Haematopoietic stem cells derived from sheep and human amniotic fluid engraft after transplantation

Introduction Mouse amniotic fluid c-Kit(+)/Lin(-) stem (AFS) cells display hematopoietic potential. We explored the haematopoietic potential of sheep and human AFS cells after in utero stem cell transplantation. Methods Human AFS cells (hAFSC) were isolated from women undergoing 3rd trimester amniodrainage. Sheep AFS cells (sAFSC) were collected under ultrasound guidance (59.5±4.5days, term=145days), and isolated using a sheep-specific CD34 antibody. hAFSC were transplanted into the peritoneal cavity of fetal mice from CD1 mothers (14dpc, n=6). The peripheral blood of recipient mice was analysed 4 weeks postnatal for engraftment by flow-cytometry using anti-human beta2-microglobin antibody. Neonatal tissues collected at 6 weeks were analysed by PCR and immuno-staining for anti-human mitochondrial antibody; bone marrow (BM) was assayed for colony-forming cells. sAFSC were transduced overnight using a lentivirus vector (HIV-SFFV-eGFP, MOI=50) and injected either intravenously into NOD-SCID-gamma (NSG) mice (3x10∧5, N=4 per group) or by ultrasound-guided peritoneal injection back into donor sheep fetuses (n=7; 2x10∧4 sAFSC). Results hAFS cells were detectable in the peripheral blood, liver, spleen and BM of neonatal mice at 6 weeks postnatal; harvested BM generated colonies of human origin. GFP+ve cells were detected in the peripheral blood, spleen, liver, and BM of NSG mice 3 months after transplantation of transduced sAFSC. Five lambs injected with autologous transduced GFP+CD34+ cells survived to birth (71.4%); peripheral blood of all lambs contained GFP+ cells (1.9-3.8%) maintained at 6 months postnatal. GFP+ cells were detected in the liver and BM of lambs. Conclusion AFSC have haematopoietic potential and be useful for autologous transplantation.