Optimization of Targeted Cell Replacement Therapy: A New Approach for Lung Disease
Authors: Pascal Duchesneau, Amy P Wong, Thomas K Waddell
Journal: Molecular Therapy (2010)

Cell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis, although rates of engraftment need to be improved. We previously showed improved cell retention in the lung using transtracheal delivery compared to intravenous injection. Here, we optimized other parameters of cell delivery using 7-day cultured bone marrow cells (BMCs). Retention of BMC in the lung was dose-dependent. Naphthalene treatment had maximal effects on BMC retention when given 2 days before cell delivery. Naphthalene treatment of the donor amplified a CCSP+ population and increased retention efficiency in the recipient. Repeated naphthalene treatment and repeated cell delivery both resulted in greater retention. The contribution of the second cell dose was minimal suggesting that a second delivery of BMC promotes proliferation of the first. Busulfan-induced myelosuppression augmented retention of exogenous BMC by up to 20-fold. These BMC helped CCSP reconstitution. Using the optimal delivery techniques and cytokeratin-18-driven green fluorescent protein (GFP) reporter mice, we detected threefold more GFP suggesting more BMC differentiated to epithelial cells. We propose that improved engraftment in the lung will increase cell replacement and thus be a more efficient therapeutic approach for various lung diseases. Cell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis, although rates of engraftment need to be improved. We previously showed improved cell retention in the lung using transtracheal delivery compared to intravenous injection. Here, we optimized other parameters of cell delivery using 7-day cultured bone marrow cells (BMCs). Retention of BMC in the lung was dose-dependent. Naphthalene treatment had maximal effects on BMC retention when given 2 days before cell delivery. Naphthalene treatment of the donor amplified a CCSP+ population and increased retention efficiency in the recipient. Repeated naphthalene treatment and repeated cell delivery both resulted in greater retention. The contribution of the second cell dose was minimal suggesting that a second delivery of BMC promotes proliferation of the first. Busulfan-induced myelosuppression augmented retention of exogenous BMC by up to 20-fold. These BMC helped CCSP reconstitution. Using the optimal delivery techniques and cytokeratin-18-driven green fluorescent protein (GFP) reporter mice, we detected threefold more GFP suggesting more BMC differentiated to epithelial cells. We propose that improved engraftment in the lung will increase cell replacement and thus be a more efficient therapeutic approach for various lung diseases. IntroductionCell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis. It differs from traditional regenerative medicine approaches in that there are two components. As for bone marrow transplantation, the respective local endogenous stem cells must first be removed or inactivated. Following this preconditioning regimen, exogenous cells with a preferred genotype can be supplied. Cystic fibrosis would seem to be an ideal candidate disease for such an approach, where the structure of the lung is relatively intact at the time of diagnosis. Moreover, the genetic defect is functionally limited to the bronchiolar epithelium, and partial replacement of the CFTR genotype would allow significant functional changes in chloride transport.1Johnson LG Olsen JC Sarkadi B Moore KL Swanstrom R Boucher RC Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis.Nat Genet. 1992; 2: 21-25Crossref PubMed Scopus (375) Google Scholar This contrasts with the more significant hurdles facing regenerative medicine approaches to other lung diseases, such as emphysema and pulmonary fibrosis, where repair or regeneration of the epithelial, stromal, and vascular components might all be required. However, previous studies have reported low retention of the delivered cells that prevents full assessment of the function of transplanted cells. Procedures and conditions to increase retention have received little attention to date.A variety of sources of exogenous cells could be utilized for cell replacement therapy. An ideal source would be autologous to the intended recipient, to avoid rejection by the immune system. It would also be readily accessible and able to be manipulated ex vivo, for example, to undergo gene correction of the defective CFTR locus. Finally, there would need to be at least some stem or long-lasting progenitor cells within the population to allow permanent correction of the cystic fibrosis defect. There has been substantial progress in identification of the stem cell hierarchies within the airway epithelium, at least in mice.2Rawlins EL Okubo T Xue Y Brass DM Auten RL Hasegawa H et al.The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium.Cell Stem Cell. 2009; 4: 525-534Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar,3Rock JR Onaitis MW Rawlins EL Lu Y Clark CP Xue Y et al.Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc Natl Acad Sci USA. 2009; 106: 12771-12775Crossref PubMed Scopus (956) Google Scholar However, there is no easy technique at present to isolate and expand bronchial airway stem cells.4McQualter JL Brouard N Williams B Baird BN Sims-Lucas S Yuen K et al.Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction.Stem Cells. 2009; 27: 623-633Crossref PubMed Scopus (141) Google Scholar Embryonic and induced pluripotent stem cells offer great promises, but there are currently no protocols for the robust differentiation in vitro to bronchiolar epithelium. Therefore, we have focused our efforts on bone marrow–derived progenitor populations.Although admittedly controversial, available data suggest that bone marrow–derived populations can express proteins normally expressed by the lung epithelium, especially CFTR.5Albera C Polak JM Janes S Griffiths MJ Alison MR Wright NA et al.Repopulation of human pulmonary epithelium by bone marrow cells: a potential means to promote repair.Tissue Eng. 2005; 11: 1115-1121Crossref PubMed Scopus (67) Google Scholar,6Dooner M Cerny J Colvin G Demers D Pimentel J Greer D et al.Homing and conversion of murine hematopoietic stem cells to lung.Blood Cells Mol Dis. 2004; 32: 47-51Crossref PubMed Scopus (28) Google Scholar,7Kleeberger W Versmold A Rothämel T Glöckner S Bredt M Haverich A et al.Increased chimerism of bronchial and alveolar epithelium in human lung allografts undergoing chronic injury.Am J Pathol. 2003; 162: 1487-1494Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar,8Krause DS Theise ND Collector MI Henegariu O Hwang S Gardner R et al.Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell.Cell. 2001; 105: 369-377Abstract Full Text Full Text PDF PubMed Scopus (2456) Google Scholar,9Macpherson H Keir P Webb S Samuel K Boyle S Bickmore W et al.Bone marrow-derived SP cells can contribute to the respiratory tract of mice in vivo.J Cell Sci. 2005; 118 (Pt 11): 2441-2450Crossref PubMed Scopus (71) Google Scholar,10Mattsson J Jansson M Wernerson A Hassan M Lung epithelial cells and type II pneumocytes of donor origin after allogeneic hematopoietic stem cell transplantation.Transplantation. 2004; 78: 154-157Crossref PubMed Scopus (73) Google Scholar,11Wong AP Dutly AE Sacher A Lee H Hwang DM Liu M et al.Targeted cell replacement with bone marrow cells for airway epithelial regeneration.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L740-L752Crossref PubMed Scopus (78) Google Scholar,12Wong AP Keating A Lu WY Duchesneau P Wang X Sacher A et al.Identification of a bone marrow-derived epithelial-like population capable of repopulating injured mouse airway epithelium.J Clin Invest. 2009; 119: 336-348PubMed Google Scholar As proof of principle, Krause's group was able to demonstrate partial restoration of nasal potential difference measurements following neonatal bone marrow transplantation of wild-type marrow into CFTR knockout mice.13Bruscia EM Price JE Cheng EC Weiner S Caputo C Ferreira EC et al.Assessment of cystic fibrosis transmembrane conductance regulator (CFTR) activity in CFTR-null mice after bone marrow transplantation.Proc Natl Acad Sci USA. 2006; 103: 2965-2970Crossref PubMed Scopus (62) Google Scholar Use of bone marrow–derived cells at this point in the development of cell replacement therapy offers several advantages. The technology to isolate and expand bone marrow populations is widespread and relatively well accepted. These cells can be expanded ex vivo and gene corrected, using hematologic disorders as a clinical precedent.We have recently made substantial progress toward the goal of clinically relevant cell replacement therapy. For example, we identified that the transtracheal delivery route offered substantial improvements compared to intravenous delivery.11Wong AP Dutly AE Sacher A Lee H Hwang DM Liu M et al.Targeted cell replacement with bone marrow cells for airway epithelial regeneration.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L740-L752Crossref PubMed Scopus (78) Google Scholar We have also identified a novel population of bone marrow cells (BMCs), with significantly increased propensity for both retention in the lung and expression of epithelial genes, at the mRNA, protein, and electrophysiological functional levels.12Wong AP Keating A Lu WY Duchesneau P Wang X Sacher A et al.Identification of a bone marrow-derived epithelial-like population capable of repopulating injured mouse airway epithelium.J Clin Invest. 