Neonatal Donors Research Articles

Abstract 2147: Impact Of Donor Age On Microvasculature Self-assembly Of Induced Pluripotent Stem Cell Derived Endothelial Progenitors

Introduction: Current vascular tissue-engineering using induced pluripotent stem cells (iPSCs) predominantly relies on somatic cells obtained from newborn or fetal donors. However, iPSCs sourced from aged individuals exhibit epigenetic differences in angiogenic genes that may hinder their utility in iPSC derived vasculature. We hypothesized that, due to these differences, iPSCs from aged donors will exhibit decreased vasculogenic potential. Methods: We differentiated iPSCs into endothelial progenitors (iEPs) and encapsulated them in an interpenetrating polymer network hydrogel containing norbornene-functionalized hyaluronic acid and Collagen I at cell densities and hydrogel stiffness optimal for vasculogenesis. Seven days after encapsulation, the hydrogels were fixed, and the resulting microvasculature imaged. We then utilized a previously developed computational pipeline to quantify several vasculogenic properties. Results: We derived iEPs from four separate iPSC lines: 2 sourced from neonatal donors (ND) and 2 from mature donors (MD). Irrespective of donor age, differentiation yielded ~15% (n=4) iEPs of the total cell population. The two ND lines developed a similar plexus, whereas this structure was diminished in one adult line and absent in the second. Between iEPs matched for sex and somatic cell origin, there is a 50% decrease in vessel volume fraction and number of links between vessels, a 66% reduction in the percentage of vessels connected to the largest vessel, and a 43% reduction in branch points between vessels ( Fig. 1 ). Conclusion: Overall, these data suggest MD iEPs are less likely to form dense interconnected microvasculature as compared to ND iEPs. Interestingly, the average diameter did not change across all cell lines. This suggests that while MD iEPs exhibited cell clustering, they were less likely to branch out and interconnect with other clusters of iPSC-EPs to form interconnected networks

Cell Senescence-Independent Changes of Human Skin Fibroblasts with Age.

Skin ageing is defined, in part, by collagen depletion and fragmentation that leads to a loss of mechanical tension. This is currently believed to reflect, in part, the accumulation of senescent cells. We compared the expression of genes and proteins for components of the extracellular matrix (ECM) as well as their regulators and found that in vitro senescent cells produced more matrix metalloproteinases (MMPs) than proliferating cells from adult and neonatal donors. This was consistent with previous reports of senescent cells contributing to increased matrix degradation with age; however, cells from adult donors proved significantly less capable of producing new collagen than neonatal or senescent cells, and they showed significantly lower myofibroblast activation as determined by the marker α-SMA. Functionally, adult cells also showed slower migration than neonatal cells. We concluded that the increased collagen degradation of aged fibroblasts might reflect senescence, the reduced collagen production likely reflects senescence-independent processes.

Biomimetic human skin model patterned with rete ridges

Rete ridges consist of undulations between the epidermis and dermis that enhance the mechanical properties and biological function of human skin. However, most human skin models are fabricated with a flat interface between the epidermal and dermal layers. Here, we report a micro-stamping method for producing human skin models patterned with rete ridges of controlled geometry. To mitigate keratinocyte-induced matrix degradation, telocollagen–fibrin matrices with and without crosslinks enable these micropatterned features to persist during longitudinal culture. Our human skin model exhibits an epidermis that includes the following markers: cytokeratin 14, p63, and Ki67 in the basal layer, cytokeratin 10 in the suprabasal layer, and laminin and collagen IV in the basement membrane. We demonstrated that two keratinocyte cell lines, one from a neonatal donor and another from an adult diabetic donor, are compatible with this model. We tested this model using an irritation test and showed that the epidermis prevents rapid penetration of sodium dodecyl sulfate. Gene expression analysis revealed differences in keratinocytes obtained from the two donors as well as between 2D (control) and 3D culture conditions. Our human skin model may find potential application for drug and cosmetic testing, disease and wound healing modeling, and aging studies.

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition.

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium. To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model. A neonatal rat heart is transplanted into an adolescent rat's abdomen and connected to the recipient's infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients. This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Potential of neonatal organ donation and outcome after transplantation.

