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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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HLEC-iPS can be highly efficiently induced to express lens progenitor cell markers with defined chemical factors.Real-time RT-PCR analysis of 7 lens differentiation markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP) in iPSCs and ESCs from d 0 to 30 following the initial plating. The results were calculated relative to the average Ct value of GAPDH. For details on the 3-step induction procedure, see Materials and Methods.
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pone-0032612-g004: HLEC-iPS can be highly efficiently induced to express lens progenitor cell markers with defined chemical factors.Real-time RT-PCR analysis of 7 lens differentiation markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP) in iPSCs and ESCs from d 0 to 30 following the initial plating. The results were calculated relative to the average Ct value of GAPDH. For details on the 3-step induction procedure, see Materials and Methods.

Mentions: Our next goal was to determine whether these HLEC-derived iPSCs could be further differentiated into lens progenitor cells. To achieve lens-specific differentiation, we applied a 3-step induction procedure, as reported by Yang et al [6]. We also analyzed 7 lens progenitor and differentiation markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1 and MIP) in HLEC-iPSCs and hESCs from d 0 to d 30 (Fig. 4). The results indicated that several markers (PAX6, BFSP1 and MIP) were upregulated in HLEC-iPSCs more rapidly than in hESCs. However, the expression of the hESCs marker SOX2 decreased rapidly in the HLEC-iPSCs. The immunocytochemical analyses confirmed that the HLEC-iPSCs have the capacity to differentiate into lens progenitor and primary lens fibroblast cells using a 3-step induction procedure. This finding was determined by the expression of PAX6 (Fig. 5 A, B) α- crystallin (Fig. 5 C, D) and β-crystallin proteins (Fig. 5 E, F). By using western blots to detect the expression levels of these markers, we found comparable levels of these proteins in human lens and iPS-induced cells (Fig. 5 G).From these data, we conclude that this 3-step induction procedure is highly efficient for generating lens cells from patient-specific iPSCs.


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)

HLEC-iPS can be highly efficiently induced to express lens progenitor cell markers with defined chemical factors.Real-time RT-PCR analysis of 7 lens differentiation markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP) in iPSCs and ESCs from d 0 to 30 following the initial plating. The results were calculated relative to the average Ct value of GAPDH. For details on the 3-step induction procedure, see Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032612-g004: HLEC-iPS can be highly efficiently induced to express lens progenitor cell markers with defined chemical factors.Real-time RT-PCR analysis of 7 lens differentiation markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP) in iPSCs and ESCs from d 0 to 30 following the initial plating. The results were calculated relative to the average Ct value of GAPDH. For details on the 3-step induction procedure, see Materials and Methods.
Mentions: Our next goal was to determine whether these HLEC-derived iPSCs could be further differentiated into lens progenitor cells. To achieve lens-specific differentiation, we applied a 3-step induction procedure, as reported by Yang et al [6]. We also analyzed 7 lens progenitor and differentiation markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1 and MIP) in HLEC-iPSCs and hESCs from d 0 to d 30 (Fig. 4). The results indicated that several markers (PAX6, BFSP1 and MIP) were upregulated in HLEC-iPSCs more rapidly than in hESCs. However, the expression of the hESCs marker SOX2 decreased rapidly in the HLEC-iPSCs. The immunocytochemical analyses confirmed that the HLEC-iPSCs have the capacity to differentiate into lens progenitor and primary lens fibroblast cells using a 3-step induction procedure. This finding was determined by the expression of PAX6 (Fig. 5 A, B) α- crystallin (Fig. 5 C, D) and β-crystallin proteins (Fig. 5 E, F). By using western blots to detect the expression levels of these markers, we found comparable levels of these proteins in human lens and iPS-induced cells (Fig. 5 G).From these data, we conclude that this 3-step induction procedure is highly efficient for generating lens cells from patient-specific iPSCs.

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