2009; 119: 336-348PubMed Google Scholar Here, we have developed a robust quantitative PCR-based assay for retention within the lung. We demonstrate that several components of the entire cell replacement procedure can be improved, with an overall increase of cells remaining in the lung at a medium-term follow-up of 30 days of as much as 20-fold. We also show that delivered cells might proliferate locally in the lung, and this proliferation can be augmented by a second delivery of cells. Delivered BMC can also accelerate airway regeneration and directly or indirectly improve survival. These delivered cells can differentiate in vivo into epithelial cells and this is also augmented by a second delivery. At the very least, these techniques should allow robust functional assessment of any delivered cells, whether bone marrow–derived, endogenous lung stem cells, or ultimately iPS-differentiated lung epithelial cells.ResultsEffect of BMC dose, BMC culture, and naphthalene treatment on cell retentionBMCs have been suggested to engraft in various organs of the body, but reported levels are very low. In few cases, functional integration into the host tissue has been formally demonstrated, and we have therefore chosen to use the term "retention" instead. The type of cells used and the available niche might have an impact on retention. Naphthalene treatment was used to deplete Clara cells and allow for partial removal of endogenous cells occupying a putative lung "niche." First, we explored whether the size of the space available for retention might limit numbers of retained cells. Increasing numbers of 7-day cultured BMC were delivered transtracheally following our standard protocol with naphthalene treatment at day −2 (ref. 11Wong AP Dutly AE Sacher A Lee H Hwang DM Liu M et al.Targeted cell replacement with bone marrow cells for airway epithelial regeneration.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L740-L752Crossref PubMed Scopus (78) Google Scholar). Retention was assessed by quantitative real-time PCR for the Y chromosome–specific gene, SRY, 30 days after cell delivery. The PCR result was compared to a standard curve generated by evaluation of a female lung injected with increasing numbers of male BMC in vitro. Overall retention of BMC was increased in a dose-dependent manner with cell number (Figure 1a). Retention efficiency, that is, the percentage of the delivered cell number still detectable after 30 days, remained similar at around 2–3% (data not shown). Next, we explored the effect of BMC culture on retention efficiency. BMC culture for 7 days resulted in an increased retention efficiency in the lung compared to fresh cells, but longer culture had no beneficial effects (Figure 1b). We previously reported a small population of BMC that express CCSP with distinct functional properties.12Wong AP Keating A Lu WY Duchesneau P Wang X Sacher A et al.Identification of a bone marrow-derived epithelial-like population capable of repopulating injured mouse airway epithelium.J Clin Invest. 2009; 119: 336-348PubMed Google Scholar This population is amplified by culture and remains high for up to 28 days (Figure 1c). Finally, the optimal timing of naphthalene treatment was examined. Retention efficiency was optimal when BMCs were delivered 2 days after naphthalene treatment (Figure 1d) that corresponds to maximal Clara cell depletion (data not shown).Effect of primed BMC on retention in the lungWe previously showed that the CCSP+ population in fresh bone marrow increased after naphthalene treatment of donor mice from ~2% to ~10% with a peak at 2 days after treatment.12Wong AP Keating A Lu WY Duchesneau P Wang X Sacher A et al.Identification of a bone marrow-derived epithelial-like population capable of repopulating injured mouse airway epithelium.J Clin Invest. 2009; 119: 336-348PubMed Google Scholar Here, we confirmed this effect of naphthalene whereby CCSP increases almost fourfold (Figure 2a). 7-day Cultured BMCs from untreated donors were FACS-sorted into CCSP+ or CCSP− cells prior to transtracheal delivery. This confirmed the enhanced retention efficiency of the CCSP+ subset (Figure 2b). To test whether BMC from naphthalene-treated donors would also have better retention efficiency, we used fresh or 7-day cultured BMC from normal and naphthalene-treated mice for transtracheal delivery. Fresh BMC retention efficiency was not improved by pretreatment of the donor with naphthalene at day −2 or −10 (Figure 2c). However, retention efficiency of 7-day cultured BMC from primed donor was slightly increased by naphthalene treatment of the donor at day −2 or −10 (Figure 2d). All 7-day cultured BMC, primed or nonprimed, expressed 25% CCSP despite higher starting population in primed donor (data not shown).Figure 2Effect