Faculty Bibliography

Abstract Objective: The placenta of mid-gestation mice is a known rich source of hematopoietic stem cells. We hypothesized that it is also a source of other multipotent stem cells. Methods: We isolated fetal cells from the murine placenta across the second half of gestation and characterized their expression of surface antigens known to be associated with mesenchymal stem cells (MSCs) on a subset of hematopoietic lineage-negative cells. Using real-time reverse-transcriptase quantitative polymerase chain reaction, we also evaluated the expression of intracellular transcription factors (TFs) known to be associated with renal development and/or multipotent stem cells. Results: Cell phenotypes with surface marker and TF expression consistent with multipotent stem cells of a mesenchymal lineage as well as renal cell progenitors were found in the placenta. The expression of MSC and renal progenitor surface markers varied throughout gestation, but was highest on E12-15 where such cells represented a small but significant percentage of the population. Of the studied TFs, 10 of 11 renal TFs were found at moderate to high levels, and all stem cell TFs were found. Conclusion: The mid-gestation murine placenta may serve as a source of multipotent stem cells and also contains cells which may be renal cell progenitors.

Amniotic fluid contains a mixture of cells with capacity to differentiate into all germ layers. These cells are present in large numbers in midtrimester samples obtained for cytogenetic diagnosis, and have been identified by stem cell surface markers and transcription factors. We studied cultured samples from patients who had both direct cultures and matched cultures obtained 2 weeks later from the cytogenetics laboratory as well as patients with cytogenetics material only. Samples were cryogenically frozen, thawed, expanded in culture with excellent viability. There was considerable individual variation unrelated to gestational age or telomere length. Phenotype for embryonic markers was assessed by flow cytometry and by quantitative polymerase chain reaction. The most consistently present stem cell markers in substantial amounts were CD90, SSEA-4, & TRA-1-60. Cells with CD90, SSEA-4 & TRA-1-60 double and triple labeled also could be identified and subcultured, confirming the heterogeneity of the amniotic fluid stem cell population

Amniotic fluid stem cells(AFSC) are noncontroversial and abundant potentially transplantable cells. They are derived from fetal and amniotic sources and show osteogenic, neuronal, and adipogenic differentiation in culture. Clinical material must be expanded in culture and sorted if clinical use is desired. Cellular senescence and replicative potential for AFSC cultures has had limited study, with scant data on gene expression over time. We report changes in samples from 17 patients over multiple passages form 10 to 81 population doublings. Longevity was unrelated to telomere length in these cells. It was related to upregulation of TWIST1, which is highly expressed in stem cells, and downregulation of genes associated with apoptosis

If maternal atopy and environmental exposure affect prenatal Th cell development, the maternal and fetal immune systems should display common Th1/Th2 phenotypes. To test this hypothesis, we studied maternal and neonatal blood samples from mothers with total serum IgE <300 IU/mL. Basal levels of IFN-gamma, IL-4, and eotaxin in paired maternal and fetal sera were tightly correlated. Polyclonal T cell activation in vitro by Staphylococcal exotoxin B induced co-ordinate IFN-gamma production from paired maternal and fetal mononuclear cells, accompanied by co-ordinate increases in activated CD4+CD69+ cells that display the CCR4+Th2 and CXCR3+ Th1 phenotypes. Maternal and fetal CD4+CXCR3+ T cells were subsequently identified as the major producers of IFN-gamma. The data established that a transplacental nexus exists during normal pregnancy and that fetal Th cell responses may be biased by the maternal immune system.

OBJECTIVE: We sought to characterize markers of pluripotency in amniotic fluid derived cells as a non-controversial and readily available source of potentially therapeutic stem cells. STUDY DESIGN: Amniotic fluid stem cells (AFSC) express stem cell surface markers as well as transcription factors. Isolation and enrichment of the stem cell population is essential to their therapeutic potential. We studied expression of stem cell surface markers CD 117, CD 133, SSEA3, SSEA4, TRA 160, TRA 181 and CD90; as well as transcription factors OCT4, SOX2, NANOG and REX 1 by magnetic bead separation, flow cytometry analysis and PCR for transcription factors. Samples were obtained after cytogenetic analysis following routine amniocentesis for age or maternal anxiety in 10 normal patients. Cells were cultured for up to seven passages with analysis after confluence. RESULTS: There was great variation in different samples ability to express the different markers. The most prevalent marker was CD90, a mesenchymal stem cell factor; followed by SSEA4 and TRA160, both embryonic stem cell markers. The other markers were significantly present as well (SSEA3, TRA 160, TRA 181,CD90,OCT4, SOX2, NANOG and REX 1), however CD 117 and CD 133 were often undetectable or present in small amounts. CONCLUSION: There was considerable variation among samples, possibly gestational age related. In Amniotic fluid derived cells, stem cell markers CD90, SSEA4 and TRA160 are expressed in the largest amount with CD90 being the most abundantly expressed marker. Therefore these markers might be used for identification, isolation and enrichment of amniotic fluid stem cells for potential clinical use, since amniotic fluid is widely available and non-controversial as a source of multipotent stem cells.(Table persented)

OBJECTIVE: To study if maternal atopy and environmental exposure might affect fetal immune response. STUDY DESIGN: We studied paired maternal and neonatal blood samples from 38 unlabored normal mothers delivered by cesarean section, without recent infection as evidenced by total serum IgE<300 IU/ml. At delivery, 10 ml of fetal blood (FB) was withdrawn from the umbilical cord. At the same time, 10 ml of maternal blood (MB) was obtained by venipuncture. Specimens were processed and analyzed sideby- side within 12 hours. The samples were incubated with 10mcg/ml Staphylococcal Enterotoxin B (SEB). Th phenotypes were analyzed by flow cytometry. Cytokines were assayed by multiplex analysis (Luminex 200) RESULTS: Basal levels of IFN-, IL-4, and eotaxin in paired maternal and fetal sera were tightly correlated. Polyclonal T cell activation in vitro induced co-ordinate IFN- production from paired maternal and fetal mononuclear cells, accompanied by co-ordinate increases in activated CD4+CD69+ cells that display the CCR4+ Th2 and CXCR3+Th1 phenotypes. Maternal and fetalCD4+CXCR3+Tcells were subsequently identified as the major producers of IFN-. CONCLUSION: The data established that a transplacental nexus exists during normal pregnancy such that fetal Th responses to an immune stimulus are biased by the maternal immune system. (Table Presented)