Graham Jenkin

Human Amnion Epithelial Cell Transplantation Abrogates Lung Fibrosis and Augments Repair

RATIONALE Chronic lung disease characterized by loss of lung tissue, inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. OBJECTIVES To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell-like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. METHODS Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. MEASUREMENTS AND MAIN RESULTS hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A-D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks post-transplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-alpha, IL-1 and -6, and profibrotic transforming growth factor-beta in mouse lungs. In addition, lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors of matrix metalloproteinase-1 and 2. CONCLUSIONS hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis.

Melatonin provides neuroprotection in the late-gestation fetal sheep brain in response to umbilical cord occlusion.

Oxygen free radicals, including the highly toxic hydroxyl radical (·OH), initiate lipid peroxidation and DNA/RNA fragmentation and damage cells. The pineal hormone melatonin is an antioxidant and powerful scavenger of ·OH. We hypothesized that maternally administered melatonin could reduce ·OH formation, lipid peroxidation, and DNA/RNA damage in the fetal brain in response to asphyxia. In 15 fetal sheep, extracellular ·OH was measured by microdialysis in white and gray matter of the parasagittal cortex. In 10 fetuses, asphyxia was induced by umbilical cord occlusion for 10 min using an inflatable cuff – the ewes of these fetuses received either intravenous melatonin (1 mg bolus, then 1 mg/h for 2 h; n = 5) or vehicle (1% ethanol in saline; n = 5), and results were compared to fetuses with sham cord occlusion and vehicle-infused ewes (n = 5). Hypoxemia, acidemia, hypertension and bradycardia produced by cord occlusion was similar in the melatonin- and vehicle-treated groups. In the vehicle-treated group, cord occlusion resulted in a significant increase in ·OH in gray matter at 8–9.5 h after occlusion (p < 0.05); in contrast, there was no ·OH change in the melatonin-treated group. After cord occlusion, lipid peroxidation (4-hydroxynonenal immunoreactivity) found throughout the brain of vehicle-infused ewes was significantly less in the melatonin-infused group. Melatonin had no significant effect on the distribution of DNA/RNA fragmentation, as shown by 8-hydroxydeoxyguanosine immunoreactivity. Thus, brief asphyxia results in significant and delayed entry of ·OH into the extracellular space of cortical gray matter in the fetal sheep brain, and melatonin given to the mother at the time of the insult abrogates this increase. Melatonin, in reducing O2 free radical production, may be an effective neuroprotective treatment for the fetus.

Human amnion epithelial cells prevent bleomycin-induced lung injury and preserve lung function

Human amnion epithelial cells (hAECs) have attracted recent attention as a promising source of cells for regenerative therapies, with reports that cells derived from human term amnion possess multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties. Specifically, in animal models of lung disease characterized by significant loss of lung tissue secondary to chronic inflammation and fibrosis, the transplantation of hAECs has been shown to reduce both inflammation and subsequent fibrosis. To further explore the mechanisms by which hAECs reduce pulmonary fibrosis and enhance lung regeneration, we utilized a bleomycin-induced model of pulmonary fibrosis and investigated the ability of hAECs to reduce fibrosis and thereby improve pulmonary function. We aimed to determine if hAECs, injected into the peritoneal cavity could migrate to the lung, engraft, and form functional lung epithelium, and whether hAECs could modulate the inflammatory environment in the bleomycin-injured lung. We demonstrated that, compared to bleomycin alone, IP administration of hAECs 24 h after bleomcyin, decreased gene expression of the proinflammatory cytokines TNF-α, TGF-β, IFN-γ, and IL-6 and decreased subsequent pulmonary fibrosis with less pulmonary collagen deposition, reduced levels of α-smooth muscle actin and decreased inflammatory cell infiltrate. We also showed that hAECs are able to prevent a decline in pulmonary function associated with bleomycin-induced lung damage. We were unable to detect any significant engraftment of hAECs in injured, or uninjured, lung after administration. The findings from this study support the further investigation of hAECs as a potential cell therapy for inflammatory and fibrogenic diseases.

