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Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors. The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source-peripheral blood-has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34(+) cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3(-)/CD19(-) cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Which are the Yamanaka factors?

53137541e3eabad021000010_001

Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors. The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source-peripheral blood-has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34(+) cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3(-)/CD19(-) cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Which are the Yamanaka factors?

53137541e3eabad021000010_002

Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors. The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source-peripheral blood-has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34(+) cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3(-)/CD19(-) cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Which are the Yamanaka factors?

53137541e3eabad021000010_003

Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors. The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source-peripheral blood-has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34(+) cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3(-)/CD19(-) cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Which are the Yamanaka factors?

53137541e3eabad021000010_004

Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells. The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

Which are the Yamanaka factors?

53137541e3eabad021000010_005

Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells. The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

Which are the Yamanaka factors?

53137541e3eabad021000010_006

Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells. The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

Which are the Yamanaka factors?

53137541e3eabad021000010_007

Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells. The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

Which are the Yamanaka factors?

53137541e3eabad021000010_009

Artd1/Parp1 regulates reprogramming by transcriptional regulation of Fgf4 via Sox2 ADP-ribosylation. The recently established reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka represents a valuable tool for future therapeutic applications. To date, the mechanisms underlying this process are still largely unknown. In particular, the mechanisms how the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) directly drive reprogramming and which additional components are involved are still not yet understood. In this study, we aimed at analyzing the role of ADP-ribosyltransferase diphtheria toxin-like one (Artd1; formerly called poly(ADP-ribose) polymerase 1 [Parp1]) during reprogramming. We found that poly(ADP-ribosylation) (PARylation) of the reprogramming factor Sox2 by Artd1 plays an important role during the first days upon transduction with the reprogramming factors. A process that happens before Artd1 in conjunction with 10-11 translocation-2 (Tet2) mediates the histone modifications necessary for the establishment of an activated chromatin state at pluripotency loci (e.g., Nanog and Essrb) [Nature 2012;488:652-655]. Wild-type (WT) fibroblasts treated with an Artd1 inhibitor as well as fibroblasts deficient for Artd1 (Artd1-/-) show strongly decreased reprogramming capacity. Our data indicate that Artd1-mediated PARylation of Sox2 favors its binding to the fibroblast growth factor 4 (Fgf4) enhancer, thereby activating Fgf4 expression. The importance of Fgf4 during the first 4 days upon initiation of reprogramming was also highlighted by the observation that exogenous addition of Fgf4 was sufficient to restore the reprogramming capacity of Artd1-/- fibroblast to WT levels. In conclusion, our data clearly show that the interaction between Artd1 and Sox2 is crucial for the first steps of the reprogramming process and that early expression of Fgf4 (day 2 to day 4) is an essential component for the successful generation of iPSCs.

Which are the Yamanaka factors?

53137541e3eabad021000010_010

Artd1/Parp1 regulates reprogramming by transcriptional regulation of Fgf4 via Sox2 ADP-ribosylation. The recently established reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka represents a valuable tool for future therapeutic applications. To date, the mechanisms underlying this process are still largely unknown. In particular, the mechanisms how the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) directly drive reprogramming and which additional components are involved are still not yet understood. In this study, we aimed at analyzing the role of ADP-ribosyltransferase diphtheria toxin-like one (Artd1; formerly called poly(ADP-ribose) polymerase 1 [Parp1]) during reprogramming. We found that poly(ADP-ribosylation) (PARylation) of the reprogramming factor Sox2 by Artd1 plays an important role during the first days upon transduction with the reprogramming factors. A process that happens before Artd1 in conjunction with 10-11 translocation-2 (Tet2) mediates the histone modifications necessary for the establishment of an activated chromatin state at pluripotency loci (e.g., Nanog and Essrb) [Nature 2012;488:652-655]. Wild-type (WT) fibroblasts treated with an Artd1 inhibitor as well as fibroblasts deficient for Artd1 (Artd1-/-) show strongly decreased reprogramming capacity. Our data indicate that Artd1-mediated PARylation of Sox2 favors its binding to the fibroblast growth factor 4 (Fgf4) enhancer, thereby activating Fgf4 expression. The importance of Fgf4 during the first 4 days upon initiation of reprogramming was also highlighted by the observation that exogenous addition of Fgf4 was sufficient to restore the reprogramming capacity of Artd1-/- fibroblast to WT levels. In conclusion, our data clearly show that the interaction between Artd1 and Sox2 is crucial for the first steps of the reprogramming process and that early expression of Fgf4 (day 2 to day 4) is an essential component for the successful generation of iPSCs.

Which are the Yamanaka factors?

53137541e3eabad021000010_011

Artd1/Parp1 regulates reprogramming by transcriptional regulation of Fgf4 via Sox2 ADP-ribosylation. The recently established reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka represents a valuable tool for future therapeutic applications. To date, the mechanisms underlying this process are still largely unknown. In particular, the mechanisms how the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) directly drive reprogramming and which additional components are involved are still not yet understood. In this study, we aimed at analyzing the role of ADP-ribosyltransferase diphtheria toxin-like one (Artd1; formerly called poly(ADP-ribose) polymerase 1 [Parp1]) during reprogramming. We found that poly(ADP-ribosylation) (PARylation) of the reprogramming factor Sox2 by Artd1 plays an important role during the first days upon transduction with the reprogramming factors. A process that happens before Artd1 in conjunction with 10-11 translocation-2 (Tet2) mediates the histone modifications necessary for the establishment of an activated chromatin state at pluripotency loci (e.g., Nanog and Essrb) [Nature 2012;488:652-655]. Wild-type (WT) fibroblasts treated with an Artd1 inhibitor as well as fibroblasts deficient for Artd1 (Artd1-/-) show strongly decreased reprogramming capacity. Our data indicate that Artd1-mediated PARylation of Sox2 favors its binding to the fibroblast growth factor 4 (Fgf4) enhancer, thereby activating Fgf4 expression. The importance of Fgf4 during the first 4 days upon initiation of reprogramming was also highlighted by the observation that exogenous addition of Fgf4 was sufficient to restore the reprogramming capacity of Artd1-/- fibroblast to WT levels. In conclusion, our data clearly show that the interaction between Artd1 and Sox2 is crucial for the first steps of the reprogramming process and that early expression of Fgf4 (day 2 to day 4) is an essential component for the successful generation of iPSCs.

