Failure of bone marrow to reconstitute lung epithelium

A new paradigm of epithelial tissue reconstitution has been suggested whereby circulating cells derived from bone marrow contribute to a variety of epithelial cell types. With regard to the lung, several recent reports have used immunofluorescence microscopy to demonstrate engraftment of bone marrow-derived cells as type II pneumocytes, the endogenous progenitors of the lung alveolus. We show here that immunofluorescence microscopy, as has been used in previous reports, cannot reliably identify rare engrafted cells in lung tissue sections after transplantation of bone marrow cells or purified hematopoietic stem cells tracked with ubiquitous labels. We have employed a lineage-specific reporter system based on transgenic mice that express the GFP reporter gene only in lung epithelial cells (surfactant protein C-GFP) to assay for engrafted cells by flow cytometry, histology, and molecular methods. Using this approach to evaluate transplant recipients, including those subjected to bleomycin-induced lung injury, we demonstrate that when autofluorescence, dead cells, and contaminating blood cells are excluded from analysis, there is no detectable reconstitution of lung alveolar epithelial cells by unfractionated bone marrow cells or purified hematopoietic stem cells.     

Engraftment of donor-derived epithelial cells in multiple organs following bone marrow transplantation into newborn mice

Bone marrow-derived cells (BMDCs) can engraft as epithelial cells throughout the body, including in the lung, liver, and gastrointestinal (GI) tract following transplantation into lethally irradiated adult recipients. Except for rare disease models in which marrow-derived epithelial cells have a survival advantage over endogenous cells, the currently attained levels of epithelial engraftment of BMDCs are too low to be of therapeutic benefit. Here we tested whether the degree of bone marrow to epithelial engraftment would be higher if bone marrow transplantation (BMT) were performed on 1-day-old mice, when tissues are undergoing rapid growth and remodeling. BMT into newborn mice after multiple different regimens allowed for robust hematopoietic engraftment, as well as the development of rare donor-derived epithelial cells in the GI tract and lung but not in the liver. The highest epithelial engraftment (0.02%) was obtained in mice that received a preparative regimen of two doses of busulfan in utero. When BMDCs were transplanted into myelosuppressed newborn mice that lacked expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, the chloride channel that is not functional in patients with cystic fibrosis, the engrafted mice showed partial restoration of CFTR channel activity, suggesting that marrow-derived epithelial cells in the GI tract were functional. However, BMT into newborn mice, regardless of the myeloablative regimen used, did not increase the number of bone marrow-derived epithelial cells over that which occurs after BMT into lethally irradiated adult mice.     

Threshold of lung injury required for the appearance of marrow-derived lung epithelia

Bone marrow-derived cells (BMDCs) can adopt an epithelial phenotype in the lung following bone marrow transplantation (BMT). This phenomenon has been assumed to result from the lung injury that occurs with myeloablative radiation. To date, no study has related the degree of epithelial chimerism following bone marrow transplantation to the lung damage induced by preconditioning for BMT. Such a goal is crucial to understanding the local host factors that promote the engraftment of BMDCs as lung epithelia. We undertook this aim by performing sex-mismatched bone marrow transplantation using a variety of preconditioning regimens and comparing measurements of lung injury (bronchoalveolar lavage [BAL] cell count, alveolar-capillary leak assayed by BAL protein levels, and terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis on epithelial cells) with rigorous methods to quantify bone marrow-derived lung epithelia (costaining for epithelial and donor markers on tissue sections and isolated lung epithelia in recipient mice). We found that only at doses that induced lung injury could marrow derived lung epithelium be identified following BMT. With irradiation doses less than 1,000 centigray (cGy), there was little to no apparent injury to the lung, and there were no marrow-derived pneumocytes despite high levels of hematopoietic chimerism. In contrast, 4 days after either split or single-dose 1,000 cGy irradiation, nearly 15% of lung epithelia were apoptotic, and with this dose, marrow-derived type II pneumocytes (0.2%) were present at 28 days. These data indicate a critical relationship between lung injury and the phenotypic change from BMDCs to lung epithelial cells.     