Organ transplantation is limited by access to suitable organs. Infant recipient waitlist mortality is increased due to the scarcity of size-matched organs. Neonatal organ donors have been proposed as an underutilized source of donor organs. However, the literature on the actual prevalence and outcome of neonatal organ donation and transplantation is fragmented and not well analyzed. This literature review aims to summarize the available literature on the potential of neonatal organ donation and to analyze published cases of neonatal organ transplantation. A systematic search of the Medline and Cochrane databases yielded 2964 articles, which were screened for eligibility. In total, 86 articles were considered eligible, of which 34 were included in the literature review: 8 articles describing the potential of neonatal organ donation programs, and 26 articles describing clinical transplantation. Current evidence suggests there is a large pool of potential neonatal organ donors. In contrast, the literature on neonatal organ donor utilization is sparse. However, case series of successful kidney, heart, liver, hepatocyte, and multivisceral transplantation using organs from neonatal donors are summarized. Although good posttransplant organ function was achieved, the use of neonatal organs is associated with increased risk of thrombosis in both kidney and liver transplantation. Neonatal organ donation is a promising alternative for expanding the current donor pool. Experience is limited, but reported patient and graft survival are acceptable and more research on the subject is warranted.

In vitro responses of human dermal fibroblasts to mechanical strain: A systematic review and meta-analysis

In vitro research in the field of mechanotransducive regulation of dermal fibroblasts is characterized by highly variable methodology and contradictory results. The primary objective of this systematic review was to establish how in vitro mechanical stretch affects human dermal fibroblast function, by means of a quantitative synthesis of all available evidence. The secondary objectives were to examine the effects of covariates related to donor age, fibroblast origin, experimental treatments, and mechanical stimulation parameters on dermal fibroblast responsiveness to mechanical strain. Summary outcomes for fibroblast proliferation and collagen production were combined using a fixed-effects meta-analytical model. Subgroup analysis and meta-regression were used to investigate the effects of different conditions on the summary outcomes. Mechanical strain was found to not affect fibroblast proliferation in neonatal fibroblasts, while adult fibroblasts proliferation was significantly increased. Collagen production was significantly increased in response to mechanical stimulation, with Vitamin C stimulation as the most important covariate. Stretching frequency emerged as positively associated with fibroblast proliferation and negatively associated with collagen production. We conclude from this study that distinct differences exist in the effects of mechanical stretching between dermal fibroblasts from neonatal and adult donors, which will help to further elucidate the pathophysiological mechanism behind tension-induced scarring.

Abstract 14959: Mitochondrial Transplantation in Pediatric and Neonatal Porcine Hearts in a Model of DCD Heart Transplantation

Introduction: Heart transplantation in pediatric and neonatal patients is limited by the donor pool. Donation after circulatory death (DCD) could potentially expand the donor pool by offering an additional source of cardiac allografts. In this study, we investigated the efficacy of mitochondrial transplantation to enhance myocardial function in pediatric and neonatal DCD hearts. Hypothesis: Autologous mitochondrial transplantation enhances the viability and function of pediatric and neonatal DCD donor hearts. Methods: Circulatory death was induced by extubation in neonatal (25 - 50g heart weight, representing 4-16 month human) and pediatric (68 - 98g heart weight, representing 4-6 year human) swine model. Hearts were subjected to 20 min of warm ischemia and 10 min of cold cardioplegic arrest and then harvested. The aorta and pulmonary artery were cannulated, inferior and superior venae cavae ligated and a balloon was inserted in the left ventricle. The hearts were then mounted on the ex situ perfusion device. After 15 minutes of reperfusion, hearts received either vehicle alone ([VEH], 10 mL n = 6) or vehicle containing autologous mitochondria ([MT] 5x10 9 in 10 mL; n = 6). A Sham non-ischemic group (n = 4) did not undergo warm ischemia, mimicking donor after brain death (DBD). Hearts were machine perfused for 2 hours unloaded and 2 hours of loaded conditions. Results: Following 4 hours of reperfusion, neonatal and pediatric DCD hearts receiving VEH showed significantly decreased myocardial function and viability as compared to Sham non-ischemic control hearts. In contrast, in both neonatal and pediatric DCD hearts receiving MT, LVDP, dP/dt max, and fractional shortening were significantly increased (p < 0.001 for each) and were equal or better than that observed in Sham non-ischemic hearts. Infarct size was significantly decreased in both neonatal and pediatric DCD hearts receiving MT as compared to VEH (p < 0.001). Conclusions: Mitochondrial transplantation provides for significantly enhanced preservation of myocardial function and viability in neonatal and pediatric DCD hearts that is equivalent to or better than that observed in DBD hearts. Mitochondrial transplantation provides a possible option to expand the pediatric and neonatal heart donor pool.

