Differentiation of xenografted human fetal lung parenchyma

Abstract

The goal of this study was to characterize xenografted human fetal lung tissue with respect to developmental stage-specific cytodifferentiation. Human fetal lung tissue (pseudoglandular stage) was grafted either beneath the renal capsule or the skin of athymic mice (NCr-nu). Tissues were analyzed from 3 to 42 days post-engraftment for morphological alterations by light and electron microscopy (EM), and for surfactant protein mRNA and protein by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC), respectively. The changes observed resemble those seen in human lung development in utero in many respects, including the differentiation of epithelium to the saccular stage. Each stage occurred over approximately one week in the graft in contrast to the eight weeks of normal in utero development. At all time points examined, all four surfactant proteins (SP-A, SP-B, SP-C, and SP-D) were detected in the epithelium by ICC. Lamellar bodies were first identified by EM in 14 day xenografts. By day 21, a significant increase in lamellar body expression was observed. Cellular proliferation, as marked by PCNA ICC and elastic fiber deposition resembled those of canalicular and saccular in utero development. This model in which xenografted lung tissue in different stages of development is available may facilitate the study of human fetal lung development and the impact of various pharmacological agents on this process.

Figure 1. Both types of grafts recapitulate stages of in utero lung development

Panel A: Starting fetal lung tissue (16 weeks gestation). Panel B: Epithelial thinning and future air space enlargement seen after 3 days of grafting. Panel C: Grafts between 6 and 14 days show progression of differentiation. Panel D: Post 14 day grafts with flattening of epithelium. Panel E: Distended saccules with flattened epithelium are observed after 28 days of grafting. Panel A is characteristic of pseudoglandular stage of development. Changes in Panel B are consistent with the late pseudoglandular stage. In Panel C grafts resemble the canalicular stage of development, forming acini and increased number of capillaries. In Panel D grafts resemble saccular stage of in utero development display flattening of the epithelium, and differentiation of pre-Type II cells into Type II cells, and further differentiation onto Type I cells. Alveolar spaces are considerably enlarged, and prominent alveolar septation is evident. In Panel E, differentiation of the epithelium to Type I cells, thinning of alveolar walls, and neovascularization are observed. Legend: I: low magnification (4×) of subcapsular grafts; II: low magnification (4×) of subcutaneous grafts; III: high magnification (A, B, C, and D at 40×; E at 20×) of subcutaneous grafts.

Figure 2. Cellular proliferation during graft development shown by ICC of PCNA

Panel A: negative control - mouse kidney of subcapsular graft. Panel B: limited PCNA immunopositive cells at 16 weeks development. Panel C: 6 day graft with majority of PCNA immunopositive cells in cannalicular stage of development (marked with *). Panel D and Panel E: 10 and 14 day grafts, respectively, abundant with PCNA immunopositive cells. Panel F: 21 day graft. In Panel A there is no staining within mouse tissue section. No brown cells are detectable indicating complete inhibition of endogenous peroxidase and lack of specific PCNA staining. Therefore, the PCNA positive cells in the other sections are of human and not mouse origin. In Panel C, PCNA immunopositive cells were observed within the tissue sections entering the cannalicular stage of development (marked with *), rather than those still in pseudoglandular stage (marked ps). In Panel D and E changes in the morphology of tissue are represented by the increasing number of PCNA immunopositive cells. Panel F shows a 21 day graft entering the saccular stage of development with sparsely distributed PCNA positive cells. Pseudoglandular and cannalicular stages are defined in Figure legend 1. Figures are representative of three experiments. All pictures are at 20× magnification.

Figure 3. Elastic fiber proliferation is detectable in saccular stage

Panel A: 6 day graft lacking elastic fiber staining as would be expected in pseudoglandular/cannalicular stage of development. Panel B: 14 day graft with elastic fibers visible as black punctate dots (arrows) at the tips of developing alveolar crests. Panel C: adult lung tissue (positive control) has an abundance of layered elastic fibers within the thin alveolar walls (arrows). The inset in panel B (dotted line) shows a high magnification (40×) of an alveolar crest with elastic staining (arrows). Hardly any staining was observed in 21 day graft (not shown). There is an abundance of collagen fibers in all tissues (red). Figures are representative of three experiments. Panels A, B, and C are at 20× magnification.

Figure 4. Lamellar bodies found in grafts, consistent with in utero development

Panel A1 and A2: Lamellar bodies are absent in starting fetal lung tissue. Pre- Panel B1 and B2: Lamellar bodies are first seen in 14 day grafts. Panel C1 and C2: 21 days post-grafting, the number of lamellar body positive cells and lamellar bodies per cell is sharply increased. (first 60 characters). In Panel A, Pre-Type II columnar cells are rich with glycogen. As shown in Panel B, 14 day grafts grafts are in early saccular stage of development, as evidenced by flattening of epithelial cells and scarce microvilli. In Panel C, numerous secreted lamellar bodies are seen in the alveolar spaces, but no tubular myelin-like structures are observed.

Figure 5. Surfactant proteins are present in starting tissues as shown by ICC

The primordia of alveoli are lined by cuboidal pre-type II cells that are immunopositive for SPs. Localization of SP-A (Panel A), SP-B (Panel B), and SP-C (Panel C) is uniform within the cells. Localization of SP-D (Panel D) is limited to the apical surface of the cells. Panels are shown at medium magnification (20×).

Figure 6. Surfactant proteins are present during graft development by ICC

Surfactant proteins are abundant in 21 day grafts shown in Figure. Immunostaining for SP-A is presented in Panel A, for SP-B in Panel B, for SP-C in Panel C, and for SP-D in Panel D. Localization of SP-A, SP-B, and SP-C (insert) is uniform within the cell. Localization of SP-D is limited to the apical surface of the cells (insert). After longer periods of grafting, cyto-differentiation is observed and fewer cells found to stain positive for surfactant proteins. This would be expected in a developing epithelium, where immuno positive pre-Type II cells are differentiating into Type II cells (immuno positive) which can then become Type I cells (immuno negative) (not shown). Panels are shown in low magnification (4×) and inserts are in high magnification (40×).

Figure 7. SP-A is present throughout graft development by ICC

Panel A: 6 day graft with pre-type II cuboidal cells immunopositive for SP-A. Panel B: 10 day graft with thinning epithelial cells predominantly SP-A immunopositive. Panel C: 14 day graft with saccular-like structures and differentiation of pre-type II into type II cells (both SP-A immunopositive). Panel D: 28 day graft with thinning saccules and septae. Panels shown at medium magnification (20×).

Figure 8. Human PECAM-1 is detectable by immunocytochemistry in early stage grafts

Panel A: Presence of blood vessels of human origin within the mesenchyme of a 3 day graft is indicated by PECAM-1 immunopositive cells (brown, arrows). Panel B: Branching of the PECAM-1 immunopositive clusters of cells (arrow) and formation of blood vessels (arrowhead) is evident in 6 day graft. Panel C: As differentiation progresses PECAM-1 staining becomes significantly fainter and mostly visible in the subpleural areas of the 14 day graft (as indicated by arrows). All panels are shown at medium magnification (20×).