical cord (UC) Wharton jelly and from the umbilical cord blood (UCB) have been studied extensively in experimental BPD models. Similar to the therapeutic

MSC

Normoxia

Placebo

100 µm

100 µm

100 µm

Hyperoxia

100 µm

Fig. 21.3  Mesenchymal stem cells (MSCs) improve hyperoxia-induced alveolar simplification. The lung sections demonstrate improved alveolar structure following administration of MSCs to neonatal rats with hyperoxia-induced lung injury.

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benefit reported for BM-MSCs, UC- and UCB-derived MSCs demonstrated comparable effects in hyperoxia-induced neonatal lung injury in rodents81–85: improved alveolar and lung vascular structure and even restoration of alveolar growth after established lung injury, attenuation of lung fibrosis, reduced lung inflammation, and improved exercise capacity. The therapeutic benefit persisted into adulthood with no evidence of tumor formation or adverse effects on lung architecture.81,85 Since then numerous investigators have attempted to optimize the use of MSCs by exploring the best timing, dose, and route of administration as outlined further in the following text. A consistent finding, however, was the low rate of lung engraftment of MSCs. Increasing evidence suggests that MSCs act via a paracrine mechanism to protect the developing lung from injury.86 Table 21.2 provides a summary of the preclinical evidence. 

Mechanisms of Repair Similar observation of low organ engraftment of transplanted MSCs had already been observed in the heart and the central nervous system. The working hypothesis became that MSCs release bioactive molecules that modulate organ repair. Proofof-concept experiments in vitro showed that cell-free conditioned media derived from MSCs prevent hyperoxia-induced alveolar epithelial cell apoptosis, accelerate alveolar epithelial cell wound healing, and preserve endothelial cord formation on Matrigel Matrix (Corning, Oneonta, New York) during hyperoxia.64 This paracrine effect was then confirmed in vivo. The efficacy of intraperitoneal administration of MSCs in protecting the neonatal rodent lung already suggested some paracrine activity of these cells.82,87 Direct in vivo evidence was provided by studies in which a single IV injection of cell-free MSC-derived conditioned media improved lung function, alveolar injury, and pulmonary hypertension.80,88 A single IT injection of BM-MSCs or BM-MSC-free conditioned media protected from oxygen-­induced alveolar and vascular injury with a persistent benefit up to 3 months.89 Likewise, cell-free conditioned media derived from human UCB-MSCs injected daily intraperitoneally prevented and reversed arrested alveolar growth and lung function in hyperoxia-exposed rats with persistent safety and therapeutic benefit up to 6 months.81 Thus rather than replacing injured cells and differentiating into lung cells, MSCs release factors that protect resident lung cells from injury or modulate the function of inflammatory cells. For example, BM-MSCs and BM-MSC-derived conditioned media increase the number of BASCs in neonatal mice exposed to hyperoxia.90 In addition, MSCs can modulate the phenotype of macrophages from a M1 (proinflammatory) to an M2 (healer) phenotype in various disease conditions,91,92 but this has not yet been investigated in neonates. Also, the interactions between MSCs and other immune cells remain unexplored in neonates. The identification of soluble factors in the conditioned media may allow the discovery of novel repair mechanisms and therapeutic interventions.93 Likewise, the variety of factors released by MSCs explains their pleiotropic effects and further underscores the rationale for MSC therapy for BPD. MSCs produce factors known to promote lung growth and repair such as keratinocyte growth factors,94 vascular endothelial growth factor,95,96 or adiponectin.97 Novel molecules secreted by MSCs have already been identified and have shown therapeutic benefit in various disease models, such as stanniocalcin-198 (a potent antioxidant) or tumor necrosis factor α (TNF-α)–stimulated gene/protein 6 (TSG-6)99 (a potent antiinflammatory protein). These discoveries open interesting therapeutic avenues into manufacturing superior therapeutic cells. For example, preconditioning of BM-MSCs ex vivo in hyperoxia for 24 hours enhanced the release of stanniocalcin-1 in the conditioned media and boosted the therapeutic benefit of preconditioned MSCs on lung architecture in hyperoxic neonatal rodents compared with non-preconditioned media.100 The recognition of the paracrine effect of MSCs has also renewed the excitement regarding extracellular vesicles, which are crucial for cell-to-cell communication. In particular, MSCs, like many other cells, release membrane-derived nanosized

21

5 × 104

1 × 105

5 × 103 5 × 104 5 × 105 2 × 106

Mouse BM MSCs or CM

Rat BM MSCs

Human UCB MSCs

5 × 105 (IT) 2 × 106 (IV)