Implementation of a neonatal donation protocol in the neonatal intensive care unit: A single-center experience.

Lack of specific protocols for neonatal donation contributes to the rarity of neonatal donors. In this study, we evaluate the impact of the implementation of a neonatal donation protocol in our NICU. In this single-center study, we conducted a retrospective chart review of neonatal deaths in our NICU from January 2013 to January 2022. The study was divided into two periods: before and after the implementation of a neonatal donation protocol. The referral rates of potential neonatal donors to the OPO in the two periods were compared using the chi-square test. A p value < .05 was considered statistically significant. Sixty-four infants were reviewed. Seven (10.9%) met the inclusion criteria for potential neonatal donors after DCC. The referral rate of potential neonatal donors increased from 2.5% to 16.7% after the implementation of this protocol (p = .041), and one infant (4.1%) became an effective heart-valve donor. The implementation of a local neonatal donation protocol could have contributed to increase the referral rate of potential neonatal donors in our NICU. Following the implementation of a local neonatal donation protocol, we were able to perform a heart-valve donation for the first time in our unit.

P8.050: Pediatric Donor Transplants to Adult Recipients: Follow-up And Outcome

Introduction: The use of kidneys from pediatric donors, neonates in adults, is an option considered possible in teams dedicated to kidney transplantation. We present our experience with transplantation of separately kidneys from i neonatal donors implanted in adult recipients. Patients: Four adult patients aged 25 to 35 years were transplanted with kidneys from neonatal donors, less than 1 year old, with the usual technique. They received immunosuppression with Cyclosorine, Azathioprine and Prednisone. Results: During the first 15 days it was necessary to practice hemodialysis. From then on, the renal function of the graft improved until plasma creatinine of 1.7±0.5 mg/dl was reached. After the first year of follow-up, creatinine stabilized at around 1 mg/dl. In the first 3 - 4 weeks the graft reached the size of an adult kidney, controlled by ultrasound. These patients, after 25 years, keep the graft functioning, with plasma creatinine less than 1 mg/dl. They do not have high blood pressure and have not had acute rejection. Conclusion: Kidneys from child donors can be grafted in isolation with excellent results. Only technical-surgical reasons can prevent the use of organs from donors under 1 year of age. The result in our hands with this type of donor shows us that it is possible to use it.

Post-exposure persistence of nitric oxide upregulation in skin cells irradiated by UV-A.

Evidence suggests that exposure to UV-A radiation can liberate nitric oxide from skin cells eliciting vasodilation in-vivo. However, the duration of nitric oxide release in skin cells after UV exposure is not well studied, with emphasis on UV-B mediated iNOS upregulation. The current study demonstrated persistence of nitric oxide release in a dark reaction after moderate UV-A exposure, peaking around 48 h post exposure; this effect was shown in keratinocytes, fibroblasts and endothelial cells from neonatal donors and keratinocytes from aged donors and confirmed the hypothesis that UV-A exposure appeared to upregulate cNOS alongside iNOS. Release of nitric oxide in the skin cells induced by a moderate exposure to UV-A in sunlight may be especially beneficial for some demographic groups such as the elderly, hypertensive patients or those with impaired nitric oxide function, not only during exposure but many hours and days after that.

