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The generation of induced pluripotent stem cells for macular degeneration as a drug screening platform: identification of curcumin as a protective agent for retinal pigment epithelial cells against oxidative stress.

Chang YC, Chang WC, Hung KH, Yang DM, Cheng YH, Liao YW, Woung LC, Tsai CY, Hsu CC, Lin TC, Liu JH, Chiou SH, Peng CH, Chen SJ - Front Aging Neurosci (2014)

Bottom Line: These patient-derived RPEs with the AMD-associated background (AMD-RPEs) exhibited reduced antioxidant ability, compared with normal RPE cells.Among several screened candidate drugs, curcumin caused most significant reduction of ROS in AMD-RPEs.Curcumin represented an ideal drug that can effectively restore the neuronal functions in AMD patient-derived RPE cells, rendering this drug an effective option for macular degeneration therapy and an agent against aging-associated oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan ; School of Medicine, Institute of Pharmacology, National Yang-Ming University Taipei, Taiwan ; Department of Ophthalmology, Taipei Veterans General Hospital Taipei, Taiwan.

ABSTRACT
Age-related macular degeneration (AMD) is one retinal aging process that may lead to irreversible vision loss in the elderly. Its pathogenesis remains unclear, but oxidative stress inducing retinal pigment epithelial (RPE) cells damage is perhaps responsible for the aging sequence of retina and may play an important role in macular degeneration. In this study, we have reprogrammed T cells from patients with dry type AMD into induced pluripotent stem cells (iPSCs) via integration-free episomal vectors and differentiated them into RPE cells that were used as an expandable platform for investigating pathogenesis of the AMD and in-vitro drug screening. These patient-derived RPEs with the AMD-associated background (AMD-RPEs) exhibited reduced antioxidant ability, compared with normal RPE cells. Among several screened candidate drugs, curcumin caused most significant reduction of ROS in AMD-RPEs. Pre-treatment of curcumin protected these AMD-RPEs from H2O2-induced cell death and also increased the cytoprotective effect against the oxidative stress of H2O2 through the reduction of ROS levels. In addition, curcumin with its versatile activities modulated the expression of many oxidative stress-regulating genes such as PDGF, VEGF, IGFBP-2, HO1, SOD2, and GPX1. Our findings indicated that the RPE cells derived from AMD patients have decreased antioxidative defense, making RPE cells more susceptible to oxidative damage and thereby leading to AMD formation. Curcumin represented an ideal drug that can effectively restore the neuronal functions in AMD patient-derived RPE cells, rendering this drug an effective option for macular degeneration therapy and an agent against aging-associated oxidative stress.

No MeSH data available.


Related in: MedlinePlus

Generation and characterization of iPSCs derived from patients with AMD and unaffected control. (A) Schematic diagram of the reprogramming with episomal plasmids via electroporation. (B) Phase-contrast photomicrograph and alkaline phosphatase activity of undifferentiated control iPSCs (Ctrl-iPSCs) and AMD iPSCs (AMD-iPSCs). Scale bar = 100 μm. (C) Immunofluorescence staining demonstrated the expression of pluripotency markers (OCT4, TRA-1-61, NANOG, and TRA-1-81) in undifferentiated iPSCs from patients and unaffected. Nuclei were counterstained with DAPI (blue). Scale bar = 30 μm. (D) The RT–PCR results indicated an ESC-like gene expression pattern in representative colonies of iPSCs. T cells were used as a negative control, and human ES H9 cells were used as a positive control.
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Figure 2: Generation and characterization of iPSCs derived from patients with AMD and unaffected control. (A) Schematic diagram of the reprogramming with episomal plasmids via electroporation. (B) Phase-contrast photomicrograph and alkaline phosphatase activity of undifferentiated control iPSCs (Ctrl-iPSCs) and AMD iPSCs (AMD-iPSCs). Scale bar = 100 μm. (C) Immunofluorescence staining demonstrated the expression of pluripotency markers (OCT4, TRA-1-61, NANOG, and TRA-1-81) in undifferentiated iPSCs from patients and unaffected. Nuclei were counterstained with DAPI (blue). Scale bar = 30 μm. (D) The RT–PCR results indicated an ESC-like gene expression pattern in representative colonies of iPSCs. T cells were used as a negative control, and human ES H9 cells were used as a positive control.

