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Generation of Naivetropic Induced Pluripotent Stem Cells from Parkinson's Disease Patients for High-Efficiency Genetic Manipulation and Disease Modeling.

Hu Z, Pu J, Jiang H, Zhong P, Qiu J, Li F, Wang X, Zhang B, Yan Z, Feng J - Stem Cells Dev. (2015)

Bottom Line: The high clonal efficiency and proliferation rate of naivetropic iPSCs enabled very efficient gene targeting of GFP to the PITX3 locus by transcription activator-like effector nuclease.The naivetropic iPSCs could be readily reverted to the primed state upon the withdrawal of DOX, 2iL, and the switch to primed-state hESC culture conditions.Midbrain DA neurons differentiated from the reverted iPSCs retained the original phenotypes caused by parkin mutations, attesting to the robustness of these phenotypes and the usefulness of genomic engineering in patient-specific naivetropic iPSCs for studying PD.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Physiology and Biophysics, State University of New York at Buffalo , Buffalo, New York.

ABSTRACT
The lack of robust Parkinson's disease (PD) phenotype in parkin knockout rodents and the identification of defective dopaminergic (DA) neurotransmission in midbrain DA neurons derived from induced pluripotent stem cells (iPSC) of PD patients with parkin mutations demonstrate the utility of patient-specific iPSCs as an effective system to model the unique vulnerabilities of midbrain DA neurons in PD. Significant efforts have been directed at developing efficient genomic engineering technologies in human iPSCs to study diseases such as PD. In the present study, we converted patient-specific iPSCs from the primed state to a naivetropic state by DOX-induced expression of transgenes (Oct4, Sox2, Klf4, c-Myc, and Nanog) and the use of 2iL (MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021, and human LIF). These patient-specific naivetropic iPSCs were pluripotent in terms of marker expression, spontaneous differentiation in vitro, and teratoma formation in vivo. They exhibited morphological, proliferative, and clonogenic characteristics very similar to naive mouse embryonic stem cells (ESC). The high clonal efficiency and proliferation rate of naivetropic iPSCs enabled very efficient gene targeting of GFP to the PITX3 locus by transcription activator-like effector nuclease. The naivetropic iPSCs could be readily reverted to the primed state upon the withdrawal of DOX, 2iL, and the switch to primed-state hESC culture conditions. Midbrain DA neurons differentiated from the reverted iPSCs retained the original phenotypes caused by parkin mutations, attesting to the robustness of these phenotypes and the usefulness of genomic engineering in patient-specific naivetropic iPSCs for studying PD.

No MeSH data available.


Related in: MedlinePlus

Characterization of iPSCs reverted from naivetropic iPSCs. (A–G) Phase contrast image (A) and immunostaining of rP002 reverted iPSCs, which were converted from the naivetropic state back to the primed state, for ESC markers Nanog (B), OCT4 (C), SSEA3 (D), SSEA4 (E), TRA-1-81 (F), and TRA-1-60 (G). (H–J) Spontaneous differentiation of rP002 in vitro to cells expressing α-fetoprotein (AFP, endoderm) (H), β-tubulin (TuJ1, ectoderm) (I), or α-smooth muscle actin (SMA, mesoderm) (J). Scale bars, 100 μm
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f5: Characterization of iPSCs reverted from naivetropic iPSCs. (A–G) Phase contrast image (A) and immunostaining of rP002 reverted iPSCs, which were converted from the naivetropic state back to the primed state, for ESC markers Nanog (B), OCT4 (C), SSEA3 (D), SSEA4 (E), TRA-1-81 (F), and TRA-1-60 (G). (H–J) Spontaneous differentiation of rP002 in vitro to cells expressing α-fetoprotein (AFP, endoderm) (H), β-tubulin (TuJ1, ectoderm) (I), or α-smooth muscle actin (SMA, mesoderm) (J). Scale bars, 100 μm

Mentions: Previous studies on transgene-induced naivetropic pluripotency showed that maintenance of the naivetropic state is dependent on the transgenes [17]. We tested whether patient-specific naivetropic iPSCs can be reverted to their original primed state when DOX-mediated transgene expression was withdrawn. Several colonies of nP002 naivetropic iPSCs were manually picked and reseeded on MEF feeders in hES medium with bFGF (4 ng/mL). After 7–10 days, round and flat colonies with typical iPSC morphology emerged (Fig. 5A and Supplementary Fig. S5A, K, U). These iPSCs were designated as reverted iPSCs (riPSC) and the corresponding lines were named rC001, rC002, rP001, and rP002. Immunostaining of these riPSC lines showed that they expressed pluripotency markers, such as Nanog, Oct4, SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60 (Fig. 5B–G for rP002 and Supplementary Fig. S5 for the other three riPSC lines). Spontaneous differentiation assay mediated by EB showed that riPSCs could be differentiated to cells of all three germ layers (Fig. 5H–J for rP002 and Supplementary Fig. S5 for the other three lines). The riPSCs were cultured for over 15 passages in standard hES medium without any significant change in morphology or growth characteristics.


