Endothelial colony forming cell administration promotes neurovascular unit development in growth restricted and appropriately grown fetal lambs.

BACKGROUND: Fetal growth restriction (FGR) is associated with deficits in the developing brain, including neurovascular unit (NVU) dysfunction. Endothelial colony forming cells (ECFC) can mediate improved vascular stability, and have demonstrated potential to enhance vascular development and protection. This investigation examined whether ECFCs from human umbilical cord blood (UCB) enhanced NVU development in FGR and appropriate for gestational age (AGA) fetal sheep. METHODS: Twin-bearing ewes had surgery performed at 88-90 days' gestation, inducing FGR in one fetus. At 113 days, ECFCs (1 × 107 cells) cultured from human UCB were administered intravenously to fetal sheep in utero. At 127 days, ewes and their fetuses were euthanised, fetal brains collected, and NVU components analysed by immunohistochemistry. RESULTS: Twenty-four fetal lambs, arranged in four groups: AGA (n = 7), FGR (n = 5), AGA + ECFC (n = 6), and FGR + ECFC (n = 6), were included in analyses. FGR resulted in lower body weight than AGA (P = 0.002) with higher brain/body weight ratio (P = 0.003). ECFC treatment was associated with increased vascular density throughout the brain in both AGA + ECFC and FGR + ECFC groups, as well as increased vascular-astrocyte coverage and VEGF expression in the cortex (P = 0.003, P = 0.0006, respectively) and in the subcortical white matter (P = 0.01, P = 0.0002, respectively) when compared with the untreated groups. CONCLUSIONS: ECFC administration enhanced development of NVU components in both the AGA and FGR fetal brain. Further investigation is required to assess how to optimise the enhanced angiogenic capabilities of ECFCs to provide a therapeutic strategy to protect the developing NVU against vulnerabilities associated with FGR.

Effect of expansion of human umbilical cord blood CD34 + cells on neurotrophic and angiogenic factor expression and function.

The use of CD34 + cell-based therapies has largely been focused on haematological conditions. However, there is increasing evidence that umbilical cord blood (UCB) CD34 + -derived cells have neuroregenerative properties. Due to low cell numbers of CD34 + cells present in UCB, expansion is required to produce sufficient cells for therapeutic purposes, especially in adults or when frequent applications are required. However, it is not known whether expansion of CD34 + cells has an impact on their function and neuroregenerative capacity. We addressed this knowledge gap in this study, via expansion of UCB-derived CD34 + cells using combinations of LDL, UM171 and SR-1 to yield large numbers of cells and then tested their functionality. CD34 + cells expanded for 14 days in media containing UM171 and SR-1 resulted in over 1000-fold expansion. The expanded cells showed an up-regulation of the neurotrophic factor genes BDNF, GDNF, NTF-3 and NTF-4, as well as the angiogenic factors VEGF and ANG. In vitro functionality testing showed that these expanded cells promoted angiogenesis and, in brain glial cells, promoted cell proliferation and reduced production of reactive oxygen species (ROS) during oxidative stress. Collectively, this study showed that our 14-day expansion protocol provided a robust expansion that could produce enough cells for therapeutic purposes. These expanded cells, when tested in in vitro, maintained functionality as demonstrated through promotion of cell proliferation, attenuation of ROS production caused by oxidative stress and promotion of angiogenesis.

Insulin regulates human mammosphere development and function.

Assessing the role of lactogenic hormones in human mammary gland development is limited due to issues accessing tissue samples and so development of a human in vitro three-dimensional mammosphere model with functions similar to secretory alveoli in the mammary gland can aid to overcome this shortfall. In this study, a mammosphere model has been characterised using human mammary epithelial cells grown on either mouse extracellular matrix or agarose and showed insulin is essential for formation of mammospheres. Insulin was shown to up-regulate extracellular matrix genes. Microarray analysis of these mammospheres revealed an up-regulation of differentiation, cell-cell junctions, and cytoskeleton organisation functions, suggesting mammosphere formation may be regulated through ILK signalling. Comparison of insulin and IGF-1 effects on mammosphere signalling showed that although IGF-1 could induce spherical structures, the cells did not polarise correctly as shown by the absence of up-regulation of polarisation genes and did not induce the expression of milk protein genes. This study demonstrated a major role for insulin in mammary acinar development for secretory differentiation and function indicating the potential for reduced lactational efficiency in women with obesity and gestational diabetes.

