Cocultivation of umbilical cord blood cells with endothelial cells leads to extensive amplification of competent CD34+CD38- cells.

OBJECTIVE: In this report, methods to expand the number of human cord blood hematopoietic stem cells were explored. MATERIALS AND METHODS: CD34+ cord blood cells were expanded in the presence of various cytokine combinations in either a stroma-free cell culture system or a preformed porcine microvascular endothelial cell layer. After 7 to 21 days, stem cell number and function were monitored. In addition, the replicative history of stem cells was tracked using the fluorescent dye, PKH26. RESULTS: With the addition of various cytokine combinations, total cellular expansion was equivalent for both culture systems, although the endothelial cell-based system contained statistically greater numbers of CD34+ cells. By day 21, the endothelial-based system receiving the FLT3L, SCF, IL-6, and GM-CSF cytokine combination contained five-fold greater numbers of CD34+ than the stroma cell-free culture cell system. Endothelial-based cultures receiving these four cytokines plus megakaryocyte growth and development factor produced a 640-fold expansion of CD34+CD38- cells as compared to a four-fold expansion in the stroma-free system. The number of progenitor cells generated was similar with both systems, yet the greatest degree of expansion of cobblestone area-forming cells was observed in the endothelial based cultures (11-fold vs four-fold). Virtually all CD34+ and CD34+CD38+ cells expanded in the presence of endothelial cells had undergone self replication by day 10, yet stromal cell-free cultures contained a significant number (4.8%) of quiescent cells. Identical numbers of re-isolated cord blood CD34+ cells expanded in both systems exhibited a similar ability to engraft and generate cells belonging to multiple hematopoietic lineages in human fetal bones implanted in immunodeficient mice. CONCLUSIONS: These results suggest that the use of preformed endothelial cell monolayers might permit the ex vivo generation of sufficient numbers of cord stem cells to serve as successful grafts for adult transplant recipients.

Characterization and differentiation of human embryonic stem cells.

Cell replacement therapies have been limited by the availability of sufficient quantities of cells for transplantation. Human ES (hES) cell lines have recently been generated by several laboratories. When maintained for over 1 year in vitro, they remain karyotypically and phenotypically stable and may therefore provide an excellent source material for cell therapies. Currently, data is available for 26 hES cell lines. Although limited characterization has been performed on most of these lines, there are remarkable similarities in expression of markers. hES cell lines derived in different laboratories show similar expression profiles of surface markers, including SSEA-4, Tra-1-60, and Tra-1-81. In addition, markers associated with pluripotent cells such as OCT-4 are expressed at in all cell lines tested. These cells express high levels of telomerase and appear to have indefinite growth potential. The generation of the large quantities of cells necessary for cell replacement therapies will require a cell population which is stable over long term culture. We have characterized the properties of multiple hES cell lines that have been maintained in culture for extended periods. Quantitative analyses demonstrate that all of the cell lines examined show consistent marker expression and retain a normal karyotype after long-term culture. hES cells have been differentiated into the derivatives of all three germ layers. Specifically this includes cardiomyocytes, neural cells, hepatocyte-like cells, endothelial cells and hematopoietic progenitor cells. These data demonstrating the karyotypic and phenotypic stability of hES cells and their extensive differentiative capacity indicate that they may be an appropriate source of cells for multiple regenerative medicine applications.

[The menopause--a sociopsychological interpretation]. / Die Wechseljahre--eine sozial-psychologische Interpretation.

The psychological situation of climacteric women is determined by social economic and cultural changes. The educational backgrounds and family life are factors to a high degree of life stress, too. Psychosocial analysis and more informations about the conflicts in this time are preconditions for a successful treatment of menopausal complaints before hormone replacement in the gynecological praxis.

[Perioperative chemoprevention with metronidazole in cesarean section]. / Perioperative Chemoprophylaxe mit Metronidazol beim Kaiserschnitt.

96 caesarean section patients received 2.5 g of metronidazole rectally to prevent infection 6 or more hours after rupture of membranes. The treatment was continued until 30 hours after operation. The febrile morbidity rate was 34.3% in spite of prophylaxis. We conclude that this form of prophylaxis is not appropriate for high-risk caesarean sections.

