A New Methodology of Fetal Stem Cell Isolation, Purification, and Expansion: Preliminary Results for Noninvasive Prenatal Diagnosis

Abstract

We developed a combined methodological approach to enrich and to proliferate in vitro fetal CD34+ stem progenitor cells. Using a magnetic cell-sorting technique, CD34+ cells from pregnant women at the early-second trimester were isolated and enriched and compared to those isolated from blood of nonpregnant women. The number and frequency of CD34+ cells were significantly higher (p < 0.001) in the pregnant women. Unenriched peripheral blood mononuclear cells (PBMC) and enriched CD34+ cells were cultured in a methylcellulose system to evaluate the cloning potential of progenitor cells. After culture, the numbers of burst-forming units erythroid/colony-forming units erythroid (BFU-E/CFU-E) and colony-forming units granulocyte-macrophage (CFU-GM) colonies were increased by 33 and 16 times, respectively. Finally, to distinguish between fetal and maternal cells, four cases of cultured cells were hybridized with specific probes for X and Y chromosomes and two cases with a specific probe for chromosome 21. In normal pregnancies, we identified a high number of male fetal cells and an elevated fetal/maternal ratio. When we analyzed blood samples from pregnancies with trisomic fetuses, we scored a high ratio of trisomic cells respect to maternal cells that was significantly different from the ratio of pregnancies with normal fetuses. Our results demonstrate fetal progenitor cells may be cultured and detected successfully with an appropriate combined methodological approach, which may significantly increase the feasibility of noninvasive prenatal diagnosis.

Prenatal Diagnosis of Genetic Abnormalities Using Fetal CD34+Stem Cells in Maternal Circulation and Evidence They Do Not Affect Diagnosis in Later Pregnancies

“…Isolated and washed PBMCs were split into two equal parts: one was cultured in a semisolid methylcellulose medium and the other underwent CD34 + stem cell enrichment as reported previously [31]. Briefly, CD34 + stem cells were enriched using MiniMACS magnetically activated cell sorting (Miltenyi Biotech; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com).…”

“…Briefly, CD34 + stem cells were enriched using MiniMACS magnetically activated cell sorting (Miltenyi Biotech; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com). CD34 + cells were sorted by positive selection of CD34-expressing cells (clone: QBEND/10, isotype, mouse IgG1) as previously described [31].…”

“…1 × 10 5 enriched PBMCs and 1 × 10 4 CD34 + stem cells were cultured in Iscove's modified Dulbecco's medium (Methocult GF H4435, Stem Cell Technology; Vancouver, Canada; http://www.stemcell.com) as previously reported [31]. Colonies were then observed under an inverted microscope and were classified as BFU-E/CFU-E (colony-forming unit-erythroid) and CFU-GM (colony-forming unit-granulocyte, macrophage).…”

“…Several studies have tried to expand "in vitro" fetal erythroblasts and fetal stem cells, isolated from maternal blood [27][28][29][30][31]. Results with erythroblasts, which proliferate to a limited extent, have been disappointing because there is no clear consensus about the degree of amplification of fetal erythroblasts, and because Chen et al [32] recently reported that culturing fetal/maternal erythroblasts from maternal blood mainly produced erythroid colonies of maternal erythroid progenitor origin.…”

Are we facing a revolution in non-invasive prenatal genetic diagnosis?

“…Regarding the question of the rarity of fetal cells in maternal blood samples (2-6 fetal cells per mL of blood), it has been partially resolved by cell in vitro expansion on a suitable medium; initially, this strategy was applied to hematopoietic erythroid progenitor cells and to hematopoietic stem cells [11,17], because it was believed that they were the unique typologies of cells with proliferative potential. Guetta et al [18] then succeeded in expanding trophoblast cells in vitro.…”