Umbilical cord blood therapy to prevent progression of COVID-19 related pneumonia: a structured summary of a study protocol for a pilot randomised controlled trial.

OBJECTIVES: Objective: To undertake a pilot, feasibility RCT of umbilical cord blood derived cell therapy for treatment of adult patients infected with SARS-CoV-2 virus related moderate-to-severe pneumonia to prevent progression to severe ARDS. HYPOTHESIS: Expanded cord blood derived cell therapy will be feasible, well tolerated and show potential efficacy in the treatment of acute COVID-19 related moderate to severe pneumonia in adult patients because of their powerful anti-inflammatory and immunomodulatory properties. TRIAL DESIGN: Pilot, parallel design randomised controlled trial. PARTICIPANTS: The trial will recruit 24 hospitalised patients with confirmed SARS-CoV-2 infection and pneumonia from July to December 2020 at Monash Medical Centre in Melbourne, Australia. INTERVENTION AND COMPARATOR: Intervention: Intravenous injection of expanded umbilical cord blood cells at a dose of 5 million cells/kg (maximum dose - 500 million cells). Cell infusion will occur over 30-60 minutes through a peripheral intravenous cannula. Standard supportive care will continue as needed. Comparator: Standard supportive care. MAIN OUTCOMES: Safety and tolerability of cell administration within first 24 hours of administration; clinical improvement on a seven-category clinical improvement ordinal scale. RANDOMISATION: Randomisation will be done using computer generated allocation to intervention/ control groups in a 1:1 ratio (in blocks of 6) using sealed opaque envelopes. BLINDING (MASKING): This will be an unblinded study, given that it is the first study using expanded cord blood cells in COVID-19 patients. There will be no placebo infusion. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): Twelve participants in each group. Total n=24. TRIAL STATUS: CBC-19 protocol v2, dated 23rd April 2020. Recruitment has not started yet. Estimated recruitment timeline is between 1st July - 31st December 2020. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12620000478910, registered 16th April 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Guiding Development of the Neonate: Lessons from Mammalia.

Significantly preterm and low-birthweight (LBW) babies have diminished lung and gut development, generally fail to thrive, have increased mortality and higher frequency of mature-onset disease. Mothers often cannot breastfeed, and babies receive either formula or pasteurized donor milk, which may further limit the baby's recovery. New approaches are required to manage the early stages of neonatal development. The tammar wallaby, an Australian marsupial, has a short gestation and a simple placenta, and gives birth to an altricial young equivalent to a final trimester human embryo. The neonate remains in the pouch and attached to the teat for 100 days postpartum. The mother slows growth of the young and progressively changes the composition of the milk to deliver signals for organ development, including the lung and gut. This closely resembles the relationship between the human fetus and delivery of placental and uterine bioactives. Datasets comprised of differentially expressed genes coding for secreted proteins in early lactation in the tammar mammary gland have been compared to databases produced from human placenta, amniotic fluid, colostrum and milk to identify human homologues for the putative signaling molecules for organ development. These data will be used to develop milk fortifiers for treatment of preterm and LBW babies in both the developed and the developing world.

The tammar wallaby: A marsupial model to examine the timed delivery and role of bioactives in milk.

It is now clear that milk has multiple functions; it provides the most appropriate nutrition for growth of the newborn, it delivers a range of bioactives with the potential to stimulate development of the young, it has the capacity to remodel the mammary gland (stimulate growth or signal cell death) and finally milk can provide protection from infection and inflammation when the mammary gland is susceptible to these challenges. There is increasing evidence to support studies using an Australian marsupial, the tammar wallaby (Macropus eugenii), as an interesting and unique model to study milk bioactives. Reproduction in the tammar wallaby is characterized by a short gestation, birth of immature young and a long lactation. All the major milk constituents change substantially and progressively during lactation and these changes have been shown to regulate growth and development of the tammar pouch young and to have roles in mammary gland biology. This review will focus on recent reports examining the control of lactation in the tammar wallaby and the timed delivery of milk bioactivity.

