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Keywords:

  • acute lung injury;
  • cell-based therapy;
  • endothelial progenitor cell;
  • mesenchymal stem cell;
  • paracrine soluble factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

Acute lung injury (ALI) or acute respiratory distress syndrome remains a major cause of morbidity and mortality in hospitalized patients. The pathophysiology of ALI involves complex interactions between the inciting event, such as pneumonia, sepsis or aspiration, and the host immune response resulting in lung protein permeability, impaired resolution of pulmonary oedema, an intense inflammatory response in the injured alveolus and hypoxemia. In multiple preclinical studies, adult stem cells have been shown to be therapeutic due to both the ability to mitigate injury and inflammation through paracrine mechanisms and perhaps to regenerate tissue by virtue of their multi-potency. These characteristics have stimulated intensive research efforts to explore the possibility of using stem or progenitor cells for the treatment of lung injury. A variety of stem or progenitor cells have been isolated, characterized and tested experimentally in preclinical animal models of ALI. However, questions remain concerning the optimal dose, route and the adult stem or progenitor cell to use. Here, the current mechanisms underlying the therapeutic effect of stem cells in ALI as well as the questions that will arise as clinical trials for ALI are planned are reviewed.


Abbreviations
ALI

acute lung injury

ARDS

acute respiratory distress syndrome

BM

bone marrow

EPC

endothelial progenitor cells

HSC

haematopoietic stem cells

KGF

keratinocyte growth factor

LPS

lipopolysaccharides;

MSC

mesenchymal stem cells

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are the most common causes of acute respiratory failure in hospitalized patients. Despite extensive research into the pathophysiology, the mortality associated with ALI/ARDS has remained up to 40%, depending on the aetiology.[1, 2] Current treatment is primarily supportive with lung-protective ventilation and a fluid conservative strategy.[3, 4] Pharmacological therapies that reduce the severity of lung injury in preclinical models have not yet been translated to effective clinical treatment options. Earlier concepts of distinct disease phases underlying the pathophysiology of ALI/ARDS, from an early pro-inflammatory to a later pro-fibrotic phase now appear to be an oversimplification.[5] Consequently, strategies that targeted one aspect of the disease process have not been successful in limiting overall morbidity and mortality. Innovative therapies are needed.

Stem cells are undifferentiated precursor cells with the capacity for self-renewal and the ability to differentiate into cells of multiple lineages. They can be broadly classified by their potency (pluri-potent vs multi-potent) and origin (adult vs embryonic). Adult stem cells are multi-potent postnatal stem cells that remain in body tissues throughout life and have the potential to differentiate into a more limited range of mature cell types. In general, they include haematopoietic stem cells (HSC), mesenchymal stem cells (MSC), endothelial progenitor cells (EPC) and organ-specific stem cells such as endogenous lung stem cells. In 1998, Ferrari et al.[6] reported in a landmark manuscript that the transplantation of bone marrow-derived adult stem cells into injured muscle tissue mitigated damage and regenerated healthy muscle fibres, generating tremendous enthusiasm for stem cell-based therapy.

Much of the initial interest in stem cell-based therapy for lung injury arose initially from the multi-potent properties of the cells. Krause et al.[7] reported up to 20% engraftment of bone marrow (BM)-derived cells in the lung, including epithelial cells, from a single haematopoietic precursor. However, subsequent studies suggested that the major therapeutic effect of adult stem cells in ALI were primarily from their ability to secrete paracrine factors such as growth factors, factors regulating endothelial and epithelial permeability, anti-inflammatory cytokines and antimicrobial peptides/proteins, not from significant engraftment. To date, due to their ease of isolation and extensive preclinical studies, MSC may offer the best hope for clinical trial. However, other progenitor and adult stem cells[5, 8-11] have shown some promise as potential therapeutic candidates for ALI/ARDS as well.

Despite these encouraging results, several issues remain which must be addressed.[12] (i) The isolation and classification of stem cells need to be defined further, particularly concerning the issue of potency; (ii) a more precise understanding of the mechanisms underlying the therapeutic effect is needed, such as the role of the constituents of the conditioned medium, of cell-contact-dependent or independent effects, and whether the phenotype of the cells changes depending on the alveolar milieu; (iii) the optimal dose and route of cell delivery remains to be determined; (iv) although most of the current focus is on the use of MSC, we still need to determine the optimal ‘stem cells’ for cell-based therapy and (v) despite promising preclinical results and public enthusiasm, clinician-scientists involved in the translation of stem cell research into clinical trials must always keep in mind the lessons learned from the field of gene therapy.[13] Above all, we must do no harm.

This review will present the most recent advances in the field of adult stem cell therapy for ALI, focusing specifically on MSC and EPC, and to issues that will arise in preparation for clinical trial. To accomplish this goal, we searched PubMed for relevant studies published up to Dec 2012. We also searched the proceedings of major relevant conferences, trial databases, the reference lists of identified trials and major reviews. We identified 192 studies from electronic databases, of which 36 studies were eligible for inclusion, based on the title and abstract. After independent assessment of the full text, 29 articles were finally considered to be eligible for inclusion in the analyses after we contacted authors for additional data. Figure 1 shows the search leading to the selection of the final 29 articles.

figure

Figure 1. Research strategy for selection of mesenchymal stem cells preclinical studies.

