Jingang Li

Could cord blood cell therapy reduce preterm brain injury

Major advances in neonatal care have led to significant improvements in survival rates for preterm infants, but this occurs at a cost, with a strong causal link between preterm birth and neurological deficits, including cerebral palsy (CP). Indeed, in high-income countries, up to 50% of children with CP were born preterm. The pathways that link preterm birth and brain injury are complex and multifactorial, but it is clear that preterm birth is strongly associated with damage to the white matter of the developing brain. Nearly 90% of preterm infants who later develop spastic CP have evidence of periventricular white matter injury. There are currently no treatments targeted at protecting the immature preterm brain. Umbilical cord blood (UCB) contains a diverse mix of stem and progenitor cells, and is a particularly promising source of cells for clinical applications, due to ethical and practical advantages over other potential therapeutic cell types. Recent studies have documented the potential benefits of UCB cells in reducing brain injury, particularly in rodent models of term neonatal hypoxia–ischemia. These studies indicate that UCB cells act via anti-inflammatory and immuno-modulatory effects, and release neurotrophic growth factors to support the damaged and surrounding brain tissue. The etiology of brain injury in preterm-born infants is less well understood than in term infants, but likely results from episodes of hypoperfusion, hypoxia–ischemia, and/or inflammation over a developmental period of white matter vulnerability. This review will explore current knowledge about the neuroprotective actions of UCB cells and their potential to ameliorate preterm brain injury through neonatal cell administration. We will also discuss the characteristics of UCB-derived from preterm and term infants for use in clinical applications.

Preterm white matter brain injury is prevented by early administration of umbilical cord blood cells

Infants born very preterm are at high risk for neurological deficits including cerebral palsy. In this study we assessed the neuroprotective effects of umbilical cord blood cells (UCBCs) and optimal administration timing in a fetal sheep model of preterm brain injury. 50 million allogeneic UCBCs were intravenously administered to fetal sheep (0.7 gestation) at 12h or 5d after acute hypoxia-ischemia (HI) induced by umbilical cord occlusion. The fetal brains were collected at 10d after HI. HI (n=7) was associated with reduced number of oligodendrocytes (Olig2+) and myelin density (CNPase+), and increased density of activated microglia (Iba-1+) in cerebral white matter compared to control fetuses (P<0.05). UCBCs administered at 12h, but not 5d after HI, significantly protected white matter structures and suppressed cerebral inflammation. Activated microglial density showed a correlation with decreasing oligodendrocyte number (P<0.001). HI caused cell death (TUNEL+) in the internal capsule and cell proliferation (Ki-67+) in the subventricular zone compared to control (P<0.05), while UCBCs at 12h or 5d ameliorated these effects. Additionally, UCBCs at 12h induced a significant systemic increase in interleukin-10 at 10d, and reduced oxidative stress (malondialdehyde) following HI (P<0.05). UCBC administration at 12h after HI reduces preterm white matter injury, via anti-inflammatory and antioxidant actions.

Preterm Hypoxic-Ischemic Encephalopathy.

Hypoxic–ischemic encephalopathy (HIE) is a recognizable and defined clinical syndrome in term infants that results from a severe or prolonged hypoxic–ischemic episode before or during birth. However, in the preterm infant, defining hypoxic–ischemic injury (HII), its clinical course, monitoring, and outcomes remains complex. Few studies examine preterm HIE, and these are heterogeneous, with variable inclusion criteria and outcomes reported. We examine the available evidence that implies that the incidence of hypoxic–ischemic insult in preterm infants is probably higher than recognized and follows a more complex clinical course, with higher rates of adverse neurological outcomes, compared to term infants. This review aims to elucidate the causes and consequences of preterm hypoxia–ischemia, the subsequent clinical encephalopathy syndrome, diagnostic tools, and outcomes. Finally, we suggest a uniform definition for preterm HIE that may help in identifying infants most at risk of adverse outcomes and amenable to neuroprotective therapies.