Hepatocyte nuclear factor-1 as marker of epithelial phenotype reveals marrow-derived hepatocytes, but not duct cells, after liver injury in mice

The potential bone marrow origin of hepatocytes, cholangiocytes, and ductal progenitor cells in the liver was examined in female mice after transplantation of bone marrow cells from male green fluorescent protein (GFP) transgenic donors. Following stable hematopoietic engraftment, the livers of the recipients were injured with carbon tetrachloride (CCl(4), with or without local irradiation of the liver) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, with or without local irradiation of the liver). The presence of numerous marrow-derived, GFP-positive inflammatory cells had the potential to lead to erroneous interpretation of marrow-derived hepatocytes, cholangiocytes, and ductal progenitor cells. Identification of marrow-derived ductal progenitor or cholangiocyte phenotype using colocalization of GFP or Y chromosome with pancytokeratin staining also failed to distinguish epithelial cells from closely apposed inflammatory cells. To address this inadequacy, we developed a rigorous new immunofluorescence protocol to identify marrow-derived epithelial cells in the liver using Y chromosome (donor marker) and hepatocyte nuclear factor-1 (HNF1, a nuclear marker of liver epithelial, nonhematopoietic phenotype). Using the Y/HNF1 method, rare (approximately one in 20,000) hepatocytes in female mice transplanted with male bone marrow contained a donor-derived Y chromosome. On the other hand, no Y chromosomes were found in cholangiocytes or ductal progenitor cells in mice with liver injury due to DDC or CCl(4). The use of a nuclear marker of mature hepatocytes or cholangiocytes, such as HNF1, improves discrimination of marrow-derived epithelial cells in tissue sections.     

Formation of pancreatic duct epithelium from bone marrow during neonatal development

Recent reports suggest that bone marrow-derived cells engraft and differentiate into pancreatic tissue at very low frequency after pancreatic injury. All such studies have used adult recipients. The aim of our studies was to investigate the potential of bone marrow to contribute to the exocrine and endocrine components of the pancreas during the normal rapid growth of the organ that occurs during the neonatal period. Five to ten million bone marrow cells from adult, male, transgenic, green fluorescent protein (GFP) mice were injected into neonatal nonobese diabetic/severely compromised immunodeficient/beta2microglobulin-null mice 24 hours after birth. Two months after bone marrow transplantation, pancreas tissue was analyzed with fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH). Co-staining of GFP, with anticytokeratin antibody, and with FISH for the presence of donor Y chromosome indicated that up to 40% of ducts (median 4.6%) contained epithelial cells derived from donor bone marrow. In some of these donor-derived ducts, there were clusters of large and small ducts, all comprised of GFP+ epithelium, suggesting that whole branching structures were derived from donor bone marrow. In addition, rare cells that coexpressed GFP and insulin were found within islets. Unlike pancreatic damage models, no bone marrow-derived vascular endothelial cells were found. In contrast to the neonatal recipients, bone marrow transplanted into adult mice rarely generated ductal epithelium or islet cells (p<.05 difference between adult and neonate transplants). These findings demonstrate the existence in bone marrow of pluripotent stem cells or epithelial precursors that can migrate to the pancreas and differentiate into complex organ-specific structures during the neonatal period.     

Alteration of marrow cell gene expression, protein production, and engraftment into lung by lung-derived microvesicles: a novel mechanism for phenotype modulation

Numerous animal studies have demonstrated that adult marrow-derived cells can contribute to the cellular component of the lung. Lung injury is a major variable in this process; however, the mechanism remains unknown. We hypothesize that injured lung is capable of inducing epigenetic modifications of marrow cells, influencing them to assume phenotypic characteristics of lung cells. We report that under certain conditions, radiation-injured lung induced expression of pulmonary epithelial cell-specific genes and prosurfactant B protein in cocultured whole bone marrow cells separated by a cell-impermeable membrane. Lung-conditioned media had a similar effect on cocultured whole bone marrow cells and was found to contain pulmonary epithelial cell-specific RNA-filled microvesicles that entered whole bone marrow cells in culture. Also, whole bone marrow cells cocultured with lung had a greater propensity to produce type II pneumocytes after transplantation into irradiated mice. These findings demonstrate alterations of marrow cell phenotype by lung-derived microvesicles and suggest a novel mechanism for marrow cell-directed repair of injured tissue.     

Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells

Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.     

Macrophage response to peripheral nerve injury: the quantitative contribution of resident and hematogenous macrophages

Whereas local microglial cells of the CNS rapidly respond to injury, little is known about the functional role of resident macrophages of the peripheral nervous system in nerve pathology. Using bone marrow chimeric rats, we recently identified individual resident endoneurial macrophages that rapidly became activated after nerve injury. However, the extent of local macrophage activation and its quantitative contribution to the total macrophage response is unknown. We now have created chimeric mice by transplanting bone marrow from green fluorescent protein (GFP)-transgenic mice into irradiated wild-type mice, allowing easy differentiation and quantification of hematogenous and resident endoneurial macrophages. After sciatic nerve crush injury, both GFP(-) and GFP(+) resident macrophages, the latter having undergone physiological turnover from the blood before injury, rapidly underwent morphological alterations and increased in number. Proliferating GFP(-) and GFP(+) resident macrophages were abundant and peaked 3 days after injury. A major lesion-induced influx of hematogenous macrophages with a disproportionate increase of GFP(+) macrophages was not observed until Day 4. Throughout all time points examined, GFP(-) resident macrophages were strikingly frequent, reaching maximum numbers 9.5-fold above baseline. There was also a notable proportion of GFP(-) resident endoneurial macrophages phagocytosing myelin and expressing major histocompatibility complex class II. Our results demonstrate for the first time that the rapid response of resident endoneurial macrophages to nerve injury is quantitatively important and that local macrophages contribute significantly to the total endoneurial macrophage pool during Wallerian degeneration.     

Optimal timing to repopulation of resident alveolar macrophages with donor cells following total body irradiation and bone marrow transplantation in mice

To determine the time required to repopulate mouse lungs with donor alveolar macrophages following total body irradiation (TBI) and bone marrow transplantation (BMT), C57Bl/6 mice were subjected to TBI with 900 cGy, followed by transplantation of bone marrow cells from mice expressing green fluorescent protein (GFP) in their somatic cells. The mice were euthanized at either 30 (n=5), 60 (n=5) or 90 (n=5) days following BMT. Thirty days following transplantation, 87.8 +/- 3.9% (mean +/- S.E.M.) circulating leukocytes in recipient mice were derived from the donor, as determined by fluorescence activated cell sorting (FACS) analysis for GFP. However, only 46.9 +/- 7.4% of the resident alveolar cells expressed GFP, indicating incomplete repopulation. By day 60 post-transplantation, the percentage of bronchoalveolar lavage fluid (BALF) cells expressing GFP reached 74.5 +/- 2.4%, remaining stable 90 days after transplantation (80.4 +/- 1.9%). We conclude that 60 days after TBI with 900 cGy and bone marrow transplantation, the majority of the lung resident alveolar macrophages is of donor origin. This study provides useful information regarding the time of reconstitution with donor alveolar macrophages in the pulmonary airspaces of recipient mice following marrow transplantation.     

Donor-derived type II pneumocytes are rare in the lungs of allogeneic hematopoietic cell transplant recipients

Lung injury is a common cause of death and disability. Stem cell-related therapies are widely viewed as offering promise for people suffering from various types of pulmonary diseases, and gender-mismatched bone marrow transplant recipients serve as natural populations in which to study the role of bone marrow-derived stem cells in recovery from pulmonary injury. We evaluated the extent of lung repopulation by type II pneumocyte descendents of adult bone marrow-derived stem cells in allogeneic hematopoietic cell transplant recipients. Recut sections were obtained from five lung biopsy specimens and autopsy lung tissues from four female recipients of transplanted mobilized peripheral blood stem cells or bone marrow from male donors. Sequential immunohistochemistry and fluorescence in situ hybridization was performed on each section to evaluate for Y-chromosome-containing type II pneumocytes. A single Y-chromosome-containing type II pneumocyte was found in one lung biopsy from one hematopoietic cell transplant recipient. After adjustment for the effects of incomplete nuclear sampling, this pneumocyte represented 1.75% of all type II pneumocytes in the biopsy sample. There was no evidence of polyploidy to suggest cell-to-cell fusion. No donor-derived type II pneumocytes were found in samples from the other three patients. In conclusion, repopulation by bone marrow-derived stem cells or their progeny occurs at a low frequency in the lungs of hematopoietic cell transplant recipients. Conversely, proliferation by local stem cell populations appears to be more important for recovery from alveolar injury.     

