Long-term (postnatal day 70) outcome and safety of intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells in neonatal hyperoxic lung injury

Abstract

Purpose: This study was performed to evaluate the long-term effects and safety of intratracheal (IT) transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in neonatal hyperoxic lung injury at postnatal day (P)70 in a rat model.

Materials and methods: Newborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (90% oxygen) within 10 hours after birth and allowed to recover at room air until sacrificed at P70. In the transplantation groups, hUCB-MSCs (5×10⁵) were administered intratracheally at P5. At P70, various organs including the heart, lung, liver, and spleen were histologically examined, and the harvested lungs were assessed for morphometric analyses of alveolarization. ED-1, von Willebrand factor, and human-specific nuclear mitotic apparatus protein (NuMA) staining in the lungs and the hematologic profile of blood were evaluated.

Results: Impaired alveolar and vascular growth, which evidenced by an increased mean linear intercept and decreased amount of von Willebrand factor, respectively, and the hyperoxia-induced inflammatory responses, as evidenced by inflammatory foci and ED-1 positive alveolar macrophages, were attenuated in the P70 rat lungs by IT transplantation of hUCB-MSCs. Although rare, donor cells with human specific NuMA staining were persistently present in the P70 rat lungs. There were no gross or microscopic abnormal findings in the heart, liver, or spleen, related to the MSCs transplantation.

Conclusion: The protective and beneficial effects of IT transplantation of hUCB-MSCs in neonatal hyperoxic lung injuries were sustained for a prolonged recovery period without any long-term adverse effects up to P70.

Fig. 1

Histology and morphometric analysis of the surviving P70 rat lung. (A) Representative optical microscopic photographs of the lungs stained with hematoxylin and eosin (original magnification; ×100, scale bars; 50 um). (B) Degree of alveolarization measured by mean linear intercept. Data are expressed as mean±SEM; ^*p<0.05 versus NC; ^†p<0.05 versus HC. NC, normoxia control group; HC, hyperoxia control group; HM, hyperoxia with human UCB-derived MSCs transplantation group; MSCs, mesenchymal stem cells; SEM, standard error of the mean.

Fig. 2

(A) Representative immunofluorescence photomicrographs of von Willebrand factor (vWF) staining in the lungs of P70 rats. vWF was labeled with green fluorescent marker 5(6)-carboxyfluoresceindiacetate N-succinimidyl ester and the nuclei were labeled with 4',6-diamidino-2-phenylindole (blue) (scale bars; 25 um). (B) The immunofluorescence density of vWF per lung section. Data are expressed as mean±SEM; ^*p<0.05 versus NC; ^†p<0.05 versus HC. NC, normoxia control group; HC, hyperoxia control group; HM, hyperoxia with human UCB-derived MSCs transplantation group; MSCs, mesenchymal stem cells; SEM, standard error of the mean.

Fig. 3

(A) Representative photomicrographs of ED-1 positive cells in the lungs of P70 rats. ED-1 positive alveolar macrophages were labeled with CFSE (green), and the nuclei were labeled with DAPI (blue) (scale bar; 25 um). (B) Number of ED-1 positive cells per high power field in P70 rat lungs. (C) Representative optical microscopic photographs of the inflammatory foci grade in the P70 rat lungs stained with hematoxylin and eosin (scale bar; 100 um). (D) Severity scores of the inflammatory focis in the in P70 rat lungs. Data are expressed as mean±SEM; ^*p<0.05 versus NC. NC, normoxia control group; HC, hyperoxia control group; HM, hyperoxia with human UCB-derived MSCs transplantation group; CFSE, 5(6)-carboxyfluoresceindiacetate N-succinimidyl ester; DAPI, 4',6-diamidino-2-phenylindole; MSCs, mesenchymal stem cells; HPF, high power field; SEM, standard error of the mean.

Fig. 4

Donor cell localization in the lung of the P70 rats. Human specific nuclear mitotic apparatus protein (NuMA) positive human UCB-derived MSCs were labeled with CFSE (green), and the nuclei were labeled with DAPI (blue) (scale bars; 25 um). UCB, umbilical cord blood; MSCs, mesenchymal stem cells; CFSE, 5(6)-carboxyfluoresceindiacetate N-succinimidyl ester; DAPI, 4',6-diamidino-2-phenylindole.

Fig. 5

The number of white blood cells (WBC) (A), neutrophil (B), monocyte (C), and the number of lymphocyte with T cell (CD4 positive and CD8 positive T cell) (D) in each experimental group at P70. Although WBC counts were significantly increased in HC compared to NC, there were no significant differences in lymphocyte counts and subset between the experimental groups. Data are expressed as mean±SEM; ^*p<0.05 versus NC. NC, normoxia control group; HC, hyperoxia control group; HM, hyperoxia with human UCB-derived MSCs transplantation group; MSCs, mesenchymal stem cells; SEM, standard error of the mean.