Hyun-Young Ahn

Proangiogenic features of Wharton's jelly-derived mesenchymal stromal/stem cells and their ability to form functional vessels.

Mesenchymal stromal/stem cells derived from human Whartons jelly (WJ-MSC) have emerged as a favorable source for autologous and allogenic cell therapy. Here, we characterized the proangiogenic features of WJ-MSCs and examined their ability to form functional vessels in in vivo models. First, we examined whether WJ-MSCs express endothelial and smooth muscle cell specific markers after culture in endothelial growth media. WJ-MSCs expressed an endothelial specific marker, VEGFR1, at mRNA and protein levels, but did not express other specific markers (VEGFR2, Tie2, vWF, CD31, and VE-cadherin). Rather, WJ-MSCs expressed smooth muscle cell specific markers, α-SMA, PDGFR-β and calponin, and were unable to form tube-like structures with lumen on Matrigel. WJ-MSCs secreted growth factors including angiogenin, IGFBP-3, MCP-1, and IL-8, which stimulated endothelial proliferation, migration, and tube formation. When WJ-MSCs suspended in Matrigel were implanted into nude mice, it led to formation of functional vessels containing erythrocytes after 7 days. However, implantation of endothelial cell-suspended Matrigel resulted in no perfused vessels. The implanted WJ-MSCs were stained positively for calponin or PDGFR-β and were located adjacent to the lining of mouse endothelial cells that were stained with labeled BS-lectin B4. In a murine hindlimb ischemia model, the transplantation of MSCs (5×10(5)cells) into the ischemic limbs improved perfusion recovery and neovascularization of the limbs compared to control group. Therefore, the results suggest that WJ-MSCs promote neovascularization and perfusion by secreting paracrine factors and by functioning as perivascular precursor cells, and that WJ-MSCs can be used efficiently for cell therapy of ischemic disease.

Efficient nonadhesive ex vivo expansion of early endothelial progenitor cells derived from CD34+ human cord blood fraction for effective therapeutic vascularization

Endothelial progenitor cells (EPCs) have been shown to have therapeutic potential in ischemic disease. However, the number of EPCs for cell therapy is limited. In this study, instead of the typical adherent culture method, we investigated a more efficient, clinically applicable nonadhesive expansion method for early EPCs using cord blood‐derived cells to overcome rapid cellular senescence. After a suspension culture of isolated CD34+ cells in serum‐free medium containing each cytokine combination was maintained for 9 d, the number of expanded functional EPCs was assessed by an adherent culture assay. Compared to mononuclear cells, the CD34+ fraction was superior in its expansion of functional EPCs that could differentiate into acLDL/UEA‐1+ cells without significant cellular senescence, whereas the CD34‐ fraction showed no EPC expansion. Among the cytokine combinations tested for the CD34+ fraction, a combination (SFIb) consisting of stem cell factor (SCF), FMS‐like tyrosine kinase 3 ligand, interleukin‐3, and basic fibroblast growth factor resulted in a reproducible 64‐ to 1468‐fold EPC expansion from various cord blood origins. Interestingly, the SFIb combination displayed markedly increased EPC expansion (2.43‐fold), with a higher percentage of CD34+ cells (2.17‐fold), undifferentiated blasts (2.38‐fold) and CXCR4+ cells (1.68‐fold) compared to another cytokine combination (SCF, thrombopoietin, and granulocyte colony‐stimulating factor), although the two cytokine combinations had a similar level of total mononucleated cell expansion (~10% difference). Accordingly, the cells expanded in the SFIb combination were more effective in recovery of blood flow and neovascularization in hind‐limb ischemia in vivo. Taken together, these results suggest that the nonadhesive serum‐free culture conditions of the CD34+ fraction provide an effective EPC expansion method for cell therapy, and an expansion condition leading to high percentages of CD34+ cells and blasts is likely important in EPC expansion.—O, E., Lee, B. H., Ahn, H.‐Y., Shin, J.‐C., Kim, H.‐Y., Kim, M., Park, I. Y., Park, Y.‐G., Joe, Y. A. Efficient nonadhesive ex vivo expansion of early endothelial progenitor cells derived from CD34+ human cord blood fraction for effective therapeutic vascularization. FASEB J. 25, 159–169 (2011). www.fasebj.org

Outgrowing endothelial progenitor-derived cells display high sensitivity to angiogenesis modulators and delayed senescence

Outgrowing endothelial progenitor‐derived cells (EPDCs) originate from a novel hierarchy of endothelial progenitor cells. In this study, EPDCs isolated from human cord blood were examined for phenotype and functional features upon aging. Young or aged EPDCs were similar to human umbilical vein endothelial cells (HUVECs), in exhibiting typical endothelial phenotypes. However, EPDCs were more sensitive to angiogenesis inducers or inhibitors in proliferation and migration. In addition, EPDCs underwent senescence markedly slowly with sustained endothelial NO synthase expression and activation, and their ability to undergo capillary morphogenesis was retained throughout longterm culture. Thus, these results suggest that a homogenous population of EPDCs derived from clonogenic expansion may provide an effective vasculogenesis tool.

