umbilical cord stem cells induce glial fate in neural progenitor cells

Abstracts / Placenta 35 (2014) A1eA112 A93

Objectives: The Wharton’s jelly (WJ) of the umbilical cord is derived fromthe mesoderm of the primitive streak which invades the connecting stalkand primary chorionic villi. Mesenchymal stem cells in the mesodermdifferentiate and participate in neo-vasculogenesis of the umbilical andplacental circulation. Whether these cells have a continued role inplacental/umbilical vascular growth and maturity is not known. Fetal stemcells have been shown to traffic and repair injured myocardial endothe-lium in mice (Kara et al. 2012). Their ability to interact with the uterineendothelium has not been investigated. The aim of this study was toinvestigate whether umbilical stem cells (WJMSC) can interact and influ-ence umbilical vein and uterine endothelial monolayers.

Methods: Human umbilical vein (HUVEC) and uterine microvascularendothelial cells (HUtMEC) were grown to 70% confluence in endothelialmedia and to full confluence in mixed media (endothelial: stem cell me-dia). Isolated WJMSC, labelled with the fluorescent tracker, PKH26 wasadded on top. Cell-cell interactions were monitored with fluorescencetime-lapse microscopy. Key events were analysed by confocal microscopyafter vascular endothelial (VE) cadherin immunocytochemistry.Results: WJMSC displayed non-apoptotic blebbing, amoeboid movementand shedding of exosomes as early events. There was a time lag of 60 minbefore transmigration of WJMSC via the paracellular pathway to sub-endothelial positions. This was concomitant with disruption of VE-cad-herin (statistically significant reduction of continuous junctional profiles).By 16h, a majority of WJMSC were resident underneath the endothelialmonolayers; junctional profiles returned to normal values. At 22h, the co-cultures showed a higher % of paracellular clefts with continuous VE-cadherin labelling, indicative of increased junctional maturity.Conclusion: Our data suggests that umbilical mesenchymal stem cells caninteract with and influence junctional organisation of both fetal anduterine endothelial cells. Their role, if any, in uterine vascular re-modellingduring pregnancy requires investigation.

P2.102-N.UMBILICAL CORD STEM CELLS INDUCE GLIAL FATE IN NEURALPROGENITOR CELLS

Byron Oppliger a,b, Marianne J€orger-Messerli a, Camilla Marini a,b, UrsulaReinhart a, Daniel V. Surbek a, Andreina Schoeberlein a aDepartment ofObstetrics and Gynecology, University Hospital, Department of ClinicalResearch, University of Bern, Bern, Bern, Switzerland; bGraduate School forCellular and Biomedical Sciences (GCB), University of Bern, Bern, Bern,Switzerland

Objectives: UsingWharton’s jelly mesenchymal stem cells (WJ-MSC) fromthe umbilical cord in terms of a regenerative approach to cure perinatalbrain damage and other central nervous system disorders may be thetherapy of the future. The secretome of WJ-MSC ought to contain severalbeneficial factors to boost the endogenous neuroregenerative and remye-linating capacity. Therefore, the objective of this study is to assess the effectof the neurotrophic factors fromWJ-MSC on neuroregeneration in vitro.

Methods: The secretome of WJ-MSC was assessed by mass spectroscopyand with a membrane-based antibody array. The effect of these factors onneural progenitor cells (NPC) was analyzed in vitro in conditioned mediumand co-culture experiments by measuring the expression of neuroglialmarkers with immunocytochemistry, real-time PCR and western blot.Further the differences between WJ-MSC derived from term or pretermpregnancies were evaluated.Results: Over 500 secreted proteins were identified. Several of thoseproteins are involved in glio- and neurogenesis. WJ-MSC derived fromterm pregnancies had a more prominent effect on the expression ofoligodendrocyte markers in NPC than preterm. The conditioned mediumshowed significantly less effect on the expression of neuroglial markersthan the co-culture.Conclusion: WJ-MSC derived from term pregnancies have a differentsecretome than those from preterm pregnancies. This was demonstratedby mass spectroscopy as well as by in vitro experiments. Moreover, cellcontact may be necessary to induce neuroglial differentiation on residentNPC. In conclusion transplanting WJ-MSC into damaged brains of neonatal

infants may enhance and support endogenous remyelination and neuro-regeneration.

