Workpackage 1:

Circulatory Fetal Cells

The aim of this workpackage is to examine fetal cell-based approaches for the prenatal analysis of fetal genetic traits. The reason for pursuing this activity is that isolated fetal cells offer a potentially pure source of fetal genetic material, unlike the analysis of cell-free DNA in maternal plasma, where the bulk of the material is of maternal origin. A pure source of fetal DNA will greatly enable the analysis of fetal aneuploidies or Mendelian disorders such as b-thalassemia or cystic fibrosis.

In order to achieve this goal a main task of WP1 is to develop parameters in collaboration with WP2, which will permit the rapid identification of fetal cells by the use of automated imaging systems. The major problem concerning the retrieval of fetal cells from maternal blood samples is their scarcity (generally less than 1 in 106 maternal nucleated cells), lack of efficient enrichment systems and inability to reliably identify retrieved fetal cells, as well as unclear knowledge which fetal cell type presents the best target cell. These issues need to be carefully addressed in order to ensure an optimal interaction with WP2, a feature which was requested in the last review.

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Description of work

 Deliverables and Milestones

M1.1a Multi-centre re-evaluation of anti-erythroblast antibodies (Dec 08)
M1.1b Pilot study examining the use of proprietary magnetic tips for the enrichment of fetal cells.(Aug 08)
M.1.1c Pilot study examining the use of proprietary centrifugation device for the enrichment of fetal cells. (Aug 08)
M1.2a Multi-centre analysis of enriched fetal erythroblasts by sc-PCR (Aug 08)
M1.2b Analysis of the feasibility of using sc-CGH and Advalytix method.(Aug 08)
M1.3 Examination of novel trophoblast mAbs (Aug 08)
M1.4 Exploration for new trophoblast antigens (Aug 08)
M1.5a Examination of the feasibility of expanding circulating fetal cells populations in-vitro (Aug 08)
M1.5b Examination of the presence of persisting circulatory fetal cells (Aug 08)
M1.6 Continued exploration of the use of single micro-manipulated fetal cells for NIPD (Aug 08)
M1.7 Evaluation of two novel approaches for the enrichment of fetal erythroblasts (Aug 08)
M1.8 Evaluation of the suitability of new MSC markers for NIPD (Aug 08)

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Work in Progress and Achievements

1. Improvement of NRBC enrichment: the novel CD71 antibody developed by partner 22 (Prof. Alvarez, HUCA) previously tested by WP1 partners, has been modified in order to enable a direct comparison with the parallel commercial product.
2. Novel Trophoblast Antibodies: Antibodies directed against placental trophoblasts developed by partners 8b and 13 have been tested in trophoblast-like cell lines in order to standardize the protocol for downstream testing in maternal blood samples. In period 4, following validation in placental tissues, the most promising antibodies were tested in maternal blood samples for feasibility of fetal cell detection.
3. Validation of Novel Trophoblast Markers: Proteins revealed by comparative proteomics in period 3 in period were validated in placental tissues and were tested in maternal blood samples.
4. Trophoblast enrichment from maternal blood: In period 4 the earliest possible time-point for maternal blood sampling at which fetal (male) cells could be detected was determined. The results were compared to results obtained with real-time PCR in cell-free fetal DNA.
5. Targeting fetal mesenchymal stem cells: The goals for period 4 were to confirm epitopes suitable for rare-event selection immunocytochemically in non-cultured fMSC, evaluate novel selection strategies in model materno-fetal mixtures and clinical models of fetomaternal haemorrhage and confirm ubiquitous feto maternal MSC traffic in experimental models.
6. Persistence of fetal cells: Feasibility of culture enrichment of trophoblasts and CD34+ progenitor cells sorted from maternal blood has been demonstrated as evidenced by previous publications (partners 21 and). However it has also been demonstrated that CD34+ cells persist in the maternal blood stream after delivery, possibly for years. Thus, it was decided to explore this issue in period 4. In addition, post-delivery of persistence of trophoblasts was also explored.
7. Single-cell PCR/CGH: Systems for Single-cell PCR/CGH established in the previous period are being applied in rare cell simulation systems and maternal blood samples.
8. Automation of fetal cell enrichment: Protocols for NRBC enrichment are being developed through labelling of cells with magnetic particles and separation of those cells with magnetic tips, towards downstream application in an x-y-z programmable workstation.
   

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