Mentions: AMD results from RPE dysfunction or loss associated with photoreceptor fallout, Bruch's membrane thickening, and choriocapillary hypoperfusion (Young, 1987). However, obtaining a number of suitable RPE cells for in vitro study is still a problem. Although fibroblasts from skin biopsy or other sources (such as dental pulp cells) were used in many studies for the generation of iPSCs, peripheral blood mononuclear cells (PBMCs) have been widely accepted as a more convenient and an almost unlimited resource for cell reprogramming (Staerk et al., 2010; Seki et al., 2012). In this study, we used EBNA1-based episomal vectors, a non-viral system that can reprogram somatic cells into iPSCs in both feeder-dependent and feeder-free conditions, to generated iPSCs from T cells via electroporation (Figure 2A). These integration-free iPSCs have the capability to be utilized for a broad range of applications, including pre-clinical research and human gene therapy, thus further delivering on the promise of iPSCs. These reprogrammed cells progressively formed colonies with increasing size during the reprogramming process. These colonies were stained positive for alkaline phosphate (ALP), and exhibited morphology indistinguishable from that of human ESCs (Figure 2B). As shown by immunofluorescence, selected clones exhibited the stamens signature and revealed the strong expression of Oct4, Nanog, Tra1-60, and Tra1-81 in the 20th-passage iPSCs (Figure 2C). RT-PCR also showed that both Ctrl- and AMD-iPSCs expressed various stemness genes, such as Oct4, Sox2, klf4, Nanog, REX, DPPA2, and GDF3, identical to those observed in H9 human ESC lines (Figure 2D). Using differentiation protocols for tridermal lineages, both the 20th-passage Ctrl-iPSC-derived embryoid bodies (EBs) and AMD-iPSC-derived EBs could be induced to differentiate into neuron-like cells, smooth muscle cells, and hepatocyte-like cells (ectoderm, mesoderm, and endoderm) (data not shown). These results demonstrated that AMD patient-derived iPSCs exhibited pluripotent properties and capabilities of multi-lineage differentiation. We further employed the culture protocol described by Osakada et al., with brief modifications, for RPE differentiation (Osakada et al., 2009). We used patient-specific iPSC-derived RPE cells to model the pathophysiological features of macular degeneration (Figure 3A). Using this protocol, both Ctrl- and AMD-iPSCs underwent RPE-specific morphological changes and pigmentation and differentiated into RPE-like cells (Figure 3B). RT-PCR confirmed the expression of several RPE-specific markers, including RLBP1, RPE65, MITF, and PAX6, in both Ctrl-RPEs and BD-RPEs (Figure 3C). Immunofluorescence assays indicated that AMD-RPEs exhibited significantly lower ZO-1 and RPE65 expression than Ctrl-RPEs, suggesting tight-junction in AMD-RPEs were defective (Figure 3D). These data demonstrated that both Ctrl- and AMD-iPSCs were competent to differentiate into pigment cells with typical RPE characteristics.


The generation of induced pluripotent stem cells for macular degeneration as a drug screening platform: identification of curcumin as a protective agent for retinal pigment epithelial cells against oxidative stress.

Chang YC, Chang WC, Hung KH, Yang DM, Cheng YH, Liao YW, Woung LC, Tsai CY, Hsu CC, Lin TC, Liu JH, Chiou SH, Peng CH, Chen SJ - Front Aging Neurosci (2014)

Generation and characterization of iPSCs derived from patients with AMD and unaffected control. (A) Schematic diagram of the reprogramming with episomal plasmids via electroporation. (B) Phase-contrast photomicrograph and alkaline phosphatase activity of undifferentiated control iPSCs (Ctrl-iPSCs) and AMD iPSCs (AMD-iPSCs). Scale bar = 100 μm. (C) Immunofluorescence staining demonstrated the expression of pluripotency markers (OCT4, TRA-1-61, NANOG, and TRA-1-81) in undifferentiated iPSCs from patients and unaffected. Nuclei were counterstained with DAPI (blue). Scale bar = 30 μm. (D) The RT–PCR results indicated an ESC-like gene expression pattern in representative colonies of iPSCs. T cells were used as a negative control, and human ES H9 cells were used as a positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4117985&req=5

