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Efficient generation of lens progenitor cells from cataract patient-specific induced pluripotent stem cells.

Qiu X, Yang J, Liu T, Jiang Y, Le Q, Lu Y - PLoS ONE (2012)

Bottom Line: The resulting HLEC-derived iPS (HLE-iPS) colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers.In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT) markers compared with human embryonic stem cells (hESCs) and fibroblast-derived iPSCs.These patient-derived pluripotent cells provide a valuable model for studying the developmental and molecular biological mechanisms that underlie cell determination in lens development and cataract pathophysiology.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People's Republic of China.

ABSTRACT
The development of a technique to induce the transformation of somatic cells to a pluripotent state via the ectopic expression of defined transcription factors was a transformational event in the field of regenerative medicine. The development of this technique also impacted ophthalmology, as patient-specific induced pluripotent stemcells (iPSCs) may be useful resources for some ophthalmological diseases. The lens is a key refractive element in the eye that focuses images of the visual world onto the retina. To establish a new model for drug screening to treat lens diseases and investigating lens aging and development, we examined whether human lens epithelial cells (HLECs) could be induced into iPSCs and if lens-specific differentiation of these cells could be achieved under defined chemical conditions. We first efficiently reprogrammed HLECs from age-related cataract patients to iPSCs with OCT-4, SOX-2, and KLF-4. The resulting HLEC-derived iPS (HLE-iPS) colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers. Next, we performed a 3-step induction procedure: HLE-iPS cells were differentiated into large numbers of lens progenitor-like cells with defined factors (Noggin, BMP and FGF2), and we determined that these cells expressed lens-specific markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP). In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT) markers compared with human embryonic stem cells (hESCs) and fibroblast-derived iPSCs. Our study describes a highly efficient procedure for generating lens progenitor cells from cataract patient HLEC-derived iPSCs. These patient-derived pluripotent cells provide a valuable model for studying the developmental and molecular biological mechanisms that underlie cell determination in lens development and cataract pathophysiology.

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The derivation of induced pluripotent cells from HLECs.HLECs were infected with lentiviruses carrying DNA constructs encoding KLF-4, OCT-4 and SOX-2 to generate induced pluripotent stem cells (HLEC-iPS). (A) A schematic representation of the experimental strategy used to reprogram HLECs. (B) The infected HLECs were plated onto mouse embryonic fibroblasts (MEFs). Colonies with an ES cell-like morphology (C), which appeared as early as 12 days following infection, and (D) H9 ES cell lines. (E) HLEC-iPSCs express pluripotency markers alkaline phosphatase (AP). (F) HLECs can be induced to iPSCs with high efficiency compared with human fibroblast cells: HLEC and human fibroblast cells were collected from three age-related cataract patients. After the iPS factors had been added for 14 days, AP staining was performed, revealing approximately 25 AP-positive clones from 1×106 HLEC cells and approximately 15 clones formed from 1×106 human fibroblast cells.
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pone-0032612-g001: The derivation of induced pluripotent cells from HLECs.HLECs were infected with lentiviruses carrying DNA constructs encoding KLF-4, OCT-4 and SOX-2 to generate induced pluripotent stem cells (HLEC-iPS). (A) A schematic representation of the experimental strategy used to reprogram HLECs. (B) The infected HLECs were plated onto mouse embryonic fibroblasts (MEFs). Colonies with an ES cell-like morphology (C), which appeared as early as 12 days following infection, and (D) H9 ES cell lines. (E) HLEC-iPSCs express pluripotency markers alkaline phosphatase (AP). (F) HLECs can be induced to iPSCs with high efficiency compared with human fibroblast cells: HLEC and human fibroblast cells were collected from three age-related cataract patients. After the iPS factors had been added for 14 days, AP staining was performed, revealing approximately 25 AP-positive clones from 1×106 HLEC cells and approximately 15 clones formed from 1×106 human fibroblast cells.

Mentions: To identify a strategy for generating iPSCs from HLECs, the cultured HLECs from the cataract patient were infected with lentiviruses carrying DNA constructs encoding KLF-4, OCT-4 and SOX-2 to generate induced pluripotent stem cells (HLEC-iPSCs). This was a 56-year-old male patient diagnosed as age-related cataract and there is no role of traumatic, metabolic or genetic factors influencing the generation and differentiation of this patient's iPSCs. A schematic representation of the experimental strategy used to reprogram the HLECs is shown in Fig. 1A. The infected HLECs were plated onto MEFs on the fifth day following infection (Fig. 1B). Colonies with an ES cell-like morphology formed 12 days following infection (Fig. 1C). The human H9 ES cell line was used as a positive control for pluripotent stem cells (Fig. 1D). These HLEC-iPSCs were labeled with the pluripotency marker alkaline phosphatase (AP) (Fig. 1E). To compare iPS clone formation efficiency, HLEC and human fibroblast cells were collected from three age-related cataract patients. After the iPS factors had been added for 14 days, AP staining was performed, revealing approximately 25 AP-positive clones from 1×106 HLEC cells and approximately 15 clones formed from 1×106 human fibroblast cells (Fig. 1F).