P2.103.HEMANGIOBLASTIC FOCI IN FIRST TRIMESTER PLACENTA:DISTRIBUTION AND GESTATIONAL PROFILE

John D. Aplin, Hannah Whittaker, Jayne Charnock, Carolyn JP.Jones University of Manchester, Manchester, UK

Objectives: Descriptions of vasculogenesis and hematopoiesis in the earlyhuman placenta are incomplete. We report the incidence and distributionof hemangioblastic foci (HAFs).

Methods: 22 specimens from the Manchester early pregnancy tissue bankand 18 early in situ placentas from Cambridge collections were analysedhistologically and cytochemically.Results: At 4 weeks, cleft-shaped HAFs containing tightly packed, mainlyerythroid lineage cells are prominent in the chorionic plate (CP) and in pre-sumptive stemvilli near sites of branching fromtheCP.HAFs are also found inmesenchymal villi alongside simple vessel profiles, and often in close appo-sition to trophoblast at one end. By 6weeks, all vessels in the central CP haveacquired open lumena. HAF lumina in villi open towards the CP, withadherent cells at the opposite pole. Distally and laterally with respect to thecentral villous placenta, avascular villi are always present. By 10 weeks mostvillous vessels are open and lack adherent cells. Endothelial cells are CD34+/CD31+/FLK-1+/UEA-1+ and mainly nestin+/smooth muscle actin+. Gaps inthe endotheliumpersist up to8weeks. Some large roundednucleated cells inHAFsarebrachyury+upto6weeks, consistentwithaprimitivemesenchymalcell. CD68+ Hofbauer cells are found in villous stroma at all gestational agesbut not in HAFs. Rarely CD45+ cells are present in the clefts.Conclusion: HAFs are found in first trimester placenta from 4-9 weeks,with erythroid cells and endothelium being the two major derivatives. Wehypothesise that Hofbauer cells develop directly from mesenchymal stemcells in the villous stroma. The CP and adjacent stem villi, structuresproximal to the embryo, are particularly important sites of erythropoiesis.Simple vasculogenic networks coexist with HAFs in the villous placenta.

P2.104.EQUINE YOLK SAC CELLS FOR ANGIOGENESIS STUDY: A NEW TOOL FORVETERINARY MEDICINE REGENERATIVE

Andre Luis Rezende Franciolli a, Rafael Cardoso Carvalho a, MarcioNogueira Rodrigues a, Maria Angelica Miglino a, Paula Fratini a, AnaClaudia Oliveira Carreira b aUniversity of Sao Paulo - FMVZ, Sao Paulo,Brazil; bUniversity of Sao Paulo - NUCEL, Sao Paulo, Brazil

Recently the fetal membranes are regarded to be abundant, ethicallyacceptable and easily accessible sources of mesenchymal stem cells, whichcause only minor immunogenic troubles. In horses, stem cells are shownvery promising in the treatment of various diseases, for example, nerveinjuries, arthritis, injuries of tendons and suspensory ligament rupture,cardiovascular disease, ischemia, laminitis, rotation of the third flange andthe wall of the diseases vessels. The yolk sac (YS) is a source of endothelialprecursor cells. However, data about the full differentiation potential of YScells and their suitability for cell therapies are rare once that therapieswhere neovascularization is required seems to be of great value. Wecharacterized canine YS cells through expression of vascular endothelialgrowth factor (VEGF) markers in flow cytometry, immunocytochemistryand real time PCR, and analyzed their potential to enrich progenitorendothelial cells after transduction with VEGF. Immunocytochemistryshowed expression for CD105, PCNA, VEGFA and vWF, flow cytometry forCD105, VEGF, PCNA, OCT-4 and qPCR for VEGF, CD31, CD105, and PCNA.After transduction with PLV-VEGF lentivirus, the undifferentiated cellschanged their morphology into endothelial-like cells similar to thoseoccurring near developing capillaries. In conclusion, treatment with VEGFresulted as an appropriate method to enrich endothelial cells derived fromisolated canine YS cells, suggesting that these cells represents a promising