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Mesenchymal Stem Cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

Mesenchymal stem or stromal cells are adult non-haematopoietic precursor cells derived from a variety of tissues such as the BM, adipose tissue and placenta which have been used as therapy in multiple diseases and syndromes such as myocardial infarction, renal failure and graft-versus-host disease. The International Society of Cellular Therapy has defined MSC in 2006 by three criteria: (i) MSC must be adherent to plastic under standard tissue culture conditions; (ii) MSC must express certain cell surface markers such as CD73, CD90 and CD105, but must not express other markers including CD45, CD34, CD14 or CD11b; and (iii) MSC must have the capacity to differentiate into mesenchymal lineages including osteoblasts, adipocytes and chrondoblasts under in vitro conditions.[14]

Mechanisms of action

Although the precise mechanisms of action of MSC remain unclear, a number of important insights from recent preclinical studies have emerged[10] (Fig. 2).

figure

Figure 2. Potential therapeutic mechanisms of mesenchymal stem cells (MSC) in lung injury. The therapeutic response of MSC in lung injury appears to be primarily based on immunomodulation of inflammatory cells such as alveolar macrophages or immune cells like CD4+CD25+forkhead box P3+ regulatory T cells (CD4+CD25+foxp3+ Treg cells), suppression of lung protein permeability, improvement in alveolar fluid clearance and the secretion of antimicrobial peptides/proteins. Despite the low engraftment rate, however, specific subpopulation of modified MSC may increase lung engraftment and/or regeneration in vivo. Ang-1, angiopoietin-1; CCSP, Clara cell secretory protein; CFTR, cystic fibrosis trans-membrane conductance regulator; ENaC, epithelial sodium channel; IL-10, interleukin-10; KGF, keratinocyte growth factor; MSC, mesenchymal stem cells; PGE2, prostaglandin E2; MIP-2, macrophage inflammatory protein 2; SP-C, surfactant protein-C; TSG-6, tumour necrosis factor-inducible gene 6; VCAM-1, vascular cell adhesion molecule 1.

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Engraftment

Despite initial interest in MSC's multi-potent properties, engraftment in the lung now does not appear to play a major beneficial role.[15-17] This perception was initially based on earlier studies that demonstrated that intravenous administration of MSC resulted in high pulmonary engraftment because MSC expressed adhesion molecules, such as vascular cell adhesion molecule 1 and P-selectin, which facilitated their retention in the lung[18] and which was enhanced by the presence of hyaluronan degradation products in the inflamed tissue.[19, 20] These results were subsequently questioned by multiple groups, who observed only engraftment of leucocyte lineages[15] or low engraftment rates in lung injury models with observed rates of <1%.[16, 17] Yet, several new publications[21-23] have highlighted the potential of in vitro modification of MSC, which may increase lung engraftment and/or regeneration. These authors found specific subpopulations of MSC which expressed functional markers of lung epithelial cells such as Clara cell secretory protein, surfactant protein-C, cystic fibrosis trans-membrane conductance regulator and epithelial sodium channel when cultured in mouse tracheal epithelial cell medium, small airway growth medium or with keratinocyte growth factor (KGF) and retinoic acid respectively. In addition, several groups[24-26] have identified adult stem cells within the lung, some with the features of MSC. Tropea et al.[27] demonstrated that systemic treatment with MSC or MSC condition medium had a direct effect on stimulating bronchioalveolar stem cells to repair and restore injured lung epithelium.

Immunomodulation

Multiple studies have demonstrated that MSC possess potent immunosuppressive effects by inhibiting the activity of both innate and adaptive immune cells.[28-31] This immunosuppression has shown to be mediated by cell-contact-dependent and independent mechanisms through the release of soluble factors such as tumour necrosis factor-inducible gene 6,[32] prostaglandin E2,[33] interleukin-10[33, 34] and interleukin-1 receptor antagonist[35] among others. For example, we[36] and other investigators[37-39] have demonstrated that MSC may switch the phenotype of alveolar macrophages from a M1 (inflammatory) to a M2 (anti-inflammatory). Also, in an endotoxin-induced ALI model treated with umbilical cord-derived MSC, Sun et al.[40] found that MSC suppressed lung injury by upregulating CD4+CD25+forkhead box P3+ regulatory T cells, reduced the levels of the pro-inflammatory cytokines Interferon-γ, macrophage inflammatory protein 2 and tumour necrosis factor-α and increased the anti-inflammatory cytokine, interleukin-10. Treg cells play a central role in the prevention of autoimmunity and in the control of immune responses by downregulating the function of effector CD4+ or CD8+ T cells;[41-43] these findings are consistent with other reports[28, 36] that MSC can decrease T-cell responses by shifting from a T-helper1- to T-helper2-type response.

