Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation.

In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived

Generation of Germline-Competent Rat Induced Pluripotent Stem Cells

Recent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.We generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.Our data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras

Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation

Summary: In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5–9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived. : In this article, Yamaguchi and colleagues generated vascular endothelial cells and hematopoietic cells from pluripotent stem cells in vivo by blastocyst complementation technique. This approach could be used to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are pluripotent stem cell derived. Keywords: tissue regeneration, vascular endothelial cells, hematopoietic cells, blastocyst complementation, pluripotent stem cell

Development of an All-in-One Inducible Lentiviral Vector for Gene Specific Analysis of Reprogramming

<div><p>Fair comparison of reprogramming efficiencies and <em>in vitro</em> differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (<em>Oct3/4</em>, <em>Klf4</em> and <em>Sox2</em>). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of <em>c-Myc</em> compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.</p> </div

Reprogramming efficiency of somatic cells in the presence of c-Myc.

<p>(A) Reprogramming efficiency of fibroblasts (MEF, NB fb, 1wk fb and Adult fb), hematopoietic cells (FL CD45, HSC, HPC, MP and Mac) by four reprogramming factors including <i>c-Myc</i>. (B) Cell proliferation rate of fibroblasts (MEF, NB fb, 1wk fb and Adult fb) at three, four and five days after seeded in the absence of Dox (left) and in the presence of Dox (right). (C) Reprogramming efficiency of MEF were compared between reprogrammed by three factors (Oct4, Klf4 and Sox2), four factors (<i>Oct4</i>, <i>Klf4</i>, <i>Sox2</i> and <i>c-Myc</i>) and three factors plus VPA (0.5 mM, 1 mM and 2 mM).</p

Reprogramming efficiency of rat somatic cells.

<p>(A) Reprogramming efficiency of rat fibroblasts (REF, r1wk fb and rAdult fb) by three reprogramming factors (<i>Oct4</i>, <i>Klf4</i> and <i>Sox2</i>) (left) and by four reprogramming factors (<i>Oct4</i>, <i>Klf4</i>, <i>Sox2</i> and <i>c-Myc</i>) (right). (B) Cell proliferation rate of rat fibroblasts in the presence of Dox.</p