Role of the SDF-1/CXCR4 axis in the pathogenesis of lung injury and fibrosis

Stromal cell-derived factor-1 (SDF-1) participates in mobilizing bone marrow-derived stem cells, via its receptor CXCR4. We studied the role of the SDF-1/CXCR4 axis in a rodent model of bleomycin-induced lung injury in C57BL/6 wild-type and matrix metalloproteinase (MMP)-9 knockout mice. After intratracheal instillation of bleomycin, SDF-1 levels in serum and bronchial alveolar lavage fluid increased. These changes were accompanied by increased numbers of CXCR4(+) cells in the lung and a decrease in a population of CXCR4(+) cells in the bone marrow that did not occur in MMP-9(-)/(-) mice. Both SDF-1 and lung lysates from bleomycin-treated mice induced migration of bone marrow-derived stem cells in vitro that was blocked by a CXCR4 antagonist, TN14003. Treatment of mice with TN14003 with bleomycin-induced lung injury significantly attenuated lung fibrosis. Lung tissue from patients with idiopathic pulmonary fibrosis had higher numbers of cells expressing both SDF-1 and CXCR4 than did normal lungs. Our data suggest that the SDF-1/CXCR4 axis is important in the complex sequence of events triggered by bleomycin exposure that eventuates in lung repair. SDF-1 participates in mobilizing bone marrow-derived stem cells, via its receptor CXCR4.     

Bone marrow-derived progenitor cells contribute to lung remodelling after myocardial infarction

BACKGROUND: Congestive heart failure (CHF) causes structural modifications of the lungs that contribute to the functional limitations of affected subjects. We hypothesized that bone marrow-derived progenitor cells could contribute to lung structural remodelling after myocardial infarction (MI). METHODS: Wistar rats were irradiated and received a bone marrow transplant (BMT) from green fluorescent protein (GFP) transgenic rats, followed 5 weeks later by coronary artery ligation or sham operation. Five weeks after MI, lung immunofluorescence studies were performed and GFP expression evaluated by Western immunoblotting. RESULTS: After MI, rats developed lung structural remodelling characterized by myofibroblast (MF) proliferation in the alveolar septa. After BMT, some GFP+ cells were found in the lungs of sham animals. The amount of GFP+ cells in the lungs of MI rats was greatly increased with evidence of differentiation into MFs, as evaluated by co-localization correlation analysis with smooth muscle alpha-actin (P<.01). These cells were particularly abundant in the perivenular regions where they incorporated into the wall of blood vessels. There was a threefold increase in lung GFP protein expression after MI (P=.01). CONCLUSIONS: After MI, bone marrow-derived progenitor differentiates into lung MFs. This novel pathophysiologic process may contribute to the pulmonary manifestations of CHF and could have significant therapeutic implications.     

Conversion potential of marrow cells into lung cells fluctuates with cytokine-induced cell cycle

Green fluorescent protein (GFP)-labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung-specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit the cell cycle by exposure to interleukin-3 (IL-3), IL-6, IL-11, and Steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G(1)/S interface of the cell cycle have a three-fold increase in cells that assume a nonhematopoietic or pulmonary epithelial cell phenotype and that this increase is no longer seen in late S/G(2). These cells have been characterized as GFP(+) CD45(-) and GFP(+) cytokeratin(+). Thus, marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine-induced cell cycle transit. Previous studies have shown that the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse the cell cycle, leading to a continuum model of stem cell regulation. The present study indicates that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.     

