Analysis of Engraftment of CB-CD 34 Cells in Newborn Mouse Lungs

The role of umbilical cord blood (CB)-derived stem cell therapy in neonatal lung injury remains undetermined. We investigated the capacity of human CB-derived CD34 hematopoietic progenitor cells to regenerate injured alveolar epithelium in newborn mice. Doubletransgenic mice with doxycycline (Dox)-dependent lung-specific Fas ligand (FasL) overexpression, treated with Dox between embryonal day 15 and postnatal day 3, served as a model of neonatal lung injury. Singletransgenic non-Dox-responsive littermates were controls. CD34 cells (1 10 to 5 10) were administered at postnatal day 5 by intranasal inoculation. Engraftment, respiratory epithelial differentiation, proliferation, and cell fusion were studied at 8 weeks after inoculation. Engrafted cells were readily detected in all recipients and showed a higher incidence of surfactant immunoreactivity and proliferative activity in FasL-overexpressing animals compared with non-FasL-injured littermates. Cord blood-derived cells surrounding surfactant-immunoreactive type II-like cells frequently showed a transitional phenotype between type II and type I cells and/or type I cell-specific podoplanin immunoreactivity. Lack of nuclear colocalization of human and murine genomic material suggested the absence of fusion. In conclusion, human CB-derived CD34 cells are capable of longterm pulmonary engraftment, replication, clonal expansion, and reconstitution of injured respiratory epithelium by fusion-independent mechanisms. Cord blood–derived surfactant-positive epithelial cells appear to act as progenitors of the distal respiratory unit, analogous to resident type II cells. Graft proliferation and alveolar epithelial differentiation are promoted by lung injury. (Am J Pathol 2011, 178:1329–1339; DOI: 10.1016/j.ajpath.2010.11.062) Premature infants treated with supplemental oxygen and mechanical ventilation are at risk for bronchopulmonary dysplasia (BPD) or chronic lung disease of the preterm newborn, a complex condition characterized by an arrest of alveolar development. Although surfactant therapy, antenatal steroids, and changes in neonatal intensive care have modified its phenotype, BPD remains a significant complication of premature birth. The main pathological hallmark of BPD is an arrest of alveolar development, characterized by large and simplified distal airspaces. In addition, several reports have shown that the lungs of ventilated preterm infants with early BPD show markedly increased levels of alveolar epithelial cell death. Recently, it was demonstrated that increased alveolar epithelial apoptosis induced by Fas ligand (FasL) overexpression in newborn mice is sufficient to disrupt alveolar remodeling, supporting the notion that the loss of alveolar epithelial cells plays a critical role in the arrested alveolar development seen in BPD. These findings suggest that cell-based therapy aimed at restoring or protecting the alveolar epithelium in injured newborn lungs may be beneficial. Multiple publications during the past decade have suggested that bone marrow–derived stem and progenitor cells can structurally engraft as mature differentiated airway and alveolar epithelial cells. Epithelial engraftment is suggested by some investigators to be a rare event, regardless of the marrow-derived cell type used or the type of antecedent lung injury. In fact, it has recently been questioned whether engraftment and transdifferentiation can occur at all, based on failure to duplicate these results using state-of-the-art morphological techniques. Even if several studies have reported seemingly unequivocal engraftment of donor-derived airway and/or alveolar epithelium after adult stem cell administration, functional reconsti-