Impaired alveolarization and vascular growth had adverse pulmonary consequences, as indicated by abnormal pulmonary function tests in premature infants suffered from BPD, and these can extend into and beyond childhood.13
Previous studies have reported that newborn rat pups exposed to hyperoxia showed markedly and persistently inhibited distal lung growth and had an increased distal air space size, which closely mimic the histology observed in human infants with new BPD.14,15
Furthermore, we have demonstrated that exposure of newborn rat pups to 90-95% hyperoxia for 2 weeks resulted in increased mortality, delayed growth, and lung injuries similar to those seen in premature human infants with BPD.5,6
In previous studies, we demonstrated that local intratracheal administration rather than systemic intraperitoneal transplantation was a more optimal route, when the MSCs were given at P5,6
and this short-term protective effects of UCB-MSCs were dose-dependent.5
As follow up-preclinical translational study, same optimal dose, timing and route for MSCs transplantation were chosen in the present study to further evaluate the long-term effects and safety of MSC transplantation in the same experimental settings with the previous studies.5,6
A recent study reported long-term (P65) failure of alveologenesis after an early short-term (P1-7) exposure to a PDGF-receptor antagonist.16
This implies that an insult in the early critical period can result in long-lasting changes in lung architecture, and suggests that permanent inhibition of alveolar formation could occur in BPD infants. These previous data are consistent well with the findings from the present study. In the present study, although somatic catch-up growth was observed during the recovery period, the alveolar and vascular growth measured by MLI and vWF, respectively, were still delayed in HC compared to NC, and these hyperoxia-induced lung injuries were attenuated in HM at P70. These findings suggest that hyperoxia-induced lung injuries persist even after a prolonged recovery period, and that the protective effects of human UCB-derived MSCs transplantation, as evidenced by improved alveolarization and angiogenesis, also are sustained up to P70.
Pulmonary inflammatory responses have been known to play a pivotal role in the development of BPD.1,14,17-19
In our previous studies,5,6
we have shown that the protective effects of human UCB-derived MSCs transplantation against hyperoxia-induced lung injuries are mediated mainly by their anti-inflammatory effects and not by their regeneration capacity.
In the current study, we found that the pulmonary inflammatory foci and ED-1 positive alveolar macrophages were persistently increased at P70 in HC than NC. The augmented number of alveolar macrophages, observed mainly in the inflammatory foci, was related to an increase in the inflammatory foci score. In a previous study,20
increased lymphocyte infiltration, a type of inflammation, was also observed in the chronic phase of BPD in the rabbit lungs, which is consistent with our data. These hyperoxia-induced inflammatory responses appeared to be attenuated in HM at P70. These findings suggest that hyperoxia-induced inflammatory responses persist even after a prolonged recovery period, and that the anti-inflammatory effects of stem cell transplantation are sustained up to P70.
It has been shown that MSCs do not elicit alloreactive lymphocyte proliferative responses,21
and the implantation of allogeneic, major-histocompatibility-mismatched MSCs have been well tolerated in baboons.22,23
This low immunogenic property of MSCs is thought to be related with the secretion of various factors that create an immunosuppressive environment.24,25
MSCs can be obtained from various sources, including bone marrow, UCB and adipose tissue.26
Among these sources, UCB is considered a promising source for human MSCs due to the low immunogenicity,27
easy availability and high proliferation capacity.28
The low expression of HLA major histocompatibility complex (MHC) class I and lack of MHC class II molecules in UCB-derived MSCs, indicates that these MSCs may evade the immune system, even in allogeneic transplantation.29
In our previous5,6
and present studies, despite xenotransplantation to the immunocompetent wild type rat, we found no apparent gross or microscopic findings consistent with abnormal immunologic reactions after local intratracheal transplantation of hUCB-MSCs. Furthermore, a hyperoxia-induced increase in WBC count (mainly neutrophil) in the blood was not significantly noticed in HM and the lymphocyte counts and its subset were not different between the experimental groups in the present study. However, further detailed studies are needed to determine whether these results are derived from the known low-immnunogenecity of MSCs or unknown poor immune responses in newborn rats.
We have previously shown the presence of human UCB-derived MSCs which were transplanted to the wild type rat pups at P14.5,6
In the present study, although they were rare, donor stem cells were persistently present in the P70 rat lungs. However, whether the donor cells observed in the lungs held stem cell properties is not clear; further investigation is needed.
The long-term safety of MSCs transplantation is not yet known. In the present study, no gross or histological abnormal findings, such as tumors, were observed in various organs in P70 rats. Rats raised up to P70 could be considered to be as old as human adults. Furthermore, the measured proportional weight (organ/body weight) of organs was not different between the NC, HC, and HM groups. These findings suggest that IT transplantation of human UCB-derived MSCs is safe without any long-term adverse effects up to P70.
Several clinical studies have demonstrated that the incidence of wheezing or asthma, or of bronchial hyper-responsiveness, is higher in BPD survivors30
and that persistent detrimental pulmonary function is correlated with the severity of BPD at diagnosis.31
Our present study demonstrated chronic ongoing lung impairment after an initial hyperoxic insult, which could be considered as supporting evidence for long-term respiratory morbidity of BPD infants. Furthermore, transplantation of UCB-derived MSCs in the acute critical period attenuated hyperoxic lung injury at P14, and this seemed to contribute to the long-term improvement evaluated at P70. These data support the possibility that transplantation of human UCB-derived MSCs may modify the long-term respiratory morbidities, if BPD infants are treated at an early critical time point.
In summary, the hyperoxia-induced retardation in alveolar and vascular growth and inflammatory responses in newborn rats were still persistent after a prolonged recovery period. The protective effects of intratracheal transplantation of human UCB-derived MSCs against neonatal hyperoxic lung injuries, such as improved alveolarization with angiogenesis and anti-inflammatory effects, were also sustained without any long-term adverse effects. Our data warrant the future translation of the transplantation of human UCB-derived MSCs into clinical studies for the treatment of BPD.