High-resolution imaging and antitumor effects of GFP(+) bone marrow-derived cells homing to syngeneic mouse colon tumors

Bone marrow-derived cells (BMDCs) participate in the growth and spread of tumors of the breast, brain, lung, and stomach. To date, there are limited reports of bone marrow involvement in colon cancer pathogenesis, but such findings would have the potential to generate novel treatments for colon cancer patients. We have established a mouse model for imaging BMDCs from whole tumor to single-cell resolution, whereby the bone marrow of lethally irradiated host animals is reconstituted with EGFP-expressing bone marrow cells from matched TgActb(EGFP) donors. The BM transplants yield mice with fluorescently labeled bone marrow, and so BMDCs can subsequently be monitored within a tumor through optical imaging. Successful BM reconstitution was confirmed at 8 weeks after transplantation, when surviving BALB/c mice were injected with CT26 mouse colon cancer cells. We find that up to 45% of cells dissociated from the tumors are GFP(+) and approximately 50% of Lin(+), CD11b(+), and CD3(+) cells express high levels of GFP. Notably, tumor growth is reduced in BM transplanted animals, compared with untransplanted host mice or EGFP-expressing BM donor mice. A needed next step is to separate the molecular and cellular (eg, T cells, NK cells, macrophages) bases of the antitumor effect of the BMDCs from any protumorigenic effect that could be subverted for therapeutic gain.     

骨髓源干细胞参与的血管内皮更新

背景：研究表明骨髓源干细胞可塑性强,但其参与组织更新及修复的机制在转分化及细胞融合间尚存在争议。 目的：通过性别交叉骨髓移植建立雌雄嵌合体小鼠模型,观察骨髓源性干细胞是否参与内皮细胞的更新并探讨其可能的机制。 方法：采用雌性C57BL/6-GFP小鼠为供体,雄性C57BL/6小鼠为受体进行骨髓移植建立嵌合体小鼠,并应用流式细胞术分析骨髓嵌合情况。骨髓移植后20周采用Y染色体荧光原位杂交法对脑、肾、肝、脾及心脏组织Y染色体进行标记,荧光显微镜观察各组织器官中血管内皮细胞Y染色体及绿荧光蛋白表达情况。 结果与结论：①嵌合体小鼠骨髓细胞流式细胞学检测显示移植骨髓后1周骨髓GFP⁺细胞为(7.48±1.38)%、4周时达(73.92±5.57)%,结果表明成功建立性别交叉骨髓移植模型。②骨髓移植后20周嵌合体小鼠脑、肾、肝和脾组织血管壁可见绿荧光蛋白表达,且部分细胞同时表达Y染色体,表明发生骨髓源细胞和血管内皮细胞融合。脑实质及心肌组织未见绿荧光蛋白表达。结果提示骨髓源干细胞可能通过细胞融合机制参与不同组织器官中血管内皮细胞的更新。     

Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis

There is a rapid onset of organizing alveolitis/fibrosis at 120-140 d after whole lung irradiation of C57BL/6J mice. To test the hypothesis that circulating cells of bone marrow origin contribute to irradiation fibrosis, irradiated chimeric green fluorescent protein (GFP)+ C57BL/6J mice were followed for GFP+ cells in areas of lung fibrosis. In a second experimental model, C57BL/6J female mice received 20 Gy total lung irradiation, and after 60 or 80 d were intravenously injected with cells from a clonal GFP+ male bone marrow stromal cell line or male GFP+ whole bone marrow, respectively. The mice were then followed for the development of pulmonary fibrosis, and the contribution of Y-probe-positive, GFP+ cells to fibrotic areas was quantitated. Bromodeoxyuridine labeling of developing fibrotic areas showed that the cell division occurred predominantly in GFP+, Y-probe-positive, and vimentin-positive cells. Immunohistochemistry demonstrated that these cells were macrophages and fibroblasts, not endothelial cells. Mice that received manganese superoxide dismutase-plasmid/liposome intratracheal injection 24 h before total lung irradiation demonstrated a decrease in GFP+ fibroblastic cells in the lung. Thus, pulmonary irradiation fibrosis contains proliferating cells of bone marrow origin, and gene therapy prevention of this condition acts in part by decreasing the migration and proliferation of marrow origin cells.     