Alveolar fluid clearance

We and other investigators have reported that ALI pulmonary oedema fluid contained high levels of pro-inflammatory cytokines which downregulated alveolar fluid clearance (i.e. the resolution of pulmonary oedema).[44, 45] Interestingly, MSC are known to produce several epithelial specific growth factors, such as KGF, the seventh member of the fibroblast growth factor family, which are known to increase alveolar fluid clearance. We[46] and other researchers[38, 47, 48] have reported that KGF can reduce lung injury in animal and human models of pulmonary oedema whether from direct, indirect or infectious causes. KGF improved alveolar fluid clearance in part by upregulating α epithelial sodium channel gene expression and Na-K-ATPase activity or through increased trafficking of sodium transport proteins to the cell surface.[46, 49-51]

Lung permeability

The integrity of the lung microvascular endothelium is essential to prevent the influx of protein-rich fluid and inflammatory cells from the plasma, which may aggravate the ability of the lung epithelium to reduce pulmonary oedema. Several MSC paracrine soluble factors, such as angiopoietin-1, are potentially important in these effects. angiopoietin-1, a ligand for the endothelial Tie2 receptor, is a known endothelial survival and vascular stabilization factor that reduces endothelial permeability and inhibits leucocyte-endothelium interactions by modifying cell adhesion molecules and cell junctions.[52, 53] We and other investigators have found that allogeneic human MSC secreted a significant amount of angiopoietin-1, which was essential to prevent the increase in lung protein permeability.[54-56]

Antimicrobial properties

MSC have toll-like and formyl peptide-like receptors and become activated in response to different bacterial products, suggesting that MSC may be directly involved in the innate immune response.[36, 57] Recently, we found that human MSC can inhibit bacterial growth directly in part through the secretion of antimicrobial peptides, such as LL-37, which was upregulated upon bacterial stimulation,[58] and antibacterial proteins, such as lipocalin-2, which improved bacterial clearance in a mouse model of Escherichia coli pneumonia.[59] Mei et al.[60] also recently showed that the improvement in bacterial clearance in MSC-treated septic mice following caecal ligation and puncture could be in part explained by enhanced phagocytic activity of host immune cells.

Recent Experimental Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

To date, MSC have been studied extensively in preclinical lung injury models (Table 1). Most of the injury[32, 36-38, 40, 46, 55, 56, 64-69] (48%, 14 of 29) involved intra-tracheal endotoxin exposure. Rodents were the predominant small animals studied. Over 58% (17 of 29) of studies[32, 35, 36, 38-40, 47, 55, 56, 58, 59, 61, 63-66, 69] used mice while 38% (11 of 29) used rats.[34, 37, 48, 62, 67, 68, 70-74] Interestingly, Lee et al.[46] developed an E. coli endotoxin-induced ALI model in an ex vivo human lung preparation perfused partially with whole blood. Although the preparation was short term (4 h) and did not include the perfusion of other systemic organs such as the liver or spleen, which may mount a significant inflammatory response, this human model replicated most of the early clinical features of patients with ALI.

Table 1. Descriptive details of mesenchymal stem cell preclinical study
StudyLung injury modelSpeciesOriginRouteDosage (×106 cells/kg)Given time after exposureMeasured time pointsTherapeutic effectPossible mechanism
  1. a

    Result is present in total cell amount because no weight is available. Ang-1, angiopoietin-1; ALI, acute lung injury; BM, bone marrow; CCL2, chemokine (C-C motif) ligand 2; CD4+CD25+foxp3+ Treg, CD4+CD25+forkhead box P3+ regulatory T cells; HIMF, hypoxia-induced mitogenic factor; HO-1, heme oxygenase-1; i.t., intra-tracheal; i.v., intravenous; ICAM-1, intercellular adhesion molecule 1; ICR, imprinting control region, IGF-I, insulin-like growth factor I; IL-10, interleukin-10; IL-1RN, interleukin-1 receptor antagonist; IPO, ischaemic post-conditioning; I/R, ischaemia reperfusion; KGF, keratinocyte growth factor; LL-37, human cathelicidin; LPS, lipopolysaccharides; MDA, malondialdehyde; SD, Sprague Dawley; SDF-1, stromal cell-derived factor-1; TSG-6, tumour necrosis factor-inducible gene 6; VCAM-1, vascular cell adhesion molecule 1; VE, vascular endothelial; VEGF, vascular endothelial growth factor.