0.1 × 106 0.5 × 106 1 × 106

5 × 105

3 ×105 (early) 6 × 105 (late)

Human UCB MSCs

Human UCT MSCs

Human UCB MSCs

Human cord PCs or UCB MSCs

P4 (early) P14 (late)

P3 (early) P10 (late) P3 + P10

P5

P5

P7

P9

P5

P4 (prevention) P14 (treatment)

Postnatal day (P)4

Neonatal rats in hyperoxia P4–P14; evaluated at P22 (prevention) or P35 (regeneration)

Newborn SCID mice in hyperoxia Birth–P7; evaluated 8 weeks after MSC transplantation Neonatal rats in hyperoxia 90% for 2 weeks and 60% for 1 week

Neonatal rats in hyperoxia Birth–P14

Neonatal rats in hyperoxia P2–P21

Neonatal rats in hyperoxia P2–P16; evaluated at P16, P30, and P100

Neonatal rats in hyperoxia Birth–P14

Neonatal rats in hyperoxia P1–P14, studied on P21 (prevention) or P45 (treatment)

Neonatal rats in hyperoxia P1–P14

Timing of Administration Model

IT

IT

Intranasally or IP

IT or IV

IT

IT

IT

Intratracheal (IT)

Intravenous (IV)

Route of Delivery

High-dose IP MSCs restore lung compliance, elastance, and pressure-volume loops. Intranasal or low-dose MSCs have no significant effects More pronounced improvement in alveolar structure, oxidative stress, inflammation with early compared with late MSCs Both early and late MSCs and PCs improved alveolar structure and lung function. No tumors evident at 6 months

CM have more pronounced improvement in alveolar and vascular structure than MSCs Early but not late MSC administration preserves lung architecture and survival. Both early and late administration improve exercise capacity Higher-dose MSCs have more pronounced effects on alveolar structure MSC and CM similarly improve lung architecture at P16 and P30; however, MSCs have more pronounced effects than CM at P100 Improved alveolar structure, increased vascular density, reduced pulmonary hypertension (PH) and remodeling IT administration improves body weight and is better than IV in improving alveolar structure

Effects of Therapy

Paracrine-mediated

Paracrine-mediated

Female MSCs have greater antiinflammatory and pro-angiogenic effects than male MSCs IT administration has greater MSC retention and is better than IV in reducing inflammation and apoptosis Paracrine-mediated

Modulation of host inflammatory and antioxidant responses Reduced inflammation

Paracrine-mediated

Paracrine-mediated

Suggested Mechanisms of Repair

116

84

124

118

104

89

83

123

122

References

BM, Bone marrow; CM, conditioned medium; IP, intraperitoneal; MSC, mesenchymal stem cell; PC, perivascular cell; SCID, severe combined immunodeficiency syndrome; UCB, umbilical cord blood; UCT, umbilical cord tissue.

1 × 106

Male and female rat BM MSCs

Male rat BM MSCs or CM

Cell Dose

B

Cell Population

Table 21.2 MESENCHYMAL STEM CELLS FOR BRONCHOPULMONARY DYSPLASIA: SUMMARY OF PRECLINICAL EVIDENCE

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exosomes, acting as cargoes that contain not only the combination of bioactive molecules but also microRNA (miRNA).101 miRNAs are small noncoding RNA molecules involved in transcriptional regulation of gene expression, and this may explain the long-term effect of single injections of MSC-derived conditioned media. miRNA may represent interesting therapeutic targets in the prevention of BPD through activation or silencing of specific genes with beneficial or deleterious effects on lung development.102 In adult lung injury models, MSC-derived exosomes attenuate lung macrophage influx, decrease proinflammatory cytokine levels in the bronchoalveolar lavage, and prevent pulmonary vascular remodeling and hypoxic-induced pulmonary hypertension in mice.103 The therapeutic benefit of exosomes is currently being investigated in neonatal lung injury models. The impact of gender in MSC efficacy is also being explored. Female BM-MSCs secrete more pro-angiogenic and antiinflammatory factors than male BM-MSCs.104 In neonatal rats with hyperoxia-induced lung injury, female BM-MSCs are superior to male BM-MSCs in reducing lung inflammation, improving pulmonary hypertension, and attenuating vascular remodeling.104 These beneficial effects are particularly more pronounced in male recipients, suggesting that female cells104 may be a potent BM-MSC population for BPD complicated by severe pulmonary hypertension.