Human amnion epithelial cells protect against white matter brain injury after repeated endotoxin exposure in the preterm ovine fetus

Intrauterine inflammation is a significant cause of injury to the developing fetal brain. Using a preterm fetal sheep model of in utero infection, we asked whether human amnion epithelial cells (hAECs) were able to reduce inflammation-induced fetal brain injury. Surgery was undertaken on pregnant sheep at ~105 days gestation (term is 147 days) for implantation of vascular catheters. Lipopolysaccharide (LPS; 150 ng/kg bolus) or saline was administered IV at 109, 110, and 111 days. Sixty million fluorescent-labeled hAECs were administered at 110, 111, and 112 days gestation via the brachial artery catheter. Brains were collected at 114 days for histological assessment. hAECs were observed within the cortex, white matter, and hippocampus. Compared to control lambs, LPS administration was associated with significant and widespread fetal brain inflammation and injury as evidenced by increased number of activated microglia in the periventricular white matter (p = 0.02), increased pyknosis, cell degeneration (p = 0.01), and a nonsignificant trend of fewer oligodendrocytes in the subcortical and periventricular white matter. Administration of hAECs to LPS-treated animals was associated with a significant mitigation in both inflammation and injury as evidenced by fewer activated microglia (p = 0.03) and pyknotic cells (p = 0.03), significantly more oligodendrocytes in the subcortical and periventricular white matter (p = 0.01 and 0.02, respectively), and more myelin basic protein-positive cells within the periventricular white matter (p = 0.02). hAEC administration to fetal sheep exposed to multiple doses of LPS dampens the resultant fetal inflammatory response and mitigates associated brain injury.

Nucleated red blood cell counts: an early predictor of brain injury and 2-year outcome in neonates with hypoxic-ischemic encephalopathy in the era of cooling-based treatment

BACKGROUND Raised nucleated red blood cell (NRBC) counts in neonates may indicate in utero hypoxia and brain damage. OBJECTIVE The study aimed to examine the use of NRBC counts as a predictor of brain injury and neurodevelopmental outcomes in neonates with hypoxic-ischemic encephalopathy (HIE) treated under current cooling-based strategy. METHODS Forty-three neonates with asphyxia between 2004 and 2010 were retrospectively investigated. Twenty neonates with moderate/severe HIE underwent hypothermia (HT), and 23 with mild HIE were treated in normothermia (NT). Neonates were divided into groups according to the presence of cerebral parenchymal lesions on magnetic resonance imaging (MRI) at 2 weeks after birth. All patients were followed-up neurologically for ⩾ 24 months. NRBC counts during the first 3 days were compared between groups. RESULTS Eleven HT (HT-N) and 21 NT (NT-N) neonates had normal MRI, and 9 HT (HT-L) and 2 NT (NT-L) neonates had parenchymal lesions. NRBC counts, both absolute and /100 white blood cells (WBC) counts, during the first 3 days in HT-L and NT-L were significantly higher than those in HT-N and NT-N, particularly within 6 hours after birth (HT-N: 502 [0-3060]/mm(3) vs HT-L: 2765 [496-6192]; 0 [0-3417] vs NT-L: 4384 [3978-4789], median [range]). Neonates with /100 white blood cells ⩾ 6/mm(3) and absolute NRBC counts ⩾ 1324/mm(3) within 6 hours of birth had high risks of abnormal MRIs and 2-year outcomes. CONCLUSIONS NRBC counts can predict brain injury and neurological outcomes in cooled and non-cooled asphyxiated neonates.