Rojas et al. 2005[61]Bleomycin-induced lung injuryC57BL/6Bone marrowi.v.22.26 hDay 14+Engraftment
Paracrine soluble factor
Ortiz et al. 2007[35]Bleomycin-induced lung injuryC57BL/6Bone marrowi.v.22.20 hDay 3+IL-1RN
Day 7++
Day 14+
Zhao et al. 2008[62]Bleomycin-induced lung injurySD RatBone marrowi.v.22.212 hDay 14+Engraftment
Aguilar et al. 2009[47]Bleomycin-induced lung injuryC57BL/6Bone marrowi.v.20 (40 total)8 hDay 14+KGF
72 h
Saito et al. 2011[63]Bleomycin-induced lung injuryC57BL/6Bone marrowi.v.22.224 hDay 2CCL2
Day 3+
Day 7+
 Day 14+
Mei et al. 2007[55]LPS-induced ALIC57BL/6Bone marrowi.v.12.60.5 h15 minAng-1
Day 3+
Gupta et al. 2007[36]LPS-induced ALIC57BL/6Bone marrowi.t.33.34 h24 h+Paracrine soluble factor
48 h++
Xu et al. 2008[56]LPS-induced ALIC57BL/6Bone marrowi.v.44.42 hDay 3Ang-1
Day 7+
Day 14+
Lee et al. 2009[46]LPS-induced ALIHuman LungAllogeneic human BMIntrapulmonary5 × 106 cellsa1 h4 h+KGF
Araújo et al. 2010[64]LPS-induced ALIC57BL/6Bone marrowi.v.88.96 hDay 1+Engraftment
Day 7+Paracrine soluble factor
Prota et al. 2010[65]LPS-induced ALIC57BL/6Bone marrowi.v.888.91 hDay 28+Alveolar-capillary membrane repair
Sun et al. 2011[40]LPS-induced ALIBALB/CHuman umbilical cordi.t.46.54 hDay 1CD4+CD25+ foxp3+ Treg
Day 2+
Day 3+
Day 7
Liang et al. 2011[37]LPS-induced ALIWistar RatBone marrowi.v.7.12 h6 hEngraftment
24 h+Paracrine soluble factors
Day 4++
Day 7+
Day 21
Danchuk et al. 2011[32]LPS-induced ALIBALB/CHuman BMi.t.11.1 (22.2 total)4 h24 h+TSG-6
4.5 h48 h+
Song et al. 2012[66]LPS-induced ALIBALB/CBone marrowi.v.22.20 hDay 3+HIMF (−)
Day 7+
Day 14
Li et al. 2012[67]LPS-induced ALISD ratHuman umbilical cordi.v.1.91 h6 h+MDA
24 h++HO-1
48 h++Oxidative stress
Qin et al. 2012[68]LPS-induced ALISD ratBone marrowIntra-pleural40 hDay 1Paracrine soluble factor
Day 3++
Day 7+
Ionescu et al. 2012[38]LPS-induced ALIC57BL/6Bone marrowi.t.11.14 h48 h+IGF-I
Chien et al. 2012[69]LPS-induced ALIBALB/COrbital fat tissuei.v.13.30.3 hDay 3+Immunomodulation
Manning et al. 2010[34]I/R lung injuryLewis ratBone marrowi.v.750 h4 h+IL-10
24 h++
Day 3+
Day 7+
Sun et al. 2011[70]I/R lung injurySD ratAutologous Adipose tissuei.v.4.8 (14.4 total)1 hDay 3+VCAM-1
6 hICAM-1
24 hOxidative stress
Chen et al. 2012[71]I/R lung injurySD ratBone marrowi.v.3.60 h24 h+VEGF
SDF-1
IPO
Krasnodembskaya et al. 2010[58]E. coli pneumoniaC57BL/6Allogeneic human BMi.t.44.44 h18 h+LL-37
Kim et al. 2011[39]E. coli pneumoniaICR mouseHuman umbilical veinsi.t.3.33 hDay 1Paracrine soluble factor
Day 3++
Day 7+
Gupta et al. 2012[59]E. coli pneumoniaC57BL/6Bone marrowi.t.304h4 h+Lipocalin-2
8 h+
24 h+
48 h+
Curley et al. 2012[48]Ventilator-induced lung injurySD ratAllogeneic BMi.v.7.3 (14.5 total)0 h48 h+KGF
24 hParacrine soluble factor
Chimenti et al. 2012[72]Ventilator-induced lung injurySD ratLewis rat BMi.v./18.2-0.5 h3 h+VCAM-1
i.t.P-selectin
Pati et al. 2011[73]Haemorrhagic shock-induced ALISD ratHuman BMi.v.6.41 hDay 4+VE-cadherin
(12.8 total)24 hClaudin-1
Occludin-1
Yang et al. 2011[74]Paraquat poisoning-induced lung injurySD ratBone marrowi.v.506 hDay 3+Reducing lung oedema and lipid peroxidation
Day 7++Inhibiting the release of inflammatory mediators
Day 14+

The BM remained the most common source of MSC[32, 34-38, 46-48, 55, 56, 58, 59, 61-66, 68, 71-74] (83%, 24 of 29), and most current clinical trials used an allogeneic source from the BM. It was believed that MSC are able to evade clearance by the host immune system through a variety of mechanisms including low expression of the MHC I and II proteins and lack of the T-cell costimulatory molecules, CD80 and CD86; this is often referred to as being ‘immunoprivileged’. However, recent studies have shown that MSC can express higher levels of the MHC class proteins than originally thought. In addition, Nauta et al.[75] demonstrated that infusion of allogeneic MSC elicited a host response and led to graft rejection. It has now become apparent that MSC have complex interactions with the immune system. However, the alternative approach of harvesting the BM to isolate and culture autologous MSC may be problematic in acute illnesses such as ALI.