In vitroevaluation of anti‐aging and regenerative properties of dermatan sulfate for skin care

IntroductionDermatan sulfate (DS) is a linear polysaccharide classified as a sulfated glycosaminoglycan (GAGs). GAGs form an integral part of the skin and make up 0.1–0.3% of total skin weight. DS is covalently attached to the core proteins to form proteoglycans (PGs). PGs predominates in dermis presenting higher level of DS than other sulfated GAGs.The intrinsic aging processes of the skin entails the reduction of total sulfated GAGs levels causing a thinning of the dermis and modifying the quality of the skin. DS possess strong biological activity that can be modulated to improve skin quality.This study was performed to determine the effects of DS on Human Dermal Fibroblasts (HDF) to evaluate its anti‐aging and regenerative properties.MethodsHuman Dermal Fibroblasts (HDF) were isolated from a sample of the foreskin of a neonatal caucasian donor (Lonza). The effect of the DS on cell proliferation was assessed at 24 h and 48 h in HDF, using the method bromodeoxyuridine (BrdU) incorporation, detected by ELISA. The cell migration was evaluated in HDF at 48 h using the Oris™ Cell Migration Assay (Platypus Technologies). To monitor the migration progress, the cells were labelled with calcein. The wound healing capacity of DS was evaluated in HDF using the scratch test. The inductive capacity on Extracellular matrix proteins (ECMp) synthesis was evaluated by immunolocalization‐ELISA quantifying type I and type III collagen and elastin.ResultsThe results showed that DS induced HDF proliferation after 24 h and 48 h of treatment at all tested concentrations. A dose‐dependent increase in proliferation was observed after 48 h of treatment of these cells, reaching a maximum increase of 5.10 (± 0.82) fold at the highest concentration tested (5 mg/mL) compared to uHDF. Regarding HDF cell migration, no significant differences were observed at 24 h after treatment with DS.The results showed a significant improvement in the in vitro wound healing capacity of HDF treated with 0.25 and 1 mg/mL of DS, with an increase in the healed area of 30.28% (± 4.27%) and 23.76% (± 8.87%) respectively, compared to uHDF.Regarding the ECMp synthesis‐stimulating capacity of DS, the results showed a significant increase of both type III Collagen (50.98 ± 34.48% increase) and Elastin (32.25 ± 2.61%) levels in HDF treated for 72 h with 1 mg/mL of DS compared to uHDF. HDF treated with 0.25 mg/mL also showed higher type I Collagen production than uHDF, although the increase (30.96 ±7.04%) was not statistically significant.ConclusionsThe results showed that DS improves the proliferation and regeneration of HDF present in dermis. In addition, DS increases the synthesis of ECMp as collagen type III and elastin, promoting the maintenance and functionality of the dermis. All these properties support the regenerative and anti‐aging activity of DS acting against intrinsic aging in dermis.

The Effect of Neonatal, Juvenile, and Adult Donors on Rejuvenated Neocartilage Functional Properties.

Cartilage does not naturally heal, and cartilage lesions from trauma and wear-and-tear can lead to eventual osteoarthritis. To address long-term repair, tissue engineering of functional biologic implants to treat cartilage lesions is desirable, but the development of such implants is hindered by several limitations, including (1) donor tissue scarcity due to the presence of diseased tissues in joints, (2) dedifferentiation of chondrocytes during expansion, and (3) differences in functional output of cells dependent on donor age. Toward overcoming these challenges, (1) costal cartilage has been explored as a donor tissue, and (2) methods have been developed to rejuvenate the chondrogenic phenotype of passaged chondrocytes for generating self-assembled neocartilage. However, it remains unclear how the rejuvenation processes are influenced by donor age and, thus, how to develop strategies that specifically target age-related differences. Using histological, biochemical, proteomic, and mechanical assays, this study sought to determine the differences among neocartilage generated from neonatal, juvenile, and adult donors using the Yucatan minipig, a clinically relevant large animal model. Based on the literature, a relatively young adult population of animals was chosen due to a reduction in functional output of human articular chondrocytes after 40 years of age. After isolation, costal chondrocytes were expanded, rejuvenated, and self-assembled, and the neocartilages were assessed. The aggregate modulus values of neonatal constructs were at least 1.65-fold of those from the juvenile or adult constructs. Poisson's ratio also significantly differed among all groups, with neonatal constructs exhibiting values 49% higher than adult constructs. Surprisingly, other functional properties such as tensile modulus and glycosaminoglycan content did not significantly differ among groups. Total collagen content was slightly elevated in the adult constructs compared to neonatal and juvenile constructs. A more nuanced view using bottom-up mass spectrometry showed that Col2a1 protein was not significantly different among groups, but protein content of several other collagen subtypes (i.e., Col1a1, Col9a1, Col11a2, and Col12a1) was modulated by donor age. For example, Col12a1 protein content in adult constructs was found to be 102.9% higher than neonatal-derived constructs. Despite these differences, this study shows that different aged donors can be used to generate neocartilages of similar functional properties. Impact statement Tissue-engineered neocartilage can be generated with functional properties that mimic native cartilage tissue. However, cell sourcing challenges hinder clinical translation of tissue-engineered cartilage. Chondrocytes can be expanded and rejuvenated for the generation of functional self-assembled cartilage, making an allogeneic approach feasible. However, it is currently unclear if donor age impacts functional properties. In this study, using the Yucatan minipig as a clinically relevant large animal model, we demonstrate that functional properties of self-assembled neocartilage are relatively consistent regardless of donor age, suggesting that a wider range of donor ages may be used for cartilage tissue engineering than previously expected.