The Beneficial Effects of Melatonin Administration Following Hypoxia-Ischemia in Preterm Fetal Sheep

Melatonin (MLT) is an endogenous hormone that controls circadian cycle. MLT has additional important properties that make it appealing as a neuroprotective agent—it is a potent anti-oxidant, with anti-apoptotic and anti-inflammatory properties. MLT is safe for administration during pregnancy or to the newborn after birth, and can reduce white matter brain injury under conditions of chronic fetal hypoxia. Accordingly, in the current study, we examined whether an intermediate dose of MLT could restore white matter brain development when administered after an acute hypoxic ischemic (HI) insult in preterm fetal sheep. Fifteen fetal sheep at 95–98 days gestation were instrumented with femoral artery and vein catheters, and a silastic cuff placed around the umbilical cord. At 102 days gestation, the cuff was inflated, causing complete umbilical cord occlusion for 25 min in 10 fetuses, to induce acute severe HI. Five HI fetuses received intravenous MLT for 24 h beginning at 2 h after HI. The remaining five fetuses were administered saline alone. Ten days after HI, the fetal brain was collected from each animal and white and gray matter neuropathology assessed. HI caused a significant increase in apoptotic cell death (TUNEL+), activated microglia (Iba-1+), and oxidative stress (8-OHdG+) within the subventricular and subcortical white matter. HI reduced the total number of oligodendrocytes and CNPase+ myelin density. MLT administration following HI decreased apoptosis, inflammation and oxidative stress within the white matter. MLT had intermediate benefits for the developing white matter: it increased oligodendrocyte cell number within the periventricular white matter only, and improved CNPase+ myelin density within the subcortical but not the striatal white matter. MLT administration following HI was also associated with improved neuronal survival within the cortex. Neuropathology in preterm infants is complex and mediated by multiple mechanisms, including inflammation, oxidative stress and apoptotic pathways. Treatment with MLT presents a safe approach to neuroprotective therapy in preterm infants but appears to have brain region-specific benefits within the white matter.

Term vs. preterm cord blood cells for the prevention of preterm brain injury

BACKGROUNDWhite matter brain injury in preterm infants can induce neurodevelopmental deficits. Umbilical cord blood (UCB) cells demonstrate neuroprotective properties, but it is unknown whether cells obtained from preterm cord blood (PCB) vs. term cord blood (TCB) have similar efficacy. This study compared the ability of TCB vs. PCB cells to reduce white matter injury in preterm fetal sheep.METHODSHypoxia–ischemia (HI) was induced in fetal sheep (0.7 gestation) by 25 min umbilical cord occlusion. Allogeneic UCB cells from term or preterm sheep, or saline, were administered to the fetus at 12 h after HI. The fetal brain was collected at 10-day post HI for assessment of white matter neuropathology.RESULTSHI (n=7) induced cell death and microglial activation and reduced total oligodendrocytes and CNPase+myelin protein in the periventricular white matter and internal capsule when compared with control (n=10). Administration of TCB or PCB cells normalized white matter density and reduced cell death and microgliosis (P<0.05). PCB prevented upregulation of plasma tumor necrosis factor (TNF)-a, whereas TCB increased anti-inflammatory interleukin (IL)-10 (P<0.05). TCB, but not PCB, reduced circulating oxidative stress.CONCLUSIONSTCB and PCB cells reduced preterm HI-induced white matter injury, primarily via anti-inflammatory actions. The secondary mechanisms of neuroprotection appear different following TCB vs. PCB administration.

Preterm umbilical cord blood derived mesenchymal stem/stromal cells protect preterm white matter brain development against hypoxia-ischemia