Figure 2: Generation and characterization of iPSCs derived from patients with AMD and unaffected control. (A) Schematic diagram of the reprogramming with episomal plasmids via electroporation. (B) Phase-contrast photomicrograph and alkaline phosphatase activity of undifferentiated control iPSCs (Ctrl-iPSCs) and AMD iPSCs (AMD-iPSCs). Scale bar = 100 μm. (C) Immunofluorescence staining demonstrated the expression of pluripotency markers (OCT4, TRA-1-61, NANOG, and TRA-1-81) in undifferentiated iPSCs from patients and unaffected. Nuclei were counterstained with DAPI (blue). Scale bar = 30 μm. (D) The RT–PCR results indicated an ESC-like gene expression pattern in representative colonies of iPSCs. T cells were used as a negative control, and human ES H9 cells were used as a positive control.
Mentions: AMD results from RPE dysfunction or loss associated with photoreceptor fallout, Bruch's membrane thickening, and choriocapillary hypoperfusion (Young, 1987). However, obtaining a number of suitable RPE cells for in vitro study is still a problem. Although fibroblasts from skin biopsy or other sources (such as dental pulp cells) were used in many studies for the generation of iPSCs, peripheral blood mononuclear cells (PBMCs) have been widely accepted as a more convenient and an almost unlimited resource for cell reprogramming (Staerk et al., 2010; Seki et al., 2012). In this study, we used EBNA1-based episomal vectors, a non-viral system that can reprogram somatic cells into iPSCs in both feeder-dependent and feeder-free conditions, to generated iPSCs from T cells via electroporation (Figure 2A). These integration-free iPSCs have the capability to be utilized for a broad range of applications, including pre-clinical research and human gene therapy, thus further delivering on the promise of iPSCs. These reprogrammed cells progressively formed colonies with increasing size during the reprogramming process. These colonies were stained positive for alkaline phosphate (ALP), and exhibited morphology indistinguishable from that of human ESCs (Figure 2B). As shown by immunofluorescence, selected clones exhibited the stamens signature and revealed the strong expression of Oct4, Nanog, Tra1-60, and Tra1-81 in the 20th-passage iPSCs (Figure 2C). RT-PCR also showed that both Ctrl- and AMD-iPSCs expressed various stemness genes, such as Oct4, Sox2, klf4, Nanog, REX, DPPA2, and GDF3, identical to those observed in H9 human ESC lines (Figure 2D). Using differentiation protocols for tridermal lineages, both the 20th-passage Ctrl-iPSC-derived embryoid bodies (EBs) and AMD-iPSC-derived EBs could be induced to differentiate into neuron-like cells, smooth muscle cells, and hepatocyte-like cells (ectoderm, mesoderm, and endoderm) (data not shown). These results demonstrated that AMD patient-derived iPSCs exhibited pluripotent properties and capabilities of multi-lineage differentiation. We further employed the culture protocol described by Osakada et al., with brief modifications, for RPE differentiation (Osakada et al., 2009). We used patient-specific iPSC-derived RPE cells to model the pathophysiological features of macular degeneration (Figure 3A). Using this protocol, both Ctrl- and AMD-iPSCs underwent RPE-specific morphological changes and pigmentation and differentiated into RPE-like cells (Figure 3B). RT-PCR confirmed the expression of several RPE-specific markers, including RLBP1, RPE65, MITF, and PAX6, in both Ctrl-RPEs and BD-RPEs (Figure 3C). Immunofluorescence assays indicated that AMD-RPEs exhibited significantly lower ZO-1 and RPE65 expression than Ctrl-RPEs, suggesting tight-junction in AMD-RPEs were defective (Figure 3D). These data demonstrated that both Ctrl- and AMD-iPSCs were competent to differentiate into pigment cells with typical RPE characteristics.

Bottom Line: These patient-derived RPEs with the AMD-associated background (AMD-RPEs) exhibited reduced antioxidant ability, compared with normal RPE cells.Among several screened candidate drugs, curcumin caused most significant reduction of ROS in AMD-RPEs.Curcumin represented an ideal drug that can effectively restore the neuronal functions in AMD patient-derived RPE cells, rendering this drug an effective option for macular degeneration therapy and an agent against aging-associated oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan ; School of Medicine, Institute of Pharmacology, National Yang-Ming University Taipei, Taiwan ; Department of Ophthalmology, Taipei Veterans General Hospital Taipei, Taiwan.

ABSTRACT
Age-related macular degeneration (AMD) is one retinal aging process that may lead to irreversible vision loss in the elderly. Its pathogenesis remains unclear, but oxidative stress inducing retinal pigment epithelial (RPE) cells damage is perhaps responsible for the aging sequence of retina and may play an important role in macular degeneration. In this study, we have reprogrammed T cells from patients with dry type AMD into induced pluripotent stem cells (iPSCs) via integration-free episomal vectors and differentiated them into RPE cells that were used as an expandable platform for investigating pathogenesis of the AMD and in-vitro drug screening. These patient-derived RPEs with the AMD-associated background (AMD-RPEs) exhibited reduced antioxidant ability, compared with normal RPE cells. Among several screened candidate drugs, curcumin caused most significant reduction of ROS in AMD-RPEs. Pre-treatment of curcumin protected these AMD-RPEs from H2O2-induced cell death and also increased the cytoprotective effect against the oxidative stress of H2O2 through the reduction of ROS levels. In addition, curcumin with its versatile activities modulated the expression of many oxidative stress-regulating genes such as PDGF, VEGF, IGFBP-2, HO1, SOD2, and GPX1. Our findings indicated that the RPE cells derived from AMD patients have decreased antioxidative defense, making RPE cells more susceptible to oxidative damage and thereby leading to AMD formation. Curcumin represented an ideal drug that can effectively restore the neuronal functions in AMD patient-derived RPE cells, rendering this drug an effective option for macular degeneration therapy and an agent against aging-associated oxidative stress.

No MeSH data available.


Related in: MedlinePlus