From Bench to Bedside: Evidence from a Phase I Clinical Trial The above-mentioned preclinical evidence supporting the role of MSC transplantation as a potential therapy for BPD without short- and long-term toxicity or tumorigenicity81,82,84,85,105 provided the foundation for the first clinical trial in preterm newborns.106 This study was an open-label, single-center clinical trial that assessed the safety and feasibility of a single IT transplantation of allogenic human UCBderived MSCs for BPD. MSCs were transplanted into 9 very preterm infants at high risk for developing BPD (mean gestational age 25.3 ± 0.9 weeks, range 24–26 weeks; mean birth weight 793 ± 127 g, range 630–1030 g) at a mean age of 10.4 ± 2.6 days (range 7–14 days) after birth. All of the infants were receiving ventilator support, the settings of which could not be decreased owing to significant respiratory distress within 24 hours before enrollment. The first three patients received a low dose of MSCs (1 × 107 cells/kg in 2 mL/kg of saline solution), while the remaining six patients received a high dose (2 × 107 cells/kg in 4 mL/kg of saline solution). The MSCs were administered IT into the left and right lungs in two fractions via a feeding tube, the same method used to administer surfactant. The treatment was well tolerated without any serious adverse effects or dose-limiting toxicity up to 84 days following transplantation. The incidence of serious adverse effects did not significantly differ in the low- and high-dose groups. The levels of interleukin 6 (IL-6), IL-8, matrix metalloproteinase-9, TNF-α, and transforming growth factor beta 1 in the tracheal aspirates collected on day 7 were significantly reduced compared with baseline and day 3 posttransplantation levels. Furthermore, compared with a historical control group matched for gestational age, birth weight, and respiratory severity score, BPD severity was significantly lower in transplant recipients. Overall, these findings suggest that IT allogenic human UCB-derived MSC transplantation in very preterm infants at high risk for developing BPD is safe and feasible. Long-term follow-up studies extending up to 2 years (clinical trial No. NCT01632475) and 5 years of age (clinical trial No. NCT02023788) are currently underway to assess the long-term safety of MSC treatment in the subjects of these phase I trials. Furthermore, a phase II double-blind, randomized, multicenter, controlled clinical trial investigating the efficacy and safety of low-dose MSC transplantation compared with a control group for the treatment of BPD is also in progress (clinical trial No. NCT0182957). Another study following these phase II clinical trial patients to 5 years of age has also begun with the goal of gathering further long-term safety and efficacy data (clinical trial No. NCT01897987). Favorable results from these current clinical trials are greatly anticipated and are expected to pave the way for future clinical translation of stem cell therapies for BPD. 

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Barriers to Implementation Clinical introduction of cell-based therapies could be a paradigm shift in neonatal medicine and in the treatment of BPD. However, barriers to clinical implementation, including optimization of the cells delivered to the right patients via the right route, at the right time, and right dose need to be overcome for the success of future clinical translation.106–113 