Procurement and Evaluation of Hepatocytes for Transplantation From Neonatal Donors After Circulatory Death

Hepatocyte transplantation is a promising treatment for liver failure and inborn metabolic liver diseases, but progress has been hampered by a scarcity of available organs. Here, hepatocytes isolated from livers procured for a neonatal hepatocyte donation program within a research setting were assessed for metabolic function and suitability for transplantation. Organ donation was considered for infants who died in neonatal intensive care in the Stockholm region during 2015–2021. Inclusion was assessed when a decision to discontinue life-sustaining treatment had been made and hepatectomy performed after declaration of death. Hepatocyte isolation was performed by three-step collagenase perfusion. Hepatocyte viability, yield, and function were assessed using fresh and cryopreserved cells. Engraftment and maturation of cryopreserved neonatal hepatocytes were assessed by transplantation into an immunodeficient mouse model and analysis of the gene expression of phase I, phase II, and liver-specific enzymes and proteins. Twelve livers were procured. Median warm ischemia time (WIT) was 190 [interquartile range (IQR): 80–210] minutes. Median viability was 86% (IQR: 71%–91%). Median yield was 6.9 (IQR: 3.4–12.8) x106 viable hepatocytes/g. Transplantation into immunodeficient mice resulted in good engraftment and maturation of hepatocyte-specific proteins and enzymes. A neonatal organ donation program including preterm born infants was found to be feasible. Hepatocytes isolated from neonatal donors had good viability, function, and engraftment despite prolonged WIT. Therefore, neonatal livers should be considered as a donor source for clinical hepatocyte transplantation, even in cases with extended WIT.

Eligible neonatal donors after circulatory determination of death (Maastricht type III): A national survey of level III NICUs.

Organ donation continues to increase worldwide, but in general paediatric patients remain less likely to receive a transplant. The inclusion of neonates as donors after cDCD should be considered in an effort to increase donation rates. The survey for a cross-sectional national study of potential cDCD neonatal donors (Maastricht type III) was sent to all 90level III Spanish neonatal units to explore: 1) protocols, education, and specific opinions on donation and 2) potential cDCD that could have been eligible over a 2-year period (2014-2015). Forty-five centers (50%) completed the survey, and 38/45gave information about potential eligible donors. In 16% of the centers specific protocols on neonatal donation exist. All hospitals demanded more specific training, and 65% noted that the donation process could be a problem in the family's dismissal of the child. During the study period 46805 neonates were admitted in the 38 centers, and 625 neonates died. Ninety-five born at a gestational age ≥34weeks and above 2000gr died after an EoL decision, 38 (40%) and 13 (14%) of them due to neonatal encephalopathy and multiple congenital anomalies, respectively. There were 31 (33%) elegible infants who died in less than 120min due to pathologies that did not contraindicate donation. Neonatal cDCD could help to reduce the gap between the supply of and demand for organs according to the potentially eligible patients emerging from this study. Training in EoL and donation processes should be provided to healthcare professionals.

