Anthony Atala

Enhanced re-endothelialization of acellular kidney scaffolds for whole organ engineering via antibody conjugation of vasculatures

Decellularization of whole organs, such as the kidney hold great promise in addressing donor shortage for transplantation. However, successful implantation of engineered whole kidney constructs has been challenged by the inability to maintain endothelial cell coverage of the vasculature matrix, resulting in excessive blood clots, loss of vascular patency, and cell death within the construct. In this study, we describe an endothelial cell seeding approach that permits effective coating of the vascular matrix of the decellularized porcine kidney scaffold using a combination of static and ramping perfusion cell seeding. Furthermore, conjugation of CD31 antibodies to the vascular matrix improved endothelial cell retention on the vasculatures, which enhanced vascular patency of the implanted scaffold. These results demonstrate that our endothelial cell seeding method combined with antibody conjugation improves endothelial cell attachment and retention leading to vascular patency of tissue-engineered whole kidney in vivo.

Amniotic Fluid-Derived Stem Cells for Bone Tissue Engineering

Implantation of bone substitute materials and autologous bone grafting have been used for the treatment of extensive bone defects. Recent advances in tissue engineering have led to integration of viable, biological bone grafts composed of osteogenic cells proliferating within three-dimensional (3D) scaffolds. Not only could these novel grafts be used for implantation, but also they could serve in basic and translational studies of bone development, disease, and drug discovery. The ability to isolate human cells, expand them to a large density, and differentiate them into bone-forming cells remains critical to the success of human bone graft engineering. This chapter will focus on the characteristics and limitations of human amniotic stem cells and their application in bone tissue engineering.

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.