Priming Wharton's Jelly‐Derived Mesenchymal Stromal/Stem Cells With ROCK Inhibitor Improves Recovery in an Intracerebral Hemorrhage Model

Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron‐like cells under specific conditions and to secrete paracrine factors for neuroprotection and regeneration. Previously, Rho‐kinase inhibitors have been reported to potentiate differentiation of rodent bone marrow MSCs into neuron‐like cells induced by CoCl2 (HIF‐1α activation‐mimicking agent). Here, a strategy of priming MSCs with fasudil, a Rho‐kinase inhibitor, was investigated using Whartons jelly‐derived MSCs (WJ‐MSCs) to improve recovery in a rat model of intracranial hemorrhage (ICH). In vitro culture of WJ‐MSCs by co‐treatment with fasudil (30 μM) and CoCl2 provoked morphological changes of WJ‐MSCs into neuron‐like cells and increased the expression of neuronal markers. Assessment of the secretion profiles showed that fasudil (30 μM) specifically increased glial cell line‐derived neurotrophic factor (GDNF) among the secreted proteins at the transcription and secretion levels. For in vivo experiments, WJ‐MSCs primed with fasudil (10 μM, exposure for 6 h) were transplanted into ICH rats with HIF‐1α upregulation 1 week after injury, and neurological function was assessed via rotarod and limb placement tests for 7 weeks after transplantation. The group with WJ‐MSCs primed with fasudil showed improved functional performance compared with the non‐primed group. Accordingly, the primed group showed stronger expression of GDNF and higher levels of microtubule‐associated protein 2 and neurofilament‐H positive‐grafted cells in the ICH lesion 3 weeks after transplantation compared with the non‐primed group. Therefore, this work suggests that priming WJ‐MSCs with fasudil is a possible application for enhanced cell therapy in stroke, with additional beneficial effect of up‐regulation of GDNF. J. Cell. Biochem. 116: 310–319, 2015.

The Rho kinase inhibitor fasudil augments the number of functional endothelial progenitor cells in ex vivo cultures.

Rho kinase (ROCK) has been implicated in the regulation of vascular tone, endothelial dysfunction, inflammation and remodeling. Endothelial progenitor cells (EPC) have been proven to have the efficacy of therapeutic neovascularization in ischemia. However, the scarcity of EPCs limits cell therapy. Using an in vitro EPC culture assay, Y27632 was found to increase the number of adherent EPCs. In this study, we investigated the effect of fasudil, another ROCK inhibitor being used in the clinic, on EPC number and examined whether EPCs expanded by fasudil are functional in vitro and in vivo. In ex vivo cultures of EPCs, fasudil effectively increased the number of ac-LDL/UEA-1 positive cells as well as adherent cells, in contrast to H89, a less selective ROCK inhibitor. Fasudil also increased EPC numbers in culture up to 10 µM, in a dose-dependent manner. When EPCs expanded with fasudil were examined for the migratory activity toward stromal cell-derived factor-1 and vascular endothelial growth factor, these cells retained functional properties in migration, albeit with some decrease. Fasudil-cultured EPCs labeled with PKH26 showed an activity similar to non-treated EPCs for cellular adhesion into an endothelial cell (EC) monolayer and incorporation into capillary-like structures formed by ECs. Finally, when EPCs cultured with fasudil (106 cells/mouse) were injected into ischemic limbs, these cells showed a blood flow recovery at a level comparable to non-treated control EPCs and increased neovascularization. Therefore, these data suggest that the ROCK inhibitor fasudil can provide a beneficial effect in the treatment of ischemic diseases by increasing EPC numbers.

Enhancement of Endothelial Progenitor Cell Numbers and Migration by H1152, a Rho Kinase Specific Inhibitor

Endothelial progenitor cells (EPCs) are applied in the treatment of ischemic diseases. In ex vivo culture of human cord-blood derived EPCs, H1152, (S)-(+)-2-methyl-1-[(4-methyl-5-iso-quinolinyl) sulfonyl]-homopiperazine, markedly increased the number of EPCs. It also induced EPC migration, stimulated the phosphorylation of AKT, and reduced the expression of p27 in the EPCs. Thus H1152 can be used effectively in ex vivo expansion of EPCs.

