Human mesenchymal stem cells (MSCs) are multipotent stem cells, able to differentiate into multiple mesenchymal lineages.1-3 Previously, we have shown that human fetal lung–derived MSCs enhance the engraftment of human umbilical cord blood (UCB)–derived CD34+ hematopoietic cells in nonobese diabetic–severe combined immunodeficiency mice.1 Here we show that second-trimester 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 second-trimester 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 (ethylenediaminetetra-acetic 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 × 106 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 second-trimester fetal tissues and adult BM1,2 (Table 1). Amniotic fluid–derived 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).

View this table:
Table 1.

Immunophenotype of culture-expanded second-trimester MSCs: expression of antigens on cultured MSC-derived from amniotic fluid

Figure 1.

The Dynal Reli SSO (Dynal Biotech, Hamburg, Germany) reverse line blot strip assay was used for molecular typing of the HLA-A and HLA-B locus alleles of maternal cells, fetal cells, and culture-expanded amniotic fluid–derived MSCs from the same sample. The HLA-A and HLA-B type of the culture-expanded amniotic fluid–derived MSCs is identical to the fetal HLA-A and HLA-B type and mismatched with the maternal HLA-A and HLA-B type. Upward arrows indicate maternal specific HLA antigens, and downward arrows indicate fetal-specific HLA antigens.

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

Cotransplantation of UCB and haploidentical MSCs derived from parental BM is proposed as a strategy to reduce the delay in engraftment that is associated with UCB transplantation. The presence of MSCs in second-trimester amniotic fluid enables the possibility of cotransplantation of hematopoietic stem cells and MSCs from the same donor. This could be particularly useful in the setting of UCB transplantation between siblings.


We thank W. Beekhuizen of the Center of Human Reproduction in Leiden for collecting the amniotic fluid.


  • Correspondence: W. E. Fibbe, Department of Hematology, C2R, PO Box 9600, 2333 AA Leiden, The Netherlands; e-mail: fibbe.hematology{at}