of "priming" bone marrow. (a) Freshly isolated bone marrow cell (BMC) from untreated animals showed a small population of CCSP+ cells that were four times greater when BMCs were isolated from mice treated with naphthalene 2 days prior to cell isolation. The gate was set to include 1% of cells for isotype antibody staining. (b) 7-day Cultured BMCs were sorted by fluorescence-activated cell sorting prior to being delivered into recipients conditioned with naphthalene 2 days earlier. CCSP+ BMC demonstrated significantly superior retention than CCSP− BMC. (c) Fresh male BMC isolated from naphthalene-treated mice did not show greater retention efficiency in the lung of female recipients after 30 days. (d) 7-day Cultured BMC isolated from naphthalene-treated mice had slightly better retention efficiency than the control BMC from untreated donor. Each bar represents mean ± SEM; (n ≥ 4) mice per group. *P < 0.05 by one-way analysis of variance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Repeated naphthalene treatment and repeated cell dose increase retention efficiencyBecause Clara cell depletion by naphthalene treatment increased BMC retention efficiency in the lung, we sought to explore ways that could increase Clara cell depletion and therefore potentially create more space for delivered cells. Repeated naphthalene treatments at days −5 and −2; and −9 and −2 appear to denude the airways more completely than a single treatment at day −2 where occasional semidetached cells can be found (Figure 3a). For naphthalene treatment at days −9 and −5; and −9, −5, and −2, Clara cell regeneration could be detected. Repeated naphthalene treatments at various time points increased BMC retention efficiency by up to ninefold (Figure 3b). Increasing cell number (Figure 1a) was not limiting and so repeated BMC delivery was also evaluated. Multiple cell delivery treatments increased retention efficiency in the lung by up to fourfold (Figure 3c). The increased retention was more than additive. Therefore, we investigated the effect of each delivery separately by using male BMC for either the first or second cell treatment only. Retention of the second BMC dose appeared to be minimal, whereas more BMCs from the first dose were detected in the presence of two doses (Figure 3d).Figure 3Retreatment increases retention efficiency. (a) Female mice were treated with repeated doses of naphthalene and killed at day 0. Formalin-fixed paraffin-embedded lung sections were stained for CCSP by immunoperoxidase to observe depletion levels. Double naphthalene treatment at days −5 and −2; and −9 and −2 resulted in a more complete depletion of CCSP than single treatment. Arrows are pointing to CCSP+ cells. The inset represents isotype antibody staining. (b) 7-day Cultured male bone marrow cell (BMC) isolated from untreated donor mice were delivered to female recipient mice treated with naphthalene in various preparatory regimens. Double or triple naphthalene treatment increased male BMC retention efficiency as measured by SRY. (c) One million total male BMCs were delivered in two or three repeated dose combinations into naphthalene-treated female recipient mice (single dose at day −2). Repeated cell delivery of fractionated doses showed greater male cell retention efficiency compared to a one-time dose and was optimal for days 0 and 3. (d) Sex-mismatch BMC delivery at days 0 and 3 were used to assess retention efficiency for each dose separately. Retention of the first cell dose is greatly increased in the presence of a second, whereas retention efficiency of the latter dose is minimal. Bar = 100 µm. Each bar represents mean ± SEM; (n ≥4) mice per group. *P < 0.05 by one-way analysis of variance. **P value <0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Busulfan increases exogenous BMC retention efficiencyFreshly isolated BMC can migrate to factors released by naphthalene-treated lung in vitro (Figure 4a). This suggested that endogenous BMCs could compete with delivered cells and reduce retention in the lung. We first confirmed 7-day cultured BMC and CCSP+ sorted cells could also migrate to naphthalene-treated lung (Figure 4b). Next, we used busulfan, which has been suggested by others to suppress the endogenous bone marrow contribution,14Rojas M Xu J Woods CR Mora AL Spears W Roman J et al.Bone marrow-derived mesenchymal stem cells in repair of the injured lung.Am J Respir Cell Mol Biol. 2005; 33: 145-152Crossref PubMed Scopus (697) Google Scholar in recipients receiving BMC transtracheally. Busulfan treatment increased BMC retention efficiency in a dose-dependent manner (Figure 4c). At the highest dose of 50 mg/kg, busulfan in combination with naphthalene treatment was lethal for 50% of the mice, whereas all mice also receiving BMC transtracheally