Expression of hepatocyte growth factor and its receptor in the placental basal plate in pre-eclamptic pregnancies.

Pre-eclampsia remains one of the leading causes of maternal and fetal morbidity and mortality. Although the etiology of pre-eclampsia is unclear, there is accumulating evidence that insufficient trophoblast invasion of the spiral arteries of the placental bed leads to inadequate perfusion of the placenta and to hypoxia in pre-eclampsia w1,2x. Hepatocyte growth factor (HGF) and c-met interactions seem to play an important role in regulating trophoblast invasion in pregnancy w3x. This study was designed to determine whether the expression of HGF and yor c-met are altered in pre-eclamptic placentas, including their differential expression, according to the severity of the condition, and to elucidate the possible relationship between their expression and the pathogenesis of pre-eclampsia. The investigative protocol for this study was approved by the Institutional Human Subject Committee of the Catholic University Medical Center. Placental tissue was obtained during cesarean delivery from five normotensive, nine mildly preeclamptic, and five severely pre-eclamptic women

P02.16: Selective transarterial embolization of uterine arteriovenous malformation complicated in previous Cesarean section scar pregnancy

The incidence of ectopic pregnancy is 2% of all pregnancies and has increased. It is reported that a recurrent ectopic pregnancy rate is 10–15% after salpingotomy compared with 10% after salpingectomy. Corneal pregnancy after ipsilateral total salpingectomy or remnant tubal pregnancy after ipsilateral partial salpingectomy is reported but recurrent ectopic pregnancy in the tubal remnant stump after ipsilateral total salpingectomy is rare and exact incidence is unknown. We present a case of spontaneous ectopic pregnancy occurring in the stump of a remnant fallopian tube following ipsilateral total salpingectomy for an ectopic pregnancy.

P27.13: Transabdominal amnioinfusion does not improve perinatal outcome in uncomplicated term pregnancies with oligohydramnios

Objectives: Oligohydramnios occurring early during the second trimester has to be considered a severe sign of poor prognosis. Amnioinfusion has been reported improving the prognosis in case of reduction of amniotic fluid volume due to premature rupture of membranes (PROM). We present our experience with amnioinfusion for oligohydramnios with or without preterm PROM. Methods: We prospectively enrolled pregnant women presenting oligohydramnios from January 2009 to December 2009. Second level scan was performed after each amnioinfusion. Further analysis such as magnetic resonance or genetic tests were performed after counselling. Results: Twenty-six cases were included in our study. Ten cases presented PROM and eight out of ten performed more than one amnioinfusion. One case of PROM revealed sonographic sign of fetal aneuploidy confirmed at karyotype. Sixteen cases were enrolled without any anamnestic or clinical sign of PROM. Ten cases presented sign of renal dysplasia before amnioinfusion confirmed after the procedure, and in two cases the second level scan revealed further anomalies. The five remaining fetuses did not presented either PROM of renal anomalies. One case out of six demonstrate intrauterine growth retardation (IUGR) and congenital heart disease, two cases presented IUGR associated to altered uterine artery Doppler. The three remaining cases remained unexplained. Conclusions: Amnioinfusion might provide benefits such as confirmation of rupture of membranes and detailed sonography examination. This procedure has to be considered in cases with oligohydramnios due to further counselling during pregnancy.

P01.16: A case of sacrococcygeal teratoma (type II) prenatal diagnosed by ultrasonography and fetal MRI

Results: We identified 84 cases. 24 had associated gastro-intestinal tract and respiratory anomalies; 22 had abnormal karyotypes (10 Trisomy 18s, 5 Trisomy 21s and 1 each of other aneuploidies or other karyotypical abnormalities); 6 had neuromuscular syndromes; 3 had major central nervous system anomalies; 7 had renal anomalies causing anhydramnios; 5 had other genetic syndromes; 2 had placental insufficiency leading to anhydramnios; 8 were found to have normal stomach appearances and had normal outcomes and 7 had a normal outcome despite persistently absent stomach on ultrasound examination. Of these 84: 26 underwent termination of pregnancy; 9 suffered in utero fetal demise; 8 died in the neonatal period; 3 died in infancy; 44 had a live birth and survived infancy and 5 outcomes were not obtained. Conclusions: A persistently absent stomach on ultrasound scanning is associated with a guarded prognosis – with an incidence of abnormal karyotype of 29% and a high incidence of associated structural abnormalities. In only 9.2% of persistently absent stomachs was the outcome normal. We have not been able to explain why these normal fetuses did not demonstrate stomach ‘bubbles’ on ultrasound.