Potential Heart, Liver, and Kidney Donation after Circulatory Determination of Death in a Neonatal Intensive Care Unit

Background: Pediatric organ donation after circulatory determination of death (DCD) has increased in recent years; however, there are few data reporting the number of neonatal potential DCD organ donors and no Canadian-specific reports. Objective: The main objective of this study was to estimate the number of patients who may have become actual DCD organ donors from a single, tertiary neonatal intensive care unit (NICU) over 5 years. Methods: We reviewed all medical charts of newborns ≥2.5 kg, who died in our center’s NICU from January 2013 to December 2017. We determined how many could have become actual organ donors after brain death (DBD) or DCD based on 3 sets of organ-specific eligibility criteria defined as conservative, standard, and liberal. Results: Of the 39 deceased patients, none met the criteria for DBD. Twenty-nine (75%) died after the withdrawal of life-sustaining therapies. According to the conservative criteria, 1 patient would have been eligible for kidneys and liver donation. Three patients met standard criteria for kidneys and 1 for liver. Eight patients would have been eligible donors for kidneys, 7 for liver, and 2 for heart according to liberal criteria. Only 2 patients were evaluated for DCD, and no organ donation was performed. Conclusions: While uncommon, we identified potential DCD organ donors in the NICU population for kidney, heart, and liver transplants. The substantial variability in the number of potential donors depending on the selected eligibility criteria emphasizes the need for a standardized definition adapted to local capacities.

Neonatal donation: are newborns too young to be recognized?

Neonatal organ and tissue donation is not common practice in the Netherlands. At the same time, there is a transplant waiting list for small size-matched organs and tissues. Multiple factors may contribute to low neonatal donation rates, including a lack of awareness of this option. This study provides insight into potential neonatal organ and tissue donors and reports on how many donors were actually reported to the procurement organization. We performed a retrospective analysis of the mortality database and medical records of two largest neonatal intensive care units (NICUs) in the Netherlands. This study reviewed records of neonates with a gestational age >37 weeks and weight >3000g who died in the period from January 1, 2005 through December 31, 2016. During the study period, 259 term-born neonates died in the two NICUs. In total, 132 neonates with general contra-indications for donation were excluded. The medical records of 127 neonates were examined for donation suitability. We identified five neonates with documented brain death who were not recognized as potential organ and/or tissue donors. Of the remaining neonates, 27 were found suitable for tissue donation. One potential tissue donor had been reported to the procurement organization. In three cases, the possibility of donation was brought up by parents.Conclusion: A low proportion (2%) of neonates who died in the NICUs were found suitable for organ donation, and a higher proportion (12%) were found suitable for tissue donation. We suggest that increased awareness concerning the possibility of neonatal donation would likely increase the identification of potential neonatal donors.What is Known:• There is an urgent need for very small organs and tissues from neonatal donorsWhat is New:• A number of neonates who died in the NICU were suitable organ or/and tissue donors but were not recognized as donors.• Knowledge on neonatal donation possibilities is also important for proper counseling of parents who sometimes inquire for the possibility of organ and tissue donation.

Single-Nucleus and In Situ RNA–Sequencing Reveal Cell Topographies in the Human Pancreas

Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell sequencing approaches. We innovated single-nucleus RNA-sequencing protocols and profiled more than 120,000 cells from pancreata of adult and neonatal human donors. We validated the single-nucleus findings using RNA fluorescence in situ hybridization, in situ sequencing, and computational approaches. We created the first comprehensive atlas of human pancreas cells including epithelial and nonepithelial constituents, and uncovered 3 distinct acinar cell types, with possible implications for homeostatic and inflammatory processes of the pancreas. The comparison with neonatal single-nucleus sequencing data showed a different cellular composition of the endocrine tissue, highlighting the tissue dynamics occurring during development. By applying spatial cartography, involving cell proximity mapping through in situ sequencing, we found evidence of specific cell type neighborhoods, dynamic topographies in the endocrine and exocrine pancreas, and principles of morphologic organization of the organ. Furthermore, similar analyses in chronic pancreatitis biopsy samples showed the presence of acinar-REG+ cells, a reciprocal association between macrophages and activated stellate cells, and a new potential role of tuft cells in this disease. Our human pancreas cell atlas can be interrogated to understand pancreatic cell biology and provides a crucial reference set for comparisons with diseased tissue samples to map the cellular foundations of pancreatic diseases.