Compared with the BM, human umbilical cord-derived MSC have a faster population doubling time than BM-MSC, and such proliferation characteristics do not change even after 30 passages. In addition, umbilical cord-derived MSC showed lower expression of CD106 and human leucocyte antigens in comparison with BM-MSC.[76] Therefore, umbilical cord-derived MSC are currently being studied for clinical application due to their accessibility, ease of procurement from donors and lack of ethical concerns.[39, 40, 67] Another adult source is fatty tissue. Human orbital fat-derived stem cells are advantageous over BM-MSC because they can be isolated from minimal volume of redundant orbital fat tissue.[69, 77] They also exhibit a higher epithelial differentiation potential than adipose-derived stem cells isolated from subcutaneous fatty tissues.[70, 77] However, the anti-inflammatory ability of orbital fat-derived stem cells needs to be evaluated in other clinically relevant models such as pneumonia/sepsis. Clearly, further studies in the optimal source of MSC are needed.

Optimal Dosage and Route of Cell Delivery

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

The dose and route of MSC varies substantially based on different preclinical animal studies, and the optimal treatment remains to be determined. The mean dose[32, 35, 36, 38-40, 47, 55, 56, 58, 59, 61, 63, 64, 66, 69] instilled during the early phase of lung injury in mice is (29.9 ± 20.4) × 106 cells/kg, which is slightly higher than that of rats, (20.3 ± 22.5) × 106 cells/kg (Fig. 3a, P = 0.26), suggesting that the effective administration dose is about 20∼30 × 106 cells/kg. One study[65] using an ALI model in C57BL/6 mice gave a particularly large dose (888.9 × 106 cells/kg) of MSC just 1 h after exposure. To our knowledge, this is the only study showing the delayed effects of BM-MSC on day 28 because all others therapies were followed up to day 14. Although the administration of MSC was able to repair the lung epithelium and endothelium, reduce the amount of alveolar collapse and led to an improvement in lung mechanics, it is still unknown whether the beneficial effects reported at day 28 persist if the MSC were given later in the course of lung injury. In terms of tissue origin, the average dose of MSC derived from the BM was (28.3 ± 21.8) × 106 cells/kg. No significant differences were found between the dose of MSC from the BM or other tissues such as umbilical cord[39, 40, 67] ((17.2 ± 25.4) × 106 cells/kg) (Fig. 3b, P = 0.42) or fat tissue[69, 70] ((13.9 ± 0.8) × 106 cells/kg) (Fig. 3b, P = 0.37). For most clinical trials utilizing MSC in lung disease such as for idiopathic pulmonary fibrosis (NCT01385644) or bronchopulmonary dysplasia (NCT01632475), the dose administered is approximately 1∼20 × 106 cells/kg, which appears to be largely based on earlier clinical trials in GVHD, myocardial infarction, etc. Therefore, a current pilot clinical trial conducted by University of California San Francisco (NCT01775774) is underway to assess the safety of intravenous infusion of allogeneic human BM-MSC in patients with ALI/ARDS by using a dose-escalation protocol from 1 to 10 × 106 cells/kg. What remains unclear is whether there is a dose effect or a therapeutic ceiling in the use of MSC for lung diseases or whether the dose is limited by the safety concern of the effect of the infusion on pulmonary vascular resistance. And perhaps more importantly, it is unclear whether a second dose of MSC is needed for the later phase of ALI (the resolution), and whether the functional phenotype of the stem cells is therapeutic at this point. Although many believe that higher doses will give a prolonged response, no actual dose response has been reported in the literature.

figure

Figure 3. Average dose of mesenchymal stem cells (MSC) administered in acute lung injury (ALI) models. (a) No significant difference was found in the average dose of MSC used between mouse and rat models of ALI. Data are shown as mean ± standard deviation (SD), n = 16 studies in mice and n = 11 studies in rat, P = 0.26. (b) No significant difference was found in the average dose used among bone marrow-, umbilical cord- or fat tissue-derived MSC for ALI. Data are shown as mean ± SD, n = 22 studies using bone marrow derived, n = 3 studies using umbilical cord derived and n = 2 studies using fat tissue derived. P = 0.42, bone marrow versus umbilical cord; P = 0.37, bone marrow versus fat tissue.

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Most of the studies[39, 58, 59] using an E. coli endotoxin or bacterial pneumonia models of ALI administrated MSC intra-tracheally while those using bleomycin-induced,[35, 47, 61-63] ischaemia reperfusion-induced,[34, 70, 71] ventilator-induced[48, 72] or other[73, 74] lung injury models delivered MSC intravenously. Although for practical reasons it may not be feasible to instil stem cells intra-bronchially in ALI patients who are hypoxemic, the optimal route of delivery is unclear. However, for patients with ALI from pneumonia, it is now known that BM-MSC possess direct antimicrobial activity through the secretion of antimicrobial peptides/proteins such as LL-37 or lipocalin-2 as well as the ability to enhance macrophage/monocyte phagocytosis of bacteria. Thus, the intrapulmonary delivery may be the most effective route to enhance bacteria clearance. On the other hand, intravenous delivery of MSC was used by the majority of studies[34, 35, 37, 47, 48, 55, 56, 61-67, 69-74] (69%, 20 of 29) and may be more relevant to clinical practice; it may be more practical to give large amount of MSC suspension through intravenously.

