<tt lang="NYYLKTqF"></tt> VSports手机版 - Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in VSports app下载. gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2018 Nov 13;11(5):1051-1060.
doi: 10.1016/j.stemcr.2018.09.010. Epub 2018 Oct 18.

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells

Friederike Philipp  1 , Anton Selich  2 , Michael Rothe  3 , Dirk Hoffmann  3 , Susanne Rittinghausen  4 , Michael A Morgan  3 , Denise Klatt  3 , Silke Glage  5 , Stefan Lienenklaus  6 , Vanessa Neuhaus  4 , Katherina Sewald  4 , Armin Braun  4 , Axel Schambach  7
Affiliations

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells

Friederike Philipp et al. Stem Cell Reports. .

Abstract

Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells VSports手机版. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model. .

Keywords: EHT; HUVECs; embryogenesis; genetic barcoding; hematopoiesis; hiPSC; teratoma. V体育安卓版.

PubMed Disclaimer

"VSports注册入口" Figures

None
Graphical abstract
Figure 1
Figure 1
Hematopoiesis in hiPSC-Derived Teratomas Is Improved by Steady Cytokine Supply in NSGS Mice and by Co-injection of HUVECs (A) Exemplary flow cytometry (FC) detecting hematopoietic marker CD45 and progenitor marker CD34 in a teratoma generated in NSG mice. (B) Immunohistochemistry on teratomas generated in NSG mice. (C) FC summary of hematopoietic populations in teratomas generated with or without hematopoietic supporter cells. Median, quartiles, and outer values are depicted. (D) Summary of FC analyses of teratoma hematopoietic populations generated in NSGS mice that express IL-3, GM-CSF, and SCF (median, quartiles, and outer values). (E) Summary of FC results of teratoma samples generated with co-injection of OP9 or HUVECs expressing DLL1, DLL4, or WNT3A (mean and SD). (F) Fold change of all CD45+ cells in teratomas generated with hiPSC and different supporter cell types in NSG or NSGS mice. The median CD45+ population of teratomas generated in NSG with hiPSC + OP9 was set to a value of 1. Graph depicts median and range. Statistics of (C), (D), and (F) were calculated by Kruskal-Wallis and Dunn's multiple comparisons tests (∗p < 0.05).
Figure 2
Figure 2
Hematopoiesis in hiPSC-Derived Teratomas Is Improved by Steady Cytokine Supply in NSGS Mice and by Co-injection of HUVECs (A) Bioluminescent signal of HUVEC-firefly luciferase (Fluc) and red fluorescent signal of hiPSC-Katushka2S (Kat) during teratoma growth in an exemplary NSGS mouse. (B) Summary of longitudinal study showing bioluminescent signal of HUVEC-Fluc on left y axis and fluorescence signal of hiPSC-Kat on right y axis. Signal intensities were analyzed in regions of interest covering the teratomas at their largest size. Graph displays mean and SD (until day 31 n = 10, day 47 n = 8). (C) Summary of clonogenic assays for myeloid and erythroid lineages. CD34+/CD45+ cells were isolated from teratomas (n = 6), generated with hiPSC and HUVECs in NSGS mice. CD34+ cord blood cells were used as control (n = 3). Graph shows mean and SD. (D) Pappenheim stain of isolated colonies types described in (C). (E) Pappenheim staining of teratoma-derived CD45+ cells. G, granulocyte; M, macrophage; Mo, monocyte; N, normoblast.
Figure 3
Figure 3
Human Hematopoietic Cells Localize inside and in Proximity to Vascular Structures of Teratoma Fluorescent IHC analysis of teratomas generated with hiPSCs in NSGS mice. (A and B) CD45+/CD43+ cells embedded in CD34+/CD43−/CD45− vascular tissue (A); CD43+ cells detected inside or in proximity to CD34+ cell clusters (B). (C) CD34+ vascular structure contained erythrocytes (white arrows) and human hematopoietic cells (CD43+). (D) Vascularization visualized by staining of CD31 (PECAM-1) and CD34. (E) z-stack image of a 150-μm stained teratoma slice recorded with a confocal microscope. 3D model was calculated with Imaris software (Bitplane).
Figure 4
Figure 4
Genetic Barcoding Determined Teratoma Formation and Subsequent Hematopoiesis as Highly Polyclonal Events (A) Design of the lentiviral construct used to transduce hiPSC prior to teratoma formation. Fluorescent reporters Venus, Cerulean, or mCherry are expressed under an EFS promoter. (B) Micrographs of fluorescent reporter expression during expansion of hiPSC prior to teratoma induction and after teratoma isolation (38 days). (C) Experimental scheme to access clonality of teratomas and teratoma-derived hematopoiesis. (D) Barcode variety in hiPSC cultures and teratoma samples. (E) Venn diagram depicting barcodes detected in CD45+ samples isolated from teratoma. (F) Venn diagram depicting barcodes detected in teratoma samples.
See this image and copyright information in PMC

References

    1. Amabile G., Welner R.S., Nombela-Arrieta C., D’Alise A.M., Di Ruscio A., Ebralidze A.K., Kraytsberg Y., Ye M., Kocher O., Neuberg D.S. In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells. Blood. 2013;121:1255–1264. - V体育ios版 - PMC - PubMed
    1. Bertrand J.Y., Chi N.C., Santoso B., Teng S., Stainier D.Y.R., Traver D. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature. 2010;464:108–111. - PMC - PubMed
    1. Bigas A., Espinosa L. Hematopoietic stem cells: to be or Notch to be. Blood. 2012;119:3226–3235. - PubMed
    1. Blum B., Benvenisty N. Clonal analysis of human embryonic stem cell differentiation into teratomas. Stem Cells. 2007;25:1924–1930. - PubMed
    1. Chen X., Zhao Q., Li C., Geng Y., Huang K., Zhang J., Wang X., Yang J., Wang T., Xia C. OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo. Stem Cell Res. 2015;15:395–402. - PubMed

Publication types

MeSH terms