Evaluation of the long-term function of cryopreserved ovarian grafts in the mouse, implications for human applications

Ovarian tissue storage has several potentially very valuable clinical applications, including the management of young female patients that are at risk of premature menopause. Ovarian tissue collection, used alone or in combination with oocyte and embryo cryopreservation, may help these patients safeguard their own future fertility. All available evidence from animal studies indicates that grafting of frozen ovarian tissue should be feasible in the human. This study on the mouse shows that frozen thawed ovarian tissue grafts can restore long term fertility to previously ovariectomised recipients. This, and other available evidence, indicates that ovarian tissue collection and storage, used alone or in combination with oocyte or embryo collection, may help safeguard the fertility of patients at risk of premature menopause.

Gene expression profiling of human oocytes following in vivo or in vitro maturation

BACKGROUND Immature human oocytes matured in vitro, particularly those from gonadotrophin stimulated ovaries, are developmentally incompetent when compared with oocytes matured in vivo. This developmental incompetence has been explained as poor oocyte cytoplasmic maturation without any determination of the likely molecular basis of this observation. METHODS Replicate whole human genome arrays were generated for immature and mature oocytes (matured in vivo and in vitro, prior to exposure to sperm) recovered from women undertaking gonadotrophin treatment for assisted reproduction. RESULTS More than 2000 genes were identified as expressed at more than 2-fold higher levels in oocytes matured in vitro than those matured in vivo (P < 0.05, range 4.98 x 10(-2) -2.22 x 10(-4)) and 162 of these are expressed at 10-fold or greater levels (P < 0.05, range 4.98 x 10(-2)-1.38 x 10(-3)). Many of these genes are involved in transcription, the cell cycle and its regulation, transport and cellular protein metabolism. CONCLUSIONS Global gene expression profiling using microarrays and bioinformatics analysis has provided a molecular basis for differences in the developmental competence of oocytes matured in vitro compared with in vivo. The over-abundance of transcripts identified in immature germinal vesicle stage oocytes recovered from gonadotrophin stimulated cycles and matured in vitro is probably due to dysregulation in either gene transcription or post-transcriptional modification of genes. Either mechanism would result in an incorrect temporal utilization of genes which may culminate in developmental incompetence of any embryos derived from these oocytes.

Amnion Epithelial Cell Isolation and Characterization for Clinical Use

Human amnion epithelial cells (hAECs) are a heterologous population positive for stem cell markers; they display multilineage differentiation potential, differentiating into cells of the endoderm (liver, lung epithelium), mesoderm (bone, fat), and ectoderm (neural cells). They have a low immunogenic profile and possess potent immunosuppressive properties. Hence, hAECs may be a valuable source of cells for cell therapy. This unit describes an efficient and effective method of hAEC isolation, culture, and cryopreservation that is animal product-free and in accordance with current guidelines on preparation of cells for clinical use. Cells isolated using this method were characterized after 5 passages by analysis of karyotype, cell cycle distribution, and changes in telomere length. The differentiation potential of hAECs isolated using this animal product-free method was demonstrated by differentiation into lineages of the three primary germ layers and expression of lineage-specific markers analyzed by PCR, immunocytochemistry, and histology.

Antenatal antioxidant treatment with melatonin to decrease newborn neurodevelopmental deficits and brain injury caused by fetal growth restriction

Fetal intrauterine growth restriction (IUGR) is a serious pregnancy complication associated with increased rates of perinatal morbidity and mortality, and ultimately with long‐term neurodevelopmental impairments. No intervention currently exists that can improve the structure and function of the IUGR brain before birth. Here, we investigated whether maternal antenatal melatonin administration reduced brain injury in ovine IUGR. IUGR was induced in pregnant sheep at 0.7 gestation and a subset of ewes received melatonin via intravenous infusion until term. IUGR, IUGR + melatonin (IUGR + MLT) and control lambs were born naturally, neonatal behavioral assessment was used to examine neurological function and at 24 hr after birth the brain was collected for the examination of neuropathology. Compared to control lambs, IUGR lambs took significantly longer to achieve normal neonatal lamb behaviors, such as standing and suckling. IUGR brains showed widespread cellular and axonal lipid peroxidation, and white matter hypomyelination and axonal damage. Maternal melatonin administration ameliorated oxidative stress, normalized myelination and rescued axonopathy within IUGR lamb brains, and IUGR + MLT lambs demonstrated significant functional improvements including a reduced time taken to attach to and suckle at the udder after birth. Based on these observations, we began a pilot clinical trial of oral melatonin administration to women with an IUGR fetus. Maternal melatonin was not associated with adverse maternal or fetal effects and it significantly reduced oxidative stress, as evidenced by reduced malondialdehyde levels, in the IUGR + MLT placenta compared to IUGR alone. Melatonin should be considered for antenatal neuroprotective therapy in human IUGR.

