In vivo capacity of blood progenitor/stem cells arising in teratomas. (A) Schematic model for in vitro differentiation and in vivo transplantation of CD45⁺CD34⁺ isolated from teratoma. (B) CFU assay revealed the capability of CD45⁺CD34⁺ cells to give rise mostly to GM and M colonies. Few E− colonies were also detected. (C) Graph representing human chimerism of CD45⁺ cells in NSG recipient bone marrow. Three mice per group were transplanted with a different range of human CD45⁺CD34⁺ cells isolated from CB and teratoma as indicated. An additional 3 mice were injected with saline solution and used as negative controls. The percentage of human CD45⁺ cells over total mononuclear cells in bone marrow is shown in the y-axis. (D) Representative FACS analysis showing teratoma and cord blood human CD45⁺ and human CD34⁺CD45⁺ HSPCs populations in NSG recipient bone marrow. (E) Bar-graph showing multi-lineage reconstitution of a NSG bone marrow transplanted with human CD34⁺CD45⁺ cells derived from teratomas (Ter) and from cord blood (CB). (F-G) Primary and secondary multi-lineage reconstitution of murine organs by human CD34⁺CD45⁺ teratoma cells. For all the transplantation experiments mice injected with saline solution were used as negative control of the experiment. Isotype antibodies were used for gates settings and as additional control. Error bars represent SD. (H) Human-specific PCR of mtDNA shows that human cells are present in engrafted animals. Lane 1: DNA ladder markers. Lanes 2-4: specificity of the assay: human PCR primers efficiently amplify human DNA (lane 4) and do not amplify mouse DNA (lane 2). The latter control is essential as test samples (lanes 5-10) do contain some mouse DNA. Lane 3: validation of the mouse DNA control. The same sample as in lane 2 is successfully amplified with mouse PCR primers to confirm the presence of amplifiable mouse DNA. Lanes 5-7: amplification of FACS-purified human CD45⁺ cells derived from teratomas isolated from transplanted animals. Lanes 8-9: amplification of FACS-purified human CD34⁺CD45⁺ cells derived from teratomas isolated from transplanted animals. Lane 10: amplification of FACS-purified human CD34⁺CD45⁺ cells derived from CB isolated from transplanted animals. All the amplifications were performed using human-specific primers which confirm the presence of human cells in the engrafted animals.