survived and had normal weight (Figure 4d).Figure 4BMC migrate to injured lung and contribute to recovery. (a) Fresh BMC can migrate in vitro to injured lung. (b) BMC cultured for 7 days [as used for transtracheal delivery (black bar)] and CCSP+ cells sorted from 7-day cultured BMC (white bar) can also migrate to injured lung. (c) Mice were treated with naphthalene at day −2, and busulfan was used to suppress endogenous bone marrow at various doses. Busulfan pretreatment at day −1 increased delivered BMC retention efficiency in a dose-dependent manner. The control group was treated with busulfan at 30 mg/kg without naphthalene injury. (d) Naphthalene and busulfan (50 mg/kg) treatment caused death in 50% of the mice that did not receive BMC treatment, whereas all mice receiving BMC survived the drug treatment. Each bar represents mean ± SEM; (n ≥4) mice per group. *P < 0.05 by one-way analysis of variance. **P value <0.01. For the mortality studies, n = 12 and statistic calculated by χ2. BMC, bone marrow cell.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Contribution of BMC to Clara cell regenerationTo explore the contribution of BMC in lung regeneration, and in direct or indirect prevention of mortality, we treated mice with the highest dose of busulfan (50 mg/kg) in combination with naphthalene prior to BMC delivery, then killed the mice 10 days later before most of the mortality could be observed as in Figure 4d. The lungs were then sectioned and stained for CCSP or hematoxylin and eosin. BMC delivery appears to accelerate recovery of CCSP+ airway cells and improve the viability of the epithelium (Figure 5a). The CCSP+ cell count was significantly increased by approximately fourfold in mice that received BMC compared to no BMC treatment (Figure 5b).Figure 5BMC contribute to recovery by accelerating CCSP regeneration. (a) Naphthalene- and busulfan- (50 mg/kg) treated animals were killed at day 10 before most of the mortality could be observed as in Figure 4d. Formalin-fixed paraffin-embedded lung sections were stained with CCSP antibody and Alexa Fluor 546 secondary or by hematoxylin and eosin (H&E). Fluorescent microscopic images show more CCSP in mice that received BMC treatment compared to those that received no BMC. Control mice were treated with busulfan only. H&E staining also appears to show a more intact airway in BMC-treated lungs. Arrows are pointing at magnified area in inset. (b) CCSP+ cells were counted from four animals per group and normalized to the total airway cell numbers. Bar = 100 µm. Each bar represents mean ± SEM; (n ≥4) mice per group. *P < 0.05 by one-way analysis of variance. BMC, bone marrow cell; H&E, hematoxylin and eosin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Epithelial fate of optimally delivered cellTo determine whether these measures to increase BMC retention actually result in increased contribution of BMC to epithelial lineages within the lung, a transgenic reporter mouse was used as BMC donor. The K18GFP mouse on a CD1 outbred background carries a transgene driven by the human cytokeratin-18 promoter. We combined naphthalene and busulfan (30 mg/kg) administration with repeated cell delivery (days 0 and 3) as an optimized treatment protocol and compared it to the previous standard protocol (Figure 6a). As expected, the optimal protocol resulted in significantly increased retention as measured by SRY real-time PCR (Figure 6b). This was confirmed when measured by gDNA PCR for the K18GFP transgene (Figure 6c). Finally, to move beyond simply retention within the lung, we addressed the epithelial-like fate of these delivered BMCs. The optimal protocol resulted in significantly increased K18GFP transgene mRNA (Figure 6d) and protein (Figure 6e,f), confirming progress toward our therapeutic goal of cell replacement therapy.Figure 6Epithelial fate of optimally delivered cells. (a) Schematic representation of standard and optimal delivery. (b) Combining repeated cell treatment and busulfan (30 mg/kg) into an optimal cell replacement protocol resulted in approximately tenfold improvement in the overall level of cell retention as measured by quantitative real-time PCR for SRY. (c) Assessment of retention by gDNA for K18GFP yielded very similar results. (d) Expression of the epithelial-specific reporter transgene K18GFP was significantly increased using the "optimal" cell replacement protocol. As a comparison, the expression of K18GFP in the lungs of untreated transgenic animals is shown. (e) Frozen lung sections stained with anti-GFP followed by AlexaFluor 546 secondary (green pseudocolor). Representative image of K18GFP lung (top right panel) and K18GFP cells retained in a CD1 mouse lung by standard or optimal protocol (bottom panel). Insets are showing isotype control