[Rare finding in laparotomy--retroperitoneal para-aortic paraganglioma]. / Seltener Befund bei der Laparotomie--Retroperitoneales paraaortales Paragangliom.

This report presents one case of paraaortal paraganglioma of the retroperitoneum in a 17 years old patient. Frequency, diagnosis and the treatment are discussed.

[Recurrence of choriocarcinoma following methotrexate monotherapy]. / Rezidiv eines Chorionkarzinomas nach Methotrexat-Monotherapie.

Clinical course, diagnosis and therapy of a recurrence of an intramural choriocarcinoma in a women of 26 years are explained. Primary treatment of the patient was only methotrexate. In addition to the clinical findings the results of the HCG determinations are important for the follow-up.

[Prevention of sterility]. / Prophylaxe der Sterilität.

An overview is given about possibly preventable causes of infertility. The best chances for prevention are in tubal sterility and ectopic pregnancy. Methods of "primary prevention" are reduction of STD by health education and epidemiologic actions, by reduction and improvement of intrauterine operations in young women. The most important methods in "secondary prevention" are early and optimal treatment of PID and organ preservation in ectopic pregnancy. The difficulties in prophylaxis of endocrine infertility are discussed. For prevention of male infertility some procedures are necessary: Prevention resp. optimal treatment of genital infections, early treatment of development disorders and varicocele and elimination of chemical and physical noxes on male fertility.

PIP: An overview of possibly preventable causes of infertility is given. The best chances for prevention are in tubal sterility and ectopic pregnancy. Methods of primary prevention are reduction of sexually transmitted diseases by health education and epidemiologic actions, and by reduction and improvement of intrauterine operations in young women. The most important methods in secondary prevention are early and optimal treatment of pelvic inflammatory disease and organ preservation in ectopic pregnancy. The difficulties in prophylaxis of endocrine infertility are discussed. For prevention of male infertility, some procedures are necessary: optimal treatment of genital infections, early treatment of development disorders and varicoceles, and elimination of chemical and physical toxins.

[Aspiration cytology--endometrium diagnosis of peri- and postmenopausal bleeding disorders]. / Die Aspirationszytologie--Endometriumdiagnostik peri- und postmenopausaler Blutungsstörungen.

The aspiration of endometrial cell samples for cytological analysis in comparison to histological diagnosis with a following conventional curettage was evaluated in a symptomatic group of 100 women. The age ranged between 45 to 72 years. In 87% of the detected cytological and histological findings were comparable. In 2% the results were false negative. There was determined a specificity of 88.7% and a sensitivity of 88.2%. The usage as screening method in asymptomatic group of women may be a future interest of the endometrial cytology.

An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells.

The marrow repopulating potential (MRP) of different sources of human hematopoietic stem cells (HSCs) was directly compared using an in vivo assay in which severe combined immunodeficient disease (SCID) mice were implanted with human fetal bones. HSCs from 2 human lymphocyte antigen (HLA)-mismatched donors were injected individually or simultaneously into the fetal bones of a 3rd distinct HLA type and donor and recipient myeloid and lymphoid cells were identified after 8 to 10 weeks. The study compared the MRP of umbilical cord blood (CB) and adult bone marrow (ABM) CD34(+) cells as well as grafts of each type expanded ex vivo. Equal numbers of CB and ABM CD34(+) cells injected individually demonstrated similar abilities to establish multilineage hematopoiesis. However, when CB and ABM cells were transplanted simultaneously, the engraftment of CB cells was markedly superior to ABM. CB and ABM CD34(+) cells were expanded ex vivo using either a porcine microvascular endothelial cell (PMVEC)-based coculture system or a stroma-free expansion system. Primary CB CD34(+) cells or CD34(+) cells expanded in either culture system demonstrated a similar ability to engraft. However, the MRP of expanded grafts simultaneously injected with primary CB cells was uniformly inferior to primary CB cells. CD34(+) cell grafts expanded in the stroma-free system, furthermore, outcompeted CD34(+) cells expanded using the PMVEC coculture system. The triple HLA-mismatched SCID-hu model represents a novel in vivo stem cell assay system that permits the direct demonstration of the functional consequences of ex vivo HSC expansion and ontogeny-related differences in HSCs.