Human amniotic fluid stem cells can integrate and differentiate into epithelial lung lineages

Abstract

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein-injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10-kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung. Disclosure of potential conflicts of interest is found at the end of this article.

Figure 1

hAFSC integrate into embryonic lung epithelium and mesenchyme and express the early marker of differentiation TTF1. Embryonic lung at embryonic stage 11.5 was microinjected with 10⁴ hAFSC and cultured for 1 week. Immunohistochemistry for TTF1 (green) showed epithelial cells. hAFSC labeled with CM-Dil (red) were found in the epithelium (A), as well as the mesenchyme (B). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue) (×63). (C): Reverse transcription-polymerase chain reaction from embryonic lungs a week after the microinjection showed that hAFSC start to express TTF1. Primers were designed to discriminate human from mouse TTF1. Abbreviations: ACTB, β-actin; hAFSC, human amniotic fluid stem cells; hLung, human lung; m-eLung, mouse embryonic lung; TTF1, thyroid transcription factor 1.

Figure 2

Human amniotic fluid stem cells (hAFSC) can integrate into adult mouse lung after tail vein injection. (A–C): Bioluminescence of mice injected with amniotic fluid stem cells (AFSC) expressing luciferase. After 21 days AFSC were still detected at the lung position. (A–C) present different levels of integration. Magnified views of the thorax are shown below each panel. Relatively intense AFSC luciferase bioluminescence was detected shortly after tail vein injection (4 h). At subsequent times, persistent bioluminescence was detected, albeit requiring increased sensitivity in the settings of the detector (color scale is calibrated in arbitrary bioluminescence units). In (D), the yellow arrow shows CM-Dil-labeled hAFSC (red) in an alveolar wall, corresponding with a type I pneumocyte stained for PDPN (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). In (E), chromogenic in situ hybridization for Y Chr also showed hAFSC (yellow arrow) in the alveolar wall position. Nuclei were counterstained with hematoxylin. In (F), immunohistochemistry for the antigen F4/80 was performed to detect macrophage activity (green). The arrow points to a group of CM-Dil-labeled hAFSC (red) that were not near macrophages (×63). Abbreviations: h, hours; PDPN, podoplanin; Y Chr, Y chromosome.

Figure 3

Lung injury increases the level of integration of hAFSC. (A): Absolute quantification of Sry genes on the Y chromosome by real-time polymerase chain reaction. Data are reported as Y chromosome copy number and thus the number of hAFSC per 1,000 lung cells. A standard curve was performed using a synthetic oligonucleotide (E = 1.94; error = 0.066). The graph shows the number of hAFSC in Cntr mice and in mice after oxygen versus naphthalene injury (n = 4) at 7, 15, and 40 days. At 7 days the number of hAFSC was significantly elevated (more than sevenfold) in naphthalene-injured trachea versus fourfold in oxygen-injured parenchyma compared with uninjured Cntr lung (all p < .001). At 15 days naphthalene-injured and oxygen-injured mice reached similar numbers of hAFSC compared with uninjured Cntr lung (3- and 3.5-fold, respectively) (B): Engraftment of hAFSC into mouse adult lung increased after O₂ injury. Data are reported as percentage of donor cells on the total lung population. Integration of hAFSC was compared between untreated and O₂-exposed lungs of adult nude mice. The analysis of three time points (n = 6) showed significant increases of integration (p < .001) 7 days (3.8-fold), 15 days (3.6-fold), and 40 days (3.1-fold) after damage. Abbreviation: Cntr, control.