Which are the Yamanaka factors?

53137541e3eabad021000010_012

Artd1/Parp1 regulates reprogramming by transcriptional regulation of Fgf4 via Sox2 ADP-ribosylation. The recently established reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka represents a valuable tool for future therapeutic applications. To date, the mechanisms underlying this process are still largely unknown. In particular, the mechanisms how the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) directly drive reprogramming and which additional components are involved are still not yet understood. In this study, we aimed at analyzing the role of ADP-ribosyltransferase diphtheria toxin-like one (Artd1; formerly called poly(ADP-ribose) polymerase 1 [Parp1]) during reprogramming. We found that poly(ADP-ribosylation) (PARylation) of the reprogramming factor Sox2 by Artd1 plays an important role during the first days upon transduction with the reprogramming factors. A process that happens before Artd1 in conjunction with 10-11 translocation-2 (Tet2) mediates the histone modifications necessary for the establishment of an activated chromatin state at pluripotency loci (e.g., Nanog and Essrb) [Nature 2012;488:652-655]. Wild-type (WT) fibroblasts treated with an Artd1 inhibitor as well as fibroblasts deficient for Artd1 (Artd1-/-) show strongly decreased reprogramming capacity. Our data indicate that Artd1-mediated PARylation of Sox2 favors its binding to the fibroblast growth factor 4 (Fgf4) enhancer, thereby activating Fgf4 expression. The importance of Fgf4 during the first 4 days upon initiation of reprogramming was also highlighted by the observation that exogenous addition of Fgf4 was sufficient to restore the reprogramming capacity of Artd1-/- fibroblast to WT levels. In conclusion, our data clearly show that the interaction between Artd1 and Sox2 is crucial for the first steps of the reprogramming process and that early expression of Fgf4 (day 2 to day 4) is an essential component for the successful generation of iPSCs.

Which are the Yamanaka factors?

53137541e3eabad021000010_014

Effect of the STAT3 inhibitor STX-0119 on the proliferation of cancer stem-like cells derived from recurrent glioblastoma. Signal transducer and activator of transcription (STAT) 3, a member of a family of DNA-binding molecules, is a potential target in the treatment of cancer. The highly phosphorylated STAT3 in cancer cells contributes to numerous physiological and oncogenic signaling pathways. Furthermore, a significant association between STAT3 signaling and glioblastoma multiforme stem-like cell (GBM-SC) development and maintenance has been demonstrated in recent studies. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the present study, we focused on cancer stem-like cells derived from recurrent GBM patients and investigated the efficacy of STX-0119. Three GBM stem cell lines showed many stem cell markers such as CD133, EGFR, Nanog, Olig2, nestin and Yamanaka factors (c-myc, KLF4, Oct3/4 and SOX2) compared with parental cell lines. These cell lines also formed tumors in vivo and had similar histological to surgically resected tumors. STAT3 phosphorylation was activated more in the GBM-SC lines than serum-derived GB cell lines. The growth inhibitory effect of STX-0119 on GBM-SCs was moderate (IC50 15-44 µM) and stronger compared to that of WP1066 in two cell lines. On the other hand, the effect of temozolomide was weak in all the cell lines (IC50 53-226 µM). Notably, STX-0119 demonstrated strong inhibition of the expression of STAT3 target genes (c-myc, survivin, cyclin D1, HIF-1α and VEGF) and stem cell-associated genes (CD44, Nanog, nestin and CD133) as well as the induction of apoptosis in one stem-like cell line. Interestingly, VEGFR2 mRNA was also remarkably inhibited by STX-0119. In a model using transplantable stem-like cell lines in vivo GB-SCC010 and 026, STX-0119 inhibited the growth of GBM-SCs at 80 mg/kg. STX-0119, an inhibitor of STAT3, may serve as a novel therapeutic compound against GBM-SCs even in temozolomide-resistant GBM patients and has the potential for GBM-SC-specific therapeutics in combination with temozolomide plus radiation therapy.

Which are the Yamanaka factors?

53137541e3eabad021000010_016

Effect of the STAT3 inhibitor STX-0119 on the proliferation of cancer stem-like cells derived from recurrent glioblastoma. Signal transducer and activator of transcription (STAT) 3, a member of a family of DNA-binding molecules, is a potential target in the treatment of cancer. The highly phosphorylated STAT3 in cancer cells contributes to numerous physiological and oncogenic signaling pathways. Furthermore, a significant association between STAT3 signaling and glioblastoma multiforme stem-like cell (GBM-SC) development and maintenance has been demonstrated in recent studies. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the present study, we focused on cancer stem-like cells derived from recurrent GBM patients and investigated the efficacy of STX-0119. Three GBM stem cell lines showed many stem cell markers such as CD133, EGFR, Nanog, Olig2, nestin and Yamanaka factors (c-myc, KLF4, Oct3/4 and SOX2) compared with parental cell lines. These cell lines also formed tumors in vivo and had similar histological to surgically resected tumors. STAT3 phosphorylation was activated more in the GBM-SC lines than serum-derived GB cell lines. The growth inhibitory effect of STX-0119 on GBM-SCs was moderate (IC50 15-44 µM) and stronger compared to that of WP1066 in two cell lines. On the other hand, the effect of temozolomide was weak in all the cell lines (IC50 53-226 µM). Notably, STX-0119 demonstrated strong inhibition of the expression of STAT3 target genes (c-myc, survivin, cyclin D1, HIF-1α and VEGF) and stem cell-associated genes (CD44, Nanog, nestin and CD133) as well as the induction of apoptosis in one stem-like cell line. Interestingly, VEGFR2 mRNA was also remarkably inhibited by STX-0119. In a model using transplantable stem-like cell lines in vivo GB-SCC010 and 026, STX-0119 inhibited the growth of GBM-SCs at 80 mg/kg. STX-0119, an inhibitor of STAT3, may serve as a novel therapeutic compound against GBM-SCs even in temozolomide-resistant GBM patients and has the potential for GBM-SC-specific therapeutics in combination with temozolomide plus radiation therapy.

