Volume 168 Number I, Part 2

218· IDENTIFICATION OF PLACENTAL BED MOLECULAR WEIGHT CYTOKERATIN

USING A LOW MONOCLONAL

ANTIBODY, J. Meekinsx, R. PijnenborgX, M.Hanssensx , I.

McFadyenX, A. Van Assche. Dept. Ob/Gyn ,Univ. Hosp. Gasthuisberg,K.U.L.,Leuven, Belgium and Dept. Ob/Gyn, Royal Liverpool Univ. Hosp., Liverpool, UK. OBJECTIVE: Immunohistological staining was used in an attempt to refine the identification of placental bed biopsies compared to standard morphological methods. STUDY DESIGN: 320 placental bed biopsies were taken under direct vision at caesarean section. No trophoblast was seen in 148 (46%) using standard morphological techniques. 33 of these biopsies were randomly selected. Immunohistological staining using the avidin biotin complex method was carried out on adjacent sections with a monoclonal antibody to cytokeratin ( a marker for trophoblast and epithelial tissue). Control tissue was decidua vera and placental bed biopsies deemed positive for trophoblast by standard methods. RESULTS: 13 of the 33 sections (39%) showed cells positive to cytokeratin, thought to be trophoblast on morphological criteria and not glandular epithelium. Placental bed biopsy controls, stained with cytokeratin, showed that mononuclear trophoblast was by no means scanty nor giant cells predominant. Trophoblast morphology and staining intensity was diverse, and endometrial glands were abundant. CONCLUSION: Immunohistological staining for low molecular weight cytokeratin facilitates the detection of trophobast and refines the identification of placental bed. The absence of giant cells or presence of abundant glandular tissue may not necessarily suggest the biopsy be rejected for interpretation.

219 EXISTENCE OF ENDOTHELIN CONVERTING ENZYME IN HUMAN PLACENTAL VEINS. J....Y.... Momboulix, N. Wasserstrum, and P.M. Vanhoutte.x Ctr. Exp. Ther. & Depts. Ob/Gyn and Medicine, Baylor Coli. Med. Houston, Tx. OBJECTIVE: Endothelin-1 (ET-1) may playa role in controlling the fetal placental circulation. Endothelin converting enzyme(s) (ECE) generate ET-1 from its inactive precursor Big ET-1. Thus responsiveness to Big ET-1 serves as an index for tissue ECE activity. Therefore, we determined the responses of isolated placental veins to Big ET-1 and to ET-1. STUDY DESIGN: Immediately after vaginal delivery, veins of the placental chorionic plate were dissected free of extraneous tissue, divided into rings and suspended in conventional organ chambers filled with physiological salt solution for recording of isometric tension. A 2 hour equilibration was followed by reference contraction to potassium (60 mM K+). After return to baseline, rings were incubated (40 min) with either 10.5 M phosphoramidon (ECE-inhibitor) or vehicle. Statistical analysis of data was performed by use of Student's t test. RESUL TS. ET-1 and Big ET-1 each caused concentration­dependent contractions. The response to Big ET-1, but not that to ET -1, was blocked by the ECE-inhibitor phopshoramidon. CONCLUSIONS. The contraction of human placental veins to Big ET-1 is antagonized by inhibitors of ECE. Therefore, endothelins generated locally within the vascular wall may help to regulate tone in the human placental venous circulation.

SPO Abstracts 359

220 THE ABSENCE OF ARGININE VASOPRESSIN RECEPTORS IN ISOLATED CYTOTROPHOBLAST CELLS. Robert S E&erman and John M. Bissonnettex. Dept. Ob/Gyn, Oregon Health Sciences Univ., Portland, OR. OBJECTIVE: Arginine vasopressin (A VP) plays a vital role in water absorption by increasing membrane permeability in the distal nephron. To determine if AVP contributes to placental water homeostasis, we attempted to show the presence of A VP receptors on isolated cytotrophoblast cells through isotope binding. STUDY DESIGN: Cytotrophoblast cells were prepared from placentas through enzymatic digestion and density gradient centrifugation. Cells were then divided into two grou~. The fIrst set was incubated for two hours at 40C with 5nM [3H]AVP in Media-l99 with 2% BSA. The second set of cells was similarily prepared, but IOmcM AVP was added to determine nonspecifIc binding. Cells were centrifuged through oil to remove them from the incubation media. Additional studies were carried out using attached cells on 35mm culture plates and bound [3H]AVP was extracted with O.IN NaOH. In separate studies, LLCPKI cells [a porcine renal cell line ], were used as a positive control. RESULTS: Binding of [3H]AVP at 5nM concentration was not different from that seen in the presence of IOmcM AVP. Total binding and nonspecifIc binding were similar, reflecting the absence of A VP receptors in isolated cytotrophoblast cells. In contrast [3H]AVP showed 74% specifIc binding in LLCPKI cells. CONCLUSION: Isolated cytotrophoblasts do not contain receptors for A VP suggesting transplacental fluid regulation is not affected by AVP levels.

221 IMIILlCAl IDID BU)OO: SURFACE MARKER ANALYSIS AlII ASSAYS FOR STEM CELL AlllIlEGAlCARYOCYTE PROGEIIITOR ACTIVITY_ C.L. Cetrulo, L. Murray", L. Snipes" M. Malachowski" New England Medical Center, Tufts University School of Medicine, Boston, MAi Systemix, Palo Al to, CA. OBJECTIVE: To assess stem cell and megakaryocyte progeni tor activity in hunan unbi 1 icsl cord blood. STIllY DESIGII: Cord blood saq>les were collected in heparin and were analyzed by FACSCAN for various cell surface markers. Post-Ficoll cells contained over SOX glycophorin ' cells, which could be reduced by lysis to less than lOX. RESULTS: The mean percent CD34' cells was 1.2X (nlJIber = 7), C014' cells 18X, C03310 15' (mature myeloid) 8.2X, C033' IS' (inmature myeloid) 16X, C03' 4lX, C019' lX, glycophorin receptor' 1.8X. Three saq>les were placed at decreasing cell concentrations into llmegaculturell conditions to select for the production of megakaryocytes. All three saq>les gave positive megacolor readings at day 10-14 in both primary and secondary cul tures. Both GM and mega progenitor activity was demonstrated in agar, colony assays (55 CFU-GM/10' cells at day 13 and 12 megacolor ' colonies/10' cells at day 28). Stem cell activity was tested using Sys·1 cocultures, a long term cuLture assay for hunan stem cells. Cord blood cells prol iterated for 6-8 weeks and were able to differentiate into both myeloid and lyq>hoid cells, at a limit dilution frequency of 1 to 2100. C034' cells sorted from cord blood prol iterated in cocul tures at a frequency of greater than 1 in 222. COIICLUSIOllS: These results indicate that cord blood contains cells capable of initiating long term stem cell cultures and megakaryocyte cul tures. Based on these and other findings, we have establ ished a pi lot study to cryopreserve hunan unbil ical cord blood.