Neuroprotective effects of human umbilical cord-derived mesenchymal stem cells on periventricular leukomalacia-like brain injury in neonatal rats

Chikako Morioka, Motohiro Komaki, Atsuko Taki, Izumi Honda, Naoki Yokoyama, Kengo Iwasaki, Sachiko Iseki, Tomohiro Morio, Ikuo Morita

Periventricular leukomalacia (PVL) is a type of multifactorial brain injury that causes cerebral palsy in premature infants. To date, effective therapies for PVL have not been available. In this study, we examined whether mesenchymal stem cells (MSCs) possess neuroprotective property in a lipopolysaccharide (LPS)-induced neonatal rat PVL-like brain injury. Human umbilical cord-derived MSCs (UCMSCs) were used in this study. Four-day-old rats were intraperitoneally injected with LPS (15 mg/kg) to cause the PVL-like brain injury and were treated immediately after the LPS-injection with UCMSCs, conditioned medium prepared from MSCs (UCMSC-CM) or interferon-gamma (IFN-γ)-pretreated MSC (IFN-γ-UCMSC-CM). To assess systemic reaction to LPS-infusion, IFN-γ in sera was measured by ELISA. The brain injury was evaluated by immunostaining of myelin basic protein (MBP) and caspase-3. RT-PCR was used to quantitate pro-inflammatory cytokine levels in the brain injury, and the expression of tumor necrosis factor-stimulated gene-6 (TSG-6) or indoleamine 2,3-dioxygenase (IDO) to evaluate anti-inflammatory or immunomodulatory molecules in UCMSCs, respectively. A cytokine and growth factor array was employed to investigate the cytokine secretion profiles of UCMSCs. Elevated serum IFN-γ was observed in LPS-infused rats. The expression of IL-6, tumor necrosis factor-alpha (TNF-α), IL-1ß, and monocyte chemoattractant protein-1 (MCP-1) were increased in the brain by LPS-infusion in comparison to saline-infused control. LPS-infusion increased caspase-3-positive cells and decreased MBP-positive area in neonatal rat brains. A cytokine and growth factor array demonstrated that UCMSCs secreted various cytokines and growth factors. UCMSCs significantly suppressed IL-1ß expression in the brains and reversed LPS-caused decrease in MBP-positive area. UCMSC-CM did not reverse MBP-positive area in the injured brain, while IFN-γ-UCMSC-CM significantly increased MBP-positive area compared to control (no treatment). IFN-γ-pretreatment increased TSG-6 and IDO expression in UCMSCs. We demonstrated that bolus intraperitoneal infusion of LPS caused PVL-like brain injury in neonatal rats and UCMSCs infusion ameliorated dysmyelination in LPS-induced neonatal rat brain injury. Conditioned medium prepared from IFN-γ-pretreated UCMSCs significantly reversed the brain damage in comparison with UCMSC-CM, suggesting that the preconditioning of UCMSCs would improve their neuroprotective effects. The mechanisms underline the therapeutic effects of MSCs on PVL need continued investigation to develop a more effective treatment.