Interestingly, Qin et al.[68] developed a novel intra-pleural delivery method. They found that MSC delivered by this method can survive at least 1 month in vivo and their distribution was found to be limited to the surface of the pleurae and in the pleural cavity, forming a ‘MSC repository’ in vivo. The advantage of using the pleural cavity for MSC delivery is that it is a potential compartment that can receive larger dose of MSC without restriction. Although promising, the delivery route will need to be investigated further due to the limited number of study.

Timing of MSC Administration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

Although preclinical animal models cannot replicate the natural course of ALI, MSC were typically given within 6 h following ALI in these models. In lipopolysaccharides (LPS)-induced ALI models, the beneficial effects of MSC were typically found less than 3 days following intra-tracheal delivery but the effects were prolonged when MSC were given intravenously. What remains to be determined is whether giving MSC once lung injury is firmly established or during the resolution phase where fibrosis may occur have any therapeutic effect.

For bleomycin-induced lung injury, the most optimal time of cell administration and end-point needs to be determined. Existing preclinical studies[35, 47, 61] demonstrated that MSC were efficacious in ameliorating the resulting fibrosis, correlating with the early inflammatory steps in the pathogenesis of bleomycin-induced lesions,[78] only when administered at the time of injury and not at later time points; it may take 2 to 3 days for MSC to produce soluble factors that modulate inflammation in vivo. For example, Saito et al.[63] observed that the highest concentration of 7ND, a dominant-negative inhibitor of chemokine (C-C motif) ligand 2 (an inducer of macrophage recruitment and activation), was obtained on day 2 after MSC, transfected with the 7ND plasmid, administration. However, the serum level of 7ND was undetectable 11 days after MSC administration, suggesting that the number of MSC diminished over time in vivo.[63] Ortiz et al.[35] also showed that MSC significantly secreted interleukin-1 receptor antagonist, a competitive inhibitor of interleukin-1α, only after 72 h of stimulation by interleukin-1α, which correlated with the development of pulmonary fibrosis exposed to bleomycin. In addition, Aguilar et al.[47] demonstrated that the early benefit of MSC in bleomycin-induced lung injury was lost at a later end-point, day 14. Consequently, MSC may reach its therapeutic peak 2 to 3 days after administration and decrease over time regardless of the time of cell delivery.

Summary for MSC in ALI

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

There are currently more than >300 clinical trials registered with clinicaltrial.gov testing MSC in a variety of disorders, including GVHD, Crohn's disease, acute myocardial infarction and acute kidney failure. More recently, MSC have been tested in clinical trials for lung diseases such as chronic obstructive pulmonary disease, bronchopulmonary dysplasia, pulmonary emphysema, pulmonary hypertension and silicosis.[5, 79] However, as previously shown, existing preclinical studies to date have used relatively poorly defined MSC; the potential exists for identifying subpopulations[80, 81] of MSC with more efficacy. Although a set of criteria for defining MSC has been developed by International Society of Cellular Therapy in 2006,[14] there remains no validated method of measuring MSC bioactivity,[82] a potency assay, and the duration of benefit in vivo. It may be time to revise the definition of MSC set forth by International Society of Cellular Therapy to allow better comparisons between preclinical animal studies and efficacy and the subsequent clinical trials which are underway. How potency will be defined, whether through characterization of secretion of soluble factors or through a functional assay, remains to be determined.

Other Adult Stem Cells for ALI

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

Endothelial Progenitor Cells

Asahara et al.[83] in 1997 purified a population of cells that displayed properties of both endothelial cells and progenitor cells, which were capable of trafficking towards ischaemic sites and differentiating into mature endothelial cells. These authors termed the cells EPC. Although a complete phenotypic description of EPC remains to be determined, the most common surface markers for EPC include endothelial cell antigens such as CD34 and CD133 as well as endothelial-specific markers such as vascular endothelial growth factor receptor-2 and von Willebrand factor.[83, 84]

Mechanisms of action

EPC appear to exert their therapeutic effects via engraftment and differentiation into the endothelium of the damaged vascular site, via secretion of growth factors and cytokines inducing neovascularization and via an immunomodulatory effect. Although most previous studies indicated that the level of engraftment of EPC in lung injury was low, with observed rates of less than 5%, these authors[85-87] studied the trafficking of the EPC to the lung with immunohistochemistry or fluorescence-conjugated cell tracers for up to 14 days. Because of the short time period of injury studied and the fact that the intravenous infusion of BM-derived EPC typically are trapped in the pulmonary microcirculation, additional studies are needed to determine the contribution of engraftment in the therapeutic response of EPC. Interestingly, similar to MSC, Cao et al.[88] found that EPC modulated immune cell response following lung injury by reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokine, interleukin-10, and inhibiting the influx of inflammatory cells, specifically neutrophils.