Introduction Preterm infants are at high risk for white matter injury and subsequent neurodevelopmental impairments. Mesenchymal stem/stromal cells (MSC) have anti‐inflammatory/immunomodulatory actions and are of interest for neural repair in adults and newborns. This study examined the neuroprotective effects of allogeneic MSC, derived from preterm umbilical cord blood (UCB), in a preterm sheep model of white matter injury. Methods Quad‐lineage differentiation, clonogenicity and self‐renewal ability of UCB‐derived MSC were confirmed. Chronically instrumented fetal sheep (0.7 gestation) received either 25 min hypoxia‐ischemia (HI) to induce preterm brain injury, or sham‐HI. Ten million MSC, or saline, were administered iv to fetuses at 12 h after HI. Fetal brains were collected 10d after HI for histopathology and immunocytochemistry. Results HI induced white matter injury, as indicated by a reduction in CNPase‐positive myelin fiber density. HI also induced microglial activation (Iba‐1) in the periventricular white matter and internal capsule (P < .05 vs control). MSC administration following HI preserved myelination (P < .05), modified microglial activation, and promoted macrophage migration (CD163) and cell proliferation (Ki‐67) within cerebral white matter (P < .05). Cerebral CXCL10 concentration was increased following MSC administration (P < .05), which was likely associated with macrophage migration and cell proliferation within the preterm brain. Additionally, MSC administration reduced systemic pro‐inflammatory cytokine TNF&agr; at 3d post‐HI (P < .05). Conclusions UCB‐derived MSC therapy preserved white matter brain structure following preterm HI, mediated by a suppression of microglial activation, promotion of macrophage migration and acceleration of self‐repair within the preterm brain. UCB‐derived MSC are neuroprotective, acting via peripheral and cerebral anti‐inflammatory and immunomodulatory mechanisms. HighlightsPreterm UCB‐MSC infusion reduces HI‐induced white matter injury in the preterm brainMSC induce macrophage migration and oligodendrocyte proliferation within white matterMSC suppress systemic TNF‐a release and cerebral microglial activation following HI

Human Umbilical Cord Blood Therapy Protects Cerebral White Matter from Systemic LPS Exposure in Preterm Fetal Sheep

Background: Infants born preterm following exposure to in utero inflammation/chorioamnionitis are at high risk of brain injury and life-long neurological deficits. In this study, we assessed the efficacy of early intervention umbilical cord blood (UCB) cell therapy in a large animal model of preterm brain inflammation and injury. We hypothesised that UCB treatment would be neuroprotective for the preterm brain following subclinical fetal inflammation. Methods: Chronically instrumented fetal sheep at 0.65 gestation were administered lipopolysaccharide (LPS, 150 ng, 055:B5) intravenously over 3 consecutive days, followed by 100 million human UCB mononuclear cells 6 h after the final LPS dose. Controls were administered saline instead of LPS and cells. Ten days after the first LPS dose, the fetal brain and cerebrospinal fluid were collected for analysis of subcortical and periventricular white matter injury and inflammation. Results: LPS administration increased microglial aggregate size, neutrophil recruitment, astrogliosis and cell death compared with controls. LPS also reduced total oligodendrocyte count and decreased mature myelinating oligodendrocytes. UCB cell therapy attenuated cell death and inflammation, and recovered total and mature oligodendrocytes, compared with LPS. Conclusions: UCB cell treatment following inflammation reduces preterm white matter brain injury, likely mediated via anti-inflammatory actions.

Controlling the Effective Oxygen Tension Experienced by Cells Using a Dynamic Culture Technique for Hematopoietic Ex Vivo Expansion

Clinical hematopoietic stem/progenitor cell (HSPC) transplantation outcomes are strongly correlated with the number of cells infused. Hence, to generate sufficient HSPCs for transplantation, the best culture parameters for expansion are critical. It is generally assumed that the defined oxygen (O2 ) set for the incubator reflects the pericellular O2 to which cells are being exposed. Studies have shown that low O2 tension maintains an undifferentiated state, but the expansion rate may be constrained because of limited diffusion in a static culture system. A combination of low ambient O2 and dynamic culture conditions has been developed to increase the reconstituting capacity of human HSPCs. In this unit, the protocols for serum-free expansion of HSPCs at 5% and 20% O2 in static and dynamic nutrient flow mode are described. Finally, the impact of O2 tension on HSPC expansion in vitro by flow cytometry and colony forming assays and in vivo through engraftment using a murine model is assessed.