Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of pluripotency markers in cloned human iPSCs and their ability to form teratomas.(A) Alkaline phosphatase staining and immunofluorescence staining of pluripotency markers in cloned human iPSCs obtained with M3O-SKM on day 27 after four passages. Bar, 100 µm. (B) Hematoxylin and eosin staining of teratoma sections derived from an iPSC clone after subcutaneous injection into an NOD/SCID mouse. Bar, 200 µm. (C) Immunofluorescence staining of foreskin fibroblasts with anti-SOX2 antibody on day 2 after transduction of M3O-SKM and OSKM. Bar, 100 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316619&req=5

pone-0034149-g007: Expression of pluripotency markers in cloned human iPSCs and their ability to form teratomas.(A) Alkaline phosphatase staining and immunofluorescence staining of pluripotency markers in cloned human iPSCs obtained with M3O-SKM on day 27 after four passages. Bar, 100 µm. (B) Hematoxylin and eosin staining of teratoma sections derived from an iPSC clone after subcutaneous injection into an NOD/SCID mouse. Bar, 200 µm. (C) Immunofluorescence staining of foreskin fibroblasts with anti-SOX2 antibody on day 2 after transduction of M3O-SKM and OSKM. Bar, 100 µm.

Mentions: Previously we showed that M3O-SKM induces iPSC formation from adult human dermal fibroblasts at an efficiency of 0.30% in feeder-free culture with KSR when seeded at 17,000 cells/well of a 12-well plate, in contrast to 0.0052% with OSKM [4]. Following the success of Protocol B as described above, we seeded transduced human neonatal fibroblasts at densities ranging from 1,000 to 17,000 cells/well of a 12-well plate on feeder cells (Fig. 6A). The number of ESC-like colonies that expressed NANOG and TRA1-60 were counted as iPSC colonies (Fig. 6B). When transduced fibroblasts were seeded on feeder cells at 17,000 cells/well, the efficiency of iPSC formation was raised to 1.7%. However, as with mouse iPSCs, the highest efficiency was obtained at 2,000 cells/well. At this cell density, iPSC colonies emerged around day 5 with M3O-SKM and day 8 with OSKM (Fig. 6C, KSR). The colony number peaked on day 14 with 6.6% of fibroblasts becoming iPSCs with M3O-SKM, in contrast to 0.8% on day 16 with OSKM. Two additional serum-free media were less effective than KSR (Fig. 6C, Pluriton and TeSR1). Cloned iPSCs prepared with M3O-SKM and KSR also expressed other pluripotency markers, including SSEA-4, alkaline phosphatase, NANOG and endogenous OCT4 on day 27 (Fig. 7A). Pluripotency of an iPSC clone established with M3O-SKM and KSR was verified with teratoma formation (Fig. 7B). In these experiments we transduced M3O, OCT4, SOX2, KLF4 and c-MYC in separate viruses. Since KLF4 and c-MYC were endogenously expressed in fibroblasts [9], transduction efficiency of these genes could not be determined but transduction efficiency of SOX2 was sufficiently high (87.0% with M3O-SKM and 88.9% with O-SKM) (Fig. 7C).


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)

Expression of pluripotency markers in cloned human iPSCs and their ability to form teratomas.(A) Alkaline phosphatase staining and immunofluorescence staining of pluripotency markers in cloned human iPSCs obtained with M3O-SKM on day 27 after four passages. Bar, 100 µm. (B) Hematoxylin and eosin staining of teratoma sections derived from an iPSC clone after subcutaneous injection into an NOD/SCID mouse. Bar, 200 µm. (C) Immunofluorescence staining of foreskin fibroblasts with anti-SOX2 antibody on day 2 after transduction of M3O-SKM and OSKM. Bar, 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0034149-g007: Expression of pluripotency markers in cloned human iPSCs and their ability to form teratomas.(A) Alkaline phosphatase staining and immunofluorescence staining of pluripotency markers in cloned human iPSCs obtained with M3O-SKM on day 27 after four passages. Bar, 100 µm. (B) Hematoxylin and eosin staining of teratoma sections derived from an iPSC clone after subcutaneous injection into an NOD/SCID mouse. Bar, 200 µm. (C) Immunofluorescence staining of foreskin fibroblasts with anti-SOX2 antibody on day 2 after transduction of M3O-SKM and OSKM. Bar, 100 µm.
Mentions: Previously we showed that M3O-SKM induces iPSC formation from adult human dermal fibroblasts at an efficiency of 0.30% in feeder-free culture with KSR when seeded at 17,000 cells/well of a 12-well plate, in contrast to 0.0052% with OSKM [4]. Following the success of Protocol B as described above, we seeded transduced human neonatal fibroblasts at densities ranging from 1,000 to 17,000 cells/well of a 12-well plate on feeder cells (Fig. 6A). The number of ESC-like colonies that expressed NANOG and TRA1-60 were counted as iPSC colonies (Fig. 6B). When transduced fibroblasts were seeded on feeder cells at 17,000 cells/well, the efficiency of iPSC formation was raised to 1.7%. However, as with mouse iPSCs, the highest efficiency was obtained at 2,000 cells/well. At this cell density, iPSC colonies emerged around day 5 with M3O-SKM and day 8 with OSKM (Fig. 6C, KSR). The colony number peaked on day 14 with 6.6% of fibroblasts becoming iPSCs with M3O-SKM, in contrast to 0.8% on day 16 with OSKM. Two additional serum-free media were less effective than KSR (Fig. 6C, Pluriton and TeSR1). Cloned iPSCs prepared with M3O-SKM and KSR also expressed other pluripotency markers, including SSEA-4, alkaline phosphatase, NANOG and endogenous OCT4 on day 27 (Fig. 7A). Pluripotency of an iPSC clone established with M3O-SKM and KSR was verified with teratoma formation (Fig. 7B). In these experiments we transduced M3O, OCT4, SOX2, KLF4 and c-MYC in separate viruses. Since KLF4 and c-MYC were endogenously expressed in fibroblasts [9], transduction efficiency of these genes could not be determined but transduction efficiency of SOX2 was sufficiently high (87.0% with M3O-SKM and 88.9% with O-SKM) (Fig. 7C).

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