Recent experimental results

Based on a pilot study[89] in 2004, which showed that the suppression of BM-derived progenitor cells by irradiation before intrapulmonary LPS led to disruption of tissue structure and emphysema-like changes that was prevented by the reconstitution of the BM, a number of preclinical animal studies[85-88, 90-92] have been conducted to evaluate the effect of EPC mobilization or administration in reducing lung injury or in regenerating the lung following the initial insult. Most studies[85, 86, 88, 90, 91] (71%, 5 of 7) used an endotoxin-induced ALI model. Rabbit (57%, 4 of 7) was the predominant animal model studied,[86-88, 91] and autologous EPC isolated from peripheral blood was the primary source of the progenitor cells (Table 2). The advantages of autotransplantation with EPC included no need to use an allogeneic source, completely avoiding immunological rejection, potential use in clinical applications and a simple isolation technique using a blood cell separator.[93] The disadvantage was isolating progenitors cells in patients with ongoing inflammation and/or infection, possibly changing the phenotype of the EPC.

Table 2. Descriptive details of endothelial progenitor cell preclinical study
StudyLung injury modelSpeciesOriginRouteDosage (×105 cells/kg)Given time after exposureMeasured time pointsTherapeutic effectPossible mechanism
  1. ALI, acute lung injury; HO-1, heme oxygenase-1; i.v., intravenous; ICAM-1, intercellular adhesion molecule 1; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharides; MnSOD, manganese superoxide dismutase; SD, Sprague Dawley; VEGF, vascular endothelial growth factor.

Wary et al. 2009[90]LPS-induced ALIC57BL/6Bone marrowi.v.1454 h12 h+α4β1 α5β1 integrin
24 h+
Day 2+
Day 3++
Mao et al. 2010[85]LPS-induced ALISD ratBone marrowi.v.1820.5 hDay 3

Engraftment

Immunomodulation

Day 7+
Day 14+
Lam et al. 2011[86]LPS-induced ALINew Zealand rabbitPeripheral bloodi.v.0.360.5 hDay 2+

Alveolar-capillary membrane repair

Restore gas exchange

Gao et al. 2011[91]LPS-induced ALINew Zealand rabbitPeripheral bloodi.v.0.360.5 hDay 1+

ICAM-1

P-selectin

Day 3++
Day 5+
Cao et al. 2012[88]LPS-induced ALINew Zealand rabbitPeripheral bloodi.v.54 hDay 2+

iNOS

VEGF

Kähler et al. 2007[92]Left-sided lung transplantation-induced ALISD ratBone marrowi.v.411−2 hDay 1+

Homing

Establish endothelial integrity

Day 3 
Day 9 
Lam et al. 2008[87]Oleic acid-induced lung injuryNew Zealand rabbitPeripheral bloodi.v.0.360.5 hDay 2+

HO-1

MnSOD

Optimal dosage and route of cell delivery

Based on animal models, the primary route of cell delivery is intravenously because EPC are thought to originate primarily from the BM and circulate in the peripheral blood.[94, 95] Similar to MSC, intravenous delivery of exogenous EPC is an effective route for a pulmonary endothelial-targeted therapeutic strategy because EPC have been shown to be effectively retained in the pulmonary microcirculation.[96] For example, Mao et al.[85] studied the effect of EPC on the lung capillary endothelium following intravenous administration of LPS in order to explore the effect of EPC in vascular repair more directly. The choice of dosage, however, varied from different studies. The average dose in rabbit models was (1.52 ± 2.32) × 105 cell/kg while that in rodent models was (122.7 ± 73.1) × 105 cell/kg. It is unclear why the dosages differ among these species. In addition, what remains to be determined is whether there is a dose effect and whether the paracrine mechanisms discussed is clinically relevant.

Timing of EPC administration

A recent clinical investigation[97] provided evidence that EPC could be released from the BM in inflammatory condition, and the number of EPC at an early phase correlated with the recovery process. In preclinical animal studies, EPC are usually administered within 4 h of injury. In these studies,[85, 90] 80–95% of injected EPC are trapped in lung tissue within 20 min, and a significant population of these cells remained in the lungs during the first 12 h. The number of cells retained in the lungs declined to 40–50% by the end of 24 h, but a significant number of cells remained sequestered in lungs even for up to 8 weeks. The findings suggested that EPC homed to the pulmonary endothelium and subsequently engrafted into the injured endothelium. However, the number of EPC was very low in the pulmonary circulation and lung tissue after intravenous administration in experimental model of oleic acid-induced[87] and LPS-induced ALI.[86] These findings may have been due to the extremely large surface area of the pulmonary circulation, leading to an artificially low count in the thin histological sections. In addition, low homing efficacy of progenitors in the target organs has been reported after systemic or regional delivery, as a result of a washout effect.[98] Further investigation into the level of engraftment may offer a more effective way to measure the efficacy of EPC.

