In this study we have demonstrated for the first time that hAFSC can undergo lung-specific lineage differentiation and that these cells possess a certain level of plasticity in response to different types of lung damage. In vitro microinjection into mouse embryonic lung showed the ability of hAFSC to engraft and differentiate by expressing the early lung marker TTF1. The expression of TTF1 1 week after microinjection suggests that hAFSC can commit to differentiation into lung lineages shortly after integration and prompted us to evaluate hAFSC for longer-term experiments in adult mice.
To determine the ability of AFSC to home in adult lung, we used AFSC expressing luciferase and detected the bioluminescence after tail vein injection. For these experiments, no damage to the lung of the recipient nude mice was done, but we were able to find a basal level of engraftment after AFSC administration in uninjured lung. AFSC were concentrated in the lung shortly after injection. During the following days AFSC circulated around the body, concentrating in the liver and in the lung but having low signals from other parts, including the head in some cases. Most importantly, AFSC were detectable in the lung 3 weeks after the injection, suggesting that there was a reasonable window of time in which to study them in the adult lung. The integration and persistence of AFSC in the absence of damage to the recipient lung was confirmed with direct analysis of CM-Dil-labeled AFSC after sectioning of the injected lungs at different time points. Quantitative analysis of the Y chromosome by real-time PCR confirmed the integration of AFSC. Despite the ability of hAFSC to integrate into the lung of nude mice in absence of injury, under these conditions we were not able to identify hAFSC differentiated into epithelial cells in the absence of injury. This result is consistent with the necessity of producing damage of the recipient lung to obtain stem cell-derived epithelial cells [12
After exposure to oxygen, hAFSC exhibit significantly stronger engraftment, probably due to enhanced niche accessibility [13
]. Hyperoxia is known to cause oxidative damage of alveolar cells, acting directly because of cellular oxygen toxicity and indirectly through the accumulation of inflammatory mediators [14
]. The engraftment of hAFSC into the three-dimensional scaffold of the lung during the first days after injection showed a distribution in clusters positioned around small vessels. This pattern was previously reported for injection of marrow-derived cells into lung [15
]. The percentage of integration was analyzed, counting integrated hAFSC after sectioning and with absolute quantification of Y chromosome by real-time PCR, with comparable results. One week after injection the number of integrated cells among the total population was ~6%. This percentage decreased over time, but a detectable level of cells persisted at 2 weeks and 40 days. Combining Y chromosome detection and pan-cytokeratin as an epithelial marker on IHC, we tried to address the percentage of integrated AFSC going through differentiation. Approximately 37% of the cells integrated at 40 days were going through differentiation, indicating that 0.5% of the lung population was represented by hAFSC differentiated into epithelial lineages. Notably, human TTF1 was still found by RT-PCR and IHC after 7 months from the injection into adult mice. This result suggests that at least some of the integrated hAFSC may have retained a self-renewal capability and can maintain the expression of this early differentiation marker.
Pro-SPC expression by hAFSC has been observed during our analysis by IHC, and this was confirmed with Y chromosome on single cells, suggesting that engraftment as type II pneumocytes can occur. However, the major caveat on this conclusion is that we could not detect SPC expression with RT-PCR with human-specific primers.
After naphthalene injury, hAFSC showed localization within the bronchial tissue versus at bronchioalveolar junctions, where the damage to Clara cells had occurred, depending on the respective route of administration: i.v. versus intratracheal. Naphthalene is an aromatic hydrocarbon used to injure the lung by specifically destroying Clara cells that express the cytochrome P450 (Cyp2f2) [16
]. hAFSC given by tail vein injection after naphthalene injury accumulated in the upper airway, increased numbers compared with after oxygen treatment. This behavior suggests a certain level of plasticity of AFSC in responding to different types of lung damage.
The chemokine SDF1 is involved in progenitor cell trafficking [17
], and the CXCR4/SDF1 signaling axis is involved in the homing of circulating stem cells into bleomycin-injured lung [18
]. We found that cultured hAFSC express hCXCR4 and retain a significant amount of hCXCR4 expression 7 days after the administration of the cells to a naphthalene-injured lung. SDF1, which is the ligand of CXCR4, is constitutively expressed in the airways, and its level of expression has a peak after 7 days from naphthalene injury. Taken together, these observations are consistent with a possible involvement of the CXCR4/SDF1 signaling axis on the plasticity of hAFSC observed in this study.
hAFSC added intratracheally after naphthalene injury initially had an higher level of epithelial integration, possibly due to the direct administration (). During the following days, there was a progressive reduction of integrated cells from 15 to 40 days. After naphthalene injury, intratracheally administered hAFSC were found at bronchioalveolar positions where CC10-positive cells had been damaged. Also, FISH for Y chromosome and IHC for CC10 on tissue sections, as well as single-cell cytospin, showed that hAFSC in these conditions can start to express CC10, indicating that they may differentiate into Clara cells ().
We tried to determine whether hAFSC, once integrated into the recipient lung, were undergoing cellular fusion with endogenous resident cells (supplemental online Fig. 1
). During the analysis of tissue and cytospin samples, we never found Y chromosome-positive cells in combination with the X chromosome. However, considering the small absolute number of Y chromosome-positive cells identified, we are unable to positively exclude the possibility that hAFSC may occasionally fuse with resident cells of recipient mouse lung.