Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation

Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantationHuman mesenchymal stem cells (MSCs) are multipotent stem cells, able to differentiate into multiple mesenchymal lineages.[1][2][3] Previously, we have shown that human fetal lung-derived MSCs enhance the engraftment of human umbilical cord blood (UCB)derived CD34 ϩ hematopoietic cells in nonobese diabetic-severe combined immunodeficiency mice. 1 Here we show that secondtrimester amniotic fluid is an abundant source of fetal MSCs that exhibit a phenotype and multilineage differentiation potential similar to that of postnatal bone marrow (BM)-derived MSCs.We suggest that amniotic fluid is an attractive source of MSCs for cotransplantation in conjunction with UCB-derived hematopoietic stem cells.Amniotic fluid was collected transcervically from 6 secondtrimester legal terminations of pregnancy (mean gestational age, 19 weeks [range, 17-22 weeks]) according to a protocol approved by the medical ethical review board of our hospital.Amniotic fluid samples, without visible contamination with blood, were centrifuged for 10 minutes at 1283 rpm.Pellets were resuspended and cultured as described previously. 1 Adherent cells were detached with trypsin/EDTA (ethylenediaminetetraacetic acid) and phenotypically characterized by flow cytometry using fluorescein isothiocyanate-or phycoerythrin-conjugated antibodies.The adipogenic and osteogenic differentiation capacity of culture-expanded MSCs was determined as previously reported.1 To confirm the fetal origin of cultured cells, a molecular HLA typing was performed on DNA obtained from expanded MSCs, and fetal and maternal blood cells by polymerase chain reaction/sequence-specific oligonucleotide using a reverse dot blot method.4 MSCs were cultured from all 6 consecutive samples of second-trimester amniotic fluid.A quantity of 2 mL amniotic fluid was sufficient to culture these cells.The expansion potential of amniotic fluid-derived MSCs exceeded that of BM-derived MSCs.As a result, we were able to expand amniotic fluid MSCs to about 180 ϫ 10 6 cells within 4 weeks (3 passages).The phenotype of the culture-expanded amniotic fluid-derived cells was similar to that reported for MSCs derived from secondtrimester fetal tissues and adult BM 1,2 (Table 1).Amniotic fluidderived MSCs showed multilineage differentiation potential into fibroblasts, adipocytes, and osteocytes.Molecular HLA typing of fetal and maternal cells confirmed that the cultured cells were of fetal origin, without detectable contamination of maternal cells (Figure 1).Following allogeneic transplantation, most studies indicate that MSCs remain of host origin, 5 possibly as a result of the low frequency of these cells in stem cell grafts.The frequency of MSCs in UCB is particularly low, and most laboratories have been unable to grow MSCs from UCB. 6,7 Supplementing stem cell grafts with MSCs to promote engraftment has been proposed.Studies in mice and sheep show that engraftment can be promoted by the addition of third-party MSCs.1,8 Preliminary clinical studies support these data and suggest that cotransplantation with MSCs derived from the stem cell donor also results in a reduced incidence of graft-versus-host disease.9