Which are the Yamanaka factors?

53137541e3eabad021000010_017

Effect of the STAT3 inhibitor STX-0119 on the proliferation of cancer stem-like cells derived from recurrent glioblastoma. Signal transducer and activator of transcription (STAT) 3, a member of a family of DNA-binding molecules, is a potential target in the treatment of cancer. The highly phosphorylated STAT3 in cancer cells contributes to numerous physiological and oncogenic signaling pathways. Furthermore, a significant association between STAT3 signaling and glioblastoma multiforme stem-like cell (GBM-SC) development and maintenance has been demonstrated in recent studies. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the present study, we focused on cancer stem-like cells derived from recurrent GBM patients and investigated the efficacy of STX-0119. Three GBM stem cell lines showed many stem cell markers such as CD133, EGFR, Nanog, Olig2, nestin and Yamanaka factors (c-myc, KLF4, Oct3/4 and SOX2) compared with parental cell lines. These cell lines also formed tumors in vivo and had similar histological to surgically resected tumors. STAT3 phosphorylation was activated more in the GBM-SC lines than serum-derived GB cell lines. The growth inhibitory effect of STX-0119 on GBM-SCs was moderate (IC50 15-44 µM) and stronger compared to that of WP1066 in two cell lines. On the other hand, the effect of temozolomide was weak in all the cell lines (IC50 53-226 µM). Notably, STX-0119 demonstrated strong inhibition of the expression of STAT3 target genes (c-myc, survivin, cyclin D1, HIF-1α and VEGF) and stem cell-associated genes (CD44, Nanog, nestin and CD133) as well as the induction of apoptosis in one stem-like cell line. Interestingly, VEGFR2 mRNA was also remarkably inhibited by STX-0119. In a model using transplantable stem-like cell lines in vivo GB-SCC010 and 026, STX-0119 inhibited the growth of GBM-SCs at 80 mg/kg. STX-0119, an inhibitor of STAT3, may serve as a novel therapeutic compound against GBM-SCs even in temozolomide-resistant GBM patients and has the potential for GBM-SC-specific therapeutics in combination with temozolomide plus radiation therapy.

Which are the Yamanaka factors?

53137541e3eabad021000010_019

Blood - SeV derived fibroblast generated iPSCs. CytoTune-iPS Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins. The CytoTune-iPS Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety.

Which are the Yamanaka factors?

53137541e3eabad021000010_020

Blood - SeV derived fibroblast generated iPSCs. CytoTune-iPS Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins. The CytoTune-iPS Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety.

Which are the Yamanaka factors?

53137541e3eabad021000010_021

Blood - SeV derived fibroblast generated iPSCs. CytoTune-iPS Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins. The CytoTune-iPS Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety.

Which are the Yamanaka factors?

53137541e3eabad021000010_022

Blood - SeV derived fibroblast generated iPSCs. CytoTune-iPS Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins. The CytoTune-iPS Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety.

Which are the Yamanaka factors?

53137541e3eabad021000010_024

Generation of transgene-free induced pluripotent stem cells with non-viral methods. Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.

Which are the Yamanaka factors?

53137541e3eabad021000010_026

Generation of transgene-free induced pluripotent stem cells with non-viral methods. Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.

Which are the Yamanaka factors?

53137541e3eabad021000010_027

Generation of transgene-free induced pluripotent stem cells with non-viral methods. Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.

Which are the Yamanaka factors?

53137541e3eabad021000010_029

HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks. BACKGROUND: Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. METHODS/PRINCIPAL FINDINGS: We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.

Which are the Yamanaka factors?

53137541e3eabad021000010_030

HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks. BACKGROUND: Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. METHODS/PRINCIPAL FINDINGS: We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.

Which are the Yamanaka factors?

53137541e3eabad021000010_031

HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks. BACKGROUND: Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. METHODS/PRINCIPAL FINDINGS: We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.

Which are the Yamanaka factors?

53137541e3eabad021000010_032

HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks. BACKGROUND: Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. METHODS/PRINCIPAL FINDINGS: We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.

Which are the Yamanaka factors?

53137541e3eabad021000010_033

DNp73 improves generation efficiency of human induced pluripotent stem cells. BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Which are the Yamanaka factors?

53137541e3eabad021000010_034

DNp73 improves generation efficiency of human induced pluripotent stem cells. BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Which are the Yamanaka factors?

53137541e3eabad021000010_035

DNp73 improves generation efficiency of human induced pluripotent stem cells. BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Which are the Yamanaka factors?

53137541e3eabad021000010_036

DNp73 improves generation efficiency of human induced pluripotent stem cells. BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Which are the Yamanaka factors?

53137541e3eabad021000010_037

In vitro transformation of mouse testis cells by oncogene transfection. Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming.

Which are the Yamanaka factors?

53137541e3eabad021000010_039

In vitro transformation of mouse testis cells by oncogene transfection. Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming.

Which are the Yamanaka factors?

53137541e3eabad021000010_040

In vitro transformation of mouse testis cells by oncogene transfection. Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming.

Which are the Yamanaka factors?

