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

Abstract

Fair comparison of reprogramming efficiencies and in vitro 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 (Oct3/4, Klf4 and Sox2). 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 c-Myc 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.

In Vivo Cardiac Cellular Reprogramming Efficacy Is Enhanced by Angiogenic Preconditioning of the Infarcted Myocardium With Vascular Endothelial Growth Factor

“…Although Song et al appear to have made progress in addressing this challenge by their addition of a fourth reprograming gene, HAND2 , to their treatment “cocktail,” the need to “hit” target cells with 4 different gene transfer vectors remains a limitation of current vector strategy . Alternative “triple transgene” or other vector constructs and/or microRNA strategies as demonstrated by Jayawardena et al could potentially address this challenge and further enhance the efficacy of this proposed new therapy …”

“…[32][33][34] Alternative "triple transgene" or other vector constructs and/or microRNA strategies as demonstrated by Jayawardena et al could potentially address this challenge and further enhance the efficacy of this proposed new therapy. 34,51,52 Finally, it is conceivable that delayed administration of either the adenovirus or lentivirus vectors might likewise yield greater effect if this administration was delayed to allow amelioration of the immediate postinfarct inflammatory milieu. 52 Unfortunately, this strategy would be a prohibitive technical challenge in the current small animal model as it would involve 3 operations.…”

Gene Targeting Study Reveals Unexpected Expression of Brain-expressed X-linked 2 in Endocrine and Tissue Stem/Progenitor Cells in Mice

“…Transgenic mice expressing Venus fluorescent protein under the control of a Foxa3-enhancer promoter sequence were generated by pronuclear injection. Induced pluripotent stem cell lines were established from fibroblasts derived from the Foxa3-promoter-Venus transgenic mice using a Dox-inducible Oct3/4-Klf4-Sox2-expressing lentivirus (23). These cell lines were injected into tetraploid embryos and subsequently implanted into a pseudopregnant mouse.…”

Generation of Human Induced Pluripotent Stem Cells Using Epigenetic Regulators Reveals a Germ Cell-Like Identity in Partially Reprogrammed Colonies

“…To accomplish this, we used the neonatal human foreskin fibroblast line, HFF-1, as well as adult human fibroblasts since fibroblasts isolated from earlier developmental stages can be more easily reprogrammed [35]. In addition, we nucleofected HFF-1 and HUF5 fibroblasts with different combinations of DNMT3B-GFP, SETD7-MO as well as expression vectors for AURKB and PRMT5 (Figure 6B and Figure S3C ), the other histone-modifying enzymes also shown to be highly expressed in undifferentiated hESCs and hiPSCs that decreased upon differentiation (Figure 2C, 2D and Figure S2 ).…”