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The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.

Three major methods have been described for the isolation of fetal cells from maternal blood: fluorescence-activated cell sorting (FACS), immunomagnetic beads, and magnetic-activated cell sorting (MACS). To date, no study has directly compared fetal cell recovery using each of these methods. Here we describe our system using a "model' male fetal cell mixed into female peripheral blood mononuclear cells. Fetal cell yields and purities were assayed by a quantitative polymerase chain reaction (qPCR) using chromosomes Y- and 7-specific sequences. Fetal cell recovery was investigated by selection of CD71+ cells or depletion of CD45+ cells. Our data demonstrated variation in fetal cell recovery for all methods tested, although CD71+ selection by FACS gave the best and most consistent results.

Monoclonal antibody to fetal haemoglobin (alpha 2 gamma 2) has been proposed as a fetal-specific reagent. We developed an intracellular staining protocol that combines fluorescein isothiocyanate or phycoerythrin conjugated anti-gamma with the DNA binding dye Hoechst 33342 to identify and flow sort fetal erythroblasts from maternal blood. Our preliminary observations on anti-gamma-positive cells sorted from four different pregnant women are described here, using fluorescence in situ hybridization (FISH) with chromosome-specific probes to identify fetal cells. Our data demonstrate that far fewer candidate fetal cells are sorted with this protocol than by current cell surface staining methods that employ the monoclonal antibody CD71. This results in increased fetal cell sorting purities. With this protocol, standard FISH techniques require modification due to the rigorous fixation with 4 per cent paraformaldehyde. Our initial data indicate the promise of this approach.