53137541e3eabad021000010_041

Induction of pluripotent stem cells from fetal and adult cynomolgus monkey fibroblasts using four human transcription factors. Induced pluripotent stem (iPS) cells have the potential to become a universal resource for cell-based therapies in regenerative medicine; however, prior to the use of such iPS cell-based therapies, preclinical assessment of their safety and efficacy is essential. Non-human primates serve as valuable animal models for human diseases or biomedical research; therefore, in this study, we generated cynomolgus monkey iPS cells from adult skin and fetal fibroblast cells by the retrovirally mediated introduction of four human transcription factors: c-Myc, Klf4, Oct3/4, and Sox2 (the so-called "Yamanaka factors"). Twenty to 30 days after the introduction of these factors, several cynomolgus monkey embryonic stem (ES) cell-like colonies appeared on SNL and mouse embryonic fibroblast (MEF) feeder layers. These colonies were picked and cultivated in primate ES medium. Seven iPS cell lines were established, and we detected the expression of pluripotent markers that are also expressed in ES cells. Reverse transcription polymerase chain reaction (PCR) showed that these iPS cells expressed endogenous c-Myc, Klf4, Oct3/4, and Sox2 genes, whereas several transgenes were silenced. Embryoid body and teratoma formation showed that the cynomolgus iPS cells had the developmental potential to differentiate into cells of all three primary germ layers. In summary, we generated cynomolgus monkey iPS cells by retrovirus-mediated transduction of the human transcription factors, c-Myc, Klf4, Oct3/4, and Sox2 into adult cynomolgus monkey skin cells and fetal fibroblasts. The cynomolgus monkey is the most relevant primate model for human disease, and the highly efficient generation of monkey iPS cells would allow investigation of the treatments of various diseases in this model via therapeutic cloning.

Which are the Yamanaka factors?

53137541e3eabad021000010_043

Induction of pluripotent stem cells from fetal and adult cynomolgus monkey fibroblasts using four human transcription factors. Induced pluripotent stem (iPS) cells have the potential to become a universal resource for cell-based therapies in regenerative medicine; however, prior to the use of such iPS cell-based therapies, preclinical assessment of their safety and efficacy is essential. Non-human primates serve as valuable animal models for human diseases or biomedical research; therefore, in this study, we generated cynomolgus monkey iPS cells from adult skin and fetal fibroblast cells by the retrovirally mediated introduction of four human transcription factors: c-Myc, Klf4, Oct3/4, and Sox2 (the so-called "Yamanaka factors"). Twenty to 30 days after the introduction of these factors, several cynomolgus monkey embryonic stem (ES) cell-like colonies appeared on SNL and mouse embryonic fibroblast (MEF) feeder layers. These colonies were picked and cultivated in primate ES medium. Seven iPS cell lines were established, and we detected the expression of pluripotent markers that are also expressed in ES cells. Reverse transcription polymerase chain reaction (PCR) showed that these iPS cells expressed endogenous c-Myc, Klf4, Oct3/4, and Sox2 genes, whereas several transgenes were silenced. Embryoid body and teratoma formation showed that the cynomolgus iPS cells had the developmental potential to differentiate into cells of all three primary germ layers. In summary, we generated cynomolgus monkey iPS cells by retrovirus-mediated transduction of the human transcription factors, c-Myc, Klf4, Oct3/4, and Sox2 into adult cynomolgus monkey skin cells and fetal fibroblasts. The cynomolgus monkey is the most relevant primate model for human disease, and the highly efficient generation of monkey iPS cells would allow investigation of the treatments of various diseases in this model via therapeutic cloning.

Which are the Yamanaka factors?

53137541e3eabad021000010_044

Induction of pluripotent stem cells from fetal and adult cynomolgus monkey fibroblasts using four human transcription factors. Induced pluripotent stem (iPS) cells have the potential to become a universal resource for cell-based therapies in regenerative medicine; however, prior to the use of such iPS cell-based therapies, preclinical assessment of their safety and efficacy is essential. Non-human primates serve as valuable animal models for human diseases or biomedical research; therefore, in this study, we generated cynomolgus monkey iPS cells from adult skin and fetal fibroblast cells by the retrovirally mediated introduction of four human transcription factors: c-Myc, Klf4, Oct3/4, and Sox2 (the so-called "Yamanaka factors"). Twenty to 30 days after the introduction of these factors, several cynomolgus monkey embryonic stem (ES) cell-like colonies appeared on SNL and mouse embryonic fibroblast (MEF) feeder layers. These colonies were picked and cultivated in primate ES medium. Seven iPS cell lines were established, and we detected the expression of pluripotent markers that are also expressed in ES cells. Reverse transcription polymerase chain reaction (PCR) showed that these iPS cells expressed endogenous c-Myc, Klf4, Oct3/4, and Sox2 genes, whereas several transgenes were silenced. Embryoid body and teratoma formation showed that the cynomolgus iPS cells had the developmental potential to differentiate into cells of all three primary germ layers. In summary, we generated cynomolgus monkey iPS cells by retrovirus-mediated transduction of the human transcription factors, c-Myc, Klf4, Oct3/4, and Sox2 into adult cynomolgus monkey skin cells and fetal fibroblasts. The cynomolgus monkey is the most relevant primate model for human disease, and the highly efficient generation of monkey iPS cells would allow investigation of the treatments of various diseases in this model via therapeutic cloning.

Which are the Yamanaka factors?

53137541e3eabad021000010_046

Induction of pluripotent stem cells from autopsy donor-derived somatic cells. Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models.

Which are the Yamanaka factors?

53137541e3eabad021000010_048

Induction of pluripotent stem cells from autopsy donor-derived somatic cells. Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models.

Which are the Yamanaka factors?

53137541e3eabad021000010_049

Induction of pluripotent stem cells from autopsy donor-derived somatic cells. Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models.

Which are the Yamanaka factors?