Stem cell therapy to protect and repair the developing brain: a review of mechanisms of action of cord blood and amnion epithelial derived cells.

In the research, clinical, and wider community there is great interest in the use of stem cells to reduce the progression, or indeed repair brain injury. Perinatal brain injury may result from acute or chronic insults sustained during fetal development, during the process of birth, or in the newborn period. The most readily identifiable outcome of perinatal brain injury is cerebral palsy, however, this is just one consequence in a spectrum of mild to severe neurological deficits. As we review, there are now clinical trials taking place worldwide targeting cerebral palsy with stem cell therapies. It will likely be many years before strong evidence-based results emerge from these trials. With such trials underway, it is both appropriate and timely to address the physiological basis for the efficacy of stem-like cells in preventing damage to, or regenerating, the newborn brain. Appropriate experimental animal models are best placed to deliver this information. Cell availability, the potential for immunological rejection, ethical, and logistical considerations, together with the propensity for native cells to form teratomas, make it unlikely that embryonic or fetal stem cells will be practical. Fortunately, these issues do not pertain to the use of human amnion epithelial cells (hAECs), or umbilical cord blood (UCB) stem cells that are readily and economically obtained from the placenta and umbilical cord discarded at birth. These cells have the potential for transplantation to the newborn where brain injury is diagnosed or even suspected. We will explore the novel characteristics of hAECs and undifferentiated UCB cells, as well as UCB-derived endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs), and how immunomodulation and anti-inflammatory properties are principal mechanisms of action that are common to these cells, and which in turn may ameliorate the cerebral hypoxia and inflammation that are final pathways in the pathogenesis of perinatal brain injury.

Gonadotrophin administration can benefit ovarian tissue grafted to the body wall: implications for human ovarian grafting.

Ovarian grafting provides a strategy for clinical infertility treatment and is starting to be used in conjunction with ovarian tissue storage for patients at risk of early ovarian failure. As patients are starting to return for their frozen stored tissue we need to ascertain how to maximise follicle survival when this tissue is grafted back to the patient. For research purposes ovarian tissue is commonly grafted to the kidney capsule as the rich capillary bed at this site favours rapid graft revascularization. This is however not an ideal site for natural conceptions or for the harvest of mature oocytes for in vitro fertilization. While oocytes would be relatively easy to recover from grafts on the abdominal wall or subcutaneous tissue graft revascularization at these sites is slower and evidence indicates that fewer follicles survive. As gonadotropins can upregulate angiogenic growth factors in the ovary this study was designed to test whether the administration of exogenous gonadotropins would increase the number of surviving follicles in grafts placed at less vascularised sites. We showed that exogenous gonadotrophins, given to either the donor or the recipient, could increase the number of developing follicles but the magnitude of this effect was influenced by the timing of the injections relative to the time of grafting.

Human amnion epithelial cells reduce ventilation-induced preterm lung injury in fetal sheep

OBJECTIVE The objective of the study was to explore whether human amnion epithelial cells (hAECs) can mitigate ventilation-induced lung injury. STUDY DESIGN An established in utero ovine model of ventilation-induced lung injury was used. At day 110 of gestation, singleton fetal lambs either had sham in utero ventilation (IUV) (n = 4), 12 hours of IUV alone (n = 4), or 12 hours of IUV and hAEC administration (n = 5). The primary outcome, structural lung injury, was assessed 1 week later. RESULTS Compared with sham controls, IUV alone was associated with significant lung injury: increased collagen (P = .03), elastin (P = .02), fibrosis (P = .02), and reduced secondary-septal crests (P = .009). This effect of IUV was significantly mitigated by the administration of hAECs: less collagen (P = .03), elastin (P = .04), fibrosis (P = .02), normalized secondary-septal crests (P = .02). The hAECs were immunolocalized within the fetal lung and had differentiated into type I and II alveolar cells. CONCLUSION The hAECs mitigate ventilation-induced lung injury and differentiated into alveolar cells in vivo.