antibody staining and magnified area indicated by arrows. Non-GFP control lung represents mice treated with drug regimen than receiving non-GFP bone marrow cell and stained with GFP antibody. (f) GFP+ cells were counted from four animals per group and normalized to the total airway cell numbers. Optimal treatment resulted in a twofold increase in GFP+ cells. Bar = 50 µm. Each bar represents mean ± SEM; (n ≥4) mice per group. *P < 0.05 by one-way analysis of variance. GFP, green fluorescent protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionIn our previous work, we demonstrated that BMCs are retained in the lung, up to 120 days after naphthalene treatment.11Wong AP Dutly AE Sacher A Lee H Hwang DM Liu M et al.Targeted cell replacement with bone marrow cells for airway epithelial regeneration.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L740-L752Crossref PubMed Scopus (78) Google Scholar Here, we could detect donor BMC in the lung at 6 and 12 months after delivery (data not shown). After long-term residence in the lung, these BMCs take on many attributes of lung epithelial cells as documented by immunocolocalization of epithelial markers. Moreover, to avoid all the potential artifacts of microscopy, we developed a transgenic mouse reporter line (K18GFP) that expresses green fluorescent protein (GFP) under the control of the epithelial-specific cytokeratin-18 promoter. As previously reported, freshly isolated and cultured BMC from these mice do not express GFP (data not shown).11Wong AP Dutly AE Sacher A Lee H Hwang DM Liu M et al.Targeted cell replacement with bone marrow cells for airway epithelial regeneration.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L740-L752Crossref PubMed Scopus (78) Google Scholar Expression of the K18GFP transgene confirms the ability of BMC to contribute to epithelial lineages. In order to further advance the concept of cell replacement therapy, we explored several approaches to increasing retention of unfractionated plastic-adherent BMC in an objective quantitative assay. Increasing cell dose, increasing naphthalene pretreatment, increasing number of cell delivery, and administration of busulfan all increase retention of BMC delivered via the airway. We confirmed that increasing retention does indeed translate into increased contribution of BMC to lung epithelium by quantitative real-time PCR measurement of the CK18 transgene expression at the mRNA and protein level.Since the initial reports of Krause and Kotton, which generated tremendous excitement, there has been increasing skepticism regarding the potential contribution of BMC to the lung and other organs.8Krause DS Theise ND Collector MI Henegariu O Hwang S Gardner R et al.Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell.Cell. 2001; 105: 369-377Abstract Full Text Full Text PDF PubMed Scopus (2456) Google Scholar,15Kotton DN Ma BY Cardoso WV Sanderson EA Summer RS Williams MC et al.Bone marrow-derived cells as progenitors of lung alveolar epithelium.Development. 2001; 128: 5181-5188Crossref PubMed Google Scholar Some reports have determined that there is minimal contribution, especially in the absence of injury or that the contribution is only deleterious myofibroblasts.16Wagers AJ Sherwood RI Christensen JL Weissman IL Little evidence for developmental plasticity of adult hematopoietic stem cells.Science. 2002; 297: 2256-2259Crossref PubMed Scopus (1297) Google Scholar,17Balsam LB Wagers AJ Christensen JL Kofidis T Weissman IL Robbins RC Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature. 2004; 428: 668-673Crossref PubMed Scopus (1528) Google Scholar,18Kotton DN Fabian AJ Mulligan RC Failure of bone marrow to reconstitute lung epithelium.Am J Respir Cell Mol Biol. 2005; 33: 328-334Crossref PubMed Scopus (230) Google Scholar,19Epperly MW Guo H Gretton JE Greenberger JS Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis.Am J Respir Cell Mol Biol. 2003; 29: 213-224Crossref PubMed Scopus (239) Google Scholar Others continue to believe, based on careful state-of-the-art microscopy studies and transgenic reporter systems, that such a contribution is possible although rare.20Sueblinvong V Loi R Eisenhauer PL Bernstein IM Suratt BT Spees JL et al.Derivation of lung epithelium from human cord blood-derived mesenchymal stem cells.Am J Respir Crit Care Med. 2008; 177: 701-711Crossref PubMed Scopus (151) Google Scholar,21Loi R Beckett T Goncz KK Suratt BT Weiss DJ Limited restoration of cystic fibrosis lung epithelium in vivo with adult bone marrow-derived cells.Am J Respir Crit Care Med. 2006; 173: 171-179Crossref PubMed Scopus (173) Google Scholar,22Serikov VB Popov B Mikhailov VM Gupta N Matthay MA Evidence of temporary airway epithelial repopulation and rare clonal formation by BM-derived cells follo