Analysis of human breast milk cells: gene expression profiles during pregnancy, lactation, involution, and mastitic infection.

The molecular processes underlying human milk production and the effects of mastitic infection are largely unknown because of limitations in obtaining tissue samples. Determination of gene expression in normal lactating women would be a significant step toward understanding why some women display poor lactation outcomes. Here, we demonstrate the utility of RNA obtained directly from human milk cells to detect mammary epithelial cell (MEC)-specific gene expression. Milk cell RNA was collected from five time points (24 h prepartum during the colostrum period, midlactation, two involutions, and during a bout of mastitis) in addition to an involution series comprising three time points. Gene expression profiles were determined by use of human Affymetrix arrays. Milk cells collected during milk production showed that the most highly expressed genes were involved in milk synthesis (e.g., CEL, OLAH, FOLR1, BTN1A1, and ARG2), while milk cells collected during involution showed a significant downregulation of milk synthesis genes and activation of involution associated genes (e.g., STAT3, NF-kB, IRF5, and IRF7). Milk cells collected during mastitic infection revealed regulation of a unique set of genes specific to this disease state, while maintaining regulation of milk synthesis genes. Use of conventional epithelial cell markers was used to determine the population of MECs within each sample. This paper is the first to describe the milk cell transcriptome across the human lactation cycle and during mastitic infection, providing valuable insight into gene expression of the human mammary gland.

The tammar wallaby: a model system to examine domain-specific delivery of milk protein bioactives.

The role of milk extends beyond simply providing nutrition to the suckled young. Milk has a comprehensive role in programming and regulating growth and development of the suckled young, and provides a number of potential autocrine factors so that the mammary gland functions appropriately during the lactation cycle. This central role of milk is best studied in animal models such as marsupials that have evolved a different lactation strategy to eutherians and allow researchers to more easily identify regulatory mechanisms that are not as readily apparent in eutherian species. For example, the tammar wallaby (Macropus eugenii) has evolved with a unique reproductive strategy of a short gestation, birth of an altricial young and a relatively long lactation during which the mother progressively changes the composition of the major, and many of the minor components of milk. Consequently, in contrast to eutherians, there is a far greater investment in development of the young during lactation and it is likely that many of the signals that regulate development of eutherian embryos in utero are delivered by the milk. This requires the co-ordinated development and function of the mammary gland since inappropriate timing of these signalling events may result in either limited or abnormal development of the young, and potentially a higher incidence of mature onset disease. Milk proteins play a significant role in these processes by providing timely presentation of signalling molecules and antibacterial protection for the young and the mammary gland at times when there is increased susceptibility to infection. This review describes studies exploiting the unique reproductive strategy of the tammar wallaby to investigate the role of several proteins secreted at specific times during the lactation cycle and that are correlated with potential roles in the young and mammary gland. Interestingly, alternative splicing of some milk protein genes has been utilised by the mammary gland to deliver domain-specific functions at specific times during lactation.

WFDC2 is differentially expressed in the mammary gland of the tammar wallaby and provides immune protection to the mammary gland and the developing pouch young.

WAP four disulfide core domain 2 (WFDC2) is a four disulfide core (4-DSC) protein secreted in the milk of the tammar wallaby. It is comprised of two 4-DSC domains assigned domain III at the NH2-terminal end and domain II at the COOH-terminal end. The WFDC2 gene was expressed only during pregnancy, early lactation, towards the end of lactation and involution. The WFDC2 protein showed antibacterial activity against Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa and this activity resided with domain II. There was no antibacterial activity detected against Enterococcus faecalis. The observed expression pattern of tammar WFDC2 and its antibacterial activity suggests a role to either reduce mastitis in the mammary gland caused by S. aureus or to protect the gut of the young at a time when it is not immune-competent. The latter effect could be achieved without disturbing the balance of commensal gut flora such as E. faecalis.