Efficient generation of lens progenitor cells from cataract patient-specific induced pluripotent stem cells.

Qiu X, Yang J, Liu T, Jiang Y, Le Q, Lu Y - PLoS ONE (2012)

The derivation of induced pluripotent cells from HLECs.HLECs were infected with lentiviruses carrying DNA constructs encoding KLF-4, OCT-4 and SOX-2 to generate induced pluripotent stem cells (HLEC-iPS). (A) A schematic representation of the experimental strategy used to reprogram HLECs. (B) The infected HLECs were plated onto mouse embryonic fibroblasts (MEFs). Colonies with an ES cell-like morphology (C), which appeared as early as 12 days following infection, and (D) H9 ES cell lines. (E) HLEC-iPSCs express pluripotency markers alkaline phosphatase (AP). (F) HLECs can be induced to iPSCs with high efficiency compared with human fibroblast cells: HLEC and human fibroblast cells were collected from three age-related cataract patients. After the iPS factors had been added for 14 days, AP staining was performed, revealing approximately 25 AP-positive clones from 1×106 HLEC cells and approximately 15 clones formed from 1×106 human fibroblast cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293838&req=5

pone-0032612-g001: The derivation of induced pluripotent cells from HLECs.HLECs were infected with lentiviruses carrying DNA constructs encoding KLF-4, OCT-4 and SOX-2 to generate induced pluripotent stem cells (HLEC-iPS). (A) A schematic representation of the experimental strategy used to reprogram HLECs. (B) The infected HLECs were plated onto mouse embryonic fibroblasts (MEFs). Colonies with an ES cell-like morphology (C), which appeared as early as 12 days following infection, and (D) H9 ES cell lines. (E) HLEC-iPSCs express pluripotency markers alkaline phosphatase (AP). (F) HLECs can be induced to iPSCs with high efficiency compared with human fibroblast cells: HLEC and human fibroblast cells were collected from three age-related cataract patients. After the iPS factors had been added for 14 days, AP staining was performed, revealing approximately 25 AP-positive clones from 1×106 HLEC cells and approximately 15 clones formed from 1×106 human fibroblast cells.
Mentions: To identify a strategy for generating iPSCs from HLECs, the cultured HLECs from the cataract patient were infected with lentiviruses carrying DNA constructs encoding KLF-4, OCT-4 and SOX-2 to generate induced pluripotent stem cells (HLEC-iPSCs). This was a 56-year-old male patient diagnosed as age-related cataract and there is no role of traumatic, metabolic or genetic factors influencing the generation and differentiation of this patient's iPSCs. A schematic representation of the experimental strategy used to reprogram the HLECs is shown in Fig. 1A. The infected HLECs were plated onto MEFs on the fifth day following infection (Fig. 1B). Colonies with an ES cell-like morphology formed 12 days following infection (Fig. 1C). The human H9 ES cell line was used as a positive control for pluripotent stem cells (Fig. 1D). These HLEC-iPSCs were labeled with the pluripotency marker alkaline phosphatase (AP) (Fig. 1E). To compare iPS clone formation efficiency, HLEC and human fibroblast cells were collected from three age-related cataract patients. After the iPS factors had been added for 14 days, AP staining was performed, revealing approximately 25 AP-positive clones from 1×106 HLEC cells and approximately 15 clones formed from 1×106 human fibroblast cells (Fig. 1F).

Bottom Line: The resulting HLEC-derived iPS (HLE-iPS) colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers.In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT) markers compared with human embryonic stem cells (hESCs) and fibroblast-derived iPSCs.These patient-derived pluripotent cells provide a valuable model for studying the developmental and molecular biological mechanisms that underlie cell determination in lens development and cataract pathophysiology.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People's Republic of China.

ABSTRACT
The development of a technique to induce the transformation of somatic cells to a pluripotent state via the ectopic expression of defined transcription factors was a transformational event in the field of regenerative medicine. The development of this technique also impacted ophthalmology, as patient-specific induced pluripotent stemcells (iPSCs) may be useful resources for some ophthalmological diseases. The lens is a key refractive element in the eye that focuses images of the visual world onto the retina. To establish a new model for drug screening to treat lens diseases and investigating lens aging and development, we examined whether human lens epithelial cells (HLECs) could be induced into iPSCs and if lens-specific differentiation of these cells could be achieved under defined chemical conditions. We first efficiently reprogrammed HLECs from age-related cataract patients to iPSCs with OCT-4, SOX-2, and KLF-4. The resulting HLEC-derived iPS (HLE-iPS) colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers. Next, we performed a 3-step induction procedure: HLE-iPS cells were differentiated into large numbers of lens progenitor-like cells with defined factors (Noggin, BMP and FGF2), and we determined that these cells expressed lens-specific markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP). In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT) markers compared with human embryonic stem cells (hESCs) and fibroblast-derived iPSCs. Our study describes a highly efficient procedure for generating lens progenitor cells from cataract patient HLEC-derived iPSCs. These patient-derived pluripotent cells provide a valuable model for studying the developmental and molecular biological mechanisms that underlie cell determination in lens development and cataract pathophysiology.

Show MeSH
Related in: MedlinePlus