53137541e3eabad021000010_051

Protein intrinsic disorder and induced pluripotent stem cells. Induced pluripotent stem (iPS) cells can be obtained from terminally differentiated somatic cells by overexpression of defined sets of reprogramming transcription factors. These protein sets have been called the Yamanaka factors, namely Sox2, Oct3/4 (Pou5f1), Klf4, and c-Myc, and the Thomson factors, namely Sox2, Oct3, Lin28, and Nanog. Other sets of proteins, while not essential for the formation of iPS cells, are important for improving the efficiency of the induction and still other sets of proteins are important as markers for embryonic stem cells. Structural information about most of these important proteins is very sparse. Our bioinformatics analysis herein reveals that these reprogramming factors and most of the efficiency-improving and embryonic stem cell markers are highly enriched in intrinsic disorder. As is typical for transcription factors, these proteins are modular. Specific sites for interaction with other proteins and DNA are dispersed in the long regions of intrinsic disorder. These highly dynamic interaction sites are evidently responsible for the delicate interplay among various molecules. The bioinformatics analysis given herein should facilitate the investigation of the roles and organization of these modular interaction sites, thereby helping to shed further light on the pathways that underlie the mechanism(s) by which terminally differentiated cells are converted to iPS cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_054

Protein intrinsic disorder and induced pluripotent stem cells. Induced pluripotent stem (iPS) cells can be obtained from terminally differentiated somatic cells by overexpression of defined sets of reprogramming transcription factors. These protein sets have been called the Yamanaka factors, namely Sox2, Oct3/4 (Pou5f1), Klf4, and c-Myc, and the Thomson factors, namely Sox2, Oct3, Lin28, and Nanog. Other sets of proteins, while not essential for the formation of iPS cells, are important for improving the efficiency of the induction and still other sets of proteins are important as markers for embryonic stem cells. Structural information about most of these important proteins is very sparse. Our bioinformatics analysis herein reveals that these reprogramming factors and most of the efficiency-improving and embryonic stem cell markers are highly enriched in intrinsic disorder. As is typical for transcription factors, these proteins are modular. Specific sites for interaction with other proteins and DNA are dispersed in the long regions of intrinsic disorder. These highly dynamic interaction sites are evidently responsible for the delicate interplay among various molecules. The bioinformatics analysis given herein should facilitate the investigation of the roles and organization of these modular interaction sites, thereby helping to shed further light on the pathways that underlie the mechanism(s) by which terminally differentiated cells are converted to iPS cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_055

Protein intrinsic disorder and induced pluripotent stem cells. Induced pluripotent stem (iPS) cells can be obtained from terminally differentiated somatic cells by overexpression of defined sets of reprogramming transcription factors. These protein sets have been called the Yamanaka factors, namely Sox2, Oct3/4 (Pou5f1), Klf4, and c-Myc, and the Thomson factors, namely Sox2, Oct3, Lin28, and Nanog. Other sets of proteins, while not essential for the formation of iPS cells, are important for improving the efficiency of the induction and still other sets of proteins are important as markers for embryonic stem cells. Structural information about most of these important proteins is very sparse. Our bioinformatics analysis herein reveals that these reprogramming factors and most of the efficiency-improving and embryonic stem cell markers are highly enriched in intrinsic disorder. As is typical for transcription factors, these proteins are modular. Specific sites for interaction with other proteins and DNA are dispersed in the long regions of intrinsic disorder. These highly dynamic interaction sites are evidently responsible for the delicate interplay among various molecules. The bioinformatics analysis given herein should facilitate the investigation of the roles and organization of these modular interaction sites, thereby helping to shed further light on the pathways that underlie the mechanism(s) by which terminally differentiated cells are converted to iPS cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_057

Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system. Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.

Which are the Yamanaka factors?

53137541e3eabad021000010_058

Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system. Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.

Which are the Yamanaka factors?

53137541e3eabad021000010_059

Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system. Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.

Which are the Yamanaka factors?

53137541e3eabad021000010_060

Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system. Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.

Which are the Yamanaka factors?

53137541e3eabad021000010_062

Generation of human melanocytes from induced pluripotent stem cells. Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.

Which are the Yamanaka factors?

53137541e3eabad021000010_064

Generation of human melanocytes from induced pluripotent stem cells. Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.

Which are the Yamanaka factors?

53137541e3eabad021000010_065

Generation of human melanocytes from induced pluripotent stem cells. Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.

Which are the Yamanaka factors?

53137541e3eabad021000010_067

The efficient generation of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells. Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 +/- 2.62%, ADCs 70.61 +/- 15.4%, NSCs, 68.72 +/- 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 +/- 4.10 colonies, 0.13 +/- 0.06%) and ADCs (118.20 +/- 38.28 colonies, 1.14 +/- 0.77%) when compared with the mEF control (3.17 +/- 0.29 colonies, 0.03 +/- 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Which are the Yamanaka factors?

53137541e3eabad021000010_068

The efficient generation of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells. Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 +/- 2.62%, ADCs 70.61 +/- 15.4%, NSCs, 68.72 +/- 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 +/- 4.10 colonies, 0.13 +/- 0.06%) and ADCs (118.20 +/- 38.28 colonies, 1.14 +/- 0.77%) when compared with the mEF control (3.17 +/- 0.29 colonies, 0.03 +/- 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Which are the Yamanaka factors?

53137541e3eabad021000010_069

The efficient generation of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells. Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 +/- 2.62%, ADCs 70.61 +/- 15.4%, NSCs, 68.72 +/- 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 +/- 4.10 colonies, 0.13 +/- 0.06%) and ADCs (118.20 +/- 38.28 colonies, 1.14 +/- 0.77%) when compared with the mEF control (3.17 +/- 0.29 colonies, 0.03 +/- 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Which are the Yamanaka factors?

53137541e3eabad021000010_070

The efficient generation of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells. Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 +/- 2.62%, ADCs 70.61 +/- 15.4%, NSCs, 68.72 +/- 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 +/- 4.10 colonies, 0.13 +/- 0.06%) and ADCs (118.20 +/- 38.28 colonies, 1.14 +/- 0.77%) when compared with the mEF control (3.17 +/- 0.29 colonies, 0.03 +/- 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Which are the Yamanaka factors?