Optimal Cells B

Determining the optimal cells for transplantation is the most critical issue for successful clinical translation. MSCs have been extensively investigated, and their therapeutic efficacy has consistently been proven in BPD animal models.64,82,114–116 Because donor cells do not engraft,117 allogenic transplantation of MSCs is considered safe. Moreover, considering the time-consuming and costly isolation and expansion processes involved in the generation of autologous MSCs, allogenic therapy might have a logistic advantage for its ability to be used off the shelf in a clinical setting. MSCs are broadly distributed in the body; UCB-derived MSCs exhibited better efficacy in attenuating hyperoxic lung injuries and greater paracrine potency compared with fat-derived MSCs in newborn rats.115 Production of a high-quality, standardized, clinical-grade product using good manufacturing practice (GMP) criteria is another important factor in the success of MSCs in a clinical setting.111 Based on present evidence, both the Korean Ministry of Food and Drug Safety and the United States Food and Drug Administration approved the use of allogenic human UCB-derived MSCs manufactured with strict GMP compliance for use in phase I/II BPD clinical trials conducted in Korea106 and the United States (clinical trial No. NCT02381366). Standardization of the isolation, expansion, and production of these cells in GMP facilities would be necessary for the introduction of other cell types, such as endothelial progenitor cells and amnion epithelial cells, as well as cell-derived products such as exosomes, showing promising effects in BPD models. Right Patient Despite its potential benefits, cautious risk/benefit evaluations must be made before using MSC transplantation in the most vulnerable preterm infants. Therefore identification of the preterm infants at highest risk for BPD is a very important step in the application of these potentially beneficial rescue/preventive strategies. Because early gestational age and prolonged respiratory support must be cited as the most important predictors of BPD development among extremely preterm infants,113 infants of 24 to 2 weeks’ gestation requiring continuous ventilator support were enrolled in a phase I clinical trial conducted by Chang et al.106 However, the clinical courses of these infants may vary widely and not all progress to development of BPD. Additional clinical predictors and/or biomarkers112 will be necessary to further stratify these infants and determine who will ultimately progress to BPD, thus avoiding unnecessary treatment exposure.  Right Time Even though the therapeutic efficacy of MSC transplantation in the prevention and rescue of experimental BPD has already been shown, the optimal timing of administration is a key issue that remains to be clarified. Pierro et al.116 reported that although the long-term benefits of the rescue approach have not been investigated, MSCs or their conditioning media were not only effective for prevention at P4, their use at P14 as a rescue approach both prevented and rescued hyperoxic alveolar growth arrest. Chang and colleagues compared the therapeutic efficacy of early versus late MSC administration at P3 or P10 as well as at both of these time points.84 Their study found significant attenuation of hyperoxic lung injuries only with early but not with late MSC transplantation, and combined early and late transplantation did not have a synergistic effect. In a study by van Haaften et al.,64 MSC transplantation at P4 for prevention significantly attenuated neonatal hyperoxic lung injuries, whereas transplantation at P14 for regeneration showed no effect. Taken together, the therapeutic

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time window for MSC transplantation in the treatment of BPD appears to be narrow. Thus in a clinical setting, early identification of the highest-risk preterm infants and administration of MSCs within the first few days of life seem preferable to later administration in infants with established BPD.  Right Route Several routes, including local IT instillation,64,82 systemic IV injection80,118 and intraperitoneal administration,82 have been tested to determine the optimal route of MSC transplantation. As injured lung tissue produces chemotactic factors, systemically transplanted MSCs migrate119 and localize to the injured lung.120 The most convenient and minimally invasive systemic IV approach might be more therapeutically advantageous compared with a local IT approach, especially in very unstable preterm infants with BPD. Most preterm infants at high risk of BPD are intubated and receive ventilator support, and since MSCs can be instilled in the same way as surfactant, IT MSC transplantation during the first days of life might not be so invasive in the extremely preterm infants.106 Furthermore, despite a fourfold lower dose and more frequent donor cell engraftment, better paracrine potency and attenuation of hyperoxic lung injuries were observed with local IT MSC transplantation than with systemic administration.82,118 Based on superior therapeutic efficacy observed with local IT compared with systemic IV or intraperitoneal transplantation as demonstrated in these preclinical data, MSCs were delivered IT in the first phase I clinical trial.106  Right Dose Because previous studies82,118 have shown that the MSC therapeutic dose could be reduced more than fourfold by choosing a local IT route over systemic IV or intraperitoneal administration, the route of MSC administration might be a major determinant of optimal dosing. Chang et al.105 compared the therapeutic efficacy of three different doses of human UCB-derived MSCs (5 × 103, 5 × 104, and 5 × 105 cells per animal, reflecting 0.5 to 50 × 106 cells per kg body weight) given IT to hyperoxic newborn rat pups at P5. The best therapeutic effects were seen in the mid- and highdose treatment groups. Based on this evidence, a single IT administration of 1 × 107 cells/kg (low-dose, 3 infants) and 2 × 107 cells (high-dose, 6 infants) were used in the phase I clinical trial; no dose-limiting toxicity or serious adverse events were observed with either dose.106 In light of these findings, further preclinical and clinical studies examining the optimal MSC dose of clinical benefit to human preterm neonates with BPD are anticipated. 

Conclusion In recent years, various translational studies have broadened the knowledge and understanding of stem cell therapy in neonatal lung injury, and clinical trials are harnessing the therapeutic potential of stem cell therapies for BPD. The exciting progress in both preclinical and clinical research has brought human stem cell BPD therapy one step closer to clinical translation. However, a better understanding of the potential protective mechanism of stem cells in BPD and the resolution of several issues, such as manufacturing processes, clinical indication, timing, dose, and modes of action, are required to permit safe clinical translation of stem cell therapy for this disorder. It will be essential to proceed sequentially rather than in haste with relentless efforts to overcome all obstacles in making stem cell therapy the next treatment breakthrough for BPD. REFERENCES









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