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Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming.

Yamaguchi T, Hamanaka S, Kamiya A, Okabe M, Kawarai M, Wakiyama Y, Umino A, Hayama T, Sato H, Lee YS, Kato-Itoh M, Masaki H, Kobayashi T, Yamazaki S, Nakauchi H - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Japan Science Technology Agency, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Tokyo, Japan. tomoyama@ims.u-tokyo.ac.jp

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.

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Differentiation capacity of iPSCs to hematopoietic cells.(A) Protocol for in vitro differentiation of iPSCs to hematopoietic cells. (B) Frequency of CD41+, c-Kit+ primitive hematopoietic progenitor in dissociated EB cells at day six. *p<0.05 (left). Frequency of CD45+ hematopoietic cells in sorted CD41+, c-Kit+ cells cultured on OP9 feeder layer for four days (middle). Total induction rate of CD45+ hematopoietic cells from iPSCs were analyzed. *p<0.05 (right).
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pone-0041007-g002: Differentiation capacity of iPSCs to hematopoietic cells.(A) Protocol for in vitro differentiation of iPSCs to hematopoietic cells. (B) Frequency of CD41+, c-Kit+ primitive hematopoietic progenitor in dissociated EB cells at day six. *p<0.05 (left). Frequency of CD45+ hematopoietic cells in sorted CD41+, c-Kit+ cells cultured on OP9 feeder layer for four days (middle). Total induction rate of CD45+ hematopoietic cells from iPSCs were analyzed. *p<0.05 (right).

Mentions: Previous reports showed that iPSCs derived from murine tissues possessed residual DNA methylation signatures characteristic of their somatic tissue of origin, which tends to differentiate to lineages of the donor cell [23], [24], [25]. To analyze whether the miPSCs re-reprogrammed from chimeric mice (2nd miPSCs) posses this phenotype, we generated 2nd miPSCs from E13.5 fetal liver CD45+ hematopoietic cells (FL CD45), adult dermal fibroblasts (Adult fb), adult hepatocytes (Adult hep) and E13.5 fetal hepatoblasts (Fetal hep) and compared their efficiency of induction to hematopoietic cells by in vitro differentiation assay (Fig. 2A). As shown in Fig. 2B, differentiation efficiency of each tissue type to CD41+c-kit+ primitive hematopoietic progenitor cells (primitive HPC) was 7.3%(FL CD45), 2.1% (Adult fb), 3.7% (Adult hep) and 2.0% (Fetal hep). Although no significant differences were observed in the efficiency of differentiation from CD41+c-kit+ primitive HPC to CD45+ hematopoietic cells (14.2%, 14.4%, 13.3% and 14.9%, respectively) after 4-day culture on OP9 stromal cells, overall differentiation capacity of 2nd miPSCs derived from FL CD45 to CD45+ hematopoietic cells was over 2 folds higher than iPSCs derived from other tissues with a statistically significant difference (Fig. 2B). These results observed for our system coincide with previously described epigenetic memories in the 2nd miPSCs [25].


Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming.

Yamaguchi T, Hamanaka S, Kamiya A, Okabe M, Kawarai M, Wakiyama Y, Umino A, Hayama T, Sato H, Lee YS, Kato-Itoh M, Masaki H, Kobayashi T, Yamazaki S, Nakauchi H - PLoS ONE (2012)

Differentiation capacity of iPSCs to hematopoietic cells.(A) Protocol for in vitro differentiation of iPSCs to hematopoietic cells. (B) Frequency of CD41+, c-Kit+ primitive hematopoietic progenitor in dissociated EB cells at day six. *p<0.05 (left). Frequency of CD45+ hematopoietic cells in sorted CD41+, c-Kit+ cells cultured on OP9 feeder layer for four days (middle). Total induction rate of CD45+ hematopoietic cells from iPSCs were analyzed. *p<0.05 (right).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3399796&req=5

pone-0041007-g002: Differentiation capacity of iPSCs to hematopoietic cells.(A) Protocol for in vitro differentiation of iPSCs to hematopoietic cells. (B) Frequency of CD41+, c-Kit+ primitive hematopoietic progenitor in dissociated EB cells at day six. *p<0.05 (left). Frequency of CD45+ hematopoietic cells in sorted CD41+, c-Kit+ cells cultured on OP9 feeder layer for four days (middle). Total induction rate of CD45+ hematopoietic cells from iPSCs were analyzed. *p<0.05 (right).
Mentions: Previous reports showed that iPSCs derived from murine tissues possessed residual DNA methylation signatures characteristic of their somatic tissue of origin, which tends to differentiate to lineages of the donor cell [23], [24], [25]. To analyze whether the miPSCs re-reprogrammed from chimeric mice (2nd miPSCs) posses this phenotype, we generated 2nd miPSCs from E13.5 fetal liver CD45+ hematopoietic cells (FL CD45), adult dermal fibroblasts (Adult fb), adult hepatocytes (Adult hep) and E13.5 fetal hepatoblasts (Fetal hep) and compared their efficiency of induction to hematopoietic cells by in vitro differentiation assay (Fig. 2A). As shown in Fig. 2B, differentiation efficiency of each tissue type to CD41+c-kit+ primitive hematopoietic progenitor cells (primitive HPC) was 7.3%(FL CD45), 2.1% (Adult fb), 3.7% (Adult hep) and 2.0% (Fetal hep). Although no significant differences were observed in the efficiency of differentiation from CD41+c-kit+ primitive HPC to CD45+ hematopoietic cells (14.2%, 14.4%, 13.3% and 14.9%, respectively) after 4-day culture on OP9 stromal cells, overall differentiation capacity of 2nd miPSCs derived from FL CD45 to CD45+ hematopoietic cells was over 2 folds higher than iPSCs derived from other tissues with a statistically significant difference (Fig. 2B). These results observed for our system coincide with previously described epigenetic memories in the 2nd miPSCs [25].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Japan Science Technology Agency, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Tokyo, Japan. tomoyama@ims.u-tokyo.ac.jp

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.

Show MeSH