53137541e3eabad021000010_072

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_074

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_075

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_077

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_079

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_080

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_082

Protein intrinsic disorder and induced pluripotent stem cells. Induced pluripotent stem (iPS) cells can be obtained from terminally differentiated somatic cells by overexpression of defined sets of reprogramming transcription factors. These protein sets have been called the Yamanaka factors, namely Sox2, Oct3/4 (Pou5f1), Klf4, and c-Myc, and the Thomson factors, namely Sox2, Oct3, Lin28, and Nanog. Other sets of proteins, while not essential for the formation of iPS cells, are important for improving the efficiency of the induction and still other sets of proteins are important as markers for embryonic stem cells. Structural information about most of these important proteins is very sparse. Our bioinformatics analysis herein reveals that these reprogramming factors and most of the efficiency-improving and embryonic stem cell markers are highly enriched in intrinsic disorder. As is typical for transcription factors, these proteins are modular. Specific sites for interaction with other proteins and DNA are dispersed in the long regions of intrinsic disorder. These highly dynamic interaction sites are evidently responsible for the delicate interplay among various molecules. The bioinformatics analysis given herein should facilitate the investigation of the roles and organization of these modular interaction sites, thereby helping to shed further light on the pathways that underlie the mechanism(s) by which terminally differentiated cells are converted to iPS cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_085

Protein intrinsic disorder and induced pluripotent stem cells. Induced pluripotent stem (iPS) cells can be obtained from terminally differentiated somatic cells by overexpression of defined sets of reprogramming transcription factors. These protein sets have been called the Yamanaka factors, namely Sox2, Oct3/4 (Pou5f1), Klf4, and c-Myc, and the Thomson factors, namely Sox2, Oct3, Lin28, and Nanog. Other sets of proteins, while not essential for the formation of iPS cells, are important for improving the efficiency of the induction and still other sets of proteins are important as markers for embryonic stem cells. Structural information about most of these important proteins is very sparse. Our bioinformatics analysis herein reveals that these reprogramming factors and most of the efficiency-improving and embryonic stem cell markers are highly enriched in intrinsic disorder. As is typical for transcription factors, these proteins are modular. Specific sites for interaction with other proteins and DNA are dispersed in the long regions of intrinsic disorder. These highly dynamic interaction sites are evidently responsible for the delicate interplay among various molecules. The bioinformatics analysis given herein should facilitate the investigation of the roles and organization of these modular interaction sites, thereby helping to shed further light on the pathways that underlie the mechanism(s) by which terminally differentiated cells are converted to iPS cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_086

Protein intrinsic disorder and induced pluripotent stem cells. Induced pluripotent stem (iPS) cells can be obtained from terminally differentiated somatic cells by overexpression of defined sets of reprogramming transcription factors. These protein sets have been called the Yamanaka factors, namely Sox2, Oct3/4 (Pou5f1), Klf4, and c-Myc, and the Thomson factors, namely Sox2, Oct3, Lin28, and Nanog. Other sets of proteins, while not essential for the formation of iPS cells, are important for improving the efficiency of the induction and still other sets of proteins are important as markers for embryonic stem cells. Structural information about most of these important proteins is very sparse. Our bioinformatics analysis herein reveals that these reprogramming factors and most of the efficiency-improving and embryonic stem cell markers are highly enriched in intrinsic disorder. As is typical for transcription factors, these proteins are modular. Specific sites for interaction with other proteins and DNA are dispersed in the long regions of intrinsic disorder. These highly dynamic interaction sites are evidently responsible for the delicate interplay among various molecules. The bioinformatics analysis given herein should facilitate the investigation of the roles and organization of these modular interaction sites, thereby helping to shed further light on the pathways that underlie the mechanism(s) by which terminally differentiated cells are converted to iPS cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_087

Protein intrinsic disorder and induced pluripotent stem cells. Induced pluripotent stem (iPS) cells can be obtained from terminally differentiated somatic cells by overexpression of defined sets of reprogramming transcription factors. These protein sets have been called the Yamanaka factors, namely Sox2, Oct3/4 (Pou5f1), Klf4, and c-Myc, and the Thomson factors, namely Sox2, Oct3, Lin28, and Nanog. Other sets of proteins, while not essential for the formation of iPS cells, are important for improving the efficiency of the induction and still other sets of proteins are important as markers for embryonic stem cells. Structural information about most of these important proteins is very sparse. Our bioinformatics analysis herein reveals that these reprogramming factors and most of the efficiency-improving and embryonic stem cell markers are highly enriched in intrinsic disorder. As is typical for transcription factors, these proteins are modular. Specific sites for interaction with other proteins and DNA are dispersed in the long regions of intrinsic disorder. These highly dynamic interaction sites are evidently responsible for the delicate interplay among various molecules. The bioinformatics analysis given herein should facilitate the investigation of the roles and organization of these modular interaction sites, thereby helping to shed further light on the pathways that underlie the mechanism(s) by which terminally differentiated cells are converted to iPS cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_089

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_091

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_092

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Which are the Yamanaka factors?

53137541e3eabad021000010_094

DNp73 improves generation efficiency of human induced pluripotent stem cells. BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Which are the Yamanaka factors?

53137541e3eabad021000010_095

DNp73 improves generation efficiency of human induced pluripotent stem cells. BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Which are the Yamanaka factors?

53137541e3eabad021000010_096

DNp73 improves generation efficiency of human induced pluripotent stem cells. BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Which are the Yamanaka factors?

53137541e3eabad021000010_097

DNp73 improves generation efficiency of human induced pluripotent stem cells. BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Which are the Yamanaka factors?

53137541e3eabad021000010_098

Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system. Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.

Which are the Yamanaka factors?

53137541e3eabad021000010_099

Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system. Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.

Which are the Yamanaka factors?

53137541e3eabad021000010_100

Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system. Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.

Which are the Yamanaka factors?

53137541e3eabad021000010_101

Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system. Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.

Which are the Yamanaka factors?

53137541e3eabad021000010_103

Molecular and clinical genetics of mitochondrial diseases due to POLG mutations. Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial disease in children and adults. They are responsible for a heterogeneous group of at least 6 major phenotypes of neurodegenerative disease that include: 1) childhood Myocerebrohepatopathy Spectrum disorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time-dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. We sequenced the exons and flanking intron region from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease and found informative mutations in 61 (17%). Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG-related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. We describe the predicted structural/functional and clinical importance of the previously unreported missense variants and discuss their likelihood of being pathogenic. In conclusion, sequence analysis allows the identification of mutations responsible for POLG-related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations can provide an unequivocal diagnosis of the disease.