Summary of EPC in ALI

To date, several descriptive studies have been conducted to quantify circulating EPC in the peripheral blood of patients with ALI/ARDS. Two groups[97, 99] found a higher number of colony-forming units of EPC from patients with ALI compared with healthy control subjects, and in patients with ALI, an increased number of circulating EPC were associated with improved survival, suggesting that circulating EPC were mobilized from the BM to replenish the injured endothelium. While the earlier mentioned clinical studies only quantified EPC and correlated the count to disease outcome, two small pilot clinical trials[100, 101] (NCT00257413, NCT00641836) investigated the intravenous infusion of autologous EPC at ∼2 × 105 cell/kg in idiopathic pulmonary arterial hypertension, showing an increase in 6-min walk test by 18% and 11% in adult and children respectively after 12 weeks of follow up with no immunological reactions or adverse effects noted from EPC infusion.

However, barriers to clinical application of EPC for ALI remain. (i) It would be difficult to harvest and culture enough EPC for autologous transplantation because their phenotype and function and cell number may be impaired during a systemic inflammatory state; (ii) the number of EPC needed to target the large surface area of the pulmonary circulation is unknown. No dose response experiment has been performed; and (iii) in addition, an allogeneic source of EPC may cause an immune reaction in the host; it is unclear if EPC are ‘immunoprivileged’ in a manner similar to that of MSC. Further preclinical studies are warranted prior to any clinical trial for ALI.

Hematopoietic Stem Cells

Haematopoietic CD34+ cells are rare stem cell-derived progenitors, representing only 1 in 104 to 105 of total blood cells in the BM, with combined myeloid and lymphoid differentiation and self-renewal potential.[79, 102] Because cultured HSC rapidly lose their ability to engraft and self-renew in vivo, which limits the options to maintain, expand or manipulate HSC in vitro for therapeutic purposes, very little is known about the potential of HSC in lung injury models. Aguilar et al.[47] demonstrated a successful HSC-based KGF gene therapy by using an inducible lentiviral vector (Tet-On) in bleomycin-induced lung fibrosis. They demonstrated that transplantation of lentivirus-transduced HSC-KGF showed a significant reduction in lung fibrosis, specifically a reduction in collagen 1α1 messenger RNA and collagen content, and lung damage using a histological score. However, the beneficial effects of HSC have to be demonstrated with further preclinical studies.

Endogenous lung stem cells

The ideal cell type to regenerate the injured lung would be the lung's own endogenous stem cell population. The ability of the lung to regenerate following injury provides clear evidence for the existence of one or more native lung stem cell populations. Kajstura et al.[103] reported a human c-KIT-positive adult lung stem cell that was clonogenic and able to regenerate the architecture of the lung bronchiole, alveoli and arteriole after cryoablation injury in mice. Chapman et al.[104] also identified a novel subpopulation of mouse alveolar epithelial cells expressing the laminin receptor α6β4 that can repair the lung following ALI. Taken together, these studies offer considerable promise for a therapeutic role for endogenous lung stem/progenitor cells in lung diseases. However, this putative adult lung-derived stem cell population remains poorly characterized and needs to be replicated to determine the translational potential of these cells for ALI.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References

The major findings of this review of adult stem cells for ALI can be summarized as follows: (i) for MSC, the effective administration dose from preclinical studies in animals is approximately 20∼30 × 106 cells/kg, regardless of the origin of the cells. Whereas, from current ongoing clinical trials (>300 for a variety of diseases/syndromes), the average dose of MSC is approximately 5∼10 × 106 cells/kg; (ii) intrapulmonary delivery of MSC may be a more effective route for direct antimicrobial effect, while the intravenous route may be more practical for clinical application where the patients are hypoxic; (iii) in most preclinical studies, MSC were delivered in lung injury models early usually within 6 h following injury. Whereas, in bleomycin-induced ALI, the therapeutic effect of MSC reached its peak 2 to 3 days after administration; (iv) the beneficial effects of MSC in LPS-induced ALI model were typically found less than 3 days after intra-tracheal delivery but the effects were prolonged when MSC were given intravenously; (v) for EPC, intravenous delivery of exogenous EPC is an effective route for pulmonary endothelial-targeted therapeutic strategy, but research into the optimal dose is needed; (vi) EPC were delivered usually within 4 h following injury and (vii) although the engraftment rate was low, multiple studies suggest that EPC appear to home to the injured pulmonary endothelium and repopulate the injured tissue.

Despite some limitations in the characterization of MSC or EPC, especially concerning the issue of potency, a significant amount of preclinical data in both animal models and clinical trials suggest that stem or progenitor cell-based therapies may be beneficial in lung repair and remodelling after ALI. Although questions and concerns still remain, a novel and safe therapy for acute lung diseases might eventually emerge.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mesenchymal Stem Cells
  5. Recent Experimental Results
  6. Optimal Dosage and Route of Cell Delivery
  7. Timing of MSC Administration
  8. Summary for MSC in ALI
  9. Other Adult Stem Cells for ALI
  10. Conclusions
  11. Acknowledgements
  12. References