P-1017: Using mouse blastocyst as a reprogramming vector for human dental pulp stem cells

Abstract

ObjectiveHuman embryonic stem (hES) cells derived from inner cell mass (ICM) of mammalian blastocysts are rapidly proliferating, pluripotent cells. Due to their unique properties, hES cells have potential application in creating cells and tissues for clinical application in degenerative diseases, such as Parkinson’s, Alzheimer’s, spinal cord injury, heart disease, diabetes and etc. However, since using hES requires embryo destruction it faces many ethical objections. Therefore, adult stem cells are thought as an alternative. While some adult stem cells are pluripotent, they are usually more limited in their differentiation properties.DesignWe are reporting results of using mouse blastocyst as a reprograming vector for human dental pulp stem cells.Materials and methodsHuman immature dental pulp stem (IDPS) cells from normal non-exfoliated human deciduous teeth were isolated using our original protocol. The cells were characterized for expression of embryonic stem cell markers (oct-3/4, Nanog) by RT-PCR and FACS. Only IDPS cells which were positive for embryonic stem cell markers were used in our experiments. The cells were harvested, counted and labeled with Vybrant CM-Dil (Molecular Probes, Invitrogen). Following this, 5-6 cells were injected into mouse morulas (2.5 days post coitum) and blastocysts into perivitelin spaces using micromanipulator (Nikon Eclipse 300), and the opening of the zona pellucida was done using a laser assisted hatching techinique (Fertilise). These morulas (n = 8) and blastocysts (n=20) were cultured during 24 hours in M16 (Sigma) culture medium. The embryos were fixed by 4% paraformaldehyd (Merck) and the incorporation of IDPS cells was assessed using Confocal microscope (LSM 510, Zeiss, Jena, Germany).ResultsAll morulas and early blastocysts survived the injection and produced chimer embryos composed by IDPS and recipient mouse cells. Based on the number of cells with Vybrant fluorescence, injected cells apparently proliferated within the embryo. Confocal microscopy has shown predominant incorporation of IDPS cells into ICM while in some cases these cells were found in trophoblasts. Undifferentiated IDPS cells displayed fibroblast-like morphology prior to injection. However 24 hours following injection, cells showing fluorescent signal were not morphologically distinguishable from neighboring mouse ICM cells.ConclusionWe demonstrate that human IDPS cells may survive and proliferate within the mouse blastocyst. They either fuse with the host cells or respond to host’s reprogramming signals by changing their morphology. Further studies are necessary to determine whether the re-programming of the human stem cells took place and to identify requirements for their subsequent separation from the host. ObjectiveHuman embryonic stem (hES) cells derived from inner cell mass (ICM) of mammalian blastocysts are rapidly proliferating, pluripotent cells. Due to their unique properties, hES cells have potential application in creating cells and tissues for clinical application in degenerative diseases, such as Parkinson’s, Alzheimer’s, spinal cord injury, heart disease, diabetes and etc. However, since using hES requires embryo destruction it faces many ethical objections. Therefore, adult stem cells are thought as an alternative. While some adult stem cells are pluripotent, they are usually more limited in their differentiation properties. Human embryonic stem (hES) cells derived from inner cell mass (ICM) of mammalian blastocysts are rapidly proliferating, pluripotent cells. Due to their unique properties, hES cells have potential application in creating cells and tissues for clinical application in degenerative diseases, such as Parkinson’s, Alzheimer’s, spinal cord injury, heart disease, diabetes and etc. However, since using hES requires embryo destruction it faces many ethical objections. Therefore, adult stem cells are thought as an alternative. While some adult stem cells are pluripotent, they are usually more limited in their differentiation properties. DesignWe are reporting results of using mouse blastocyst as a reprograming vector for human dental pulp stem cells. We are reporting results of using mouse blastocyst as a reprograming vector for human dental pulp stem cells. Materials and methodsHuman immature dental pulp stem (IDPS) cells from normal non-exfoliated human deciduous teeth were isolated using our original protocol. The cells were characterized for expression of embryonic stem cell markers (oct-3/4, Nanog) by RT-PCR and FACS. Only IDPS cells which were positive for embryonic stem cell markers were used in our experiments. The cells were harvested, counted and labeled with Vybrant CM-Dil (Molecular Probes, Invitrogen). Following this, 5-6 cells were injected into mouse morulas (2.5 days post coitum) and blastocysts into perivitelin spaces using micromanipulator (Nikon Eclipse 300), and the opening of the zona pellucida was done using a laser assisted hatching techinique (Fertilise). These morulas (n = 8) and blastocysts (n=20) were cultured during 24 hours in M16 (Sigma) culture medium. The embryos were fixed by 4% paraformaldehyd (Merck) and the incorporation of IDPS cells was assessed using Confocal microscope (LSM 510, Zeiss, Jena, Germany). Human immature dental pulp stem (IDPS) cells from normal non-exfoliated human deciduous teeth were isolated using our original protocol. The cells were characterized for expression of embryonic stem cell markers (oct-3/4, Nanog) by RT-PCR and FACS. Only IDPS cells which were positive for embryonic stem cell markers were used in our experiments. The cells were harvested, counted and labeled with Vybrant CM-Dil (Molecular Probes, Invitrogen). Following this, 5-6 cells were injected into mouse morulas (2.5 days post coitum) and blastocysts into perivitelin spaces using micromanipulator (Nikon Eclipse 300), and the opening of the zona pellucida was done using a laser assisted hatching techinique (Fertilise). These morulas (n = 8) and blastocysts (n=20) were cultured during 24 hours in M16 (Sigma) culture medium. The embryos were fixed by 4% paraformaldehyd (Merck) and the incorporation of IDPS cells was assessed using Confocal microscope (LSM 510, Zeiss, Jena, Germany). ResultsAll morulas and early blastocysts survived the injection and produced chimer embryos composed by IDPS and recipient mouse cells. Based on the number of cells with Vybrant fluorescence, injected cells apparently proliferated within the embryo. Confocal microscopy has shown predominant incorporation of IDPS cells into ICM while in some cases these cells were found in trophoblasts. Undifferentiated IDPS cells displayed fibroblast-like morphology prior to injection. However 24 hours following injection, cells showing fluorescent signal were not morphologically distinguishable from neighboring mouse ICM cells. All morulas and early blastocysts survived the injection and produced chimer embryos composed by IDPS and recipient mouse cells. Based on the number of cells with Vybrant fluorescence, injected cells apparently proliferated within the embryo. Confocal microscopy has shown predominant incorporation of IDPS cells into ICM while in some cases these cells were found in trophoblasts. Undifferentiated IDPS cells displayed fibroblast-like morphology prior to injection. However 24 hours following injection, cells showing fluorescent signal were not morphologically distinguishable from neighboring mouse ICM cells. ConclusionWe demonstrate that human IDPS cells may survive and proliferate within the mouse blastocyst. They either fuse with the host cells or respond to host’s reprogramming signals by changing their morphology. Further studies are necessary to determine whether the re-programming of the human stem cells took place and to identify requirements for their subsequent separation from the host. We demonstrate that human IDPS cells may survive and proliferate within the mouse blastocyst. They either fuse with the host cells or respond to host’s reprogramming signals by changing their morphology. Further studies are necessary to determine whether the re-programming of the human stem cells took place and to identify requirements for their subsequent separation from the host.