Generation of Naivetropic Induced Pluripotent Stem Cells from Parkinson's Disease Patients for High-Efficiency Genetic Manipulation and Disease Modeling.

Hu Z, Pu J, Jiang H, Zhong P, Qiu J, Li F, Wang X, Zhang B, Yan Z, Feng J - Stem Cells Dev. (2015)

Characterization of iPSCs reverted from naivetropic iPSCs. (A–G) Phase contrast image (A) and immunostaining of rP002 reverted iPSCs, which were converted from the naivetropic state back to the primed state, for ESC markers Nanog (B), OCT4 (C), SSEA3 (D), SSEA4 (E), TRA-1-81 (F), and TRA-1-60 (G). (H–J) Spontaneous differentiation of rP002 in vitro to cells expressing α-fetoprotein (AFP, endoderm) (H), β-tubulin (TuJ1, ectoderm) (I), or α-smooth muscle actin (SMA, mesoderm) (J). Scale bars, 100 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4620536&req=5

f5: Characterization of iPSCs reverted from naivetropic iPSCs. (A–G) Phase contrast image (A) and immunostaining of rP002 reverted iPSCs, which were converted from the naivetropic state back to the primed state, for ESC markers Nanog (B), OCT4 (C), SSEA3 (D), SSEA4 (E), TRA-1-81 (F), and TRA-1-60 (G). (H–J) Spontaneous differentiation of rP002 in vitro to cells expressing α-fetoprotein (AFP, endoderm) (H), β-tubulin (TuJ1, ectoderm) (I), or α-smooth muscle actin (SMA, mesoderm) (J). Scale bars, 100 μm
Mentions: Previous studies on transgene-induced naivetropic pluripotency showed that maintenance of the naivetropic state is dependent on the transgenes [17]. We tested whether patient-specific naivetropic iPSCs can be reverted to their original primed state when DOX-mediated transgene expression was withdrawn. Several colonies of nP002 naivetropic iPSCs were manually picked and reseeded on MEF feeders in hES medium with bFGF (4 ng/mL). After 7–10 days, round and flat colonies with typical iPSC morphology emerged (Fig. 5A and Supplementary Fig. S5A, K, U). These iPSCs were designated as reverted iPSCs (riPSC) and the corresponding lines were named rC001, rC002, rP001, and rP002. Immunostaining of these riPSC lines showed that they expressed pluripotency markers, such as Nanog, Oct4, SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60 (Fig. 5B–G for rP002 and Supplementary Fig. S5 for the other three riPSC lines). Spontaneous differentiation assay mediated by EB showed that riPSCs could be differentiated to cells of all three germ layers (Fig. 5H–J for rP002 and Supplementary Fig. S5 for the other three lines). The riPSCs were cultured for over 15 passages in standard hES medium without any significant change in morphology or growth characteristics.

Bottom Line: The high clonal efficiency and proliferation rate of naivetropic iPSCs enabled very efficient gene targeting of GFP to the PITX3 locus by transcription activator-like effector nuclease.The naivetropic iPSCs could be readily reverted to the primed state upon the withdrawal of DOX, 2iL, and the switch to primed-state hESC culture conditions.Midbrain DA neurons differentiated from the reverted iPSCs retained the original phenotypes caused by parkin mutations, attesting to the robustness of these phenotypes and the usefulness of genomic engineering in patient-specific naivetropic iPSCs for studying PD.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Physiology and Biophysics, State University of New York at Buffalo , Buffalo, New York.

ABSTRACT
The lack of robust Parkinson's disease (PD) phenotype in parkin knockout rodents and the identification of defective dopaminergic (DA) neurotransmission in midbrain DA neurons derived from induced pluripotent stem cells (iPSC) of PD patients with parkin mutations demonstrate the utility of patient-specific iPSCs as an effective system to model the unique vulnerabilities of midbrain DA neurons in PD. Significant efforts have been directed at developing efficient genomic engineering technologies in human iPSCs to study diseases such as PD. In the present study, we converted patient-specific iPSCs from the primed state to a naivetropic state by DOX-induced expression of transgenes (Oct4, Sox2, Klf4, c-Myc, and Nanog) and the use of 2iL (MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021, and human LIF). These patient-specific naivetropic iPSCs were pluripotent in terms of marker expression, spontaneous differentiation in vitro, and teratoma formation in vivo. They exhibited morphological, proliferative, and clonogenic characteristics very similar to naive mouse embryonic stem cells (ESC). The high clonal efficiency and proliferation rate of naivetropic iPSCs enabled very efficient gene targeting of GFP to the PITX3 locus by transcription activator-like effector nuclease. The naivetropic iPSCs could be readily reverted to the primed state upon the withdrawal of DOX, 2iL, and the switch to primed-state hESC culture conditions. Midbrain DA neurons differentiated from the reverted iPSCs retained the original phenotypes caused by parkin mutations, attesting to the robustness of these phenotypes and the usefulness of genomic engineering in patient-specific naivetropic iPSCs for studying PD.

No MeSH data available.


Related in: MedlinePlus