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Efficient iPS cell production with the MyoD transactivation domain in serum-free culture.

Hirai H, Katoku-Kikyo N, Karian P, Firpo M, Kikyo N - PLoS ONE (2012)

Bottom Line: A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells.Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium.This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Institute, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O), along with Sox2, Klf4 and c-Myc (SKM). In addition, M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs, including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast, the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs, M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition, in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.

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Verification of pluripotency of iPSCs prepared from MEFs with FM3O-SKM using Protocol B.(A) Quantitative RT-PCR analyses of pluripotency genes comparing day 10 iPSC colonies and MEFs. The value in ESCs was defined as 1.0 for each gene. Mean+SEM obtained from ten colonies is shown. (B) Quantitative RT-PCR analyses of MEF-enriched genes comparing iPSCs and ESCs. The value in MEFs was defined as 1.0 for each gene. Ten colonies were harvested on day 10. (C) Hematoxylin and eosin staining of teratoma sections derived from an iPSC clone after subcutaneous injection into an NOD/SCID mouse. The arrow and arrowhead indicate striated muscle (mesoderm) and respiratory epithelium (endoderm), respectively. Bar, 200 µm. (D) Chimeric mice prepared with an iPSC clone. High (right) and low (left) contribution of iPSCs to the skin is shown. The host 8-cell-stage embryos used to generate mice were derived from the ICR strain with a white coat color. The mottled coat color of the chimeric mice is due to the fact that iPSCs were derived from 129/B6 mice which displayed a brown or black coat color. (E) Pups obtained from mating the chimeric mouse on the right in panel D with an ICR female (white mouse shown in this photograph). This result shows that the derivatives of iPSCs contributed to germ cells.
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pone-0034149-g003: Verification of pluripotency of iPSCs prepared from MEFs with FM3O-SKM using Protocol B.(A) Quantitative RT-PCR analyses of pluripotency genes comparing day 10 iPSC colonies and MEFs. The value in ESCs was defined as 1.0 for each gene. Mean+SEM obtained from ten colonies is shown. (B) Quantitative RT-PCR analyses of MEF-enriched genes comparing iPSCs and ESCs. The value in MEFs was defined as 1.0 for each gene. Ten colonies were harvested on day 10. (C) Hematoxylin and eosin staining of teratoma sections derived from an iPSC clone after subcutaneous injection into an NOD/SCID mouse. The arrow and arrowhead indicate striated muscle (mesoderm) and respiratory epithelium (endoderm), respectively. Bar, 200 µm. (D) Chimeric mice prepared with an iPSC clone. High (right) and low (left) contribution of iPSCs to the skin is shown. The host 8-cell-stage embryos used to generate mice were derived from the ICR strain with a white coat color. The mottled coat color of the chimeric mice is due to the fact that iPSCs were derived from 129/B6 mice which displayed a brown or black coat color. (E) Pups obtained from mating the chimeric mouse on the right in panel D with an ICR female (white mouse shown in this photograph). This result shows that the derivatives of iPSCs contributed to germ cells.

Mentions: The pluripotency of iPSCs prepared with FM3O-SKM using Protocol B were evaluated with a standard set of experiments. The iPSC colonies expressed nine key genes for pluripotency at similar levels to those in ESCs (Fig. 3A). Three genes highly expressed in MEFs — Thy1, Col6a2 and Fgf7 — were downregulated to ESC levels in iPSCs (Fig. 3B). When iPSCs were subcutaneously injected into NOD/SCID mice, they formed teratomas that contained tissues derived from all three germ layers (Fig. 3C). Furthermore, iPSCs formed chimeric mice upon aggregation with 8-cell-stage ICR mouse embryos (Fig. 3D). When one of the chimeric mice was mated with an ICR female mouse, all six pups had a brown coat color, proving that the derivatives of iPSCs contributed to germ cells (Fig. 3E).