Endothelial to mesenchymal transition during neonatal hyperoxia-induced pulmonary hypertension.

Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, results from mechanical ventilation and hyperoxia, amongst other factors. Although most BPD survivors can be weaned from supplemental oxygen, many show evidence of cardiovascular sequelae in adulthood, including pulmonary hypertension and pulmonary vascular remodeling. Endothelial-mesenchymal transition (EndoMT) plays an important role in mediating vascular remodeling in idiopathic pulmonary arterial hypertension. Whether hyperoxic exposure, a known mediator of BPD in rodent models, causes EndoMT resulting in vascular remodeling and pulmonary hypertension remains unclear. We hypothesized that neonatal hyperoxic exposure causes EndoMT, leading to the development of pulmonary hypertension in adulthood. To test this hypothesis, newborn mice were exposed to hyperoxia and then allowed to recover in room air until adulthood. Neonatal hyperoxic exposure gradually caused pulmonary vascular and right ventricle remodeling as well as pulmonary hypertension. Male mice were more susceptible to developing pulmonary hypertension compared to female mice, when exposed to hyperoxia as newborns. Hyperoxic exposure induced EndoMT in mouse lungs as well as in cultured lung microvascular endothelial cells (LMVECs) isolated from neonatal mice and human fetal donors. This was augmented in cultured LMVECs from male donors compared to those from female donors. Using primary mouse LMVECs, hyperoxic exposure increased phosphorylation of both Smad2 and Smad3, but reduced Smad7 protein levels. Treatment with a selective TGF-β inhibitor SB431542 blocked hyperoxia-induced EndoMT in vitro. Altogether, we show that neonatal hyperoxic exposure caused vascular remodeling and pulmonary hypertension in adulthood. This was associated with increased EndoMT. These novel observations provide mechanisms underlying hyperoxia-induced vascular remodeling and potential approaches to prevent BPD-associated pulmonary hypertension by targeting EndoMT. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Early dynamic accumulation of tissue resident memory T cells in the human lung during infancy

Abstract Infants exhibit increased susceptibility to viral and bacterial pathogens with pulmonary infections representing one of the top causes of infant mortality worldwide. Optimal protection from pathogens is mediated by coordinated functions of different T cell subsets, with tissue resident populations maintaining site-specific immunity in the respiratory tract. Currently, little is known of how tissue localized T cells populate and mediate mucosal immune responses in early life in humans. Here we analyzed T cell populations in the lungs obtained from infant and pediatric organ donors aged 8 days postnatal (PN) to 8 years by imaging and flow cytometry. In the earliest stage of life at 8 days PN, few αβ T cells were present in the lung, while innate-type γδ T cells were present around the airways. During the first two years of life, αβ T cells exhibiting memory phenotypes preferentially accumulated around the central airways and expressed canonical markers of tissue residency (CD69 and CD103), consistent with flow cytometry data. By contrast, the total number of γδ T cells in the lung did not change significantly with age for up to 8 years of life; for example, the ratio of αβ:γδ T cells in neonatal donors was 1.5:1, which increased to 16:1 in lungs of a 3 year old donor. Consistent with the imaging data, the frequency of γδ T cells by flow cytometry was significantly reduced after the early neonatal period. These results show that γδ T cells populate the lung during the early neonatal period and are maintained in relatively constant numbers over childhood, while tissue resident αβ T cells accumulate over first 2 years of life and predominate thereafter. Our results demonstrate the dynamic nature of lung-localized T cell immune responses in a mucosal site.

US NICUs and donor milk banks brace for COVID-19

Neonatal intensive care units (NICUs) and donor human milk programmes across the USA are bracing for the coronavirus disease 2019 (COVID-19) pandemic. NICU workers have tested positive for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19. A worker at the Children's Hospital & Medical Center NICU in Omaha (NE) was in contact with ten patients 3–4 days before testing positive for the virus, according to a March 25 report by the Associated Press. In Alabama, at least one newborn was placed in isolation after a NICU nurse tested positive.