Figure 4

hAFSC have differentiated into epithelial lung lineages 2 weeks after tail vein injection. (A): Reverse transcription-polymerase chain reaction (RT-PCR) PDPN for prior injection of hAFSC. (B): Immunohistochemistry showed that hAFSC expressed endogenous PDPN (green) (×40). (C–F): Fluorescence in situ hybridization for Y Chr showed integrated cells (white arrow) positive for TTF1 (red). Magnification: ×32 for (C, D) and ×63 for (E, F). (G): Cytospin samples showed hAFSC positive for both TTF1 (red) and Y Chr (green). Samples were counterstained with 4′,6-diamidino-2-phenylindole (blue) (×100). (H): RT-PCR for TTF1 7 months after the tail vein injection. Primers were designed to discriminate human from mouse TTF1. hACTB was used as a control for the presence of hAFSC mRNA, and mACTB was used as a loading control. Abbreviations: ACTB, β-actin; hACTB, human β-actin; hAFSC, human amniotic fluid stem cells; hLung, human lung; hTTF1, human thyroid transcription factor 1; mACTB, mouse β-actin; mLung, mouse lung; PDPN, podoplanin; TTF1, thyroid transcription factor 1; Y Chr, Y chromosome.

Figure 5

Human amniotic fluid stem cells (hAFSC) can acquire a type II pneumocyte phenotype. (A, B): Murine adult lung treated with >99% oxygen for 3 days was processed for immunohistochemistry (IHC) 15 days after tail vein injection. CM-Dil-labeled hAFSC (red) were found positive for type II pneumocyte marker pro-SPC (green). Magnification: ×25 for (A) and ×63 for (B). (C): The same lung was subjected to single-cell analysis after cytospin. A male hAFSC injected into a female mouse was localized by fluorescence in situ hybridization for Y Chr (green) and was positive for pro-SPC (red) on IHC. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue) (×100). Abbreviations: SPC, surfactant protein C; Y Chr, Y chromosome.

Figure 6

hAFSC can integrate into the upper airways and express the Clara cell-specific marker CC10. First, lungs were damaged with 275 mg/kg naphthalene, and then ∼2.5 × 10⁵ hAFSC were administered intratracheally. (A): Fifteen days after the damage, endogenous Clara cells expressing CC10 (red) were repopulating the p airways. Dotted lines (white) delineate the boundary between p and d airways at the bronchioalveolar junction (×40). (B): Male hAFSC detected by fluorescence in situ hybridization (FISH) for Y Chr (green) were found at the bronchioalveolar junction (×100, enlarged from [A]). (C): Immunohistochemistry for CC10 (red) was combined with FISH for Y Chr (green) (×40). Arrows show hAFSC integrated into the upper airways and positive for both CC10 and Y Chr. Inset is a lower-magnification view to demonstrate three examples (arrows). (D): Lungs were also processed for cytospin to detect single-cell expression. hAFSC positive for both CC10 (red) and Y Chr (green) were found (×100). All samples were nuclear counterstained with 4′,6-diamidino-2-phenylindole (blue). (E): Reverse transcription-polymerase chain reaction for human CC10 40 days after intratracheal administration of hAFSC. Primers were designed to discriminate human from mouse CC10. hACTB was used as a control for the presence of hAFSC mRNA, and mACTB was used as a loading control. Abbreviations: CC10, Clara cell 10-kDa protein; d, distal; hACTB, human β-actin; hAFSC, human amniotic fluid stem cells; hCC10, human Clara cell 10-kDa protein; hLung, human lung; mACTB, mouse β-actin; mLung, mouse lung; p, proximal; Y Chr, Y chromosome.

Figure 7

CXCR4 and SDF1 expression during naphthalene lung injury. The CXCR4/SDF1 axis may be involved in hAFSC plasticity. (A): Immunohistochemistry (IHC) showed that cultured hAFSC expressed CXCR4 (green), indicating that they may be responsive to presence of the ligand SDF1. (B): Following naphthalene injury, hAFSC were injected intratracheally. Relative quantification by real-time polymerase chain reaction (PCR) for hCXCR4 showed that hAFSC in the airway started to lose CXCR4 expression over time but that a significant 47% ± 23% of expression was still detectable 1 week after the administration of hAFSC. (C–E): IHC showed that SDF1 was constitutively expressed in the airways and that SDF1 was upregulated during naphthalene injury. (F): Real-time PCR showed that SDF1 expression was significantly increased (16-fold; p < .001) 1 week after naphthalene injury and that this persisted at fourfold for at least 2 weeks. Abbreviations: cntr, control; hAFSC, human amniotic fluid stem cells; hCXCR4, human CXCR4; SDF1, stromal cell-derived factor 1.