Maintenance and repair of the lung endothelium does not involve contributions from marrow-derived endothelial precursor cells

Lung endothelium is believed to be a quiescent tissue with the potential to exhibit rapid and effective repair after injury. Endothelial progenitor cells derived from the bone marrow have been proposed as one source of new endothelial cells that may directly contribute to pulmonary endothelial cell homeostasis and repair. Here we use bone marrow transplantation models, using purified hematopoietic stem cells (HSCs) or unfractionated whole marrow, to assess engraftment of cells in the endothelium of a variety of tissues. We find scant evidence for any contribution of bone marrow-derived cells to the pulmonary endothelium in the steady state or after recovery from hyperoxia-induced endothelial injury. Although a rare population of CD45-/CD31+/VECadherin+ bone marrow-derived cells, originating from HSCs, can be found in lung tissue after transplantation, these cells are not readily found in anatomic locations that define the pulmonary endothelium. Moreover, by tracking transplanted bone marrow cells obtained from donor transgenic mice containing endothelial lineage-selective reporters (Tie2-GFP), no contribution of bone marrow-derived cells to the adult lung, liver, pancreas, heart, and kidney endothelium can be detected, even after prolonged follow-up periods of 11 months or after recovery from hyperoxic pulmonary endothelial injury. Our findings argue against any significant engraftment of bone marrow-derived cells in the pulmonary vascular endothelium.     

Intratracheal administration of liposomal clodronate accelerates alveolar macrophage reconstitution following fetal liver transplantation

To facilitate study of alveolar macrophages in vivo, we developed a method to rapidly and efficiently replace resident alveolar macrophages with macrophages of a different (donor) genotype. Chimeric mice were generated by lethal irradiation followed by fetal liver transplantation (FLT) using green fluorescent protein (GFP) transgenic reporter mice as donors. Kinetics of peripheral blood monocyte (PBM) and alveolar macrophage reconstitution was determined 4 and 10 weeks post-FLT by quantifying the percentage of GFP+ cells. To enhance the recruitment of donor monocytes into the lung after FLT, mice were treated with intratracheal administration of liposomal clodronate to deplete host alveolar macrophages at 6 weeks post-FLT. PBM reconstitution occurred by 4 weeks after FLT (85.7+/-1.6% of CD11b+/Gr-1+ monocytes were GFP+), and minimal alveolar macrophage repopulation was observed (9.5% GFP+). By 10 weeks following FLT, 48% of alveolar macrophages were GFP+ by immunostaining of macrophages on lung tissue sections, and 55.1 +/- 1.6% of lung lavage macrophages were GFP+ by fluorescein-activated cell sorter analysis. Clodronate treatment resulted in a significant increase in GFP+ alveolar macrophages 10 weeks after FLT. By immunostaining, 90% of macrophages were GFP+ on lung tissue sections and 87.5 +/- 1.1% GFP+ in lung lavage (compared with GFP-transgenic controls). The ability of newly recruited alveolar macrophages to clear Pseudomonas aeruginosa and activate nuclear factor-kappaB in response to Eschericia coli lipopolysaccharide demonstrated normal macrophage function. Optimizing this methodology provides an important tool for the study of specific genes and their contribution to alveolar macrophage function in vivo.     

Bone marrow-derived mesenchymal stem cells in repair of the injured lung

We sought to determine whether an intact bone marrow is essential to lung repair following bleomycin-induced lung injury in mice, and the mechanisms of any protective effects conferred by bone marrow-derived mesenchymal stem cell (BMDMSC) transfer. We found that myelosupression increased susceptibility to bleomycin injury and that BMDMSC transfer was protective. Protection was associated with the differentiation of engrafted BMDMSC into specific and distinct lung cell phenotypes, with an increase in circulating levels of G-CSF and GM-CSF (known for their ability to promote the mobilization of endogenous stem cells) and with a decrease in inflammatory cytokines. In vitro, cells from injured, but not from normal, mouse lung produced soluble factors that caused BMDMSC to proliferate and migrate toward the injured lung. We conclude that bone marrow stem cells are important in the repair of bleomycin-injured lung and that transfer of mesenchymal stem cells protects against the injury. BMDMSC localize to the injured lung and assume lung cell phenotypes, but protection from injury and fibrosis also involves suppression of inflammation and triggering production of reparative growth factors.