Which currently known mitochondrial diseases have been attributed to POLG mutations?

5718bbb37de986d80d00000b_001

Molecular and clinical genetics of mitochondrial diseases due to POLG mutations. Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial disease in children and adults. They are responsible for a heterogeneous group of at least 6 major phenotypes of neurodegenerative disease that include: 1) childhood Myocerebrohepatopathy Spectrum disorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time-dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. We sequenced the exons and flanking intron region from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease and found informative mutations in 61 (17%). Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG-related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. We describe the predicted structural/functional and clinical importance of the previously unreported missense variants and discuss their likelihood of being pathogenic. In conclusion, sequence analysis allows the identification of mutations responsible for POLG-related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations can provide an unequivocal diagnosis of the disease.

Which currently known mitochondrial diseases have been attributed to POLG mutations?

5718bbb37de986d80d00000b_002

Molecular and clinical genetics of mitochondrial diseases due to POLG mutations. Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial disease in children and adults. They are responsible for a heterogeneous group of at least 6 major phenotypes of neurodegenerative disease that include: 1) childhood Myocerebrohepatopathy Spectrum disorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time-dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. We sequenced the exons and flanking intron region from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease and found informative mutations in 61 (17%). Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG-related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. We describe the predicted structural/functional and clinical importance of the previously unreported missense variants and discuss their likelihood of being pathogenic. In conclusion, sequence analysis allows the identification of mutations responsible for POLG-related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations can provide an unequivocal diagnosis of the disease.

Which currently known mitochondrial diseases have been attributed to POLG mutations?

5718bbb37de986d80d00000b_003

Molecular and clinical genetics of mitochondrial diseases due to POLG mutations. Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial disease in children and adults. They are responsible for a heterogeneous group of at least 6 major phenotypes of neurodegenerative disease that include: 1) childhood Myocerebrohepatopathy Spectrum disorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time-dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. We sequenced the exons and flanking intron region from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease and found informative mutations in 61 (17%). Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG-related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. We describe the predicted structural/functional and clinical importance of the previously unreported missense variants and discuss their likelihood of being pathogenic. In conclusion, sequence analysis allows the identification of mutations responsible for POLG-related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations can provide an unequivocal diagnosis of the disease.

Which currently known mitochondrial diseases have been attributed to POLG mutations?

5718bbb37de986d80d00000b_004

Molecular and clinical genetics of mitochondrial diseases due to POLG mutations. Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial disease in children and adults. They are responsible for a heterogeneous group of at least 6 major phenotypes of neurodegenerative disease that include: 1) childhood Myocerebrohepatopathy Spectrum disorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time-dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. We sequenced the exons and flanking intron region from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease and found informative mutations in 61 (17%). Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG-related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. We describe the predicted structural/functional and clinical importance of the previously unreported missense variants and discuss their likelihood of being pathogenic. In conclusion, sequence analysis allows the identification of mutations responsible for POLG-related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations can provide an unequivocal diagnosis of the disease.

Which currently known mitochondrial diseases have been attributed to POLG mutations?

5718bbb37de986d80d00000b_005

Molecular and clinical genetics of mitochondrial diseases due to POLG mutations. Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial disease in children and adults. They are responsible for a heterogeneous group of at least 6 major phenotypes of neurodegenerative disease that include: 1) childhood Myocerebrohepatopathy Spectrum disorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time-dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. We sequenced the exons and flanking intron region from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease and found informative mutations in 61 (17%). Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG-related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. We describe the predicted structural/functional and clinical importance of the previously unreported missense variants and discuss their likelihood of being pathogenic. In conclusion, sequence analysis allows the identification of mutations responsible for POLG-related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations can provide an unequivocal diagnosis of the disease.

Which currently known mitochondrial diseases have been attributed to POLG mutations?

5718bbb37de986d80d00000b_006

Mitochondrial DNA replication and disease: insights from DNA polymerase γ mutations. DNA polymerase γ (pol γ), encoded by POLG, is responsible for replicating human mitochondrial DNA. About 150 mutations in the human POLG have been identified in patients with mitochondrial diseases such as Alpers syndrome, progressive external ophthalmoplegia, and ataxia-neuropathy syndromes. Because many of the mutations are described in single citations with no genotypic family history, it is important to ascertain which mutations cause or contribute to mitochondrial disease. The vast majority of data about POLG mutations has been generated from biochemical characterizations of recombinant pol γ. However, recently, the study of mitochondrial dysfunction in Saccharomyces cerevisiae and mouse models provides important in vivo evidence for the role of POLG mutations in disease. Also, the published 3D-structure of the human pol γ assists in explaining some of the biochemical and genetic properties of the mutants. This review summarizes the current evidence that identifies and explains disease-causing POLG mutations.

Which currently known mitochondrial diseases have been attributed to POLG mutations?

5718bbb37de986d80d00000b_007

Modulation of SR Ca2+ release by the triadin-to-calsequestrin ratio in ventricular myocytes. Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_001

Modulation of SR Ca2+ release by the triadin-to-calsequestrin ratio in ventricular myocytes. Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_003

Modulation of SR Ca2+ release by the triadin-to-calsequestrin ratio in ventricular myocytes. Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_005

Modulation of SR Ca2+ release by the triadin-to-calsequestrin ratio in ventricular myocytes. Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_007

