Evaluation of a culture system for enrichment of CD34+ hematopoietic progenitor cells present in maternal blood.
BACKGROUND
Clonogenic expansion of fetal cells in maternal blood is one approach to overcome the very low number of target cells available for prenatal genetic analysis. However, efficient methods of enrichment, culturing conditions and subsequent analysis of fetal cells are lacking. Optimization of this technique requires more detailed evaluation of the composition and distribution of fetal cells that cross the placenta into the maternal circulation. Previous studies by others have shown that fetal blood is rich in CD34+ progenitor cells capable of expansion in cultures supplemented with hematopoeitic growth factors. Moreover, CD34+ fetal cells have been recovered from maternal blood following enrichment.


OBJECTIVE
In this study, we examine the type and frequency of hematopoietic progenitor cells detected in maternal (n = 13) and non-pregnant control (n = 4) peripheral blood specimens.


METHODS
A methylcellulose-based culture system was used to perform colony assays on CD34+-enriched or non-enriched cells. Overall, a total of 2,249 colonies were scored for colony type among the 17 samples. To determine whether fetal cells were present and expanded, all colonies present in each of the 10 confirmed male-cases (n = 1,525 colonies) were examined either by PCR or FISH.


RESULTS
With CD34+-enriched maternal samples, we observed a significantly higher number of burst-forming unit-erythroid (BFU-E) and a reduced number of colony-forming unit-granulocyte, macrophage (CFU-GM) colonies compared to the non-enriched samples. Of 1,067 colonies analyzed by PCR for the amelogenin locus on X and Y, none were found positive for the 250-bp Y-specific sequences. Of 458 colonies tested by FISH for presence of X and Y probe signals, no XY-male cells were detected.


CONCLUSIONS
We conclude that hematopoiesis is enhanced during pregnancy, but the number of fetal progenitor cells is either very low or fail to expand using the enrichment techniques and culturing conditions described in this study. Further development of methods is warranted before considering this approach for prenatal diagnosis.
Manotaya
Saknan
S
Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX 77030, USA.
Elias
Sherman
S
Lewis
Dorothy E
DE
Simpson
Joe Leigh
JL
Bischoff
Farideh Z
FZ
D018952:Antigens, CD34 / Q000032:analysis*; D002452:Cell Count; D018929:Cell Culture Techniques; D002469:Cell Separation; D003114:Colony-Forming Units Assay; D015672:Erythroid Precursor Cells / Q000166:cytology; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005865:Gestational Age; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008264:Macrophages / Q000166:cytology; D008297:Male; D008533:Megakaryocytes / Q000166:cytology; D016133:Polymerase Chain Reaction; D011247:Pregnancy
D018952:Antigens, CD34