Efficient iPS cell production with the MyoD transactivation domain in serum-free culture.

Hirai H, Katoku-Kikyo N, Karian P, Firpo M, Kikyo N - PLoS ONE (2012)

Verification of pluripotency of iPSCs prepared from MEFs with FM3O-SKM using Protocol B.(A) Quantitative RT-PCR analyses of pluripotency genes comparing day 10 iPSC colonies and MEFs. The value in ESCs was defined as 1.0 for each gene. Mean+SEM obtained from ten colonies is shown. (B) Quantitative RT-PCR analyses of MEF-enriched genes comparing iPSCs and ESCs. The value in MEFs was defined as 1.0 for each gene. Ten colonies were harvested on day 10. (C) Hematoxylin and eosin staining of teratoma sections derived from an iPSC clone after subcutaneous injection into an NOD/SCID mouse. The arrow and arrowhead indicate striated muscle (mesoderm) and respiratory epithelium (endoderm), respectively. Bar, 200 µm. (D) Chimeric mice prepared with an iPSC clone. High (right) and low (left) contribution of iPSCs to the skin is shown. The host 8-cell-stage embryos used to generate mice were derived from the ICR strain with a white coat color. The mottled coat color of the chimeric mice is due to the fact that iPSCs were derived from 129/B6 mice which displayed a brown or black coat color. (E) Pups obtained from mating the chimeric mouse on the right in panel D with an ICR female (white mouse shown in this photograph). This result shows that the derivatives of iPSCs contributed to germ cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316619&req=5

pone-0034149-g003: Verification of pluripotency of iPSCs prepared from MEFs with FM3O-SKM using Protocol B.(A) Quantitative RT-PCR analyses of pluripotency genes comparing day 10 iPSC colonies and MEFs. The value in ESCs was defined as 1.0 for each gene. Mean+SEM obtained from ten colonies is shown. (B) Quantitative RT-PCR analyses of MEF-enriched genes comparing iPSCs and ESCs. The value in MEFs was defined as 1.0 for each gene. Ten colonies were harvested on day 10. (C) Hematoxylin and eosin staining of teratoma sections derived from an iPSC clone after subcutaneous injection into an NOD/SCID mouse. The arrow and arrowhead indicate striated muscle (mesoderm) and respiratory epithelium (endoderm), respectively. Bar, 200 µm. (D) Chimeric mice prepared with an iPSC clone. High (right) and low (left) contribution of iPSCs to the skin is shown. The host 8-cell-stage embryos used to generate mice were derived from the ICR strain with a white coat color. The mottled coat color of the chimeric mice is due to the fact that iPSCs were derived from 129/B6 mice which displayed a brown or black coat color. (E) Pups obtained from mating the chimeric mouse on the right in panel D with an ICR female (white mouse shown in this photograph). This result shows that the derivatives of iPSCs contributed to germ cells.
Mentions: The pluripotency of iPSCs prepared with FM3O-SKM using Protocol B were evaluated with a standard set of experiments. The iPSC colonies expressed nine key genes for pluripotency at similar levels to those in ESCs (Fig. 3A). Three genes highly expressed in MEFs — Thy1, Col6a2 and Fgf7 — were downregulated to ESC levels in iPSCs (Fig. 3B). When iPSCs were subcutaneously injected into NOD/SCID mice, they formed teratomas that contained tissues derived from all three germ layers (Fig. 3C). Furthermore, iPSCs formed chimeric mice upon aggregation with 8-cell-stage ICR mouse embryos (Fig. 3D). When one of the chimeric mice was mated with an ICR female mouse, all six pups had a brown coat color, proving that the derivatives of iPSCs contributed to germ cells (Fig. 3E).

Bottom Line: A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells.Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium.This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Institute, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O), along with Sox2, Klf4 and c-Myc (SKM). In addition, M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs, including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast, the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs, M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition, in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.

Show MeSH
Related in: MedlinePlus