In spina bifida, the neural tube fails to close during the embryonic period. Exposure of the neural tube to the amniotic fluid during pregnancy causes additional neural damage. Intrauterine tissue engineering using a biomaterial seeded with stem cells might prevent this additional damage. For this purpose, autologous cells from the amniotic fluid are an attractive source. To close the defect, it is important that these cells deposit an extracellular matrix. However, it is not known if amniotic fluid mesenchymal cells (AFMCs) from a fetus with a neural tube defect (NTD) share the same characteristics as AFMCs from a healthy fetus. We found that cells derived from fetuses with a NTD, in contrast to healthy human amniotic fluid cells, did not deposit collagen type I. Furthermore, the NTD cells showed, compared with both healthy amniotic fluid cells and fetal fibroblasts, much lower mRNA expression levels of genes that are involved in collagen biosynthesis [procollagen C-endopeptidase enhancer proteins (PCOLCE), PCOLCE2, ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2), ADAMTS14]. This indicates that NTD-AFMCs have different characteristics compared with healthy AFMCs and might not be suitable for fetal therapy to close the defect in spina bifida patients.