Viral gene transfer rescues arrhythmogenic phenotype and ultrastructural abnormalities in adult calsequestrin-null mice with inherited arrhythmias. RATIONALE: Catecholaminergic polymorphic ventricular tachycardia is an inherited disease that predisposes to cardiac arrest and sudden death. The disease is associated with mutations in the genes encoding for the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2). CASQ2 mutations lead to a major loss of CASQ2 monomers, possibly because of enhanced degradation of the mutant protein. The decrease of CASQ2 is associated with a reduction in the levels of Triadin (TrD) and Junctin (JnC), two proteins that form, with CASQ2 and RyR2, a macromolecular complex devoted to control of calcium release from the sarcoplasmic reticulum. OBJECTIVE: We intended to evaluate whether viral gene transfer of wild-type CASQ2 may rescue the broad spectrum of abnormalities caused by mutant CASQ2. METHODS AND RESULTS: We used an adeno-associated serotype 9 viral vector to express a green fluorescent protein-tagged CASQ2 construct. Twenty weeks after intraperitoneal injection of the vector in neonate CASQ2 KO mice, we observed normalization of the levels of calsequestrin, triadin, and junctin, rescue of electrophysiological and ultrastructural abnormalities caused by CASQ2 ablation, and lack of life-threatening arrhythmias. CONCLUSIONS: We have proven the concept that induction of CASQ2 expression in knockout mice reverts the molecular, structural, and electric abnormalities and prevents life-threatening arrhythmias in CASQ2-defective catecholaminergic polymorphic ventricular tachycardia mice. These data support the view that development of CASQ2 viral gene transfer could have clinical application.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_014

Viral gene transfer rescues arrhythmogenic phenotype and ultrastructural abnormalities in adult calsequestrin-null mice with inherited arrhythmias. RATIONALE: Catecholaminergic polymorphic ventricular tachycardia is an inherited disease that predisposes to cardiac arrest and sudden death. The disease is associated with mutations in the genes encoding for the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2). CASQ2 mutations lead to a major loss of CASQ2 monomers, possibly because of enhanced degradation of the mutant protein. The decrease of CASQ2 is associated with a reduction in the levels of Triadin (TrD) and Junctin (JnC), two proteins that form, with CASQ2 and RyR2, a macromolecular complex devoted to control of calcium release from the sarcoplasmic reticulum. OBJECTIVE: We intended to evaluate whether viral gene transfer of wild-type CASQ2 may rescue the broad spectrum of abnormalities caused by mutant CASQ2. METHODS AND RESULTS: We used an adeno-associated serotype 9 viral vector to express a green fluorescent protein-tagged CASQ2 construct. Twenty weeks after intraperitoneal injection of the vector in neonate CASQ2 KO mice, we observed normalization of the levels of calsequestrin, triadin, and junctin, rescue of electrophysiological and ultrastructural abnormalities caused by CASQ2 ablation, and lack of life-threatening arrhythmias. CONCLUSIONS: We have proven the concept that induction of CASQ2 expression in knockout mice reverts the molecular, structural, and electric abnormalities and prevents life-threatening arrhythmias in CASQ2-defective catecholaminergic polymorphic ventricular tachycardia mice. These data support the view that development of CASQ2 viral gene transfer could have clinical application.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_016

Altered stored calcium release in skeletal myotubes deficient of triadin and junctin. Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_019

Altered stored calcium release in skeletal myotubes deficient of triadin and junctin. Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_021

Altered stored calcium release in skeletal myotubes deficient of triadin and junctin. Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_022

Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes. Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the junctional SR membrane. The physiological role for junctin in the luminal regulation of RyR Ca(2+) release remains unresolved, but it appears to be essential for proper cardiac function since ablation of junctin results in increased ventricular automaticity. Given that the junctin levels are severely reduced in human failing hearts, we performed an in-depth study of the mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)] ([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+) load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated [Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR) reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release, evidenced by the development of spontaneous aftercontractions. Single channel recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of junctin produces a dual effect on the normally linear response of RyRs to luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR channels are less responsive to luminal [Ca(2+)]; conversely, high luminal [Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus, junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates that junctin normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is possible that the activator-inhibitor role of junctin may be exerted under periods of prevalent parasympathetic and sympathetic activity, respectively.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_024

Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes. Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the junctional SR membrane. The physiological role for junctin in the luminal regulation of RyR Ca(2+) release remains unresolved, but it appears to be essential for proper cardiac function since ablation of junctin results in increased ventricular automaticity. Given that the junctin levels are severely reduced in human failing hearts, we performed an in-depth study of the mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)] ([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+) load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated [Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR) reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release, evidenced by the development of spontaneous aftercontractions. Single channel recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of junctin produces a dual effect on the normally linear response of RyRs to luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR channels are less responsive to luminal [Ca(2+)]; conversely, high luminal [Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus, junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates that junctin normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is possible that the activator-inhibitor role of junctin may be exerted under periods of prevalent parasympathetic and sympathetic activity, respectively.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_026

Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes. Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the junctional SR membrane. The physiological role for junctin in the luminal regulation of RyR Ca(2+) release remains unresolved, but it appears to be essential for proper cardiac function since ablation of junctin results in increased ventricular automaticity. Given that the junctin levels are severely reduced in human failing hearts, we performed an in-depth study of the mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)] ([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+) load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated [Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR) reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release, evidenced by the development of spontaneous aftercontractions. Single channel recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of junctin produces a dual effect on the normally linear response of RyRs to luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR channels are less responsive to luminal [Ca(2+)]; conversely, high luminal [Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus, junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates that junctin normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is possible that the activator-inhibitor role of junctin may be exerted under periods of prevalent parasympathetic and sympathetic activity, respectively.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_027

Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes. Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the junctional SR membrane. The physiological role for junctin in the luminal regulation of RyR Ca(2+) release remains unresolved, but it appears to be essential for proper cardiac function since ablation of junctin results in increased ventricular automaticity. Given that the junctin levels are severely reduced in human failing hearts, we performed an in-depth study of the mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)] ([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+) load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated [Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR) reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release, evidenced by the development of spontaneous aftercontractions. Single channel recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of junctin produces a dual effect on the normally linear response of RyRs to luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR channels are less responsive to luminal [Ca(2+)]; conversely, high luminal [Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus, junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates that junctin normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is possible that the activator-inhibitor role of junctin may be exerted under periods of prevalent parasympathetic and sympathetic activity, respectively.

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_028

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling. In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_031

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling. In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_034

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling. In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_035

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling. In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_036

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling. In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_037

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling. In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).

Which proteins participate in the formation of the ryanodine receptor quaternary macromolecular complex?

54f9d3eedd3fc62544000004_038