Inconsistency of fetal trophoblast cells in first trimester maternal peripheral blood prevents non-invasive fetal testing using this cell target.

We have investigated whether maternal peripheral blood from the first trimester of pregnancy is a reliable source of identifiable trophoblast cells. The cells were enriched from 30 ml of venous blood, with multiple antibodies shown previously to enrich trophoblasts and a new cocktail based on known trophoblast surface features. Three different magnetic solid phases were tested to enrich trophoblasts, and both positive and negative cell enrichment strategies were examined. The cells were identified as trophoblast by morphology coupled with immunocytochemistry to co-localize cytokeratin with one of three IGF-II, PAI-1 or hPLH proteins or by in-situ hybridization with a mixture of 50 oligos directed to eight different expressed genes, alpha-HCG, IGF-II, PAI-1, HASH2, hPLH, p57(KIP2), PP5, H-19. While these tools worked beautifully in chorionic villi cell/sprout preparations and tissue sections, we could not detect and identify any trophoblasts in maternal peripheral blood even if the maternal peripheral blood was drawn 5-20 min following termination of pregnancy or from individuals maintaining the pregnancy. Based on our own experience and that of some reports in the literature, trophoblasts do not appear to be a viable candidate for fetal screening using maternal peripheral blood as the source. It is important to note that while trophoblast deportation is a biological phenomenon that has been described repeatable, they do not provide a means to perform prenatal genetic diagnosis.

Cloning and characterization of human erythroid membrane-associated protein, human ERMAP.

We describe here the cloning and characterization of the human gene ERMAP, identified by subtractive hybridization using early and late gestation human fetal liver. By in situ hybridization, we found human ERMAP to be expressed not only in erythoid cells in fetal liver and adult bone marrow, but also in reticulocytes and circulating erythroblasts in 8-12-week fetal cord blood. The human ERMAP protein is predicted to contain a transmembrane segment and one extracellular immunoglobulin fold (IgV). The cytoplasmic region contains a highly conserved B30.2 motif, multiple consensus sequences for kinases, and post-Golgi sorting signals. The protein was localized to the cell surface as shown by an antibody specific for a peptide predicted from the IgV fold. The amino acid sequence of human ERMAP is highly homologous with that of mouse ERMAP, but differs in the number of extracellular immunoglobulin folds. Human ERMAP represents a new unique member of the rapidly growing B30.2 domain proteins.

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.

Identification of differentially expressed mRNAs in human fetal liver across gestation.

Differential gene expression, with its precise start and stop times, is believed to be critical for the programmed development of new cells and tissues. Within the developing fetus, one tissue of particular interest is fetal liver. This organ undergoes rapid changes in the pathway toward liver development in utero since it is also the major site of hematopoiesis, until bone marrow hematopoiesis predominates. Believing that patterns would emerge from the bi-weekly large-scale inspection of expressed genes in the fetal liver, we employed differential display reverse transcription-polymerase chain reaction (DDRT-PCR) as ourprimary inspection tool. Using DDRT-PCR, we isolated cDNAs differentially expressed throughout fetal liver development and in adult liver. We displayed approximately 25 000 cDNAs from 10 and 24 week fetal liver and adult liver. From this initial screen, we determined that approximately 0.1-1% of the mRNA population undergoes expression changes. We extracted, purified and sequenced 25 differentially displayed cDNA bands. Fourteen cDNAs had similarities to known genes, while 11 cDNAs were not similar to any characterized gene. The differentially expressed cDNAs from known genes present in fetal liver include alpha-fetoprotein, stem cell factor, erythroid alpha-spectrin, 2,3-bisphosphoglycerate mutase, insulin-like growth factor-2, porphobilinogen deaminase and Mac30. The differentially expressed cDNAs present in adult liver but not in 10 week fetal liver were nicotinamide deaminase, human fibrinogen-related protein and alpha-acid glycoprotein. The majority of differentially expressed genes found during this effort appear to be turned on during organogenesis, however, some genes were found that are apparently turned off completely.